U.S. patent application number 14/427593 was filed with the patent office on 2015-09-03 for methods for multiplex analytical measurements in single cells of solid tissues.
The applicant listed for this patent is PRESIDENT AND FELLOWS OF HARVARD COLLEGE. Invention is credited to Alexei Finski, Gavin MacBeath.
Application Number | 20150246335 14/427593 |
Document ID | / |
Family ID | 50278675 |
Filed Date | 2015-09-03 |
United States Patent
Application |
20150246335 |
Kind Code |
A1 |
Finski; Alexei ; et
al. |
September 3, 2015 |
METHODS FOR MULTIPLEX ANALYTICAL MEASUREMENTS IN SINGLE CELLS OF
SOLID TISSUES
Abstract
The invention provides a method for the isolation of a single
cell embedded in a tissue while preserving the state of molecules
of the cell, and therefore allows for transformation of a single
target cell in live tissue into a format that can be evaluated
using analytical methods.
Inventors: |
Finski; Alexei; (Somerville,
MA) ; MacBeath; Gavin; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PRESIDENT AND FELLOWS OF HARVARD COLLEGE |
Cambridge |
MA |
US |
|
|
Family ID: |
50278675 |
Appl. No.: |
14/427593 |
Filed: |
September 12, 2013 |
PCT Filed: |
September 12, 2013 |
PCT NO: |
PCT/US2013/059485 |
371 Date: |
March 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61700517 |
Sep 13, 2012 |
|
|
|
61729127 |
Nov 21, 2012 |
|
|
|
Current U.S.
Class: |
506/9 ; 506/14;
506/26; 506/40 |
Current CPC
Class: |
B01J 2219/00702
20130101; B01J 2219/00691 20130101; B01J 2219/00626 20130101; G01N
33/6848 20130101; B01J 19/0046 20130101; B01J 2219/00596 20130101;
B01J 2219/0061 20130101; B01J 2219/00725 20130101; B01J 2219/00695
20130101; G01N 33/6803 20130101; G01N 33/5091 20130101 |
International
Class: |
B01J 19/00 20060101
B01J019/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
RC1HG005354 awarded by National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of lysing a single cell present in a tissue, the method
comprising: a) identifying a cell from a tissue; b) contacting a
detergent-containing lysis buffer with the intracellular space of
the identified cell; c) allowing the lysis buffer to spread within
the intracellular space of the identified cell for a period of
time, wherein the cell is lysed from the inside of the cell; and d)
collecting the lysate.
2-12. (canceled)
13. The method of claim 1, wherein the method occurs in the absence
of tissue fixation and tissue disaggregation.
14. The method of claim 1, wherein the isolated cell is in an
organotypic culture.
15. The method of claim 1, wherein the lysate is collected by
suctioning the lysate using a suction channel.
16. The method of claim 15, wherein the suction channel is a bent
suction micropipette.
17. The method of claim 1, wherein the collected lysate is further
applied to a nitrocellulose pad.
18. The method of claim 17, wherein a standard is also applied to
the nitrocellulose pad.
19. The method of claim 1, wherein the lysate is evaluated using an
analytical method.
20. The method of claim 19, wherein the analytical method is
selected from the group consisting of mass spectrometry, protein
microarray, RT-qPCR, RNA-Seq, and MALDI-MS.
21. The method of claim 1, wherein the cell is part of a live solid
tissue.
22. The method of claim 1, wherein the detergent is sodium dodecyl
sulfate.
23. A method of analyzing a cell present in a tissue, the method
comprising: a) identifying a cell from a tissue; b) contacting a
detergent-containing lysis buffer to the intracellular space of the
identified cell; c) allowing the lysis buffer to spread within the
intracellular space of the identified cell for a period of time,
wherein the cell is lysed from the inside of the cell; d)
collecting the lysate; e) applying the collected lysate to a solid
support; and f) evaluating the collected lysate using an analytical
method.
24. The method of claim 1, wherein the outer cell membrane of the
identified cell is intact or mostly intact prior to lysate
collection.
25. The method of claim 23, wherein the outer cell membrane of the
identified cell is intact or mostly intact prior to lysate
collection.
26. A composition comprising a tissue, a cell present in the tissue
having an intracellular space, and a lysis buffer present in the
tissue, wherein all or most of the lysis buffer is confined within
the intracellular space.
27. The composition of claim 26, wherein the lysis buffer comprises
a detergent.
28. The composition of claim 27, wherein the detergent is sodium
dodecyl sulfate.
29. A composition comprising a tissue, at least two cells present
in the tissue, and a lysis buffer present in the tissue, wherein
each of the at least two cells has an intracellular space, and
wherein all or most of the lysis buffer is confined within the
intracellular spaces of the at least two cells.
30. The composition of claim 29, wherein the lysis buffer comprises
a detergent.
31. The composition of claim 30, wherein the detergent is sodium
dodecyl sulfate.
32. A robotic system comprising (a) a first robotic arm comprising
a first microcapillary configured to contact a lysis buffer with an
intracellular space of a cell through a first aperture, thereby
producing a lysate, (b) a second robotic arm comprising a second
microcapillary configured to collect the lysate through a second
aperture, (c) and a computer coupled to the first and second
robotic arms and programmed to control the first and second robotic
arms.
33. The robotic system of claim 32, wherein the second aperture is
larger than the first aperture.
34. The robotic system of claim 32, wherein the first aperture is
about 0.01 .mu.m to 1 .mu.m.
35. The robotic system of claim 32, wherein the second aperture is
about 1 .mu.m to 10 .mu.m.
36. A system comprising at least two robotic systems of claim 32,
wherein each of the at least two robotic systems is centrally
controlled from a single user interface.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/700,517, filed Sep. 13, 2012, and to U.S.
Provisional Application Ser. No. 61/729,127, filed Nov. 21, 2012,
the contents of each of which are incorporated by reference herein
in their entireties.
BACKGROUND OF THE INVENTION
[0003] For the past 50 years, biological research has primarily
been performed on cell lines and dissociated cell cultures, because
these experimental systems are easy to handle in laboratory
settings and provide large amounts of material for study. This
trend has led to the development of numerous analytical methods
that require large amounts of biological material and are readily
applicable to cell lines and dissociated cell cultures. For
example, Western blot, mass spectrometry, and lysate microarrays
all require relatively large amounts of biological material in
order to take full advantage of the analytical power of these
methods. Large amounts of biological material are usually obtained
by simultaneously lysing a large number of dissociated cells in a
culture dish. As a consequence of this continued trend, the
development of methods for sampling and analyzing single cells in
solid tissues has lagged far behind.
[0004] One way to potentially access the power of current
analytical methods and apply them to single cells, is to reduce the
solid-tissue sample to a dissociated culture or a cell suspension
by tissue disaggregation. Tissue disaggregation can be achieved by
applying collagenase, tripsin, pepsin, papain, elastase and/or
pronase to the solid tissue sample for hours in solution, sometimes
followed by the trituration of the disaggregated tissue sample
(Waymouth, 1974 In Vitro. 10: 97-111; Engelholm et al., 1985 S.A.,
Spang-Thomsen, M., Brunner, N., Nohr, I. and Vindelov, L.L. (1985)
Br J Cancer. 51(1): 93-98; Pallavicini, 1987 Techniques in cell
cycle analysis. 139-162). In this way, human solid tumors can be
reduced to cell suspensions for analysis by flow cytometry (Dalerba
et al., 2011, Nat. Biotechnol. 29:1120-1127). Rodent brain tissue
can be reduced to dissociated neuronal cultures, from which single
neurons can be sampled for RT-qPCR analysis (Morris et al., 2011,
J. Vis. Exp. 50: pii: 2634. doi: 10.3791/2634). However, the
information about the location of cells is lost after tissue
disaggregation. Also, disaggregation of solid tissue might not
disperse all cells of the tissue sample. Disaggregation of solid
tissue likely kills many native cells, and likely selects certain
cell populations over the others. The cell yields of tissue
disaggregation vary across tissue types. 1 g of tissue contains
approx. 1*10.sup.9 cells, whereas the typical yields of tissue
disaggregation procedures are below 1*10.sup.8 cells/g
(Pallavicini, 1987 Techniques in cell cycle analysis. 139-162).
Moreover, the cells that survive tissue disaggregation lose their
cell-type-specific biochemistry and functionality due to the lack
of the extracellular matrix and due to the changed cellular
environment in the culture dish or in the cell suspension. For
example, the cell division rates in cancerous tissues and in 3D
models are different from the cell division rates observed in 2D
cell lines (Fischbach et al., 2007 Nat Methods. 4(10): 855-860).
The malignancy of tumors formed by cells cultured in 2D is lower
than the malignancy of tumors formed by cells cultured in 3D
(Fischbach et al., 2007 Nat Methods 4(10): 855-860). It is also
well known that the extracellular matrix of solid tissue plays a
critical role in cancer (Bissell et al., 2001 Nat Rev Cancer
1(1):46-54; Hanahan et al., 2000 Cell. 100(1): 57-70). Therefore,
dissociated cell cultures and cell suspensions are not equivalent
to the original solid tissue.
[0005] Most current solid-tissue study methods require fixation.
Both laser-capture microdissection and all immunolabeling-based
methods (immunofluorescence, FACS, FISH etc.) require fixation
(Gutstein et al., 2007, Expert Rev. Proteomics 4:627-637; Espina et
al., 2006, Nat. Protoc. 1:586-603; Mouledous et al., 2002, J.
Biomol. Tech. 13:258-264; Brandtzaeg and Rognum, 1984, Histochem.
Cell Biol. 81:213-219; Micheva and Smith, 2007, Neuron 55:25-36).
The latter can provide useful information about the spatial
distribution of substrates across the tissue structure and even
within cells. The question however is not what can be done with
fixed tissue but whether fixed tissue represents the original
pre-fixed tissue. If fixed tissue does not represent the original
tissue sample, then any study that is based on fixed tissue is not
informative. Fixation processes were first documented more than 100
years ago (Fish, 1896, Transactions of the American Microscopical
Society, 17:319-330). The process of aldehyde- and alcohol-based
fixation is not well understood but is known to undermine the
molecular preservation of the original sample, thereby obfuscating
the true native differences between single cells (Schnell et al.,
2012, Nat. Meth. 9:152-158; Mouledous et al., 2002, J. Biomol.
Techniques 13:258-264; Melan and Sluder, 1992, J. Cell Sci.
101:731-743; Holtfreter and Cohen, 1990, Cytometry 11:676-685;
Tanaka et al., 2010, Nat. Methods 7:865-866; Collaud et al., 2010,
J. Biomol. Tech. 21:25-28). There exists no universal fixation
protocol and each specific fixation protocol is exclusively tuned
to certain cell types, certain molecular classes, and certain
molecules within a molecular class (Schnell et al., 2012, Nat.
Meth. 9:152-158; Mouledous et al., 2002, J. Biomol. Techniques
13:258-264; Melan and Sluder, 1992, J. Cell Sci. 101:731-743;
Holtfreter and Cohen, 1990, Cytometry 11:676-685).
[0006] In order to study the effects of common fixation and
permeabilization protocols on molecular preservation of biological
samples, Schnell and colleagues expressed cytosol-soluble GFP in
293T and MDCK cells (Schnell et al., 2012, Nat. Meth. 9:152-158).
As expected, aldehyde-based fixation led to protein cross-linking
and to antigen masking. A large number of GFP molecules in the
GFP-expressing cells could not be reached by the applied specific
GFP antibodies, even after the extensive permeabilization of these
GFP-expressing cells. The same aldehyde-based fixation protocol
also led to the spatial redistribution of GFP proteins in fixed
MDCK cells, as compared to the same set of MDCK cells imaged before
applying aldehyde-based fixation. In contrast to MDCK cells, no
spatial redistribution of GFP was observed after fixing 293T cells
with the same aldehyde-based fixation protocol. These observations
demonstrate that the effects of aldehyde-based fixation on
molecular preservation are cell-type dependent. The
permeabilization process that is required to access the
intracellular proteins in each aldehyde-fixed single cell also led
to the extensive extraction of GFP from all fixed single cells
(Schnell et al., 2012, Nat. Meth. 9:152-158). Similarly,
alcohol-based fixation extracted most GFP proteins from all single
cells, as confirmed by fluorescence and electron microscopy
(Schnell et al., 2012, Nat. Meth. 9:152-158). Importantly, in the
study by Schnell et al., the true GFP quantity differences between
single cells in a given set of single cells could not be reproduced
by antibody staining of the same set of single cells after fixation
and permeabilization, although the applied GFP antibody was
specific and correctly detected GFP, when GFP was targeted to the
endoplasmatic reticulum.
[0007] In another rigorous study by Melan and Sluder, the authors
labeled several proteins of different size and charge with
fluorescein-5-isothiocyanate (FITC) and then loaded these labeled
proteins into HeLa, 3T3, PtK1 and CHO cells (Melan and Sluder,
1992, J. Cell Sci. 101:731-743). They observed that the extent of
protein extraction, caused by aldehyde-based fixation and
permeabilization, depended both on the particular protein species
and on the particular cell type (Melan and Sluder, 1992, J. Cell
Sci. 101:731-743). These observations prove that aldehyde- and
alcohol-based fixation and permeabilization decrease the analytical
availability of native molecules in an unpredictable cell-type and
molecule-dependent manner. The results of additional studies
examining the effects of fixation and permeabilization on molecular
preservation demonstrate that aldehyde- and alcohol-based fixation
and permeabilization undermine the molecular preservation of the
original sample in an unpredictable cell-type- and
molecule-dependent manner (Schnell et al., 2012, Nat. Meth.
9:152-158; Mouledous et al., 2002, J. Biomol. Techniques
13:258-264; Melan and Sluder, 1992, J. Cell Sci. 101:731-743;
Holtfreter and Cohen, 1990, Cytometry 11:676-685).
[0008] The second major limitation of all fixation-based methods is
the difficulty and often the inability of constructing standard
curves. A standard curve maps recorded signals to quantities and
can be constructed by a concurrent titration series. A standard
curve is the basis for any analytical measurement in any
discipline. Different affinity-based probes, such as antibodies,
usually have different dissociation constants (KD) and thus also
have different slopes of their respective standard curves. A large
signal difference is meaningless without knowledge of the
corresponding standard curve, as it can be the result of a small
difference in quantity or the result of a large difference in
quantity depending on the slope of the underlying standard curve
(FIG. 1). Standard curves also enable absolute measurements, as
signals can be mapped to the corresponding absolute counts of the
targeted molecules, as well as the correction of non-linear
behavior of affinity-based probes at low substrate concentrations
in single cells. Standard curves are necessary for pooling data
points from different experiments together because the evolution of
technology and any variance of experimental procedures can be
corrected by the corresponding standard curves.
[0009] It is important to note that standard curves have to be
concurrent with the actual measurements and have to undergo the
same experimental conditions as the measured quantities of interest
in the unknown samples. In fixed samples, it is difficult or
impossible to construct standard curves. For example,
fixation-based solid tissue methods such as immunofluorescence and
array tomography (Micheva and Smith, 2007, Neuron 55:25-36) do not
allow the construction of concurrent standard curves. In fixed
samples, only signals can be seen, but the underlying quantities
and/or quantity differences generally cannot be determined.
[0010] All fixation-based methods usually suffer from the
unpredictable molecular modification of the original sample and
from the lack of standard curves. As a consequence of these two
major limitations, and as a consequence of the fact that most
solid-tissue methods are fixation-based, it has not been possible
to date to measure the quantities of native proteins in single
cells of solid tissues or to do so in a multiplex manner. It has
also not been possible to measure the quantities of metabolites in
single cells of solid tissues or to reliably multiplex transcripts
in single cells of solid tissues. Multiplexing across molecular
classes (proteins, transcripts, metabolites) in single cells of
solid tissues has also not been possible.
[0011] Although it has been suggested that it is not necessary to
measure true quantities or quantity differences to make informative
qualitative observations in biology, in reality the correct
quantities and quantity differences, as opposed to simply
"signals", are integral to making correct qualitative observations.
FIGS. 1 and 2 demonstrate the importance of having concurrent
standard curves of affinity-based probes in order to make accurate
qualitative observations about the presence or absence of
sub-populations in any population measurement (single cells, tissue
samples or patients). Given the same true hidden distribution of a
quantity of interest across a population, a linear standard curve
with a small slope will make this distribution look narrower. In
contrast, a linear standard curve with a large slope will make this
same true hidden distribution look broader. Given two different
affinity-based probes (antibodies for example) with different KD
values, and thus with different slopes of their standard curves and
given the same true hidden distribution of a quantity, the
above-described differences in the observed distributions solely
due to the different KD values of the two probes are recorded,
although the underlying true distribution of the quantity of
interest is the same. Thus, qualitative observations, whether about
how broad or how narrow different quantities are distributed in a
population, are impossible without the knowledge of the
corresponding standard curves.
[0012] At the single-cell level, many quantities of proteins,
transcripts, or metabolites are present in small numbers, which can
result in the non-linearity of the standard curves of the
corresponding affinity-based probes. Fixation-induced differential
extraction and modification of target molecules also likely result
in the non-linearities of the standard curves. FIG. 2 shows how
false qualitative observations about the presence or absence of a
sub-population can be made in a population measurement if the
underlying unknown standard curves are non-linear. Taken together,
without knowing the concurrent standard curves of the applied
affinity-based probes, it cannot be known if the observed
qualitative observations are accurate. Arguably, the lack of
concurrent standard curves is the main cause of the
irreproducibility and the mutual incompatibility of many biological
measurements.
[0013] Limited multiplexing is the main limitation of optical
methods. The limited optical spectrum leads to the inability of
separating tens of signals simultaneously and therefore makes it
hard to measure the multivariate molecular mechanisms in single
cells by live imaging methods. All live imaging methods are based
on intracellular fluorescent probes that inherently perturb the
native system of the imaged live cell. For example, one common
approach to image proteins in live single cells requires the fusion
of GFP derivatives to the protein of interest and the subsequent
expression of the resulting fused protein. This procedure is not
practical in mammal solid tissues at large scale. The fusion of GFP
derivatives to the protein of interest can change both the function
of the protein of interest and the native state of the cell (Sigal
et al., 2006, Nat. Methods 3:525-531; Landgraf et al., 2012, Nat.
Methods 9:480-482; Schnell et al., 2012, Nat. Methods, 3:825-831).
The over-expression of such fused proteins and their dimerization
are common. The diffusion coefficient and the kinetic parameters of
GFP-fused proteins also likely change. Therefore, fused GFP does
not directly report the abundances and activity of native proteins.
Fluorescent intracellular ion sensors are another example of how
intracellular fluorescent probes perturb the native system of the
imaged cell (Yasuda et al., 2004, Sci STKE. 219: p15). Fluorescent
intracellular ion sensors are chelators and thus perturb the native
system of the cell by changing the native concentrations of the
respective ions (such as Ca.sup.2+).
[0014] There is a need in the art for methods that examine single
cell components derived from live solid tissues where the methods
preserve the components of the single cell in analytically defined
or natural state. The present invention addresses this unmet need
in the art.
SUMMARY OF THE INVENTION
[0015] The present invention provides a method of lysing a single
cell present in a tissue. In one embodiment, the method comprises:
a) identifying a cell from a tissue; b) contacting a
detergent-containing lysis buffer with the intracellular space of
the identified cell; c) allowing the lysis buffer to spread within
the intracellular space of the identified cell for a period of
time, wherein the cell is lysed from the inside of the cell; and d)
collecting the lysate, wherein the lysate comprises cellular
components preserved in an analytically defined and analytically
accessible state that maps to the natural state in a known way.
[0016] In one embodiment, the method occurs in the absence of
tissue fixation and tissue disaggregation.
[0017] In one embodiment, the isolated cell is in an organotypic
culture.
[0018] In one embodiment, the lysate is collected by suctioning the
lysate using a suction channel.
[0019] In one embodiment, the suction channel is a bent suction
micropipette.
[0020] In one embodiment, the collected lysate is further applied
to a nitrocellulose pad.
[0021] In one embodiment, a standard is also applied the
nitrocellulose pad.
[0022] In one embodiment, the lysate is evaluated using an
analytical method.
[0023] In one embodiment, the analytical method is selected from
the group consisting of mass spectrometry, protein microarray,
RT-qPCR, RNA-Seq, and MALDI-MS.
[0024] In one embodiment, the cell is part of a live solid
tissue.
[0025] In one embodiment, the detergent is sodium dodecyl
sulfate.
[0026] The invention also provides a method of analyzing a cell
present in a tissue. In one embodiment, the method comprises: a)
identifying a cell from a tissue; b) contacting a
detergent-containing lysis buffer to the intracellular space of the
identified cell; c) allowing the lysis buffer to spread within the
intracellular space of the identified cell for a period of time,
wherein the cell is lysed from the inside of the cell; d)
collecting the lysate, wherein the lysate comprises cellular
components preserved in an analytically defined and analytically
accessible state that maps to the natural state in a known way; e)
applying the collected lysate to a solid support; and f) evaluating
the collected lysate using an analytical method.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The following detailed description of preferred embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, there are shown in the drawings embodiments which are
presently preferred. It should be understood, however, that the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0028] FIG. 1 is a series of graphs depicting the different slopes
of linear standard curves. The true hidden distribution is shown in
the bottom row. The distributions in the upper row were obtained by
applying the standard curves in the middle row to the true hidden
distributions in the bottom row. Different KD values of affinity
based probes, such as antibodies, led to different slopes of their
standard curves even at the same probe concentrations. The arrows
next to the standard curves indicate that the same signal
difference can be mapped to a large quantity difference or to a
small quantity difference depending on the slope of the underlying
standard curve.
[0029] FIG. 2 is a series of graphs depicting the different
non-linear standard curves. The true hidden distribution is shown
in the bottom row. The distributions in the upper row were obtained
by applying the standard curves in the middle row to the true
hidden distributions in the bottom row. Non-linearity of the
standard curves emerged when the substrate of an affinity-based
probe was present in very small amounts in single cells (first case
from the left). At low antigen concentrations, bivalent antibodies
may not find closely located antigens to be bound by the antigen
binding sites in Lysate Microarrays measurements, thereby leading
to the non-linearity scenario displayed in the first case from the
left. Fixation and permeabilization also changed the availability
of substrates, as discussed elsewhere herein, and likely induced
non-linearities.
