U.S. patent application number 14/285024 was filed with the patent office on 2016-12-01 for cell-based arrays, methods of making, and methods of using.
The applicant listed for this patent is University of Florida Research Foundation, Inc.. Invention is credited to Abhinav Prakash Acharya, Matthew Carstens, Emina Huang, Benjamin George Keselowsky, Edward William Scott.
Application Number | 20160349241 14/285024 |
Document ID | / |
Family ID | 45997358 |
Filed Date | 2016-12-01 |
United States Patent
Application |
20160349241 |
Kind Code |
A9 |
Keselowsky; Benjamin George ;
et al. |
December 1, 2016 |
CELL-BASED ARRAYS, METHODS OF MAKING, AND METHODS OF USING
Abstract
Embodiments of the present disclosure provide for arrays,
systems, and methods analyzing cells, methods of making arrays, and
the like.
Inventors: |
Keselowsky; Benjamin George;
(Gainesville, FL) ; Acharya; Abhinav Prakash;
(Gainesville, FL) ; Huang; Emina; (Gainesville,
FL) ; Scott; Edward William; (Gainesville, FL)
; Carstens; Matthew; (Gainesville, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Florida Research Foundation, Inc. |
Gainesville |
FL |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20140323353 A1 |
October 30, 2014 |
|
|
Family ID: |
45997358 |
Appl. No.: |
14/285024 |
Filed: |
May 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13373051 |
Nov 2, 2011 |
9012202 |
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14285024 |
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61826139 |
May 22, 2013 |
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61409223 |
Nov 2, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54366 20130101;
G01N 2500/10 20130101; G01N 33/5011 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention(s) was made with government support under
Grant No.: DGE-0802270 awarded by the National Science Foundation
and grant numbers RO1 CA142808 and RO1 CA157663, awarded by the
National Institute of Health (National Cancer Institute), and
R21Al094360 awarded by the National Institute of Health (National
Institute of Allergy and Infectious Diseases). The government has
certain rights in the invention(s).
Claims
1. An array, comprising: a non-fouling layer disposed in a first
area of the array, wherein cells do not substantially adhere to the
non-fouling layer; and a plurality of cell binding sites, each
being disposed in a cell binding site area of the array distinct
from the non-fouling layer, wherein the cell binding sites include
a cell adhesion layer and a timed-release polymer layer, wherein
each timed-release polymer layer corresponding to a cell binding
site comprises one or more thin films including one or more types
of an agent, wherein the timed-release polymer layer of at least
one cell binding site comprises at least one type of agent
different from at least one type of agent in the timed-release
polymer layer of at least one other cell binding site or comprises
a different concentration of agent than the concentration of agent
in the timed-release polymer layer of at least one other cell
binding site, and wherein one or more types of target cells adhere
to the cell adhesion layer, the timed-release polymer layer having
the characteristic of releasing the agent to the cell or cells
adhered to the cell binding site.
2. The array of claim 1, wherein one of type target cell, a first
target cell, has an affinity for another type of target cell, a
second target cell, wherein both the first target cell and the
second target cell are disposed on the cell binding site, wherein
the timed-release polymer having the characteristic of releasing
the agent to the first target cell and the second target cell
adhered to the cell binding site.
3. The array of claim 1, wherein the cell binding sites have an
area of about 20 .mu.m.sup.2 to 5 mm.sup.2 and wherein a pair of
cell binding sites is positioned about 10 .mu.m to 2 mm from one
another.
4. The array of claim 1, wherein the time release agent is not
DNA.
5. The array of claim 1, wherein the timed-release polymer layer of
at least one cell binding site comprises at least one type of agent
different from at least one type of agent in the timed-release
polymer layer of at least one other cell binding site.
6. The array of claim 1, wherein the timed-release polymer layer of
at least one cell binding site comprises a first concentration of
one type of agent different from a concentration of that type of
agent in the timed-release polymer layer of at least one other cell
binding site.
7. The array of claim 1, wherein multiple cell binding sites have
one or more different types of agent in the timed-release polymer
layer than the types of agent in the timed-release polymer layers
of other cell binding sites.
8. The array of claim 1, wherein multiple cell binding sites have
one or more different types of agents or combinations of agents
present in different concentrations than the types and combinations
of agents in the timed-release polymer layers of other cell binding
sites.
9. The array of claim 1, wherein the array comprises at least a
first and second type of agent, wherein the first agent is present
in at least two different concentrations in the timed-release
polymer layers of at least two different cell binding sites and the
second agent is present in at least two different concentrations in
at least two different cell binding sites, and wherein the first
agent and second agent are combined in different concentrations in
at least two different cell binding sites.
10. The array of claim 1, wherein at least one agent comprises
nutlin-3a.
11. The array of claim 1, wherein at least one agent comprises
camptothecin.
12. The array of claim 1, wherein at least one type of target cell
is a cancer stem cell.
13. The array of claim 12, wherein the cancer stem cell is a
colorectal cancer stem-like cell.
14. The array of claim 1, wherein the timed-release polymer layer
is selected from the group consisting of: a poly(lactic-co-glycolic
acid), polycaprolactone, polyglycolide, polylactic acid,
poly(vinylpyridine), chitosan; alginate, and a combination
thereof.
15. The array of claim 1, wherein the timed-release polymer layer
includes a plurality of layers each having a thickness of about 10
nm to 5 .mu.m.
16. The array of claim 1, wherein the cell adhesion layer is
selected from the group consisting of: fibronectin; polylysine;
collagen; vitronectin; intercellular adhesion molecules;
immunoglobulin superfamily Cell Adhesion Molecules (IgSF CAMs),
Neural Cell Adhesion Molecules; ICAM-1 Intercellular Cell Adhesion
Molecule, VCAM-1 Vascular Cell Adhesion Molecule, PECAM-1
Platelet-endothelial Cell Adhesion Molecule, L1, integrin;
cadherin; and a combination thereof.
17. An array, comprising: a first substrate having a first area and
a plurality of cell binding site areas, wherein the first area of
the array includes: a first bonding layer disposed on the first
area of the first substrate; a second bonding layer disposed on the
first bonding layer; a non-fouling layer disposed on the second
bonding layer, wherein cells do not adhere to the non-fouling
layer; and wherein the cell binding site areas are different areas
of the first substrate, wherein the cell binding site areas
include: an adhesive layer disposed on each of the cell binding
site areas of the first substrate; a timed-release polymer layer
disposed on the adhesive layer, wherein the timed-release polymer
layer comprises one or more thin films including one or more
agents, wherein the timed-release polymer layer of at least one
cell binding site comprises at least one type of agent different
from at least one type of agent in the timed-release polymer layer
of at least one other cell binding site or comprises a different
concentration of agent than the concentration of agent in the
timed-release polymer layer of at least one other cell binding
site; and a cell adhesion layer disposed on the timed-release
polymer layer.
18. The array of claim 17, wherein the first bonding layer is
selected from the group