[0030] FIG. 3 is a schematic illustration depicting the strategy
for resolving limitations. The platform consists of a new sampling
method (Inside-Out Lysis), a new analytical method for measuring
native proteins in single cells of solid tissues (Single-Cell
Lysate Microarrays) and a set of compatible analytical strategies
for the quantification of native transcripts and metabolites in
single cells of solid tissues. This platform resolved limitations
of current methods for sampling and analyzing single cells in solid
tissues.
[0031] FIG. 4, comprising FIGS. 4A-4B, depicts organotypic
cultures. FIG. 4A is an illustration of the method of organotypic
cultures. A dissected slice of tissue was cultured on a porous
membrane. The tissue slice took up the nutrients from underneath
the membrane by capillary action (from Stoppini et al., 1991, J
Neurosci Methods. 37(2): 173-182). FIG. 4B is a photograph
depicting an example of a successful organotypic culture of a mouse
hippocampus (GAD67-GFP strain, postnatal day 6+14 days in
vitro).
[0032] FIG. 5 is an illustration depicting the phase diagram of
SDS. The SDS solution was kept at pH 7.4 and contained 0.1 M NaCl
and 0.05 M sodium phosphate. The critical micellar temperature was
designated as CMT. The critical micellar concentration of SDS was
designated as CMC (from Helenius and Simons, 1975, Biochim Biophys.
Acta 415:29-79).
[0033] FIG. 6 is an illustration depicting a simplified model of
detergent-based lysis. A concentration greater than the CMC value
might be required to induce membrane solubilization. Also, the
extent of membrane solubilization depends on the composition of the
membrane and the number of detergent monomers integrated into the
membrane.
[0034] FIG. 7 is an illustration depicting the Inside-Out Lysis of
single cells in a live solid tissue. The upper diagram summarizes
the concept of Inside-Out Lysis. In the bottom frames,
SDS-containing lysis buffer was mixed with SR101. No suction was
applied. The organotypic culture of mouse hippocampus is displayed
(GAD67-GFP strain, postnatal day 6+7 days in vitro). In all
instances, the delivery of lysis buffer was accomplished
robotically without any human input. It was observed that the
second cell from the bottom had a small surface area and the
applied lysis buffer entered the adjacent dark cell by flowing into
it. The single-cell resolution of delivery was accurate in all
other instances (also see FIG. 32). The frame on the bottom right
also shows the initiation of the lysis buffer delivery to the upper
cell (fourth cell).
[0035] FIG. 8 is an illustration depicting the rapid denaturation
of soluble proteins. This is the same experiment as in FIG. 7. GFP
fluorescence decreased as soon as the SDS-containing lysis buffer
entered the intracellular space of each target cell. During this
decrease of GFP fluorescence, SR101 dyes stayed strictly within the
geometric boundaries of the target cells (with exception of cell 2)
implying that the membrane barrier was intact from the time point
of lysis buffer delivery to the time point of GFP fluorescence
loss. No suction was applied. Thus, the decrease of GFP
fluorescence represents the intracellular GFP denaturation. Here,
the delivery of lysis buffer was fully robotic with no human input.
The amount of lysis buffer applied in each instance was the same.
The line of the second cell (SR101 mixed with the lysis buffer)
shows a bump when the delivery to the first cell is initiated
because of the fluorescence of the delivery pipette under the
objective. Lysis buffer flow was perpendicular to the tissue
surface.
[0036] FIG. 9 is a series of photographs depicting a fully
automated sequential lysis buffer delivery within a set of
different regions of a cultured hippocampal slice (GAD67-GFP, P4+5
DIV). Lysis buffer was delivered to fifteen cells in 3 different
hippocampus regions in a fully automated manner within a 200 second
time period (automated lysis buffer delivery was triggered 40
seconds after the imaging start). The imaged area was constrained
by a diaphragm in order to ensure the planarity of the slice
surface within that area. For each region three images are
displayed: 1) an image acquired before the lysis process was
triggered, 2) the same image after image segmentation, 3) an image
acquired at a time point of the lysis buffer delivery process in
that particular region.
[0037] FIG. 10 is a series of photographs depicting cell membrane
growth during the lysis buffer delivery. LB stands for lysis
buffer. SDS was delivered to single cells by a focal stream of
lysis buffer. GFP was denatured and the membrane of each target
cell grew simultaneously. Alexa 555 dyes stayed within the
geometric boundaries of the target cells during GFP denaturation
and during membrane growth. Although not wishing to be bound by any
particular theory, this suggests that the membrane barrier was
intact from the time point of lysis buffer delivery, throughout the
phase of GFP denaturation, and to the time point of extensive
membrane growth. Also see FIG. 32.
[0038] FIG. 11 is a photograph depicting the partial and slow
solubilization of the membrane of a target cell. A smaller amount
of SDS-containing lysis buffer was applied without the subsequent
lysate uptake (no suction). SR101 dyes were observed to be slowly
diffusing out of the geometric boundaries of the single cells
following a 30 minute (upper image) or 11 minute (bottom image)
waiting period. The leftover fluorescent regions were putative
membrane patches after partial membrane solubilization. The upper
image was acquired under the 20.times. objective in a GAD67-GFP
slice (P4+4 DIV). The bottom image was acquired under the 40.times.
objective in a GAD67-GFP slice (P4+6 DIV).
[0039] FIG. 12 is a series of photographs depicting the complete
and rapid solubilization of the single-cell membrane by Inside-Out
Lysis in live solid tissue. The arrow shows the GFP-expressing
target cell (somatostatin interneuron) in a hippocampal organotypic
slice of a GIN mouse. First, a small amount of lysis buffer (Alexa
555D was used here for visualization) was applied and the cell
membrane was intact, presumably after the focal entry point of the
applied lysis buffer was sealed. More lysis buffer was applied
continuously until the membrane was completely solubilized from
inside. The lysate was up-taken by a perpendicular simultaneous
capillary-action-driven suction. The delivery micropipette was not
bent and the delivery of lysis buffer occured under an angle and
not vertically.
[0040] FIG. 13, comprising FIGS. 13A-13B, depicts printing
single-cell lysates on a glass-mounted nitrocellulose pad. FIG. 13A
is a series of images depicting the method of enriching the
proteins of a single-cell lysate within a 20-60 .mu.m spot on
nitrocellulose. The frame on the right shows the extent of solvent
spreading. However, the proteins of the single-cell lysate
partition were enriched within a much smaller region right below
the micropipette used for printing. The same micropipette was used
for capillary-action-driven suction in Inside-Out Lysis. FIG. 13B
is a series of images depicting spots of approximately 50 .mu.m
from the same single GFP-expressing somatostatin interneuron
(organotypic hippocampal slice of a GIN mouse). Spots were printed
on a nitrocellulose pad next to each other (10 depositions per
spot). The corresponding baseline spots were also printed on the
same nitrocellulose pad (10 depositions per spot). There was no
signal before antibody incubation. As expected, there were signals
only in the lysate spots after antibody incubation. The bar in low
resolution frames is 42 .mu.m.
[0041] FIG. 14 is a series of illustrations depicting the
validation of Inside-Out Lysis. Two cells were lysed in the same
slice of a GIN mouse. One cell expressed GFP (somatostatin
interneuron) and the other cell did not express GFP (CA3 pyramidal
neuron). All the concurrent negative controls were negative. All
the concurrent positive controls were positive. The entire lysate
partition of each single cell was printed in three lysate spots
respectively. The lysate spots of each single cell showed the
expected signal distribution. The baseline spots were negative.
These results demonstrate the successful use of single-cell lysate
sub-partitioning, as the lysate partition of each cell in the
experiment was subdivided into three sub-partitions.
[0042] FIG. 15 is a series of images depicting the cell sampling
and printing setup of the instant invention.
[0043] FIG. 16 is an illustration depicting two possible categories
of antibodies in the format of Lysate Microarrays. In contrast to
Western blot, proteins cannot be separated by size in Lysate
Microrrays. As a consequence, antibodies with optimal binding
probabilities (second case from the left) and thus with invariantly
low cross-reactivity are required. Once a set of antibodies in this
category is validated, analytical protein measurements can be
accomplished in unknown samples with Lysate Microarrays.
[0044] FIG. 17 is a series of graphs depicting Single-Cell Lysate
Microarrays, which combine the analytical rigor of Lysate
Microarrays with the multiplexing capacity of
lanthanide-labeling.
[0045] FIG. 18 is a series of photographs depicting additional
antibody validation. Each pan-specific antibody, previously
validated for Lysate Microarrays by comparing the signal
distribution in Lysate Microarrays with the corresponding signal
distribution in Western blots across 17 human cell lines, gave a
single dominant band in the Western blots performed on the average
lysate of GIN mouse hippocampus. The GIN mouse was of the same age
as the GIN mice used in later experiments. The second band in PKG3,
PKC.alpha., PAK1 and fl-catenin blots came from the fluorescence of
the secondary antibody against the primary .beta.-actin
antibody.
[0046] FIG. 19, comprised of FIGS. 19A-19C, depicts Single-Cell
Lysate Microarray data. In the upper segment, a555 stands for Alexa
555. FIG. 19A is a series of graphs depicting Single-Cell Lysate
Microarray data. FIG. 19B is a series of graphs depicting
Single-Cell Lysate Microarray data. FIG. 19C is a series of graphs
depicting Single-Cell Lysate Microarray data. The obtained
single-cell lysates and their baselines were printed on a
nitrocellulose pad as described elsewhere herein, but without
sub-partitioning. The integrated values are depicted in FIG.
20.
[0047] FIG. 20 is an illustration depicting integrated LA-ICP-MS
counts of the measurements in FIG. 19. The time-integrated
intensity of the baseline spot (from the 170th frame to the 370th
frame) was subtracted from the time-integrated intensity of the
single-cell lysate spot (from the 170th frame to the 370th frame)
in cells 1 to 4. In cells 5 and 6, the printing of the baseline was
not entirely successful and only the integrated instrument noise
was subtracted from the total integrated intensity over both the
baseline and the lysate spots. The maximum value within each
channel across all the single cells was assigned the value of 1.0.
The measurement of one spot on the titration series of the average
hippocampus lysate was also included.
[0048] FIG. 21 is a series of graphs depicting a concurrent
PKC.delta. and PKC.alpha. titration series revealing the standard
curves. The titration series of PKC.delta. and PKC.alpha. were
printed on the same glass-mounted nitrocellulose pad, on which the
single-cell lysates in FIG. 19 were also printed and measured. All
titration series were printed by the high-precision Aushon Arrayer.
The line delineates the dynamic range within which the recorded
signals of PKC.delta. and PKC.alpha. in the single-cell lysate
spots were confined (compared to FIG. 19). The nonlinear nature of
the standard curves also makes the inclusion of such concurrent
standards in any single-cell measurement necessary (FIG. 2).
[0049] FIG. 22, comprising FIGS. 22A-22B, depicts results from GFP
titration series and measurements. FIG. 22A is a series of graphs
depicting the same titration series (the same 4 spots), printed by
the high-precision Aushon Arrayer 2470, revealing two standard
curves with different slopes for each GFP antibody. Both GFP
antibodies were applied simultaneously in the total mixture of 8
antibodies. Because the lanthanide conjugation procedure was the
same for each GFP antibody and because the concentration of either
GFP antibody was the same during the incubation procedure, it is
believed that this difference in slopes parameterizes the
corresponding differences in the K.sub.D values of the two GFP
antibodies. FIG. 22B is a series of graphs depicting almost perfect
correlation between the in vivo GFP fluorescence and the measured
GFP levels in Single-Cell Lysate Microarrays across all the sampled
single cells of FIG. 19. The two dark CA3 neurons are represented
by the two lowest data points. The four GFP-expressing somatostatin
interneurons are represented by the other four data points. The GFP
titration series in FIG. 22A was printed on the same nitrocellulose
pad as single-cell lysates in FIG. 22B. Again, all titration series
were printed by the high-precision Aushon Arrayer 2470. All
single-cell lysates and the corresponding baselines were printed as
described elsewhere herein but without sub-partitioning.
[0050] FIG. 23 is a series of illustrations depicting Bayes Net
topologies. Eight Bayes Net topologies with increasing complexities
and the corresponding formal mathematical representations are
depicted.
[0051] FIG. 24 is a graph depicting the selection of the optimal
Learner (Bayes Net topology+ML parameter estimation). The optimal
Learner was selected from a set of Learners by running the 10-fold
cross-validation with each Learner on the data set of
dimensionality 10 and consisting of 112 data points. In each of the
10 runs of the cross-validation, the training set was used as input
to the Learner function and the resulting Bayes Net model was used
to calculate the classification accuracy in the test set and in the
training set. This procedure was performed across all the 10 runs
of cross-validation and the average cancer classification
accuracies are displayed in the figure. 10-fold cross-validation
was performed with each Learner. The optimal Learner is
AXL+MET+AKT. The simplest topology of Naive Bayes performed well
but considerably worse than AXL+MET+AKT. Increasing the complexity
of the Bayes Net topologies beyond the complexity of AXL+MET+AKT
led to the suboptimal expansion of the hypothesis space and thus to
overfitting.
[0052] FIG. 25 is a series of graphs depicting how additional
conditioning led to better test-set classification. A better
separation of Met.p data was achieved by conditioning on Axl and
Cancer random variables. Without conditioning on Axl, the
distribution of Met.p was similar both for Cancer=True and for
Cancer=False. The additional conditioning on Axl led to the
different conditional distributions of Met.p when conditioned on
Axl=High, Cancer=False and when conditioned on Axl=High,
Cancer=True. This distinction enabled for a better average
classification accuracy across the AXL+MET+AKT models as compared
to the MET+AKT models in FIG. 24. Line is the median used for
binarization of data.
[0053] FIG. 26 is an illustration depicting a framework for
Inside-Out Lysis and relations between detergent concentrations.
Relations between head-group charge and hydrocarbon chain length of
detergents and CMC and micelle size have been established in other
biophysical studies and provide the basis for parameterization of
our Inside-Out Lysis model.
[0054] FIG. 27 is a series of graphs depicting time course of
protein denaturation/cell homogenization and the breaking of the
membrane barrier with subsequent lysate uptake
[0055] FIG. 28 is a series of graphs depicting lysate uptake from
soma and proximal dendrites.
[0056] FIG. 29 is a photograph depicting the fast switching between
the uptake of the lysis product (lysate) and the deposition of the
lysate on the nitrocellulose pad in an automated experimental
set-up.
[0057] FIG. 30 is a series of photographs depicting the separation
of lysis visualization and analytical procedures. If optical
methods are used for signal detection then the visualization of
Inside-Out Lysis (sampling), necessary to ensure single-cell
resolution and to record the cell morphology (if the target cell is
not fluorescent), should not interfere with optical signal
detection. Different fluorescent dyes (SR101, Alexa 568 dextrane
(MW 10,000), Alexa 555 (MW 10,000)) were tried in order to identify
the dye that could be washed out after printing the lysate on
nitrocellulose. All dyes provided sufficient visualization
capabilities for Inside Out Lysis. Different dyes were mixed with
the lysis buffer (TrisHCl (50 mM), SDS (2%), Glycerol (5%), NaF (1
mM)). Also added were 7.4 M urea to the dye-containing lysis
buffer. In another instance Hank's Balanced Salt Solution (HBSS)
was mixed with each dye. Overall, this example demonstrates that in
contrast to the above Alexa dyes, SR101 is not suitable for optical
post-lysis signal detection because it is hard to wash out from
nitrocellulose. If signal detection is achieved by non-optical
methods (such as LA-ICP-MS), then the ability to wash out the
fluorescent dyes used for sampling visualization should not
matter.
[0058] FIG. 31 is a series of photographs depicting evidence of a
focal entry point in cells. These images were taken with very high
exposure in order to visualize the residual dye molecules (lysis
buffer) and the residual membrane fragments after Inside-Out
Lysis.
[0059] FIG. 32 is a series of photographs depicting how two
adjacent dark cells are not visibly affected during the lysis
buffer delivery stage of Inside-Out Lysis. The image on the left
was acquired with high exposure before Inside-Out Lysis in order to
capture the details of GFP distribution in the slice (GAD67-GFP
strain, postnatal day 6+7 days in vitro). The two images on the
right correspond to the same time point in the Inside-Out Lysis
process of the first cell shown in FIGS. 7 and 8. The upper image
on the right is the GFP frame. The lower image on the right is the
SR101 frame showing the delivery of the SR101-containing lysis
buffer to the target cell. The bright background section in the
upper left corner of the lower image on the right is the
fluorescence from the delivery micropipette. Two dark cells
(arrows) can be observed next to the soma and next to the apical
dendrite of the target GFP-expressing neuron. The focal point of
lysis buffer delivery is designated by the letter D. The SR101 dye
was contained strictly within the geometric boundary of the target
cell and did not enter the two adjacent dark cells, even after
extensive membrane growth.
DETAILED DESCRIPTION
[0060] The present invention relates to the development of a lysis
technology and analytical technology as well as analytical
strategies to provide the capability of sampling and evaluating
single cell lysates from a solid tissue. In one embodiment, the
invention comprises a sampling procedure that allows for obtaining
a single cell lysate and analyzing the lysate by interfacing it
with different analytical methods. Preferably, the analytical
methods are based on a lysate format. For example, the analytical
methods include but are not limited to solid support methods (e.g.,
microarrays, MALDI), non-solid support methods (e.g., qPCR or other
Mass Spec modes with direct introduction of the sample), and the
like.
[0061] One benefit of the invention is that the lysis protocol
preserves all cellular molecules in an analytically defined and
analytically accessible state that maps to the natural state in a
known way thereby allowing for a more accurate and highly multiplex
measurement of the cellular molecules as they existed in the cell.
In one embodiment, the invention provides a method for analyzing
cellular molecules from a single cell comprising: 1) lysing a
single cell (e.g., lysing step), 2) applying the lysate onto a
solid support (e.g., printing step), and 3) analyzing the lysate
using a desired technology (e.g., analytical step).
[0062] The methods of the invention also allow for the isolation of
lysate from a single cell embedded in a solid tissue while
preserving the analytically defined and analytically accessible
state of molecules that maps to the natural state within the cell
in a known way, and therefore allow for transformation of a single
target cell in a tissue sample into a format that can be evaluated
using analytical methods including, but not limited to, mass
spectrometry, lysate microarrays, protein microarrays, RT-qPCR,
RNA-Seq, LA-ICP-MS, MALDI-MS, and the like.
[0063] In one embodiment, the invention provides an "inside-out
lysis" platform comprising: 1) facilitating entry of a
detergent-containing lysis buffer into the intracellular space of a
target cell through a focal entry point in the cell membrane, 2)
allowing the lysis buffer to spread throughout the intracellular
space, 3) lysing the target cell from inside, thus providing the
highest possible single-cell resolution for the lysis of target
cells of any shape within the complex environment of a living
tissue. This lysis technology highlights the unusual directionality
of the lysis process that enables a superior spatial resolution
when compared to prior art methods. The "inside-out lysis" method
of the invention preserves the natural state of the single cell
within the lysate in a known way, which can then be examined using
a variety of analytical methods. The method of the invention also
preserves RNA transcripts, proteins and metabolites by
incorporating the technical advantages of detergent-based
lysis.
[0064] Thus, the invention described herein relates to a new method
for lysing a single cell that is derived from a solid tissue while
preserving the molecules within the cell in analytically defined
and analytically accessible state that maps to their natural state
in the cell in a known way. The method comprises a lysis step,
wherein the target cell is lysed from inside, thus providing the
highest possible single-cell resolution for the lysis of target
cells of any shape within the complex environment of living tissue,
and collecting the lysate. In one embodiment, the target cell is
encompassed in an organotypic culture. In one embodiment, the
lysate is collected by suctioning the lysate out of the system with
a suction channel. In one embodiment, the method comprises a
printing method comprising applying the lysate to a solid support
(e.g., glass-mounted nitrocellulose pad). In one embodiment, the
method comprises evaluating the printed lysate using analytical
measurements.
[0065] The invention described herein also relates to new
analytical methods for a multiplex analysis platform where the
products of the lysis method disclosed herein are analyzed. In one
embodiment, the method comprises an analytical method for
multiplexing analytical measurements of native proteins. In one
embodiment, the method comprises applying a mixture of antibodies
to the lysate printed or otherwise spotted on the solid support
(e.g., nitrocellulose). In another embodiment, the antibodies are
conjugated to lanthanide metals. In another embodiment, the method
comprises a set of compatible analytical strategies for multiplex
measurements of transcripts and metabolites in single cell
lysate.
[0066] The invention also provides methods for measuring proteins,
transcripts and/or metabolites at one time. Concurrent standard
curves can be determined in all measurements and signals can be
mapped to the corresponding quantities of the proteins, transcripts
and/or metabolites being assessed.
DEFINITIONS
[0067] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
[0068] As used herein, each of the following terms has the meaning
associated with it in this section.
[0069] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0070] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably .+-.0.1%
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0071] The term "abnormal" when used in the context of organisms,
tissues, cells or components thereof, refers to those organisms,
tissues, cells or components thereof that differ in at least one
observable or detectable characteristic (e.g., age, treatment, time
of day, etc.) from those organisms, tissues, cells or components
thereof that display the "normal" (expected) respective
characteristic. Characteristics which are normal or expected for
one cell or tissue type, might be abnormal for a different cell or
tissue type.
[0072] An "analyte," as used herein refers to any substance or
chemical constituent that is undergoing analysis. For example, an
"analyte" can refer to any atom and/or molecule; including their
complexes and fragment ions. The term may refer to a single
component or a set of components. In the case of biological
molecules/macromolecules, such analytes include but are not limited
to: polypeptides, polynucleotides, proteins, peptides, antibodies,
DNA, RNA, carbohydrates, steroids, and lipids, and any detectable
moiety thereof, e.g., immunologically detectable fragments.
[0073] "Assay," "assaying" or like terms refers to an analysis to
determine, for example, the presence, absence, quantity, extent,
kinetics, dynamics, or type of a cell's response upon stimulation
with an exogenous stimuli, such as a ligand candidate compound or a
viral particle or a pathogen.
[0074] "Biosensor" or like terms refer to a device for the
detection of an analyte that combines a biological component with a
physicochemical detector component. The biosensor typically
consists of three parts: a biological component or element (such as
tissue, microorganism, pathogen, cells, or combinations thereof), a
detector element (works in a physicochemical way such as optical,
piezoelectric, electrochemical, thermometric, or magnetic), and a
transducer associated with both components. The biological
component or element can be, for example, a living cell, a
pathogen, or combinations thereof. In embodiments, an optical
biosensor can comprise an optical transducer for converting a
molecular recognition or molecular stimulation event in a living
cell, a pathogen, or combinations thereof into a quantifiable
signal.
[0075] "Cell" or like term refers to a small usually microscopic
mass of protoplasm bounded externally by a semipermeable membrane,
optionally including one or more nuclei and various other
organelles, capable alone or interacting with other like masses of
performing all the fundamental functions of life, and forming the
smallest structural unit of living matter capable of functioning
independently including synthetic cell constructs, cell model
systems, and like artificial cellular systems.
[0076] "Cell system" or like term refers to a collection of more
than one type of cells (or differentiated forms of a single type of
cell), which interact with each other, thus performing a biological
or physiological or pathophysiological function. Such cell system
includes an organ, a tissue, a stem cell, a differentiated cell, or
the like.
[0077] As used herein, the term "cellular constituent" comprises
individual genes, proteins, mRNA, RNA, and/or any other variable
cellular component or protein activity, degree of protein
modification (e.g., phosphorylation), for example, that is
typically measured in a biological experiment by those skilled in
the art.
[0078] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to
deteriorate.
[0079] In contrast, a "disorder" in an animal is a state of health
in which the animal is able to maintain homeostasis, but in which
the animal's state of health is less favorable than it would be in
the absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0080] A disease or disorder is "alleviated" if the severity of a
symptom of the disease or disorder, the frequency with which such a
symptom is experienced by a patient, or both, is reduced.
[0081] An "effective amount" or "therapeutically effective amount"
of a compound is that amount of compound which is sufficient to
provide a beneficial effect to the subject to which the compound is
administered. An "effective amount" of a delivery vehicle is that
amount sufficient to effectively bind or deliver a compound.
[0082] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which can be used to communicate the usefulness of a
compound, composition, vector, or delivery system of the invention
in the kit for effecting alleviation of the various diseases or
disorders recited herein. Optionally, or alternately, the
instructional material can describe one or more methods of
alleviating the diseases or disorders in a cell or a tissue of a
mammal. The instructional material of the kit of the invention can,
for example, be affixed to a container which contains the
identified compound, composition, vector, or delivery system of the
invention or be shipped together with a container which contains
the identified compound, composition, vector, or delivery system.
Alternatively, the instructional material can be shipped separately
from the container with the intention that the instructional
material and the compound be used cooperatively by the
recipient.
[0083] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal, or cells
thereof whether in vitro or in situ, amenable to the methods
described herein. In certain non-limiting embodiments, the patient,
subject or individual is a human.
[0084] A "therapeutic" treatment is a treatment administered to a
subject who exhibits signs of pathology, for the purpose of
diminishing or eliminating those signs.
[0085] As used herein, "treating a disease or disorder" means
reducing the frequency with which a symptom of the disease or
disorder is experienced by a patient. Disease and disorder are used
interchangeably herein.
[0086] The phrase "therapeutically effective amount," as used
herein, refers to an amount that is sufficient or effective to
prevent or treat (delay or prevent the onset of, prevent the
progression of, inhibit, decrease or reverse) a disease or
condition, including alleviating symptoms of such diseases.
[0087] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
DESCRIPTION
[0088] One of the greatest challenges in the post-genomic era is
the development of methods that can reliably analyze the
transcripts, metabolites, proteins as well as posttranslational
modification states of different proteins in a single cell lysate
obtained with high spatial and temporal resolution from complex,
living tissue and from complex tissue in general.
[0089] The present invention relates to the development of a
microlysis technology and analytical technology as well as
analytical strategies to provide the capability of sampling and
evaluating single cell lysates from a live solid tissue while
preserving all cellular molecules in an analytically defined and
analytically accessible state that maps to the natural state in a
known way. In one embodiment, the invention provides a method for
analyzing cellular molecules from a single cell comprising: 1)
lysing a single cell (e.g., sampling), 2) applying the lysate on a
solid support (e.g., printing), and 3) analyzing the lysate using a
desired technology.
[0090] Accordingly, the invention provides compositions and methods
that enable proteomic studies on single cell lysates derived from
physiologically relevant, complex, living tissue. However, the
invention should not be construed to be limited solely to proteomic
studies. Rather, the single cell lysate can be used in any assay to
evaluate nucleic acids, polypeptides and any metabolites. That is,
after the single cell lysate is obtained and printed on a solid
support according to the invention by way of multiplexing proteins
in one single spot without sample subdivision by using a
`labeling--label detection method` pair for example the `lanthanide
labeling--LA-ICP-MS detection` pair. In some instances, the methods
of the invention include the use of cell-based assays,
protein-based assays, and DNA-based assays. In one embodiment, when
such technologies are applied to analyzing the lysate of the
invention, the results obtained therefrom depict the natural state
of the cellular components as represented in the single cell.
[0091] In one embodiment, the invention comprises an "Inside-Out
Lysis" methodology providing for a single cell in a live solid
tissue to be lysed to provide a lysate that comprises cellular
molecules of all classes of molecules that are preserved in
analytically defined and analytically accessible state that maps to
the natural state in a known way. The "Inside-Out Lysis"
methodology of the invention is contrary to the common practice in
the art where cells or tissues have been submerged in a
solubilizing/dissociation solution and thus the directionality of
solubilization/dissociation process of methodologies in the art is
from the outside. A more detailed discussion of the Inside-Out
Lysis method of the invention is discussed elsewhere herein.
[0092] In another embodiment, the invention provides an analytical
method for multiplexing analytical measurements of native proteins
present in a single cell lysate. This method is referred herein as
a "Single-Cell Lysate Microarray." A more detailed discussion of
the Single-Cell Lysate Microarray method of the invention is
discussed elsewhere herein. In one embodiment, this method provides
a set of compatible analytical strategies for multiplex
measurements of transcripts and metabolites in a single cell
lysate.
Lysis Step
[0093] The invention provides a method for obtaining lysate from a
single cell which is referred herein as a single cell lysate. In
one embodiment, the single cell lysate is of small volume (e.g.,
about 10 n1). The lysate format of the single cell can be exploited
by interfacing it with different analytical methods that are based
on the lysate format.
[0094] In one embodiment, the invention provides a method of lysing
a single cell that is embedded in a tissue, preferably a solid
tissue, more preferably a live solid tissue. In one embodiment, the
invention provides an "Inside-Out Lysis" platform for lysing a
single cell from a tissue sample wherein the lysate can be further
analyzed using any desired analytical method. For example, a single
cell is lysed according to the "inside-out lysis" method of the
invention, followed by printing or otherwise spotting the lysate on
a solid support, and the printed lysate is analyzed using a desired
protocol.
[0095] In one embodiment, the lysis method of the invention employs
detergent-based single-cell lysis buffer. An advantage of using a
detergent-based lysis buffer over a fixation method is that the
state of the proteins in the lysate is analytically defined. For
example, an SDS lysis buffer denatures proteins and kills their
activity whereas NP40 does not denature proteins. In both cases,
the state of the proteins is known because the effects of the lysis
buffer on the molecules are known and therefore the state is
analytically defined (unlike fixation methods). Also all molecules
can be accessed analytically (unlike fixation methods) using the
lysis protocol of the invention.
[0096] Detergents are amphipathic molecules, meaning they contain
both a nonpolar "tail" having aliphatic or aromatic character and a
polar "head." Ionic character of the polar head group forms the
basis for broad classification of detergents; they may be ionic
(charged, either anionic or cationic), nonionic (uncharged) or
zwitterionic (having both positively and negatively charged groups
but with a net charge of zero). In any event, detergent molecules
allow the dispersion (miscibility) of water-insoluble, hydrophobic
compounds into aqueous media, including the extraction and
solubilization of membrane proteins. Both the number of detergent
monomers per micelle (aggregation number) and the range of
detergent concentration above which micelles form (called the
critical micelle concentration, CMC) are properties specific to
each particular detergent.
[0097] In one embodiment, the lysis method involves the following
sequence of events: 1) detergent-containing lysis buffer enters the
intracellular space of the target cell through a focal entry point
in the cell membrane, 2) lysis buffer spreads throughout the
intracellular space, 3) and the target cell is lysed from the
inside, thus allowing the highest possible single-cell resolution
for the lysis of target cells of any shape within the complex
environment of living tissue.
[0098] Thus, the invention described herein relates to a new method
for lysing a single cell that is derived from a solid tissue while
preserving all cellular molecules in an analytically defined and
analytically accessible state that maps to the natural state in a
known way. The method comprises a lysis step comprising adding
detergent-containing lysis buffer to the intracellular space of the
target cell in a system through a focal entry point in the cell
membrane, allowing the lysis buffer to spread throughout the
intracellular space of the target cell over a period of time,
wherein the target cell is lysed from inside, thus providing the
highest possible single-cell resolution for the lysis of target
cells of any shape within the complex environment of living tissue,
and collecting the lysate. In one embodiment, the target cell is
encompassed in an organotypic culture.
[0099] In one embodiment, a focally directed flow of the
detergent-containing lysis buffer is applied to the cell body of
the target cell in a live tissue. Preferably, the concentration of
the detergent used is above its critical micellar concentration
(CMC) value. This property enables the applied lysis buffer to
enter the intracellular space without spilling to the adjacent
cells in the tissue. Inside the cell, the detergent is diluted to a
concentration value that is below its CMC value, allowing the lysis
buffer to accumulate in the intact intracellular space and to
diffuse and/or to flow throughout the complex shape of the target
cell without affecting its complex tissue surroundings. Once enough
lysis buffer has accumulated inside the target cell and the
detergent concentration re-approaches its critical value, the cell
membrane is lysed from inside and the lysate can be immediately
diluted and up-taken in a nearby suction channel. In one
embodiment, it is preferred that the lysate be immediately diluted
so that it does not affect other cells once the membrane barrier of
the target cell is broken. Thus the Inside-Out Lysis method is able
to convert a single live cell of any shape in a complex solid
tissue into a mixed lysate solution where all classes of molecules
are preserved.
[0100] In one embodiment, the volume of the obtained single-cell
lysate is approximately 1-500 nL, preferably 2-250 nL, more
preferably 3-100 nL, more preferably 4-50 nL, and most preferably
5-10 nL.
[0101] In one embodiment, the detergent-based single-cell lysis
buffer comprises one or more non-ionic detergents, including, but
not limited to, N-octyl-.beta.-D-glucopyranside,
N-octyl-.beta.-D-maltoside, ZWITTERGENT 3.14, deoxycholate;
n-Dodecanoylsucrose; n-Dodecyl-.beta.-D-glucopyranoside;
n-Dodecyl-.beta.-D-maltoside; n-Octyl-.beta.-D-glucopyranoside;
n-Octyl-.beta.-D-maltopyranoside;
n-Octyl-.beta.-D-thioglucopyranoside; n-Decanoylsucrose;
n-Decyl-.beta.-D-maltopyranoside; n-Decyl-.beta.-D-thiomaltoside;
n-Heptyl-.beta.-D-glucopyranoside;
n-Heptyl-3-D-thioglucopyranoside; n-Hexyl-.beta.-D-glucopyranoside;
n-Nonyl-.beta.-D-glucopyranoside; n-Octanoylsucrose;
n-Octyl-.beta.-D-glucopyranoside; n-Undecyl-.beta.-D-maltoside;
APO-10; APO-12; Big CHAP; Big CHAP, Deoxy; BRIJ.RTM. 35;
C.sub.12E.sub.5; C.sub.12E.sub.6; C.sub.12E.sub.8; C.sub.12E.sub.9;
Cyclohexyl-n-ethyl-.beta.-D-maltoside;
Cyclohexyl-n-hexyl-.beta.-D-maltoside;
Cyclohexyl-n-methyl-.beta.-D-maltoside; Digitonin; ELUGENT.TM.;
GENAPOL.RTM. C-100; GENAPOL.RTM. X-080; GENAPOL.RTM. X-100;
HECAMEG; MEGA-10; MEGA-8; MEGA-9; NOGA; NP-40; PLURONIC.RTM. F-127;
TRITON.RTM. X-100; TRITON.RTM. X-114; TWEEN.RTM. 20; or TWEEN.RTM.
80. Additionally, an ionic detergent can be used with the methods
of the invention, including, but not limited to BATC,
Cetyltrimethylammonium Bromide, Chenodeoxycholic Acid, Cholic Acid,
Deoxycholic Acid, Glycocholic Acid, Glycodeoxycholic Acid,
Glycolithocholic Acid, Lauroylsarcosine, Taurochenodeoxycholic
Acid, Taurocholic Acid, Taurodehydrocholic Acid, Taurolithocholic
Acid, Tauroursodeoxycholic Acid, and TOPPA. Zwitterionic detergents
can also be used with the compositions and methods of the
invention, including, but not limited to, amidosulfobetaines,
CHAPS, CHAPSO, carboxybetaines, and methylbetaines. Anionic
detergents can also be used with the compositions and methods of
the invention, including, but not limited to, e.g. SDS, N-lauryl
sarcosine, sodium deoxycholate, alkyl-aryl sulphonates, long chain
(fatty) alcohol sulphates, olefine sulphates and sulphonates, alpha
olefine sulphates and sulphonates, sulphated monoglycerides,
sulphated ethers, sulphosuccinates, alkane sulphonates, phosphate
esters, alkyl isethionates, and sucrose esters.
[0102] Generally any suitable liquid may be used as a solvent in
the lysis buffer of the present invention. The liquid may be
organic or inorganic and may be a pure liquid, a mixture of liquids
or a solution of substances in the liquid and may contain
additional substances to enhance the properties of the solvent. Any
liquid that is suitable for solubilizing the cellular components of
body samples in total or in parts may be regarded as a lysis buffer
as used herein.
[0103] In one embodiment, the solvent is designed, so that cells,
cell debris, nucleic acids, polypeptides, lipids and other
biomolecules potentially present in the sample are dissolved. In
further embodiments of the present invention, the solvent may be
designed to assure differential lysis of specific components of the
body sample, leaving other components undissolved.
[0104] In some instances, the lysis buffer of the invention
comprises one or more agents that prevent the degradation of
components within the sample. Such components may for example
comprise enzyme inhibitors such as proteinase inhibitors, RNAse
inhibitors, DNAse inhibitors, nuclease (e.g. endonucleases and
exonucleases) inhibitors, etc. Proteinase inhibitors may e.g.
comprise inhibitors of serine proteinases, inhibitors of cysteine
proteinases, inhibitors of aspartic proteinases, inhibitors of
acidic proteinases, inhibitors of alkaline proteinases or
inhibitors of neutral proteinases. Preferably, the lysis buffer
comprises a cocktail of irreversible and reversible protease,
phosphatase and RNAse inhibitors.
[0105] In addition one or more enzymes such as zymolyase, lyticase,
lysozyme or lysostaphin; one or more inorganic salts such as sodium
chloride, potassium chloride, or lithium chloride; one or more
acids and/or bases or buffering agents (e.g., to increase or reduce
pH); or any other compound or enzyme which may assist in the
disruption of the integrity of (i.e., lyses or causes the formation
of pores in) the cell membrane and/or cell walls (e.g., polymixin
B) can be used.
[0106] The lysis method of the invention can be applied to any
single cell type or a mixture of cell types. The invention is
suitable for use with any cell type, including primary cells,
biopsy tissue, normal and transformed cell lines, transduced cells
and cultured cells, each of which can be single cell types or cell
lines; or combinations thereof.
[0107] Preferably, the single cell is isolated from a tissue. The
tissue may be derived from all sources, particularly mammalian, and
with respect to species, e.g., human, simian, rodent, etc. The
tissue origin can be from heart, lung, liver, brain, vascular,
lymph node, spleen, pancreas, thyroid, esophageal, intestine,
stomach, thymus, etc. The invention should not be limited to the
cell type or tissue type. Rather, the invention should be construed
as being applicable to any cell and any tissue. Also, artificially
constructed 3-D tissue-like structures designed and constructed by
tissue engineering are applicable.
[0108] For example, the invention is useful to examine cell types
that include stem and progenitor cells, e.g. embryonic stem cells,
hematopoietic stem cells, mesenchymal stem cells, neural crest
cells, etc., endothelial cells, muscle cells, myocardial, smooth
and skeletal muscle cells, mesenchymal cells, epithelial cells;
hematopoietic cells, such as lymphocytes, including T-cells, such
as Th1 T cells, Th2 T cells, Th0 T cells, cytotoxic T cells; B
cells, pre-B cells, etc.; monocytes; dendritic cells; neutrophils;
and macrophages; natural killer cells; mast cells; etc.;
adipocytes, cells involved with particular organs, such as thymus,
endocrine glands, pancreas, brain, such as neurons, glia,
astrocytes, dendrocytes, etc. and genetically modified cells
thereof.
[0109] The Inside-Out Lysis method of the invention has advantages
over prior art methods in that the method does not require tissue
fixation/permeabilization and tissue disaggregation.
[0110] In one embodiment, Inside-Out Lysis is completely automated,
as described elsewhere herein. In another embodiment, Inside-Out
Lysis is user-driven with partial automation. In another
embodiment, Inside-Out Lysis is completely user-driven.
Printing Step
[0111] After the lysing step, the lysate can be collected by
suctioning the lysate using a suctioning channel and the lysate can
then be applied (i.e. printed or spotted) onto a solid support
whereby the lysate can be evaluated using the desired technology.
Prior art methods for printing (e.g., piezo-driven printing and
contact printing) are not appropriate for use with the dilute
lysates of the invention because the solvent area in prior art
methods is almost the same as the analyte area, and therefore
resulting in less solvent evaporation per unit time. Prior art
methods are also not appropriate for use with small volumes because
the prior art drop delivery process is not a continuous delivery
process. A drop delivery process results in less solvent
evaporation per unit time and therefore a longer time to print the
entire sample by repeating depositions onto the same spot, which
results in a large part of the small sample volume evaporating
before contacting solid support. That is, the prior art printing
methods are not appropriate for printing diluted single cell
lysates of small volumes (e.g., about 5-15 nl, preferably about 10
nl).
[0112] Accordingly, the invention provides a novel printing for use
with small volumes. In one embodiment, the capillary component of
the printing method of the invention does not have to necessarily
contact the solid support but has to be close enough to ensure
continuous delivery. This method is appropriate for dilute lysates
of the invention because for example, solvent area is 10-100 times
larger than the analyte area, which results in more solvent being
removed by evaporation per unit time during continuous delivery of
the lysate onto a solid support. Preferably the solid support is
porous having a higher affinity to the analyte compared to the
solvent. In another embodiment, the capillary component of the
printing method of the invention is appropriate for small volumes
because the continuous delivery within each deposition results in
more diluted sample being deposited within each deposition, thereby
fewer depositions are necessary which allows for only a small part
of the small sample volume evaporating before contacting the solid
support. Accordingly, the printing method of the invention is
appropriate for printing dilute single cell lysate of small volumes
(e.g., 5-15 nl, preferably about 10 nl).
[0113] In one embodiment, the printing method of the invention is
appropriate for use with the diluted single cell lysates of small
volume that contained limited analyte of the invention. In one
embodiment, the printing method allows for the enrichment of the
analyte on a porous solid support (e.g., nitrocellulose).
Preferably, the enrichment/printing method includes using any
porous solid support that has a higher affinity for an analyte and
a smaller affinity to the solvent or to a non-analyte.
[0114] In one embodiment, the present invention provides a method
of releasing the intracellular contents of at least one cell of a
cell-containing fluid sample for analysis. For example, the lysate
generated from using the Inside-Out Lysis of the invention can be
spotted on a substrate for analysis. In one embodiment, the
invention provides a microfluidic system for transport and lysis of
at least one cell of a cell-containing fluid sample.
[0115] In one embodiment, a customary bent glass micropipette
having about 10 .mu.m aperture is used to take up the single-cell
lysate. In some instances, the aperture is about 9 .mu.m, about 8
.mu.m, about 7 .mu.m, about 6 .mu.m, about 5 .mu.m, about 4 .mu.m,
about 3 .mu.m, about 2 .mu.m, about 1 .mu.m. In one embodiment, the
aperture is 1.5 .mu.m or smaller.
[0116] In order to analyze the protein content of each single cell,
each single-cell lysate so taken up is printed on a solid support.
In some instances, the solid support is a mobilizable material
(such as a metal sol or beads made of latex or glass) or an
immobile substrate (such as glass fibers, cellulose strips or
nitrocellulose membranes). Preferably, the solid support is a
glass-mounted nitrocellulose pad.
[0117] In one embodiment, the invention provides one or more
reservoirs for delivery or collection of a test sample, diluent,
reagent or the like. The microfluidic devices and systems used in
practicing this invention can be made using a variety of substrate
materials, including glass, fused silica and various polymeric
materials, such as PDMS or combinations of such materials.
[0118] The invention includes any surface to which the cell lysate
of the subject invention is attached, where the cell lysate or
fractions thereof are attached in a pre-determined spatial array of
arbitrary shape.
[0119] A variety of solid supports or substrates are suitable for
the purposes of the invention, including both flexible and rigid
substrates. By flexible is meant that the support is capable of
being bent, folded or similarly manipulated without breakage.
Examples of flexible solid supports include acrylamide, nylon,
nitrocellulose, polypropylene, polyester films, such as
polyethylene terephthalate, etc. Also included are gels, e.g.
collagen gels, matrigels, and ECM gels. Rigid supports do not
readily bend, and include glass, fused silica, quartz, plastics,
e.g. polytetrafluoroethylene, polypropylene, polystyrene,
polycarbonate, and blends thereof, and the like; metals, e.g. gold,
platinum, silver, and the like; etc.
[0120] The substrates can be formed in a variety of configurations,
including filters, fibers, membranes, beads, particles, dipsticks,
sheets, rods, etc. Usually, a planar or planar three-dimensional
geometry is preferred. The materials from which the substrate is
fabricated should ideally exhibit a low level of non-specific
binding during binding events, except for specific cases in which
some non-specific binding is preferred.
[0121] In some embodiments, the solid support, is porous and can
be, for example, nitrocellulose (including pure nitrocellulose and
modified nitrocellulose). The nitrocellulose can be the form of
sheets or strips. The thickness of such sheets or strips may vary
within wide limits, for example, from about 0.01 to 0.5 mm, from
about 0.02 to 0.45 mm, from about 0.05 to 0.3 mm, from about 0.075
to 0.25 mm, from about 0.1 to 0.2 mm, or from about 0.11 to 0.15
mm. The pore size of such sheets or strips may similarly vary
within wide limits, for example from about 0.025 to 15 microns, or
more specifically from about 0.1 to 3 microns; however, pore size
is not intended to be a limiting factor in selection of the solid
support.
[0122] In one embodiment, the properties of porous solid support
materials enable fast-binding of analyte molecules. In some
instances, this fast binding of analyte molecules by the porous
solid support material is used for enrichment of analyte molecules
(e.g. proteins) within the geometric confinements of a small (e.g.,
about 5-100 micron) dense spot on porous solid support. Prior art
methods do not enable single cell lysate printing on porous solid
support in dense spots (e.g., signals derived from this spot are
significantly above the noise and background levels of the solid
support material). In one embodiment of the invention, the diluted
lysate is repeatedly spotted on the same spot of solid support such
that the solvent radially spreads to an area many times larger than
the area where analyte is retained by fast binding to porous solid
support material. The solvent then evaporates while the analyte is
retained within the small dense spot of porous solid support. After
solvent evaporation, the deposition of diluted lysate is repeated
onto the same spot in the above manner. Such enrichment enables the
reconcentration of the dilute single cell lysate on porous solid
support, as described elsewhere herein. The reconcentration of the
analytes in the small dense spot makes the contributions of solid
support substance to the overall recorded signal in the analytical
step negligible. This enrichment process and thus the ability to
print single cell lysate in a dense spot on porous solid support,
allows for the analysis of analytes from a single cell lysate
(e.g., the Single-Cell Microarray format).
[0123] In one embodiment, the invention includes the use of a
standard curve. Without wishing to be bound by any particular
theory, when choosing a target, it is useful to look for a protein
that is most up-regulated or down-regulated relatively to all other
proteins in a given pathological sample. Because the slopes of
standard curves vary across different affinity-based probes (such
as antibodies), measured `signals` lead to false choices of protein
targets. Measured quantities, determined by applying the
corresponding standard curves to signals, lead to correct choices
of protein targets. Thus, qualitative observations, whether the
up-regulation or down-regulation of one molecule is more
significant than the up-regulation or down-regulation of another
molecule, are impossible without the knowledge of the corresponding
standard curves.
[0124] In one embodiment, the lysates and standard curves are
printed on a solid support (e.g., nitrocellulose pad), as described
elsewhere herein.
[0125] The standard curves of purified recombinant proteins and/or
control lysates can be printed next to the printed spots of single
cell lysates on the same nitrocellulose pad or on another solid
support. This strategy enables the construction of standard curves
for each dimension of the multiplex measurement. This strategy
enables the construction of several identical standard curves on
the same solid support in order to estimate the noise levels and to
determine the limit of detection. This strategy enables the mapping
of signals recorded from single cell lysates onto the concurrent
standard curves. In one embodiment, the standard curve is printed
with conventional Arrayer because standard curves can be
constructed from large amounts of material. Therefore in some
instances, the diluted single cell lysate is printed according to
the novel printing method of the invention and standard curves are
printed in a conventional way on the same solid support. In this
aspect, the overall printing procedure includes two printing
methods and is different from the printing method used in
conventional lysate microarrays.
[0126] In one embodiment, the method of the invention enables the
determination of the noise levels of single cell for each dimension
of a multiplex single cell measurement. This strategy enables the
determination the signals of any measured dimension of the
multiplex measurement are above the limit of detection.
[0127] If combined with the appropriate `labeling--detection
method` pair (for example lanthanide labeling and LA-ICP-MS are
`labeling--detection method` pair, as discussed elsewhere herein),
this strategy eliminates the disadvantages of prior art methods in
that the present invention does not require the sub-fractioning of
the limited single-cell material for the purpose of multiplexing.
For example, lanthanide labeling combined with LA-ICP MS eliminates
the need for subfractioning of the limited sample. This is because
in the Single-cell Lysate microarray of the invention, many
antibody labels from one spot with the precious sample deposited in
this one spot can be read out by LA-ICP-MS. This strategy also
eliminates the disadvantages of prior art methods in that the
present invention does not require tissue fixation/permeabilization
and tissue disaggregation.
[0128] At the same time, this strategy incorporates the advantages.
Namely, this strategy enables the printing of concurrent standard
curves, the rigorous validation of antibody probes and other
affinity-based probes as in a microarray, as discussed elsewhere
herein. This strategy can be combined any label-label detection
method pair applied to solid supports. For example, the pair of
lanthanide-labeling of antibodies and LA-ICP-MS detection can be
applied, as described elsewhere herein.
[0129] In one embodiment, the solid support can be pre-printed with
the matrix for MALDI analysis.
[0130] In one embodiment, the solid support (e.g., glass) is
prepared by covalently linking other molecular entities to it
before the application of the lysate. Such molecules include but
are not limited to: antibodies, enzymes, protein domains.
[0131] The lysate can also be released into a fluid for further
analysis. The lysate can then be analyzed after being released into
a fluid by analytical methods.
[0132] Lysates can be pooled together into one fluid volume. For
example, the lysates from cells of a cell type can be combined in
one fluid volume (e.g., another buffer) in order to increase the
total analyte content. By pooling many lysates together into one
fluid volume, the analysis by analytical methods with higher sample
requirements can be achieved. The fluid volume with pooled lysates
can also be concentrated by evaporation.
[0133] The lysate can be applied to an analytical device (e.g., the
mass spectrometer) without prior printing on solid support or prior
releasing it into a fluid.
Analytical Step
[0134] The Inside-Out Lysis method of the invention facilitates the
sampling of a single-cell lysate obtained from a live solid tissue
with high temporal and spatial resolution. The Inside-Out Lysis
enables analysis of the "complete molecular state" in each sampled
single cell, because the lysate format enables the analysis within
and across any classes of molecules. The mixed lysate can be
subdivided into parts, such that each part is analyzed with a
different method. The entire lysate can also be analyzed with just
one analytical method. Alternatively, the entire lysate can be
analyzed with one method and then analyzed with another method
sequentially. More than two analytical methods can be applied
sequentially to the same lysate.
[0135] In one embodiment, the native proteins of the entire single
cell lysate are enriched within a dense spot on porous solid
support (as described in the Printing Step) and are analyzed with
Single-Cell Lysate microarrays, as described elsewhere herein. More
than one single cell lysate, each derived from one cell, can be
analyzed on the same microarray. Unlike any prior art methods,
Single-Cell Lysate Microarrays enable multiplex measurements of
native proteins in single cells with concurrent standard
curves.
[0136] In the context of single cell lysates, the lysate is kept in
a single spot for the purpose of multiplexing proteins.
Accordingly, the invention is partly based on the development of a
way to multiplex proteins in one single spot without sample
subdivision by using the `lanthanide labeling--LA-ICP-MS detection`
pair as the `labeling--label detection method` pair.
[0137] In one embodiment, other `labeling--label detection method`
pairs (other than lanthanide-LA-ICP-MS) can be used in the format
of Single-Cell Lysate Microarrays for detection of proteins or
other molecules of other molecular classes. For example, nucleotide
sequences can be conjugated to antibodies instead of lanthanide
chelators and multiplexing could be achieved with RT-qPCR reaction
instead of LA-ICP-MS.
[0138] In another example, mRNA is also bound to nitrocellulose and
can be enriched in the same manner as proteins within a small dense
spot on nitrocellulose. qPCR reaction could be run to detect
abundances of mRNA.
[0139] In one embodiment, the native proteins of a partition of the
single cell lysate are analyzed with Single-Cell Lysate
microarrays. The other partition of the single cell lysate is
analyzed with another method (e.g. RT-qPCR for transcript analysis,
or MALDI for metabolite analysis) or is further subdivided into
subpartitions. This process can be continued until the required
number of subpartitions is prepared. Each subpartition can then be
analyzed with a different analytical method. Subpartition of the
lysate can be analyzed with the same analytical method in order to
establish that each subpartition reliably represents the overall
lysate. As analytical methods become more sensitive and as the
limit of detection of analytical methods improves, more
subpartitions can be generated from one single cell lysate and
analyzed with different analytical methods.
[0140] In one embodiment, the invention allows for multiplexing
across molecule classes. For example, the lysate of the invention
can be subdivided. In this situation, subdividing for the purpose
of using different analytical methods on sub-fractions is preferred
in contrast to subdividing for the sole purpose of multiplexing
within a molecule class.
[0141] In other embodiments, any other analytical methods
compatible with the lysate format can be used for analysis of the
lysate. In addition, the lysates of single cells of a cell type can
be pooled in a volume of liquid and then applied to analytical
methods. In other embodiments, the lysate of single cells of a cell
type can be pooled in a volume of solvent (e.g., buffer) and the
concentration of analyte can be increase by solvent evaporation
before applying to an analytical method. Analytical methods
compatible with the lysate format include but are not limited to:
mass spectrometry methods, PCR based methods, sequencing based
methods etc.
[0142] The methods and compositions of the invention provide, but
are not limited to one or more of the following attributes: (1)
small sample and antibody requirements and (2) scalable and
amenable to robotic automation and multiplexing. Expression and
post-translational modifications of signaling proteins can be
probed on a single support yielding quantitative expressional data
on distinct proteins and the phosphorylation levels of unique
modification sites.
[0143] Methods and kits are provided for a multiplexed protein
microarray platform, which is utilized for simultaneous monitoring
of cellular components. Of particular interest are components
affected by post-translational modification, and more particularly
signaling pathway components. The microarray comprises single cell
lysates, where the cells are from a live tissue.
[0144] The invention permits the rapid and large-scale diagnostic
screening of altered protein post translational modification (PTM)
and PTM alteration states. The methods involve, in part, applying
concentrated cell extracts or biological fluid samples from a
single cell to different analytical tests and appropriately
supplementing them to carry out one or more specific PTM or PTM
alteration reactions. Specifically, one or more PTM or PTM
alterations are then detected by labeling the modified proteins and
scanning the array.
[0145] Covalently modified proteins, such as polyubiquitinated,
ubiquitinated, phosphorylated, glycosylated, sumoylated,
acetylated, S-nitrosylated or nitrosylated, citrullinated or
deiminated, neddylated, OC1cNAc-added, ADP-ribosylated, methylated,
hydroxymethylated, fattenylated, ufmylated, prenylated,
myristoylated, S-palmitoylated, tyrosine sulfated, formylated, and
carboxylated proteins are hard to identify by the standard
biochemical technique of gel electrophoresis, because the modified
protein bands spread throughout the gel. Identifying the converse
alteration of a PTM, such as, for example, deubiquitination (DUB),
dephosphorylation, deglycosylation, desumoylation, deacetylation,
deS-nitrosylation or denitrosylation, decitrullination or
dedeimination, deneddylation, removal of OClcNAc,
de-ADP-ribosylation, demethylation, de-hydroxylation,
defattenylation, deufmylation, deprenylation, demyristoylation,
de-S-palmitoylation, tyrosine desulfation, deformylation,
decarboxylation, and deamidation is similarly difficult to detect
using such standard biochemical methods. In contrast, with the
present methods described herein, a PTM or PTM alteration reaction
is performed directly on a solid state array or the use of
multiplex formats, such as lysate microarrays, also makes possible
the simultaneous analysis of thousands of proteins. Thus, the
present invention overcomes previous obstacles for identifying
altered PTM or PTM alteration states.
[0146] A variety of mass spectrometry systems can be employed in
the methods of the invention for identifying and/or quantifying the
single cell lysate. Mass analyzers with high mass accuracy, high
sensitivity and high resolution include, but are not limited to,
ion trap, triple quadrupole, and time-of-flight, quadrupole
time-of-flight mass spectrometers and Fourier transform ion
cyclotron mass analyzers (FT-ICR-MS). Mass spectrometers are
typically equipped with matrix-assisted laser desorption (MALDI)
and electrospray ionization (ESI) ion sources, although other
methods of peptide ionization can also be used. In ion trap MS,
analytes are ionized by ESI or MALDI and then put into an ion trap.
Trapped ions can then be separately analyzed by MS upon selective
release from the ion trap. Proteins can be analyzed, for example,
by single stage mass spectrometry with a MALDI-TOF or ESI-TOF
system. Methods of mass spectrometry analysis are well known to
those skilled in the art (see, for example, Yates, J., 1998 Mass
Spect 33:1-19; Kinter and Sherman, 2000 Protein Sequencing and
Identification Using Tandem Mass Spectrometry, John Wiley &
Sons, New York; Aebersold and Goodlett, 2001 Chem. Rev.
101:269-295; Banez et al, 2005 Curr Opin Urol 15:151-156). For high
resolution protein separation, liquid chromatography ESI-MS/MS or
automated LC-MS/MS, which utilizes capillary reverse phase
chromatography as the separation method, can be used (Yates et al.,
1999 Methods Mol. Biol. 112:553-569).
[0147] In one embodiment, the assay method is mass spectroscopy.
Mass spectroscopy can include but is not limited to GC/MS, LC/MS,
LC/MS/MS, MALDI-TOF, LC-ESI-MS/MS, MALDI-MS, tandem MS, TOF/TOF,
TOF-MS, TOF-MS/MS, triple-quad MS, and triple-quad MS/MS.
[0148] In another embodiment, the single cell lysate may be
analyzed using immunoaffinity based assays such as ELISAs, Western
blots, and radioimmunoassays. Other methods useful in this context
include isotope-coded affinity tag (ICAT) followed by
multidimensional chromatography and MS/MS.
[0149] In one embodiment, the assay component of the analytical
stage of the invention may be an immunoassay such as ELISA, EIA,
RIA, lateral flow and flow-through formats.
[0150] In one embodiment, the single cell lysate can be assayed by
applying it to RT-qPCR, RNA-Seq, MALDI-MS, among others.
[0151] The methods of the invention include detection and analysis
of PTMs and as well as the expression level of the protein using
any composition or agent that can be detected by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means, thus providing a detectable signal to identify the
PTM protein level. A PTM and protein expression level can be
detected using the methods described herein, for example, if there
is a change in the average number of a given chemical group
attached per protein molecule, if there is a change in the type of
chemical group or groups attached per protein molecule, or if there
is a different mixture of protein molecules having distinct
modification patterns in a patient sample with respect to a control
sample. Alteration of a PTM state of a protein includes going from
an unmodified protein to a modified one and vice-versa, as well as
changes in the number or type of chemical moieties added to the
protein. A control sample or level is used herein to describe a
control patient, control or reference data, or data obtained from
the same patient at an earlier time. For example, in some
embodiments, a control sample is a functional cell extract obtained
from a biological sample obtained from a subject not suffering from
the disease being examined in the test sample.
[0152] Accordingly, in some embodiments, an increase in the signal
from a solid-state array compared to a background or the reaction
with a control is indicative of increased PTM or protein expression
level. The terms "increased," "increase," or "enhance" are all used
herein to generally mean an increase by a statically significant
amount; for the avoidance of any doubt, the terms "increased,"
"increase," or "enhance" mean an increase, as compared to a
reference level, of at least about 10%, of at least about 15%, of
at least about 20%, of at least about 25%, of at least about 30%,
of at least about 35%, of at least about 40%, of at least about
45%, of at least about 50%, of at least about 55%, of at least
about 60%, of at least about 65%, of at least about 70%, of at
least about 75%, of at least about 80%, of at least about 85%, of
at least about 90%, of at least about 95%, or up to and including a
100%, or at least about a 2-fold, or at least about a 3-fold, or at
least about a 4-fold, or at least about a 5-fold, at least about a
6-fold, or at least about a 7-fold, or at least about a 8-fold, at
least about a 9-fold, or at least about a 10-fold increase, or any
increase of 10-fold or greater, as compared to a control sample or
level.
EXPERIMENTAL EXAMPLES
[0153] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0154] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out the
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
Example 1
Single-Cell Sampling in Solid Tissues
[0155] The results presented herein demonstrate the successful
development of a novel sampling method that allows for obtaining
single-cell lysates from a live complex solid tissue having high
temporal and spatial resolution.
[0156] A focally directed flow of a detergent-containing lysis
buffer was applied to the cell body of the target cell in live
tissue (FIGS. 7, 26, and 31). The diameter of the flow was smaller
than the diameter of the cell body. The concentration of the
detergent was above its critical micellar concentration (CMC)
value. These two properties enabled the applied lysis buffer to
enter the intracellular space without spilling to the adjacent
cells in the tissue. Inside the cell, the detergent was diluted to
a concentration value that was below its CMC value, allowing the
lysis buffer to accumulate in the intact intracellular space and to
diffuse throughout the complex shape of the target cell without
affecting its complex tissue surroundings. Once enough lysis buffer
accumulated inside the target cell and the detergent concentration
re-approached its critical value, the cell membrane was lysed from
inside ("Inside-Out Lysis") and the lysate was immediately up-taken
by a nearby suction channel. The estimated volume of the obtained
single-cell lysate is approximately 5-10 nL. The lysis buffer
contains a cocktail of irreversible and reversible protease,
phosphatase and RNAse inhibitors. Once the lysis buffer has entered
the intracellular space, it mixes and homogenizes the intracellular
components. Within 10-20 seconds and with perfect spatial
resolution, the Inside-Out Lysis method converts a single live cell
of any shape in complex solid tissue to a mixed lysate solution
with all classes of molecules preserved (FIG. 7).
[0157] The materials and methods employed in these experiments are
now described.
Materials and Methods
[0158] Imaging Setup
[0159] The sampling setup was built on the basis of an Olympus
BX51WI fluorescence microscope, supplemented with a Hamamatsu
Orca-R2 camera and two fast filter wheels (Sutter Instrument
Company) for both the excitation channel and the emission channel.
Both filter wheels were controlled by the Lambda 10-3 controller
(Sutter Instrument Company). The light path in the excitation
channel was controller by a SmartShutter.TM. (acquired from Sutter
Instrument Company). The imaging process was controlled by the
Micromanager software.
[0160] The following water immersion objectives were used: Olympus
10.times. (UMPLFLN 10XW, NA 0.3, working distance 3.5 mm),
20.times. (UMPLFLN 20XW, NA 0.5, working distance 3.5 mm) and
40.times. (LUMPLFLN 40XW, NA 0.8, working distance 3.3 mm) These
objectives were chosen because of the acceptable working distance
given the high numerical apertures. The imaging setup was mounted
on and firmly fixed to a TMC air-pressure table to avoid any
vibrations.
[0161] Animals and Organotypic Cultures
[0162] All animal procedures were in accordance with Harvard
Medical School (HMS) regulations and under an active animal
protocol. GIN mice were purchased from Jackson labs. GAD67-GFP mice
were obtained from the Murthy lab (Harvard University). GIN mice
were used for most experiments. The standard protocol for
hippocampal organotypic cultures was used (Stoppini et al., 1991 J
Neurosci Methods. 37(2): 173-182). Mouse pups (postnatal day 5 or
6) were subject to hypothermia and were decapitated. The
hippocampus was then dissected in the Gey's Balanced Salt Solution
supplemented with 6.5 g/L glucose. 300 .mu.m thick slices of
hippocampus were obtained by using a manual tissue slicer. The
obtained hippocampus slices were then quickly transferred to the
Millipore inserts (acquired from Millipore; 0.4 .mu.m height, 30 mm
hydrophilic PTFE) and cultured in the incubator under the following
conditions: 5% CO.sub.2, 37.degree. C. The culturing medium
contained heat-inactivated horse serum and was prepared as follows:
50 ml MEM 2x, 120 mg Tris, 910 .mu.L of a 7.5% NaHCO.sub.3
solution, 50 ml heat-inactivated horse serum, 50 ml 1.times.HBSS
and ddH.sub.2O (added up to 200 ml total volume). Hippocampus
slices were serum deprived for 10 hours before lysing single cells.
Hippocampus slices were successfully cultured for periods of time
exceeding one month with excellent preservation of morphological
and tissue-organizational characteristics.
[0163] A customized recording chamber for maintaining live tissue
during the Inside-Out Lysis process, was casted from Sylgard.RTM.
184 (Dow Corning Corporation). The Millipore inserts with live
organotypic slices could be inserted and removed from this chamber
easily. The tissue slices were perfused in a heated HBSS solution.
Perfusion was stopped during and immediately after the Inside-Out
Lysis process.
[0164] Inside-Out Lysis Setup
[0165] Micropipettes were pulled from borosilicate glass tubes with
filament (Sutter Instrument Company; OD:1.0 mm, ID: 0.78 mm) with
the P-1000 Micropipette Puller (Sutter Instrument Company).
Customization of micropipettes was achieved with the assistance of
a microforge (Narishige MF-900).
[0166] Lysis buffer was prepared as described elsewhere herein.
Before loading the delivery micropipette, the cocktails of
inhibitors (Halt.TM. Protease Inhibitor Cocktail 3X, Halt.TM.
Phosphatase Inhibitor Cocktail 3X) were added to the lysis buffer
and the lysis buffer was kept on ice. AlexaD555 (MW 10,000) was
purchased from Invitrogen. SR101 was purchased from Sigma.
[0167] A set of three MP-285 and one MP-225 micromanipulators were
acquired from Sutter Instrument and were integrated on the basis of
two interconnected MPC-200 controllers (Sutter Instrument Company).
The master MPC-200 controller was then interfaced with the GUI in
Microsoft Visual Studio via the USB port and via the appropriate
C++ libraries for USB port control. All the manipulators were
arranged and aligned in such a way that quick switching between the
Inside-Out Lysis procedure and the printing procedure could be
completed within a few seconds (FIG. 29). The pressure controller
was acquired from MicroData Instrument and was interfaced with the
GUI over the parallel port and the appropriate C++ library for
parallel port control.
[0168] The recording chamber was positioned on one MP-285
manipulator. All glass slides with nitrocellulose pads were
acquired from GraceBioLabs. The glass slide with a nitrocellulose
pad was attached to another MP-285 manipulator. The delivery
micropipette was mounted on the MP-225 manipulator and the
suction/printing micropipette was mounted on the third MP-285
manipulator. A set of movements was preprogrammed to quickly switch
from the recording chamber to the nitrocellulose slide after the
completion of Inside-Out Lysis. A set of movements was
preprogrammed to quickly retract the uptake micropipette out of the
recording chamber and to position it a few microns above the
nitrocellulose pad just before printing. Printing was conducted
manually but can also be automated. All these movements were
completed within a few seconds. While imaging the Inside-Out Lysis
process on one cell, the 40.times. objective was used. When imaging
the automated version of Inside-Out Lysis on several cells, the
20.times. objective was used. The 10.times. objective was used for
printing single-cell lysates on nitrocellulose and for imaging the
overviews of whole tissue slices. The sub-micron resolution and
programmability of the used micromanipulators allowed for single
cell lysis and printing the single-cell lysates in a reproducible
fashion as described elsewhere herein.
[0169] Printing Concurrent Titration Series
[0170] Nitrocellulose slides were acquired from GraceBioLabs. 2470
Aushon Arrayer was acquired from Aushon Biosystems in order to
print the titration series on the nitrocellulose slides next to
single-cell lysates. GFP protein was acquired from Millipore
(14-392). PKC.delta. and PKC.alpha. were acquired from Invitrogen
(P2287, P2227). All purified proteins were diluted in the same
SDS-containing buffer that was used for single-cell lysis and were
printed in 1:2 dilution series on the nitrocellulose pads before
printing single-cell lysates on the same nitrocellulose pads. Eight
depositions per spot were used in the settings of the
high-precision Aushon Arrayer in order to reach a homogenous
distribution of proteins within each printed 200 .mu.m spot of the
titration series.
[0171] Antibodies
[0172] The protocol for washing nitrocellulose slides with printed
lysates and for subsequent antibody incubation as described by
Sevecka et al. was used (Sevecka and MacBeath, 2006, Nat. Methods
9:152-158; Sevecka et al., 2011, Mol. Cell Proteomics
10:M110.005363). The protocol for washing the printed
nitrocellulose slides (FIG. 30) and for subsequent antibody
incubation was used as described by Sevecka et al. (Sevecka et al.,
2011, Mol Cell Proteomics. 10(4): M110.005363; Sevecka and
MacBeath, 2006, Nat Methods. 3(10): 825-831). The nitrocellulose
slides were shortly washed in PBST first and were then washed in
Tris buffer (pH 9) for 48 hours. The slides were then blocked in 5%
BSA/PBST of the blocking solution provided by LiCor (Odyssey
Blocking Buffer) at 4.degree. C. for 1 hour. Next, the slides were
incubated with the primary antibodies. The primary .beta.-actin
antibody (A1978, Sigma) and the primary GFP antibody (2956, Cell
Signalling Technology) were incubated at 1:1000 in 5% BSA/PBST or
in Odyssey Blocking Buffer (LiCor) for 24 hours at 4.degree. C.
Subsequently, the secondary antibodies (anti-rabbit 680 and
anti-mouse 800) were applied at 1:1000 for 12-24 hours at 4.degree.
C. The nitrocellulose slides were then quickly washed in PBST
several times and were scanned on a LiCor Odyssey scanner
(LiCor).
[0173] The results of the experiments are now described.
Detergent-Based Lysis
[0174] The sampling method of the present invention is applicable
to live solid tissues. Therefore, the sampling method of the
invention circumvents the disaggregation process and eliminates the
artifacts and the biases of various fixation protocols. This
sampling method also preserves transcripts, proteins and
metabolites by incorporating the technical advantages of
detergent-based lysis (Table 1).
TABLE-US-00001 TABLE 1 Comparison Between Fixation and
Detergent-Based Lysis Fixation/Staining/Permeabilization
Detergent-Based Lysis of Live Cells Mechanism parameters are
Biophysical parameters of unknown. detergent/protein and
detergent/membrane interactions are known. Molecular preservation
varies All proteins, transcripts, metabolites across cell types are
preserved by protein denaturation across molecule classes and by
protease/RNAse/phospho across molecules within each inhibitors.
class. Incompatible with analytical Compatible with MS, Western
Blot, methods, unless reduced to Lysate Microarrays, RT-qPCR and
the lysate format. Sequencing methods across all proteins,
transcripts and metabolites.
[0175] The mixed lysate format and the preservation of all the
molecules of the target cell enables the measurement of the
`complete molecular state` in each sampled single cell by applying
RT-qPCR (or RNA-Seq), LA-ICP-MS and MALDI-MS to the sub-partitions
of the homogenous diluted lysate (FIG. 3).
[0176] The need for maintainable live tissue samples has been more
acute in neuroscience than in any other field of the life sciences.
Because of the loss of tissue context, it has been difficult to
study the relationship between neuronal morphology and neuronal
function in dissociated neuronal cultures. Dissociated neuronal
cultures also make it challenging to study the time course of
molecular and morphological development of many neuronal cell
types. Because of this acute need for maintainable live tissue
systems, organotypic cultures were developed and widely adopted in
neuroscience before the need for such live tissue systems emerged
in other areas of basic biological research.
[0177] Organotypic cultures of rodent brain regions are
maintainable for months in an incubator. After quickly dissecting
and slicing the mouse brain region of interest in an optimized
procedure, one can culture the harvested slices on a porous
membrane in a specialized dish (Stoppini et al., 1991, J. Neurosci.
Methods 37:173-182). An example of such a setup is displayed in
FIG. 4A. In this setup, the medium does not surround the tissue
slice but is up-taken from underneath the porous membrane by the
capillary action of the tissue slice. The medium can be either
serum-based or serum-free. Hippocampus, cortex, thalamus and
cerebellum can be cultured successfully in this manner (Gahwiler et
al., 1997, Trends Neurosci. 20:471-477; Banker and Goslin, 1998,
Culturing Nerve Cells, second edition).
[0178] The time course of development and the associated
molecular/morphological changes in organotypic cultures of rodent
brain regions resemble the time course of development and the
associated molecular/morphological changes in vivo (in live
rodents). For example, the onset of long term potentiation (LTP) in
vivo occurs at the end of the second postnatal week in rats (Muller
et al., 1993, Brain Res. Dev. Brain Res. 71:93-100). In a study by
Muller et al., the organotypic hippocampal cultures prepared from
8-days-old rats showed a much faster onset of LTP than the
organotypic hippocampal cultures prepared from 2-days-old rats. In
both cases however, the onset of LTP occurred at the time
equivalent to the time point of 12-14 postnatal days in vivo
(Muller et al., 1993, Brain Res. Dev. Brain Res. 71:93-100). This
observation suggests that the natural course of molecular
development is preserved in organotypic cultures. This observation
is consistent with the theory that the course of molecular
development in organotypic cultures is not a result of the
preparation procedure. The time course of synaptogenesis in
organotypic hippocampal cultures also resembles the time course of
synaptogenesis in vivo, as assessed by comparative
electrophysiological and morphological measurements in organotypic
cultures and in the acute brain slices of rats of different ages
(Gahwiler et al., 1997, Trends Neurosci. 20:471-477; Muller et al.,
1993, Brain Res. Dev. Brain Res. 71:93-100). In the same study by
Muller et al., the spatial distribution of synaptogenesis rates
across different regions of hippocampus in organotypic cultures
resembled the spatial distribution of synaptogenesis rates in vivo
(Muller et al., 1993, Brain Res. Dev. Brain Res. 71:93-100).
[0179] The organotypic cultures of human colon, lung and prostate
tumors were recently established and optimized (Vaira et al., 2010,
Proc. Natl. Acad. Sci. USA 107:8352-8356). The culturing procedure
of human tumors is similar to the above-described procedure used
for culturing rodent brain tissue. Vaira showed that the spatial
organization and morphology of cultured human tumors was preserved
in organotypic cultures for 5 days after explantation and resembled
the original tissue organization. During this culturing period of 5
days, the counts of proliferating and apoptotic cells did not
change either. Vaira also showed that pharmacological and
analytical studies can be performed in the organotypic cultures of
human tumors. The success of culturing various human tumors
demonstrates that the methodology of organotypic cultures is
general and can be applied to different organs in humans and
animals.
[0180] Detergents have widely been used for cell lysis in
analytical biochemical studies. All rigorous analytical methods
require the lysate format of the sample. Table 1 summarizes the
advantages of detergent-based cell lysis as compared to cell
fixation. There exist a large number of biophysical studies
describing the parameters of detergent/protein interactions and the
parameters of detergent/membrane interactions (Helenius and Simons,
1975, Biochim. Biophys. Acta 415:29-79; Lichtenberg et al., 1983,
Biochim. Biophys. Acta 737:285-304).
[0181] Detergents belong to the molecular class of amphiphilic
lipids. Each detergent monomer contains a polar region (head) and a
non-polar region (tail). The latter usually consists of alkyl
chains and/or aromatic groups. The size of the tail of a detergent
monomer determines its interaction area with water. The interaction
area of the tail with water determines the overall hydrophobicity
of the amphiphilic detergent due to the decrease of entropy in
water. Generally, amphiphilic lipids with high hydrophobicity
(phospholipids and cholesterol) are not soluble in water, whereas
amphiphilic lipids with lower hydrophobicity (detergents) are
soluble and have a critical micellar concentration (CMC). When the
total concentration of a given detergent reaches its CMC value, the
addition of any excess detergent molecules to the solution is
compensated by the process of micelle formation such that the
concentration of free detergent monomers does not exceed the CMC
value. The CMC value of a detergent is decreased by higher
hydrophobicity, increased by the bulkiness of the tail region, and
is also increased by the charge of the head region. Therefore,
different detergents have different CMC values. The CMC value of a
given detergent and the number of detergent molecules in micelles
(aggregation number) also depend on the temperature, the ionic
strength and the pH value of the solution.
[0182] The interactions of detergents with biological membranes
have been studied extensively with the help of the liposome model
(FIG. 6). When the total concentration of detergent molecules is
increased above the CMC value in a solution with liposomes, the
concentration of free detergent monomers stays at the level below
the CMC value because the excess detergent molecules integrate into
the phospholipid bilayer of liposomes. As the total concentration
of detergent molecules is further increased, the number of
detergent molecules in the phospholipid bilayer of liposomes
reaches the saturation level, thereby causing the formation of
mixed micelles. These mixed micelles contain liposomal
phospholipids and detergent molecules. By even further increasing
the total concentration of detergent molecules in the solution, all
liposomes are solubilized into mixed micelles. Thus, at each
concentration of detergent in the solution there is an equilibrium
between the free monomers and the monomers integrated into the
phospholipid bilayer and there is an equilibrium between the
detergent monomers integrated into the phospholipid bilayer and the
formation of mixed micelles. The micelle formation process is fast
(rate constant of 10 s.sup.-1). Empirically, it was also shown that
the presence of detergents, such as sodium dodecylsulphate (SDS),
increases the membrane area of erythrocytes and protects
erythrocytes against osmotic shock.
[0183] Micelle formation is crucial for the solubilization of
biological membranes but is not crucial for the binding of
detergents to soluble proteins. Detergents bind to soluble proteins
as monomers at concentrations below the CMC value. Ionic detergents
such as SDS generally have a denaturing character. They first bind
the hydrophobic patches on the protein surface and thereby start
unfolding the protein. The unfolding process then exposes more
hydrophobic patches. Thus, the overall process of detergent-induced
denaturation is cooperative. The detergent molecules bound to
soluble proteins are not available for micelle formation and the
detergent molecules bound to micelles are not available for protein
binding. Thus, the process of micelle formation and the process of
protein binding are competitive.
[0184] The most widely studied detergent is SDS. SDS is an anionic
detergent that denatures soluble proteins into straight polypeptide
regions and has a constant binding ratio in most proteins.
Importantly, SDS does not denature some crucial proteases even at
concentrations above the CMC value. When SDS is used for cell
lysis, it is common to add a cocktail of reversible and
irreversible protease and kinase/phosphatase inhibitors. CMC values
of SDS widely vary depending on the composition of the solution. In
pure water, the CMC value of SDS is 8.2 mM. In 0.5M NaCl solution,
its CMC value is 0.52 mM. FIG. 5 displays the
concentration-temperature phase diagram of SDS. This phase diagram
also shows that SDS is not present in the crystalline form at
biological temperatures (above critical micellar temperature).
Mechanism of Inside-Out Lysis
[0185] The following lysis buffer formulation was used: Tris-HCl50
mM, SDS 2%, Glycerol 5%, NaF 1 mM, 0.5 mM AlexaD555 (10,000),
Halt.TM. Protease Inhibitor Cocktail 3X, Halt.TM. Phosphatase
Inhibitor Cocktail 3X. In several initial experiments,
sulforhodamine 101 (SR 101) was used instead of Alexa 555 dextrans.
Two mouse strains expressing cytosol-soluble GFP in different
subsets of hippocampal interneurons were used in the experiments.
These mouse strains were: GAD67-GFP (broad set of putative
interneurons), GIN (somatostatin interneurons). Experiments were
performed in organotypic hippocampus slices of either GAD67-GFP or
GIN mice.
[0186] The Inside-Out Lysis process was imaged by acquiring the
frames of GFP and Alexa 555 fluorescent intensities with the aid of
a rapid filter wheel attached to the microscope. The delivery
process of lysis buffer was optimized by performing different
configurations of pulled glass micropipettes. The tip aperture of
the delivery pipette that was used in the experiments was 0.7
.mu.m. The lysis buffer was added to the target cell at an angle or
from above (vertical application). The latter configuration was
achieved by carefully crafting the delivery micropipette and
bending its taper such that the focal stream of lysis buffer was
perpendicular to the surface of the tissue slice. In some
instances, attempts were made to prevent the tip of the delivery
micropipette from touching the surface of the target cell at the
time of delivery initiation.
[0187] In the experiments where the final uptake of the single-cell
lysate was performed, a customary bent glass micropipette having a
10 .mu.m aperture was used. The flow vector generated by this
suction micropipette was perpendicular to the surface of the tissue
slice. The aperture of the suction micropipette was positioned 20
.mu.m above the surface of the tissue slice and directly above the
target cell. No active negative pressure was applied, as suction
was generated solely by the capillary action of the glass
capillary. Pulled glass micropipettes were used because of their
low cost and easy customization.
[0188] Rapid denaturation of GFP was observed as soon as the
SDS-containing lysis buffer entered the intracellular space of a
GFP-expressing target cell. In both mouse strains (GAD67-GFP and
GIN), GFP freely diffused throughout the cytosol. And because of
its relatively small size, GFP also entered the nucleus. GFP
fluorescence decreased to background levels immediately after the
SDS-containing lysis buffer entered the intracellular space (FIGS.
8, 10, 19, 27, and 28). In these instances, GFP fluorescence
continued to decrease while the Alexa 555 dextran (or SR101)
molecules were located strictly within the geometric boundary of
the single cell. Thus, GFP denaturation seemed to occur
intracellularly while the membrane barrier was still intact. GFP
fluorescence vanished before the cell membrane was solubilized.
FIG. 8 shows the delivery of the SDS-containing lysis buffer to
single cells without simultaneous suction (FIG. 9 depicts lysing
cells in a fully automated sequential microlysis procedure). In all
instances depicted in FIG. 8, GFP fluorescence started decreasing
at the same moment when the lysis buffer entered the target cell
and before the lysis buffer completely filled out the intracellular
space by diffusion/convection. These observations were consistent
with biophysical studies showing that detergent monomers bind and
denature soluble proteins even at concentrations below the CMC
value, which are discussed elsewhere herein.
[0189] In all instances of Inside-Out Lysis, a visible increase of
membrane area was observed as the lysis buffer was accumulating
within the geometric boundaries of the target cell (FIG. 10). This
observation was consistent with the integration of SDS monomers
into the phospholipid bilayer of the target cell before saturating
it and before causing the formation of mixed micelles. This
increase of membrane area simultaneously accompanied the decrease
of GFP fluorescence inside the growing cell. This was consistent
with the concept that protein binding by detergent monomers and
membrane solubilization are competitive processes.
[0190] Importantly, Alexa 555 dextran molecules (10,000 MW) of
SR101 molecules did not exit the geometric confinements of the
target cell when the lysis buffer was continuously applied to its
intracellular space. Although not wishing to be bound by any
particular theory, this suggests that the membrane barrier was
intact during the phase of membrane growth. The solubilization of
the cell membrane could be observed after its initial significant
growth, as Alexa 555 dyes were observed to leave the geometric
confinements of the target cell (FIG. 12). These observations were
consistent with studies describing the solubilization process of
biological membranes by detergents, which are discussed elsewhere
herein.
[0191] In some instances, the stream of lysis buffer/solubilizing
solution is preferably narrow initially and the aperture of the
application channel is around 1.5 .mu.m or smaller. Without wishing
to be bound by any particular theory, it is believed that if the
aperture of the application channel is larger, then the detergent
flow is harder to direct towards the center of the cell and to
create a focused intracellular source. Detergents are then more
likely to reach the outer membrane and to compromise it too early,
before solubilizing the nucleus.
[0192] Another important feature related to detergents is that the
outer cell membrane increases in size (grows) as detergent monomers
incorporate into it from inside. As the membrane grows, it is still
intact and still preserves the intact barrier between the inside of
the cell and the tissue surroundings. This feature effectively
increases the available volume inside the cell and allows
accumulation of the applied liquid inside the cell.
Different Modes of Membrane Solubilization
[0193] Next, the different modes of membrane solubilization were
examined. As discussed elsewhere herein, the process of
mixed-micelle formation is fast and occurs at the membrane
locations where the membrane of the target cell is saturated with
detergent monomers. It was reasoned that the total amount of the
delivered lysis buffer should be critical in determining the extent
to which the membrane of the target cell would be solubilized.
First, a smaller amount of lysis buffer was delivered to single
cells without active lysate uptake. It was observed that the
delivered SR101 dye slowly diffused out of the geometric
single-cell boundary, into the live tissue or out of the live
tissue, over the above time periods. The cell leftovers resembled
the patches of the cell membrane (FIG. 11). This observation was
consistent with the concept that the SDS-based lysis buffer can
solubilize the membrane of a single cell following its
intracellular delivery.
[0194] In the next set of experiments, it was reasoned that if
enough lysis buffer were applied continuously, the membrane of the
target cell would be solubilized more rapidly at some point during
the continuous delivery. FIG. 12 shows how when a smaller amount of
lysis buffer was first applied to the target cell, no immediate
solubilization was observed. After continuously applying more lysis
buffer to the same target cell in the later frames of FIG. 12, the
membrane was observed to be rapidly solubilized. During the
solubilization process, Alexa 555 dextrans were observed to exit
the geometric confines of the cell and were eventually up-taken by
the nearby capillary-action-driven suction channel (FIG. 12).
[0195] Single cells have different membrane areas and different
volumes. A smaller cell should require less detergent to be lysed
from inside. Importantly, the Inside-Out Lysis method first
delivers the detergent-based lysis buffer into the intracellular
space of the target cell. Thus, the Inside-Out Lysis method induces
the denaturation of soluble proteins and the homogenization of the
intracellular organelles in parallel with the detergent integration
into the phoshpolipid bilayer of the membrane that precedes the
formation of mixed micelles. There is a competition for detergent
monomers between the intracellular soluble proteins and the cell
membrane of the target cell. Thus, different levels of protein
expression across single cells will also affect the amount of lysis
buffer required for a complete and rapid solubilization of the cell
membrane.
[0196] Without wishing to be bound by any particular theory, it is
believed that any solubilization and/or chemical dissociation
processes should be compatible with this method. From the chemical
perspective, dissociation is distinct from solubilization.
Solubilization is usually accomplished with detergents. However,
different PH values, salt(s), other chemicals, and different
concentrations could influence not only the solubilization process
itself but also molecular interactions between molecules, resulting
in dissociation. For example, urea and other denaturing or reducing
(DTT) chemicals could be added. Strictly speaking, these chemicals
lead to dissociation reactions that then facilitate solubilization.
Accordingly, the present invention encompasses all processes that
dissociate and/or solubilize cell components directly, and all
processes that facilitate the aforementioned dissociation and
solubilization processes.
[0197] Sometimes it is not desirable to completely solubilize the
outer cell membrane. Instead it may be desirable to use it as a
barrier first to solubilize/dissociate the intracellular components
and then to extract the resulting liquid through a small opening in
the membrane while keeping the overall membrane barrier still
intact or mostly intact. The presence of the mostly intact overall
membrane barrier would prevent contamination of the extract by
other soluble molecules in the surrounding tissue. For a cell
located deep inside the tissue, it is much harder to move the
resulting liquid through a thick layer of tissue during collection,
which will also lead to contamination by other soluble molecules in
the tissue. Therefore, this alternative sample collection strategy
might be preferable for sampling single cells deep inside the
tissue.
[0198] The presence (at least temporary) of the intact membrane
barrier, while the intracellular components are being solubilized
and dissociated, is central to this invention. To accommodate the
increase of volume inside the cell due to influx of reagents,
detergents can be used. Detergents increase the membrane area while
still maintaining the membrane barrier. Eventually, the outer
membrane can be solubilized and dissociated or it can be kept
mostly intact during withdrawal.
Single-Cell Resolution
[0199] As described elsewhere herein, the single-cell resolution of
Inside-Out Lysis was studied by fluorescence imaging. Although not
wishing to be bound by any particular theory, the growth of cell
membranes and the simultaneous confinement of Alexa 555 dyes or
SR101 dyes strictly within the geometric boundaries of target cells
(see FIGS. 8, 10, 12 and 32) suggested that the membrane barrier of
target cells was intact from the time point of lysis buffer
delivery to the time point of visible membrane growth, and that
single-cell resolution was preserved by the intact membrane barrier
from the time point of lysis buffer delivery to the time point of
extensive membrane growth in the Inside-Out Lysis process. As Alexa
555 or SR101 dyes filled the intracellular space of GFP-expressing
target cells, strictly within their geometric boundaries, GFP
fluorescence levels decreased immediately inside these cells (FIGS.
8, 10, 14, and 32). Capillary-driven suction was not applied in the
experiment presented in FIGS. 8 and 32. Because the membrane
barrier was intact at the time point of GFP fluorescence loss, GFP
fluorescence loss likely resulted from intracellular GFP
denaturation. Without being bound by any particular theory, it
cannot be completely ruled out that some detergent monomers could
pass through gap junctions to the surrounding cells within the time
interval from the time point of lysis buffer delivery to the time
point of visible membrane growth. However, neither Alexa 555 (MW
10000) nor SR101 (MW 606) were seen passing through gap junctions
to surrounding cells at detectable levels (FIGS. 12 and 32).
[0200] At the time point of membrane solubilization, lysate was
up-taken by the simultaneous perpendicular capillary-action-driven
suction. As described elsewhere herein, the volume of the target
cell was diluted approximately 1000 fold (from a few picoliters to
5-10 nanoliters) during the perpendicular uptake into the suction
channel. In claiming single cell resolution, it was likely that
such a rapid and simultaneous dilution (eventually 1000 fold) would
eliminate any peripheral lysis in this last step of Inside-Out
Lysis.
[0201] Experiments can be performed to examine whether the
transient contact (1-4 seconds) between the diluted detergents and
the peripheral surroundings of the target cell during the lysate
uptake (last stage of Inside-Out Lysis) lead to contamination. A
slice of GAD67-GFP hippocampus can be obtained and examined for a
dark cell (not expressing GFP) surrounded by GFP-expressing cells.
This dark cell can be lysed and measured to determine whether GFP
is absent in the obtained lysate.
Printing Single-Cell Lysates
[0202] The lysate of each single cell sampled by Inside-Out Lysis
was up-taken by a nearby capillary-action-driven suction channel
and therefore is diluted in the sample solution surrounding the
tissue slice (Hank's Buffered Salt Solution (HBSS)). The volume of
the up-taken diluted lysate of each single cell was estimated to be
approximately 5-10 nL. This volume estimate was calculated as
follows: a customized bent suction micropipette was kept under
positive pressure until the time point of lysis buffer delivery in
order to ensure that it was empty before Inside-Out Lysis was
triggered. The positive pressure was then released and the vertical
suction was triggered by the capillary action of the micropipette.
At the same time, Inside-Out Lysis of the target cell was triggered
as well. After the lysate of the target cell was completely
up-taken, the suction micropipette was robotically pulled out of
the sample solution within a few seconds. Given the visible
up-taken volume and given the known approximate geometry of the
suction micropipette, the above-mentioned upper bound of 5-10 nL
was derived.
[0203] In order to analyze the protein content of each single cell,
each up-taken single-cell lysate was printed on a glass-mounted
nitrocellulose pad. A method was developed to enrich the proteins
of each diluted single-cell lysate (5-10 nL) on nitrocellulose
within the boundary of a very small spot (20-50 .mu.m). This method
is based on the biophysical property of nitrocellulose to bind the
proteins of the applied extract rapidly within a small area,
whereas the solvent freely expands in a radial manner, driven by
the capillary action of nitrocellulose, and naturally evaporates
from a much larger area on nitrocellulose (FIG. 13A). By repeatedly
applying the fractions of the total lysate volume onto the same
spot on nitrocellulose in this manner, the protein content of each
single-cell lysate was enriched within the boundary of a 20-50
.mu.m spot (FIG. 13). As the single-cell lysate was concentrated
within a small dense spot on nitrocellulose, the noise generated by
the nitrocellulose was expected to be negligible.
[0204] Live tissue surface may contain cell debris. The suction
protocol also collected the surrounding medium (HBSS) and all the
soluble factors within the tissue slice. Therefore, a protocol was
developed which ensures the specificity of the recorded signals
after printing single-cell lysates on nitrocellulose. After all the
components of Inside-Out Lysis were positioned at the target cell,
the capillary-action-driven suction was triggered by releasing its
balancing positive pressure. After one minute, the Inside-Out Lysis
process of the target cell was triggered. The only difference
between these two time intervals was the delivery of lysis buffer
to the target cell. After up-taking the lysate of the target cell,
two partitions within the total up-taken volume remained: the
baseline partition and the lysate partition (FIG. 13B). These two
partitions were then printed in separate spots next to each other
on the same nitrocellulose pad. The lysate spot(s) represented the
results of Inside-Out Lysis and the baseline spot(s) represented
all other factors that could possibly contribute to the measured
signal in the lysate spot. This protocol was used in all subsequent
measurements.
[0205] The lysate partition of one GFP-expressing somatostatin
interneuron was subdivided into 3 separate spots on the same
nitrocellulose pad (FIG. 13B). The baseline partition is also
subdivided into 3 spots on the same nitrocellulose pad. Each spot
was generated by 10 visually identical depositions, as estimated by
the visible extent of radial solvent spreading in the
nitrocellulose during each deposition. The total deposited volume
was considered to be approximately equal across all spots. However,
the corresponding GFP and .beta.-actin signals were only observed
in the lysate spots and were not observed in the baseline spots
after antibody incubation. This example in FIG. 13B demonstrates
that the lysate partition of each sampled single cell was divided
into several sub-partitions. High protein signal density was
achieved within the boundaries of even smaller spots on
nitrocellulose, making it even easier to print just one
sub-partition of the single-cell lysate partition on a
nitrocellulose pad for protein analysis and to use the other
sub-partitions of the same single-cell lysate partition for the
analysis of other molecular classes.
Validation of Inside-Out Lysis
[0206] Two spatially separated single cells from the same
organotypic hippocampus slice of a GIN mouse were lysed. One cell
expressed GFP (somatostatin interneuron) and the other cell did not
express GFP. The lysate sub-partitions of these two cells (3 spots
for each cell, covering the whole lysate partition) and the
corresponding baseline sub-partitions (3 spots for each cell) were
printed on the same nitrocellulose pad as described elsewhere
herein. Next the positive controls were added in order to assess
antibody specificity. A titration curve of purified recombinant GFP
and a titration curve of the average hippocampus lysate of a
same-age GIN mouse were printed next to the single-cell lysate
spots and next to the corresponding baseline spots on the same
nitrocellulose pad with the help of an Aushon Arrayer.
[0207] Two negative controls were also printed. In order to obtain
the first negative control, the delivery micropipette and the
suction micropipette were positioned next to each other in the
HBSS-filled sample chamber in the absence of a tissue slice.
[0208] The lysis buffer from the delivery pipette was then up-taken
by the suction micropipette in a laminar flow and was printed on
the same nitrocellulose pad in the same manner as the
above-mentioned single-cell lysates, representing the first
negative control ("no tissue, LB," FIG. 14). A stream of HBSS mixed
with Alexa 555 dextrans was applied to a single cell in the
hippocampus slice of a GIN mouse under the same settings that are
usually used for Inside-Out Lysis, resulting in the absence of any
lysis. The up-taken solution was printed on the same nitrocellulose
pad in the same manner as the above-mentioned single-cell lysates,
representing the second negative control ("tissue, no LB," FIG.
14). Overall, the baseline spots and the two above-mentioned
negative controls encompassed all the possible non-specific factors
that may contribute to the recorded signals in the lysate spots of
the two single cells. The non-specific factors covered by the
baseline spots and by the two negative controls included: lysis
buffer (Tris-HCl50 mM, SDS 2%, Glycerol 5%, NaF 1 mM, 0.5 mM
AlexaD555 (10,000), Halt.TM. Protease Inhibitor Cocktail 3.lamda.,
Halt.TM. Phosphatase Inhibitor Cocktail 3.lamda.), all
printing/procedural factors and artifacts, convective flow factors,
soluble factors in the tissue slice and cell debris on the tissue
surface. The only event that was not covered by the set of baseline
spots and the negative controls was the event of applying lysis
buffer to a live single cell, which was exclusively covered by the
lysate spots (FIG. 14).
[0209] The data in FIG. 14 show that the negative controls and the
baseline spots did not generate any GFP or .beta.-actin signals
significantly above the background level. The positive controls
were measured at the spots of the corresponding titration curves
(concurrent standard curves) with signal intensities that were in
the range of the signal intensities of the lysate spots. These
positive controls show that the antibodies were specific (FIG. 14).
As expected, the GFP levels in the average lysate were negligible
after averaging all the cells of the GIN-mouse hippocampus. The
sampled GFP-expressing single cell in FIG. 14 is the same cell
displayed in FIG. 13. As expected, the three lysate spots of the
GFP-expressing cell showed both GFP and .beta.-actin signals,
whereas the three lysate spots generated from the dark cell (CA3
neuron) showed only .beta.-actin signals. Alexa 555 traces in FIG.
14 were measured immediately after printing the samples and were
observed only in the lysate spots and the main negative control
spots, but were not observed in the baseline spots. As all the
negative controls provided negative results while all the positive
controls provided positive results, it is likely that the GFP and
.beta.-actin signals in the lysate spots were exclusively
associated with the event of applying lysis buffer to a live single
cell. Thus, these signals necessarily originated from the single
cells lysed by Inside-Out Lysis in the hippocampus.
Automation and Throughput
[0210] In order to interface the Inside-Out Lysis method with the
lysate printing procedure, an extensive setup was built from ground
up (FIG. 15). This setup facilitated the precise positioning of all
the procedural components and enabled the rapidly coordinated
movements of these components. Rapid programmable movements are
required for switching from the Inside-Out Lysis method to the
printing procedure on nitrocellulose. Because of the small liquid
volumes (approx. 10 nL lysate+approx. 30 nL baseline) and the small
apertures (10 .mu.m) involved in the lysis and the printing
processes, it was necessary to switch to the printing procedure
within just a few seconds after the final single-cell lysate
uptake. Importantly, the infrastructure for further extensive
automation has also been built. A C++ library on top of the basic
command library provided by Sutter Instrument Company was written
to encode any sequence of movements of the 4 robotic arms at
submicron resolution. This library was used for the complete
automation of the lysis delivery process (FIGS. 7 and 8). A user
interface was also built for convenience (FIG. 15).
[0211] Lysis buffer was delivered rapidly and in some instances in
a fully automated fashion (FIG. 8). Given the current formulation
of the lysis buffer, intracellular GFP denaturation was completed
within the period of approximately 10 seconds after the initiation
of lysis buffer delivery (FIG. 8). It took approximately 10 seconds
to uptake the lysate after membrane solubilization (FIG. 12).
Therefore, the whole process could take at least 10 seconds and at
most 20 seconds after optimization. Because each initial tissue
sample could provide enough material for tens of organotypic
slices, the parallelization of the entire process could result in
the effective throughput of 2 seconds per single cell for a given
tissue sample.
Uptake of all Proteins of Target Single Cells
[0212] Single-cell lysates were completely deposited on
nitrocellulose (FIGS. 13 and 14). Although not wishing to be bound
to any particular theory, FIG. 22 suggests that cytosol-soluble
proteins were completely uptaken because in vivo GFP fluorescence
perfectly correlated with the recorded GFP signals after sampling
and analyzing the lysates of four GFP-expressing cells and two dark
cells as described elsewhere herein. Experiments can be performed
to determine if all proteins of target single cells were denatured,
solubilized, and uptaken. Experiments can be performed to determine
whether all membrane proteins and all proteins clustered with
membrane proteins were up-taken in the Inside-Out Lysis process.
Different lysis buffer formulations containing stronger denaturing
agents, such as urea, can be utilized in the Inside-Out Lysis
process.
Example 2
Single-Cell Lysate Microarrays
[0213] Single-cell lysates and baselines were printed on a
nitrocellulose pad, as described elsewhere herein. A high-precision
arrayer was used to print the titration series of purified
recombinant proteins and/or control lysates next to the printed
spots of the single-cell lysates on the same nitrocellulose pad. A
mixture of lanthanide-labeled antibodies was applied and each spot
was sampled on nitrocellulose and the lanthanide signal detected
with LA-ICP-MS (the laser ablation version of ICP-MS).
[0214] This strategy eliminated the disadvantages of Lysate
Microarrays and CyTOF. Namely, this strategy did not require the
sub-fractioning of the limited single-cell material and eliminates
the auto-fluorescence of nitrocellulose, thereby resolving the
limitations of Lysate Microarrays with respect to single-cell
measurements. This strategy did not require tissue
fixation/permeabilization and tissue disaggregation, thereby
resolving the limitations of CyTOF with respect to tissue
analysis.
[0215] At the same time, this strategy incorporated the advantages
of the two methods. Namely, this strategy enabled the printing of
concurrent standard curves and the rigorous validation of antibody
probes as in Lysate Microarrays, as discussed elsewhere herein. At
the same time, this strategy also incorporated the multiplexing
capacity of lanthanide labeling.
[0216] The materials and methods employed in these experiments are
now described.
Materials and Methods
[0217] Validation and Conjugation of Antibodies
[0218] All Cell Signaling Technology antibodies were ordered both
in the standard and in the customized BSA-free formulations. The
.beta.-actin antibody acquired from Sigma and the GFP antibody
acquired from Epitomics, were available in the standard BSA-free
formulation. To measure proteins in Single-Cell Lysate Microarrays,
the BSA-free antibodies were conjugated with the polymers carrying
different lanthanides. All 8 antibodies were conjugated to
different lanthanide labels in a one-to-one manner. The polymers
and the lanthanides were acquired from DVS Sciences and their
conjugation protocol was followed. The Cell Signaling Technology
antibodies in the standard BSA-containing formulation were used for
Western blots.
[0219] In order to obtain the average hippocampal lysate of a GIN
mouse, two hippocampus samples were dissected from the same mouse
and quickly placed into 200 .mu.L of the same SDS-containing lysis
buffer used in all other procedures. The tubes remained with the
lysis buffer-submerged hippocampus samples in a 4.degree. C. cold
room for 2 hours. The hippocampus average lysate was then filtered
to remove the extracellular matrix and DNA and stored in a
-80.degree. C. freezer. The concentration of proteins in the
average lysate was measured by Micro BCA.TM. Protein Assay and was
estimated at around 3.1 mg/ml. Because the pan-specific antibodies
used in the Single-Cell Lysate Microarray experiments were
previously pre-validated for general use within the format of
Lysate Microarrays, only a Western blot experiment on the average
hippocampus lysate was run to confirm the specificity of these
antibodies in mice. The NuPAGE.RTM. SDS-PAGE Gel System (Life
Technologies) was used. Each average lysate blot was incubated with
the primary antibody of interest and with the .beta.-actin
antibody. After incubating the blots with the corresponding
fluorescently labeled secondary antibodies, the signal intensities
were acquired on a LiCor Odyssey scanner.
[0220] Detection by LA-ICP-MS
[0221] The Inside-Out Lysis procedure, the printing procedure for
single-cell lysates, the printing procedure for the titration
series, and the antibody incubation procedure described elsewhere
herein were used in the Single-Cell Lysate Microarray
experiments.
[0222] Thermo Electron X-Series ICP-MS (ICP-MS) and a New Wave 213
nm UV Laser (LA) were used for signal detection and spot sampling.
The sensitivity and stability of the LA-ICP-MS instrument were both
optimized prior to sampling the spots of interest on nitrocellulose
pads. Usually, the sensitivity of LA-ICP-MS depends on argon flow.
Argon flow is essential to sustain the inductively coupled plasma
generated by a strong oscillating magnetic field via argon
ionization. Argon flow is also used to introduce the sample into
the plasma for atomization and ionization before the ionized
elements enter the mass spectrometer. Thus, the optimization of the
argon flow was crucial to achieve high sensitivity. After turning
on the instrument, argon flow was optimized in ICP-MS with a
standard salt solution in order to achieve high sensitivity.
Subsequently, the instrument was switched to the laser ablation
mode. The diameter of all laser-ablated spots was 80 .mu.m. Laser
strength was optimized to minimize the sampling of the glass base
beneath each nitrocellulose pad.
[0223] The results of the experiments are now described.
Multiplex Measurements
[0224] By combining the advantages and by eliminating the
disadvantages of Lysate Microarrays and lanthanide-labeling/CyTOF
in one platform, multiplex analytical measurements of native
proteins in single cells of solid tissues can be performed (Table
2). This platform may be referred to as Single-Cell Lysate
Microarrays (FIG. 17).
TABLE-US-00002 TABLE 2 Single-Cell Lysate Microarrays enable
multiplex analytical protein measurements of native proteins in
single cells of solid tissues. ##STR00001## White: disadvantages,
Grey: advantages.
[0225] The Lysate Microarray technology (also referred to as
Reverse Phase Lysate Arrays) was first reported in 2001 (Paweletz
et al., 2001, Oncogene 20:1981-1989). By robotically printing small
lysate spots (.about.200 .mu.m diameter) with high signal density
on glass-mounted nitrocellulose pads, and by probing these
nitrocellulose pads with pre-validated antibodies, high sensitivity
and low sample requirements are achieved in highly multiplex
protein measurements across an extensive set of samples and
physiological conditions. Titration curves of control lysates
and/or purified target proteins can be printed next to the unknown
samples on the same nitrocellulose pads. These concurrent titration
curves reveal the standard curves for each antibody probe. Thus,
Lysate Microarrays compensate for signal non-linearity at low
substrate levels and allow to map the recorded signal differences
to the corresponding quantity differences.
[0226] The main bottleneck for the scalability of Lysate
Microarrays is the number of available antibodies with invariantly
low cross-reactivity across various lysates. Most antibodies
cross-react with unspecific antigens. In contrast to Western blot,
the cross-reactive signal component in Lysate Microarrays cannot be
separated from the specific signal component by size separation of
proteins. In Lysate Microarrays, any given lysate spot contains a
homogenous mixture of all proteins of the original sample and thus
any cross-reactive signal will also contribute to the total
recorded signal in each lysate spot. The following example
demonstrates how the cross-reactivity of antibodies can obscure the
specific signal component in Lysate Microarray measurements. [0227]
Let [0228] P(Ab binds|Target)=0.99, [0229] |P(Ab
binds|nonTarget1)=0.01, [0230] P(Ab binds|nonTarget2)=0.05, [0231]
Target=1000, [0232] # nonTarget1=70,000, [0233] #
ncanTarget2=29,000. [0234] Then [0235] P(Ab binds)=0.0314, [0236]
E(# total Ab bindings)=3140, [0237] E(# specific Ab
bindings)=990.
[0238] The above example shows that given an antibody and a
relatively low number of targets in the spotted homogenous mixture
of proteins, the cross-reactive signal component (2150) is at least
twice as large as the specific signal component (990). This example
also demonstrates that antibody cross-reactivity can be
parameterized by three parameters: 1) the proportion of target
proteins in the total mixture, 2) the distribution of various
non-target proteins in the mixture and 3) the inherent binding
probabilities of the given antibody to targets and to non-targets.
Many crucial kinases and phosphatases occur in low copy numbers
(100-1000 in single cells). The distribution of non-target proteins
also varies from cell to cell within a cell type and from cell type
to cell type. The purpose of Lysate Microarrays is to measure the
levels of crucial proteins in unknown samples. Therefore, the
binding probabilities of antibodies are the only parameter that can
be optimized in order to minimize the cross-reactivity of
antibodies in Lysate Microarrays. An extensive screening test is
required to identify the antibodies with optimal binding
probabilities.
[0239] Sevecka had designed a set of screening tests for
pan-specific antibodies (Sevecka et al., 2011, Mol. Cell Proteomics
10: M110.005363). For each tested antibody, their first test
required the presence of a single dominant band of correct size in
the Western blot across all the lysates of 17 different human cell
lines, while their second test required that the target levels
measured in Western blot should also highly correlate (>0.75)
with the corresponding levels in Lysate Microarrays across the same
17 human cell lines. Each cell line expressed different levels of
target antigens and presumably different levels of non-target
proteins. If the binding probabilities of a given antibody are
optimal, then it will be specific across different cell lines,
because the cell-line-dependent variation of the cross-reactive
component will not affect the accurate detection of the different
target levels. This will lead to a high correlation between Western
blot and Lysate Microarrays. If the binding probabilities of a
given antibody are poor, then the cell-line-dependent differences
and the noise of the cross-reactive component will obscure the true
target levels in Lysate Microarrays, leading to a low correlation
(FIG. 16). The second test by Sevecka et al. is equivalent to 17
independent Bernoulli trials, because every distinct cell line can
be considered as a Bernoulli trial and the tested antibody can be
considered as a biased coin. Given 17 or approximately 17 `good`
outcomes after 17 independent trials, the probability is high that
this particular antibody (coin) will be specific (biased=good
binding probabilities) in any other cellular context (future
trials) and thus can be used to make accurate measurements across
unknown cell types. 10% of 129 tested by Sevecka et al.
pan-specific antibodies passed these two tests. This selected set
of antibodies is generally valid for measuring proteins in unknown
samples within the format of Lysate Microarrays.
[0240] Lysate Microarrays are not appropriate for single-cell
measurements. The multiplexing capacity of Lysate Microarrays is
based on the subdivision of the original homogenous sample into its
sub-fractions and on printing these sub-fractions on multiple
spatially separated nitrocellulose pads for the subsequent
incubation of each pad with a different antibody. In the case of a
single cell, the original protein material is very limited and
should not be subdivided for the sole purpose of protein
multiplexing. Signal acquisition in Lysate Microarrays is achieved
by scanning fluorescently labeled secondary antibodies. In this
setting, auto-fluorescence of nitrocellulose can also be
prohibitively high, when low amounts of single-cell material are to
be analyzed. Nitrocellulose auto-fluorescence is another reason why
Lysate Microarrays are not appropriate for single-cell
measurements.
[0241] The possibility of detecting metal-labeled probes by atomic
mass spectrometry was first reported in 2002 (Quinn et al., 2002,
J. Anal. At Spectrom 17:892-896). By conjugating different rare
metal labels to different affinity-based probes in a one-to-one
manner, the multiplexing capacity of any probe-based measurement
can be significantly increased due to the large number of existing
rare metal elements. As the probed sample is being processed
through the 5,000-10,000.degree. C. plasma (inductively coupled
plasma mass spectrometry, ICP-MS), all molecules of this sample are
atomized and ionized before entering the mass spectrometry module.
Because rare metals do not occur in most biological systems at
significant levels, the measured counts of rare metals then
correspond to the levels of the respective labeled probes being
present in the probed sample. For example, antibodies can be
conjugated to the polymers containing lanthanide chelators. These
lanthanide-labeled antibodies enable highly multiplex detection of
proteins. Lanthanide-labeling has been developed and commercialized
in the context of the CyTOF/Mass Cytometry instrument by DVS
Sciences (Bendall et al., 2011). This instrument emulates flow
cytometry. Thus, CyTOF also shares all the limitations of flow
cytometry with respect to analyzing single cells in solid tissues
(Table 2).
Antibody Validation and Lanthanide-Labeling
[0242] A set of pan-specific antibodies that had previously been
validated by Sevecka et al. for the general use in Lysate
Microarrays as described elsewhere herein were obtained for the
development of Single Cell Lysate Microarrays. Out of 129
pan-specific antibodies tested across 17 different human cell
lines, Sevecka et al. were able to identify 12 antibodies of high
general specificity for analytical studies with Lysate Microarrays
(Sevecka et al., 2011, Mol Cell Proteomics 10:M110.005363). A
subset of these 12 antibodies was used for the development of
Single-Cell Lysate Microarrays. Because the Inside-Out Lysis method
for sampling single cells in organotypic cultures of mouse
hippocampi was to be used, each antibody in the selected subset
needed to also be antigenic in mice. Kinases were selected as
target antigens because of their crucial role in cell signaling and
their expected low relative abundances in single cells. Successful
measurements of these kinases with Single-Cell Lysate Microarrays
would constitute a confirmation of the high sensitivity of this
method. FAK, PKC.delta., PKC.alpha. and PAK1 were selected as
target kinase antigens. A validated antibody for .beta.-catenin was
also included. Because rare GFP-labeled somatostatin interneurons
in the organotypic hippocampus cultures of GIN mice were to be
lysed, two GFP-specific antibodies were added to the overall set of
antibodies. One of these GFP-specific antibodies was the same
antibody used in the validation of Inside-Out Lysis, as described
elsewhere herein.
[0243] Because the selected antibodies against kinases and
.beta.-catenin had previously been validated across 17 human cell
lines and were expected to perform well in any unknown context as
discussed elsewhere herein, it was reasoned that a simple
confirmation of their specificity in mice was sufficient. A Western
blot experiment was performed on an average lysate of the
hippocampus of a 6-day old GIN mouse. All these antibodies had a
clear dominant band of the correct size, thus confirming their
specificity against mouse antigens (FIG. 18). GFP antibodies were
not independently validated and were expected to be specific, as
confirmed in later experiments.
[0244] Next, the purified BSA-free formulations of the selected set
of antibodies were obtained for subsequent lanthanide conjugation.
Lanthanide conjugation was performed according to the protocol
provided by the manufacturer of the obtained lanthanide polymers
(DVS Sciences). The list of antibodies and the corresponding
lanthanide labels is provided in Table 3.
TABLE-US-00003 TABLE 3 Lanthanide-Labeling of the Selected Set of
Antibodies Previously Validated for Lysate Microarrays and then
Confirmed by Western Blot. Antigen Antibody Lanthanide Label
.beta.Actin A1978, Sigma Nd145 PKC.delta. 2058, CST Dy162
PKC.alpha. 2056, CST Yb174 PAK1 2602, CST Pr141 FAK 3285, CST Tb159
.beta.Catenin 9582, CST Sm147 GFP1 S2038, Epitomies Nd142 GFP2
2956, CST Sm154
Multiplex Analytical Protein Measurements of Native Proteins with
Single-Cell Lysate Microarrays
[0245] Four GFP-expressing somatostatin interneurons were sampled
from 2 slices of live organotypic hippocampus cultures of a GIN
mouse (postnatal day 5+7 days in vitro). Two putative CA3 pyramidal
neurons from the CA3 region of the same hippocampus slices were
also sampled. These 6 single-cell lysates and their corresponding
baseline spots were printed on the same glass-mounted
nitrocellulose pad. For each single-cell lysate, one spot with the
entire lysate partition and one spot with the baseline partition
were each printed, as described elsewhere herein. For each
single-cell lysate, one spot (30 depositions) with the entire
lysate partition and one spot (31-32 depositions) with the baseline
partition were printed, as described elsewhere herein. For each
cell, the deposited amount of liquid in the lysate spot was
approximately equal to the deposited amount of liquid in the
corresponding baseline spot. All in vivo GFP intensities were also
recorded prior to single-cell lysis under the same settings of the
imaging setup. Because all the imaged and sampled cells were close
to the surface of the two hippocampus slices, light scattering
should not have had any significant effect on the recorded
intensities of in vivo GFP fluorescence of these cells. The sampled
CA3 pyramidal neurons did not express any GFP at the detection
sensitivity of the imaging setup.
[0246] Next, the titration series of purified recombinant PKCa, PKC
and GFP were printed by the high-precision Aushon Arrayer on the
same nitrocellulose pad next to the printed single-cell lysates and
next to their baselines. The titration series of the average
hippocampus lysate of a GIN mouse was also printed next to these
titration series of recombinant proteins. All spots were marked by
making 10 .mu.m-large incisions on the nitrocellulose pad next to
the printed spots in order to maintain the visual coordinates of
all the printed spots throughout the subsequent washing and
antibody incubation steps.
[0247] The same set of washing and antibody incubation steps were
applied to the printed glass-mounted nitrocellulose pad as
described by Sevecka (Sevecka et al., 2011, Mol Cell Proteomics
10:M110.005363). In a different set of experiments, it was
determined that the following concentrations of the 8
lanthanide-labeled antibodies bring the corresponding signals
generated by single-cell lysates into the sensitivity range of the
LA-ICP-MS detector: 0.66 .mu.g/ml for the conjugated .beta.-actin
antibody and 3.3 .mu.g/ml for all the other conjugated antibodies.
After antibody incubation, the spots of the printed single-cell
lysates, the spots of their baselines, and the spots of all the
printed titration series were sampled with the help of a tuned and
calibrated LA-ICP-MS instrument during a single acquisition run. By
adjusting the laser strength and by recording the traces of 85Rb
and 88Sr, it was ensured that the glass base of the nitrocellulose
pad was not sampled by laser ablation to a significant extent. A
control pulse of high laser intensity was also applied to the blank
region of the nitrocellulose pad in order to ensure that by
sampling a significant amount of glass (indicated by 85Rb and 88Sr
traces) and nitrocellulose (indicated by tiny visible
nitrocellulose holes), no lanthanide traces could be detected.
Thus, after washing and antibody incubation, glass and
nitrocellulose alone did not lead to the detection of
lanthanides.
[0248] Obtained measurements by LA-ICP-MS are presented in FIG. 19.
In the first upper segment of FIG. 19A, each row represented 3 time
points of the Inside-Out Lysis process of each sampled cell
('before', lysis', `after`). For each cell, the three select time
points of the lysis process show that, as expected, GFP lost its
fluorescence after the intracellular space was filled with the
SDS-containing lysis buffer. The last time point in each row
(`after`) showed that the contents of the target cells were
eventually up-taken by the simultaneous capillary-action-driven
suction channel. In the other segments of FIG. 19, the following
LA-ICP-MS sampling procedure for sampling each spot was used. The
sampling procedure for sampling each spot on the nitrocellulose pad
by the LA-ICP-MS instrument took 3 minutes (time axis in FIG. 19 is
number of recorded time frames by the LA-ICP-MS detector within the
sampling procedure). In the first interval of each sampling
procedure, the instrument noise was measured by acquiring the noise
data prior to laser ablation. Then a sequence of laser pulses was
applied to the spot location on the nitrocellulose pad. This
sequence of fast laser pulses was applied for the duration of 60
seconds. The residual time within each sampling procedure was spent
waiting until the continuously measured counts of elements from the
most recently sampled spot returned to the noise levels of the
instrument. Using this sampling procedure for each spot, all the
spots of single-cell lysates, baselines and titration series were
sampled on the above-described nitrocellulose pad.
[0249] The baseline spots containing HBSS solution, tissue debris
and/or soluble tissue factors showed low signal in most cases
(cells 1 to 4; cells are numerated from left to right and from top
to bottom). The baseline spots of the last two somatostatin
interneurons (cell 5 and cell 6) showed significant 3-actin signals
with relatively low 3-actin signals in the corresponding lysate
spots. The baseline spots of the other 4 cells (cells 1 to 4) did
not generate any signals significantly above the instrument noise
levels across all 8 channels. The volume printed in each baseline
spot was larger or approximately equal to the volume printed in
each lysate spot, as described elsewhere herein. Each baseline spot
of the first 4 cells (cells 1 to 4) encompassed all the possible
sources of procedural noise that could originate from Inside-Out
Lysis, from printing and/or signal detection procedures. These
sources of procedural noise included: HBSS solution, tissue debris,
soluble factors in tissue, the printing process, incubation/washing
steps, and LA-ICP-MS noise. Therefore, any signal differences
recorded at the time of laser-firing in the lysate spots of the
first 4 cells did not originate from any subset of the
above-mentioned procedural noise sources. Thus, the signal levels
in the lysate spots of the first 4 cells (cells 1 to 4) did not
originate from the non-specific signal levels of the baseline
components. The signals in and the signal differences across the
lysate spots of the first 4 cells originated from the differences
in antibody binding to the printed single-cell lysates.
[0250] Inspection of the recorded signals across all 8 dimensions
suggested that there were significant differences in signals
between single cells within each dimension (FIG. 19). These
differences did not come from procedural noise sources. It was
likely that these signal differences represented the differences in
protein quantities because the data in FIG. 14 showed that lysis
buffer components had no detectable effect on antibody binding. As
all the kinase antibodies and the .beta.-catenin antibody were
validated across 17 different cell-line contexts in the format of
Lysate Microarrays, these antibodies have very low cross-reactive
components in any cell type. FIG. 21 depicts the expected signals
obtained from the concurrent titration series of purified
PKC.delta. and PKC.alpha. on the same nitrocellulose pad, on which
the single-cell lysates and their corresponding baselines were also
printed and sampled (FIG. 19). By looking at the concurrent
titration series of PKC.delta. and PKC.alpha. (FIG. 21), it was
observed that lanthanide-labeling did not affect the specificity of
the validated antibodies. The measured differences in signals
between single cells within each of the 8 dimensions (channels)
thus likely originated from the differences in the corresponding
quantities of target proteins in these lysate spots.
[0251] Because the in vivo GFP fluorescence levels of the sampled
somatostatin interneurons and CA3 neurons were recorded under the
same settings of the imaging setup prior to Inside-Out Lysis, the
recorded in vivo GFP fluorescence levels were compared with the
corresponding measurements across the same 6 sampled single cells.
FIG. 22B shows an almost perfect correlation between the in vivo
GFP fluorescence recorded before Inside-Out Lysis and the measured
GFP levels in the corresponding single-cell lysates that were
simultaneously probed with two different GFP antibodies in
Single-Cell Lysate Microarrays. This was an additional validation
of the Inside-Out Lysis method. As described elsewhere herein, and
without being bound by any particular theory, the high correlation
values suggest that most soluble proteins were uptaken from single
cells during the Inside-Out Lysis method. Four spots of the GFP
titration series were sampled and the concurrent standard curves
for each of the two GFP antibodies was obtained. Because the
conjugation procedure for each GFP antibody was the same during the
incubation procedure and because the concentrations of the two
antibodies were the same, it is likely that the different slopes of
the GFP standard curves, derived from the same four spots of the
same titration series in FIG. 22A, resulted from the corresponding
differences in the K.sub.D values between these two GFP
antibodies.
[0252] The integrated levels of .beta.-actin were similar across
all the sampled single cells except in one instance (FIG. 20). The
.beta.-actin levels of the second somatostatin interneuron (Cell4
in FIG. 20) were significantly lower than those of the other cells.
This result did not affect the high levels of GFP measured in
Single-Cell Lysate Microarrays that perfectly correlated with the
pre-lysis in vivo fluorescence of this same cell. Without wishing
to be bound by any particular theory, one explanation is that the
current lysis buffer, used in the Inside-Out Lysis procedure, is
not optimal for the solubilization of the actin network. This can
be remedied by changing the formulation of the lysis buffer or by
further optimizing the lysis procedure. An alternative explanation
is that the levels of .beta.-actin are variant across single cells
and cannot be used for normalization. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) could be used as an alternative normalizing
antigen in future measurements.
[0253] Some standard curves were non-linear within the signal range
that was relevant for single-cell measurements (FIGS. 21 and 22).
The PKC.delta., PKC.alpha. and GFP titration series (FIGS. 21 and
22) were printed by the high precision Aushon Arrayer 2470 from the
prepared dilution samples of each antigen. Eight depositions from
each prepared dilution sample were printed onto each spot of the
titration series by the Aushon Arrayer and thus the differences
between the amounts of antigen deposited in the spots within a
titration series were expected to represent the concentration
differences between the corresponding dilution samples of this
antigen (see Sevecka et al., 2011 for examples of Aushon Arrayer
printing). Aushon does not publish the numerical values of printing
noise but it is expected to be negligible in current settings. The
high number of depositions per spot (8) also ensured that the
lysate distribution within each printed spot was uniform. During
laser ablation in the LA-ICP-MS sampling procedure, a sub-spot was
sampled within each printed spot of the titration series. The
position of the sub-spot within the initial spot remained the same
in all laser ablation samplings of the titration series.
[0254] Although not wishing to be bound by any particular theory,
these experimental parameters suggest that the emergence of
non-linearity at low antigen concentrations in the PKC.delta. and
GFP standard curves (FIGS. 21 and 22) was not primarily due to the
error of printing by the Aushon Arrayer and was not primarily due
to the spot location error associated with the sampling procedure
by LA-ICP-MS. The monotonicity and the linearity of the signal
decrease in all standard curves at high antigen concentrations
(high x-axis values in FIGS. 21 and 22) also implied that printing
and pipetting errors did not primarily contribute to the emergence
of non-linearity at low antigen concentrations. The signal levels
in the last data-point of each standard curve were significantly
above the instrument noise levels and above the noise across the
four baseline spots (cells 1 to 4, FIG. 19), which were acquired
from the same nitrocellulose pad during the same LA-ICP-MS run.
Although not wishing to be bound by any particular theory, these
experimental parameters suggest that the noise of printing by the
Aushon Arrayer, the noise of pipetting, and the noise of the
sampling procedure by LA-ICP-MS did not primarily contribute to the
emergence of non-linearity at low antigen concentrations in the PKC
and GFP standard curves (FIGS. 21 and 22). It is likely that
non-linearity originated from the decrease of the probability for
antibody molecules to bind and/or to stay bound to diluted antigens
on nitrocellulose. Because the antibodies were bivalent, the
decrease of the above probability might be associated with the fact
that the binding sites of antibodies were not saturated by nearby
antigens on nitrocellulose at low antigen concentrations.
Single-Cell Lysate Microarrays enable the correction of these
non-linearities and therefore enable the accurate measurements of
quantity distributions across single-cell populations (FIGS. 1 and
2).
Example 3
Multiplexing across Molecular Classes in Human Solid Tissues
[0255] Experiments can be performed to multiplex across multiple
molecular classes. As described elsewhere herein, the lysate format
is compatible with all rigorous analytical methods. Multiplex
measurements of proteins, transcripts and/or metabolites can be
obtained from the same single cell by subdividing the lysate
partition of the sampled single cell into lysate sub-partitions as
shown in FIGS. 13 and 14. In order to further decrease the sample
requirements for Single-Cell Lysate Microarrays, the diameter of
each printed lysate spot can be decreased from 50 .mu.m to 20
.mu.m. This would ensure that the signal density of the smaller 20
.mu.m spots is approximately equivalent to the signal density of
the 50 .mu.m spots, while also maintaining a larger sub-partition
of the total single-cell lysate partition for the analysis of
transcripts (RT-qPCR) and/or metabolites (MALDI). In this setting,
the procedure of laser ablation with LA-ICP-MS can be adjusted in
order to take full advantage of the high signal density in the
smaller 20 .mu.m lysate spot, wherein the diameter of the laser
beam can be adjusted to 20 .mu.m and the energy of the laser tuned
such that the whole spot can be sampled within just 1 or 2 laser
pulses. These two adjustments generate a high signal differential
at the elemental counter of the LA-ICP-MS instrument within the
interval of just a few seconds, making it unnecessary to integrate
the signal curve over a long time interval in sampling sessions.
This can provide a high signal-to-noise ratio with respect to the
inherent noise of the LA-ICP-MS instrument and to the noise
generated by the laser-ablated nitrocellulose. Therefore,
comparable Single-Cell Lysate Microarray measurements can be
obtained with just a small sub-partition of the total single-cell
lysate partition. The other sub-partitions of the total single-cell
lysate partition can be used for the analysis of transcripts and/or
metabolites. Sampling procedures can also be optimized in order to
better mix the lysate inside the cell before its uptake by suction
and before its subdivision into sub-partitions.
[0256] After depositing the first sub-partition of the total
single-cell lysate partition into the 20 .mu.m spot on
nitrocellulose for multiplex protein measurements with Single-Cell
Lysate Microarrays, the analysis of the residual sub-partition of
the total single-cell lysate can be accomplished by amplifying the
transcripts in a RT-qPCR reaction. For example, this can be done
within the context of the Fluidigm platform (Fluidigm Inc). The
multiplex analysis of metabolites can also be achieved by MALDI.
Amantonico et al. have measured the abundances of ADP, UTP, ATP,
and GTP in the lysate amounts equivalent to approximately one half
of a single cell of Saccharomyces cerevisiae (Amantonico et al.,
2008, Angew. Chem. Int. Ed. Engl. 47:5382-5385). The median size of
a single cell of yeast is 82 .mu.m.sup.3 (Jorgensen et al., 2002,
Science 297:395-400). The size of a single mammalian COS-7 cell is
2016.+-.208 .mu.m.sup.3 (Bohil et al., 2006, Proc. Natl. Acad. Sci.
USA 103:12411-12416). Therefore, it would be expected that the
amount of single-cell material can be considerably larger in the
case of mammalian cells, thus enabling even more extensive
metabolite multiplexing.
[0257] Additionally, the platform can be used to enable single-cell
analysis in solid human tissues. Organotypic cultures of human
tumors have been shown to develop normally for one week after
biopsy harvesting and slicing (Vaira et al., 2010, Proc. Natl.
Acad. Sci. USA 107:8352-8356). The sampling and analytical methods
described herein can be used with organotypic cultures of human
tumors to define a new category of diagnostic tests, to personalize
single-cell pharmacology, and to rapidly identify mechanistic
biomarkers and drug targets.
Standard Curves
[0258] Experiments can be performed to derive the actual error bars
on the standard curves by printing titration series duplicates or
triplicates. Titration duplicates were printed in the above
experiment but were not acquired within the same LA-ICP-MS run
because of the assumption that the noise of printing by the Aushon
Arrayer, the noise of pipetting and the noise of the sampling
procedure by LA-ICP-MS were negligible. Experiments can also be
performed to determine how the variance in LA-ICP-MS tuning from
day to day operation affects the signals derived from the
duplicates of the same titration series. The duplicates or
triplicates of the same titration series can be printed on the same
nitrocellulose pad and then sampled on different days with
LA-ICP-MS.
[0259] The four baseline spots (cells 1 to 4) in FIG. 19
encompassed all the procedural sources of noise. As described
elsewhere herein, these sources of procedural noise included: HBSS
solution, tissue debris, soluble factors in tissue, the printing
process, incubation/washing steps, LA-ICP-MS noise. However, these
4 baseline spots might not measure the probabilistic aspects of
protein binding to nitrocellulose because these baseline spots did
not appear to contain much protein. There may also be some
additional noise in antibody binding to the same concentration of
protein substrate on nitrocellulose under the same antibody
concentration. Experiments can be performed to address these two
additional sources of noise. A titration curve of the average
tissue lysate can be printed in duplicates or triplicates by the
high-precision Aushon Arrayer and the noise within each of 8
recorded dimensions (.beta.-actin, .beta.-catenin, PKC.alpha.,
PKG.delta., PAK1, FAK, GFP2, GFP1) across these duplicates or
triplicates can be measured.
[0260] Experiments can also be performed to construct better
standard curves. Different known amounts of the purified antigen
can be titrated into the antigen-depleted average lysate of the
tissue of interest. Such a standard can capture the cross-reactive
component of the validated antibodies, which may contribute to the
non-linearity of standard curves.
Example 4
Data Analysis with Bayes Nets
[0261] A test for selecting the optimal Learner (Bayes Net
topology+fixed ML subroutine) was run on a related data set. The
same procedure is applied in the context of single-cell
measurements enabled by Inside-Out Lysis and by Single-Cell Lysate
Microarrays.
[0262] The materials and methods employed in these experiments are
now described.
Materials and Methods
[0263] The code was implemented in Python and was run on Python
2.6.
[0264] Each dimension of the cancer data set of dimensionality 10
consisting of 112 data points was numerated as follows: 0-Axl,
1-Met.p, 2-Stat3.p, 3-Akt.p, 4-cRaf.p, 5-Src.p, 6-Erk1.2.p,
7-S6.pl, 8-MAPK.pl, 9-Cancer Diagnosis. Each directed acyclical
graph was represented as a list of tuples in Python:
[0265] NAIVE_BAYES_TUPLES=map(tuple, [[9], [9,0], [9,1], [9,2],
[9,3], [9,4], [9,5], [9,6], [9,7], [9,8]])
[0266] AXL_MET_AKT_BAYES_TUPLES=map(tuple,[[9], [9,0], [9,0,1],
[9,2], [9,3], [9,1,3,4], [9,5], [9,6], [9,7], [9,8]])
[0267] Each tuple was used as a key in the first layer of a
dictionary of dictionaries. All possible true/false assignment
tuples for each key in the first layer were used as keys in the
second layer of the dictionary of dictionaries. The second-layer
assignment tuples then mapped to a numerical probability value. A
Bayes Net class was created to incorporate all the necessary
functions, such as a function for data import and a function for
parameter estimation by maximum likelihood. Each Bayes Net topology
was represented by the above format and could be imported into this
class. A cross-validation routine was also implemented in the Bayes
Net class. The Bayes Net class was used to evaluate all 15 Bayes
Net topologies by 10-fold cross-validation.
[0268] The results of the experiments are now described.
Bayes Net Topologies
[0269] In a highly dimensional data set, each data point is a long
vector of multiplex measurements. For example, by measuring 10
proteins in each of 100 different samples, a data set of
dimensionality 10 consisting of 100 data points is obtained. Each
dimension in such a data set can be considered as a random
variable. The entire data set can then be formally represented by
the joint probability distribution of all the random variables. It
may be difficult to visualize and to reason about the many possible
conditional independence assumptions in the joint probability
distribution of a highly dimensional data set without a convenient
graphical representation. The idea underlying Bayes Nets is that a
joint probability distribution can be represented by a directed
acyclical graph (Pearl, 1982, Proceedings, AAAI-82). Such a graph
is easy to visualize, analyze, and modify. The direction of the
edges in a Bayes Net can be used to write down the formal
mathematical representation of the corresponding joint probability
distribution (FIG. 23). A random variable in a Bayes Net is
conditionally independent from all the non-descendent random
variables if it is conditioned on all its parent random variables
(Pearl, 1982, Proceedings, AAAI-82). The topology of a Bayes Net
incorporates all the conditional independence assumptions. Given a
fixed set of random variables (vertices), the same set of
conditional independence assumptions can be represented by
different Bayes Net topologies (edges) (Pfeffer and Parkes, 2010,
CS181 Lecture Notes, Harvard University). Thus, the same set of
conditional independence assumptions can be mapped to different
Bayes Net topologies.
[0270] The graphical representation of the joint probability
distribution of a highly dimensional data set is also useful
because of the availability of standard graph algorithms in
computer science. Many interesting problems related to the joint
probability distribution of data can be reduced to the
corresponding graph problems and therefore, can be solved
efficiently by graph algorithms on large problem instances. Because
a Bayes Net represents the corresponding joint probability
distribution, it can also be used for data sampling in a generative
manner.
[0271] There are standard ways to learn the parameter values of a
given Bayes Net topology from the data set. Given a topology of a
Bayes Net, the corresponding parameter values can be estimated by
maximum likelihood (ML). If a prior distribution on parameter
values is desirable, the maximum a posteriori method (MAP) can be
used. If there are hidden random variables in the Bayes Net, then
the expectation maximization (EM) algorithm with either the ML
subroutine or the MAP subroutine can be used to learn the parameter
values from the given data set. All these methods can fit the
topology of a given Bayes Net to the data set by estimating the
fitting parameter values. However, the fit of a Bayes Net to the
data set is a poor measure of how well the parameter values and the
topology of this particular Bayes Net capture the true hidden
concept in the data set which is to be learned. A data set is a
sample from the distribution of data sets that can be generated by
the true hidden concept. In order to avoid over-interpreting the
patterns in one particular data set, the optimal Bayes Net with
minimal overfitting is found.
[0272] It is common to attribute overfitting to the presence of
noise in the data set. However, overfitting can also occur in fully
deterministic domains without noise. In deterministic domains, the
main cause of overfitting is the `curse of dimensionality` (Pfeffer
and Parkes, 2010, CS181 Lecture Notes, Harvard University; Bishop,
2006, Pattern Recognition and Machine Learning, Springer, First
Edition). The `curse of dimensionality` is also a major cause of
overfitting in non-deterministic (noisy) domains. For example, in
genomics thousands of noisy features (dimensions) are recorded in
just a few genomes (data points). Given a highly dimensional data
set consisting of just a few data points, it is quite likely that
interesting patterns will emerge across some of the many dimensions
of the data set simply by chance and solely due to the small number
of sampled data points. These patterns are spurious and do not
accurately represent the true hidden concept which is to be
learned. Such spurious patterns can be eliminated by sampling more
data points from the same true hidden concept. However, data is
generally more expensive than computation. It is preferably to
minimize overfitting caused by the noise of measurements and by the
`curse of dimensionality` without acquiring more data.
[0273] The Bayes Net topology, representing the hypothesis space
before estimating the parameter values, was distinguished from the
final Bayes Net model with fully estimated parameter values that
can be used for different inference tasks such as classification.
The best way to test the generality of a given Bayes Net model with
fully estimated parameter values is to assess the accuracy of its
predictions on an independent data set sampled from the same hidden
concept. Given an arbitrary data set, it can be subdivided it into
two disjoint sets: a training set and a test set. The parameter
values of a given Bayes Net topology can be determined by applying
the parameter estimation routine (ML, MAP or EM) to the training
set. The generality of the learned Bayes Net model can be assessed
with the estimated parameter values by testing its prediction
accuracy on the test set, which is independently and identically
sampled from the same hidden concept. However, this does not solve
the problem of overfitting, because the initial Bayes Net topology
might represent a hypothesis space that is too large or too small
in the context of the true hidden concept and/or in the context of
the given data set (Kearns and Vazirani, 1994, An Introduction to
Computational Learning Theory, MIT Press). To minimize overfitting
the optimal Bayes Net topology that represents the optimal
hypothesis space must first be selected (Pfeffer and Parkes, 2010,
CS181 Lecture Notes, Harvard University).
[0274] A learning algorithm (Learner) is defined as a function that
maps a data set to a model with fully estimated parameter values.
Clearly, one given Learner will likely produce different models on
different data sets. The learning algorithm in the case of a Bayes
Net is defined by the parameter estimation method (ML, MAP or EM)
and by the topology (conditional independence assumptions) of this
particular Bayes Net. Given a fixed parameter estimation method
such as ML, different Learners can be generated by modifying Bayes
Net topologies. The problem of comparing different Bayes Net
topologies thus reduces to the problem of selecting the optimal
Learner. The problem of selecting the optimal Learner is at the
core of Machine Learning.
[0275] One approach is to train each Learner on one training set
and to test the learned model, output by each Learner, on one
independently and identically sampled test set. Then, the Learner
that outputs the model with the best prediction accuracy on the
test set is the optimal Learner. However, the data set is a random
variable too. Only one training set and one test set will likely
result in the selection of a non-optimal Learner. Therefore, the
expected performance of each Learner is estimated and the optimal
Learner is selected based on its expected performance across many
independently and identically (iid) sampled training set and test
set pairs. The above process of training and testing is repeated on
many pairs of iid sampled training and test sets with each Learner
and then the average prediction accuracy of the models output by
each Learner is calculated across these training set and test set
pairs. This procedure will provide the expected performance
estimate of each Learner. The Learner that outputs the models with
the best average prediction accuracy across all the test sets is
the optimal Learner.
[0276] Given a data set of limited size, cross-validation is an
appropriate approximation for the selection of the optimal Learner
based on its expected performance (Pfeffer and Parkes, 2010, CS181
Lecture Notes, Harvard University). Cross-validation enables
selecting the optimal Learner on just one data set. In a k-fold
cross-validation, the data set is subdivided into k subsets of
equal size. In this way, k iid sampled test sets are obtained, each
used in only one of the k runs of the k-fold cross-validation. The
training set in each of the k runs of the k-fold cross-validation
consists of the other k-1 subsets excluding the current test set.
Thus, the training sets overlap across the k runs of the k-fold
cross-validation and are not independent (in contrast to the test
sets). However, k-fold cross-validation provides a good
approximation for selecting the optimal Learner based on its
expected performance with limited data. Eventually, after selecting
the optimal Learner the final optimal model can be learned with
minimal overfitting by applying the selected optimal Learner to the
whole data set. Importantly, the average test set accuracy of the
models, output by the optimal Learner in the above Learner
selection procedure, cannot be considered as the expected
prediction accuracy of the final optimal model. This can be done on
another independent data set that was not used for Learner
selection. In practice, however, it is often enough to know that
the selected Learner (Bayes Net topology+ML) is the optimal
Learner, before starting to use its final output model in real
applications without actually determining the expected prediction
accuracy of the final model.
[0277] In summary, the minimization of overfitting is achieved by
selecting the optimal Learner function with the optimal hypothesis
space. The optimal hypothesis space defined by the optimal Learner
minimizes the extent of overfitting in the final model. The final
model, output by the optimal Learner, is general and captures the
true hidden concept with minimal overfitting.
Data Set
[0278] A data set of multiplex protein measurements was acquired
with Lysate Microarrays. Flash-frozen human tissue samples from 56
human patients were lysed in RIPA buffer. A tumor tissue sample and
a sample of the adjacent normal tissue were collected from each
patient. In total, 56 samples of tumor tissue and 56 samples of
normal tissue were obtained. Most tumors were identified as ductal
carcinoma (48 out of 56). Each of the 112 samples was printed on
100 different glass-mounted nitrocellulose pads by an Aushon
Arrayer. Each of these pads was then incubated with a different
primary antibody, previously validated for Lysate Microarrays from
the initial set of several thousands of antibodies. This set of
validated antibodies included pan-specific antibodies and
phospho-specific antibodies. Each nitrocellulose pad was also
incubated with a validated .beta.-actin antibody for normalization
purposes. After the subsequent incubation with the corresponding
secondary antibodies, all signal intensities were recorded and
compiled in one file.
Optimal Bayes Net Topology
[0279] The first problem related to the above-described data set
was its high dimensionality (101:100 proteins+Cancer/Normal
phenotype) and a relatively low number of data points (112). Given
such a high dimensionality, it was likely that interesting patterns
could emerge simply by chance without properly representing the
true hidden concept of cancer. In order to perform the data-driven
selection of the optimal Bayes Net topology, the number of
dimensions was reduced by focusing on only one particular signaling
pathway. Out of 101 dimensions, 10 were selected: Cancer/Normal
phenotype, Axl, Met.p, Src.p, cRaf.p, Akt.p, Stat3.p, Erk1.2.p,
MAPK.p1 and S6.p1. This selection was made without any prior
inspection of the data set. In this way, the dimensionality of each
of the 112 data points was reduced to 10. Empirically, such a ratio
between the number of dimensions and the number of data points was
expected to yield meaningful results.
[0280] Given the data set of dimensionality 10 consisting of 112
data points, it was determined which set of conditional
independence assumptions optimized the hypothesis space for
capturing the true hidden concept of cancer. The data set was
binarized by choosing the median value within each dimension as its
binarization threshold. 15 different Bayes Net topologies were
empirically chosen. Each Bayes Net topology consisted of the same
10 Bernoulli random variables, but had a different set of directed
edges thus presumably representing a different set of conditional
independence assumptions (FIG. 23). Each Bernoulli random variable
represented one dimension of the data set: Axl, Met.p, Src.p,
cRaf.p, Akt.p, Stat3.p, Erk1.2.p, MAPK.pl, S6.pl and the
Cancer/Normal phenotype (FIG. 23). These 15 different Bayes Net
topologies also had different complexities determined by counting
the number of the free parameters in each topology. Because the
data set was balanced, the prior distribution for the Cancer random
variable was as follows: P(Cancer=True)=0.5, P(Cancer=False)=0.5
(FIG. 23).
[0281] The procedure of Learner selection was performed on the set
of 15 Bayes Net topologies by 10-fold cross-validation (ML
procedure was the same in all Learners). 8 of these topologies are
displayed in FIG. 23. The parameter values of each Bayes Net
topology were estimated by the maximum likelihood method on the
training set in each run of the 10-fold cross-validation. Because
all the random variables of the 15 Bayes Net topologies were
Bernoulli random variables, the maximum likelihood method was
reduced to a simple counting and normalization procedure of the
corresponding instances in the training set (Bishop, 2006, Pattern
Recognition and Machine Learning, Springer, First Edition). In
order to evaluate how well each learned Bayes Net model with
estimated parameter values captured the true hidden concept of
cancer, an appropriate measure of prediction accuracy was chosen on
the test set. It was decided that the classification accuracy of
the cancer phenotype was an appropriate measure of prediction
accuracy. In each of the 10 runs of the 10-fold cross-validation,
the parameter values of the given Bayes Net topology were estimated
on the training set and then the Bayes Net model with estimated
parameter values was tested on the test set by comparing the
predicted values of the cancer phenotype (True or False) with the
actual values of the cancer phenotype. Formally, this
classification task can be formulated as follows:
argmax.sub.c.epsilon.{True,False}P(Cancer=c,Evidence|.theta..sub.ML)
[0282] where Evidence represents all the current assignments of the
protein random variables in the currently considered data point of
the test set and .theta.ML represents the parameters estimated by
the maximum likelihood method on the training set in each run of
10-fold cross-validation. If the predicted argmax result is the
same as the actual value of the cancer phenotype in the currently
considered data point of the test set, then this case was
interpreted as a match. Otherwise, it was a mismatch. By counting
the number of matches and mismatches in the test set of the current
run of the 10-fold cross-validation, the prediction accuracy in
this test set was obtained. In total, the prediction accuracy was
determined ten times for each Learner in the 10-fold
cross-validation. The expected performance of each Learner (Bayes
Net topology) was calculated by averaging the accuracy rates of
output models across all the 10 test sets. Overall, 15 topologies
were evaluated by 10-fold cross-validation and 15 values of
expected performance were obtained.
[0283] FIG. 24 shows the results of running 10-fold
cross-validation on the 8 topologies displayed in FIG. 23. These 8
topologies were selected for demonstration because of their logical
progression. The average performance on the training set was found
to be consistently better than the average performance on the test
set for any Learner (Bayes Net topology). This result is consistent
with the output models always fitting the data in the training sets
better than the data in the unseen independent test sets.
Surprisingly, the models based on the Naive Bayes topology did
quite well on average in predicting the correct cancer phenotype.
In this data set, higher phosphorylation levels were observed
across all the proteins in cancerous samples in contrast to normal
samples. Thus, conditioning all the protein random variables on the
Cancer random variable was sufficient to capture this general
tendency for higher phosphorylation levels in the cancerous
samples. AXL+MET+AKT was the topology with the best expected
performance. This topology had three additional edges on top of the
Naive Bayes topology. By conditioning the phosphorylation levels of
Met.p on Cancer and Axl (AXL+MET+AKT), a better hypothesis space
was obtained than by conditioning the phosphorylation levels of
Met.p on Cancer alone (MET+AKT), given that cRaf.p was conditioned
on Met.p, Akt.p and Cancer in both cases (FIGS. 23 and 24). By
conditioning cRaf.p on Met.p, Akt.p and Cancer (MET+AKT), a better
hypothesis space was obtained than by conditioning cRaf.p on Cancer
alone (NA VE BAYES) or by condition cRaf.p on Cancer and Met.p
alone (MET) (FIGS. 23 and 24). Importantly, the addition of more
complexity to the AXL+MET+AKT topology expanded the hypothesis
space of the resulting Learners beyond the optimal level of
complexity and did not improve their expected performance because
of overfitting.
[0284] An explanation of why the incorporation of additional edges
into the Bayes Net topology led to a better classification accuracy
of the resulting learned models is shown in FIG. 25. The
distribution of Met.p levels in the cancerous samples was similar
to the distribution of Met.p in the non-cancerous samples. Thus,
after binarization, the probability of Met.p to be highly
phosphorylated was approximately equal to the probability of Met.p
to be phosphorylated at low levels in both the cancerous and
non-cancerous samples. However, by adding an additional
conditioning arrow from Axl to Met.p different conditional
distributions of Met.p were obtained when conditioned on Axl=High,
Cancer=False and when conditioned on Axl=High, Cancer=True (FIG.
25). Effectively, it was observed that in the samples with high Axl
levels, the phosphorylation levels of Met.p were more likely to be
low in the cancerous samples than in the non-cancerous samples.
After adding the Axl-Met.p edge to the MET+AKT topology, such a
distinction led to better average classification accuracy when all
the conditional probabilities were multiplied in the total joint
probability distribution used in the classification task (FIG.
24).
[0285] Because Bayes Nets enable the modification of graph
topologies with a clear understanding of conditional independence
assumptions by humans, the above approach can be used to search for
the conditional independence assumptions that optimize the
hypothesis space for capturing the true hidden concept of cancer.
The edges of the optimal Bayes Net topology can be interpreted
causally in the context of protein interactions. At the level of
single cells, this approach can allow for searching for the Bayes
Net topologies that enable capturing the underlying concepts of
cell types. Given a highly dimensional data set sampled from
different cell types, the Bayes Nets (topologies and the resulting
models) can be identified that accurately capture the concept
(molecular mechanism) of each cell type. Such cell-type specific
Bayes Nets can be used for data interpolation in order to predict
the mechanistic response of each cell type to previously unseen
stimulation conditions in response to drugs.
[0286] Experiments can be performed to apply the procedure of
selecting the optimal Learner (Bayes Net topology+fixed ML
subroutine) in the context of single-cell measurements enabled by
Inside-Out Lysis and by Single-Cell Lysate Microarrays. Experiments
can also be performed on single-cell measurements in animal solid
tissues or in human tumor tissues. Experiments can also be
performed to look for and identify new cell types based on single
cell data by building in hidden random variable(s) and by using EM
for parameter estimation.
Example 5
Inside Out Lysis Technology in the Investigation of Psychiatric
Diseases
[0287] The Inside Out Lysis technology described herein can be used
to investigate psychiatric diseases. In recent years, several mice
have been engineered to carry genetic modifications found in
schizophrenia and autism, and the mice are now commercially
available. The mice described elsewhere herein can be bred with
these disease models to produce mice harboring disease-relevant
genetic mutations that express GFP in defined subsets of
interneurons. Slice cultures are particularly relevant for studying
questions related to brain development. After generating the
appropriate strains of mice, Inside Out Lysis technology can be
used to obtain interneuron lysates from several developmental time
points in different regions of the brain.
[0288] Interestingly, it has been found that TrkB signaling is
significantly altered in parvalbumin expressing interneurons of
schizophrenic patients (Lewis D, et al. (2005) Nature Rev.
Neurosci. 6, 312-324). Using the technology described elesewhere
herein, the differences of TrkB signaling in different subsets of
interneurons in mouse models of schizophrenia and autism can be
investigated over the first two months of development, providing
insight into the molecular events underlying these debilitating
disorders.
Example 6
Determining Absolute Counts of Measured Proteins in a Single
Cell
[0289] Experiments can be performed to determine the absolute
counts of the measured proteins in each single cell. These counts
can be obtained when the signal for the protein, such as GFP, PKC
and PKCa, is mapped to the absolute quantity. Although it is
practically difficult to determine the exact volume deposited by
the Aushon Arrayer in each spot of the printed concurrent titration
series, this can be resolved by manually printing a well-defined
amount of the respective purified proteins of control lysates next
to the Aushon-printed titration series. By measuring the antigen
amounts in the spots with the known antigen quantities, the spots
of the Aushon Arrayer-printed titration series can be mapped to
their respective absolute quantities. Consequently, the absolute
standard curves can be derived and the absolute levels of proteins
in each printed single-cell lysate can be determined.
[0290] A somatostatin interneuron can be lysed from a living brain
slice culture displayed in and spotted onto nitrocellulose as
described elsewhere herein. The nitrocellulose plate can be
prepared with an extensive dilution series (1:1.25 resolution) of
purified GFP titrated across different levels of actin background
(average hippocampus lysate). The high resolution of dilution
series can allow for reliable fitting of nonlinear functions
without overfitting (titration data constrains the hypothesis
within its hypothesis space). Error values can be estimated
reliably as well, and cross-reactivities can be filtered out.
Nonlinearities of the signal-to-quantity function that are crucial
for correct population size estimations can be accounted for with
high resolution titration series and with nonlinear fitting.
Printing of the titrations of phoshpo-peptides (blocking peptides
for phospho-specific antibodies) can be carried out. The absolute
phoshporylation levels combined with the absolute total amounts of
proteins can also be determined in the above manner.
Example 7
Analytical Chemistry Technology in Neurobiology
[0291] According to recent estimates, the amount of ATP in a single
neuron is within the sensitivity range of a mass spectrometer.
Experiments can be carried out to determine the amount of ATP in a
single neuron. Experiments can also be carried out to provide
extensive multiplexing with the help of Dynal beads or other
methods. Breeding homozygous GFP carriers with non-carriers can
create mice with half the amount of GFP in interneurons. The
specificity of absolute measurements can then be further assessed.
These experiments can determine the current experimental bounds on
what type of data can be acquired. Given these bounds, a
pathology-related investigation can be accomplished in order to
enable the scaling of analytical chemistry technology in
neurobiology.
[0292] Whereas most human diseases occur in solid tissues (the
exception being for example blood-based diseases like leukemia),
most molecular profiling studies in neuroscience and systems
biology have so far been performed in cell lines and dissociated
cultures, grown in petri dishes outside the native tissue context.
Native heterogeneity of cell types is not preserved in these
samples.
[0293] The sampling technology of the present invention uses
complex chemistry to solubilize single cells in solid tissues from
inside the intracellular space. The "from inside" directionality of
the solubilization process enables perfect spatial and high
temporal single-cell resolution in complex tissues. The
solubilization chemistry enables collection of all molecules of
each sample single cell and analysis of these molecules with most
sensitive quantitative profiling methods.
[0294] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
* * * * *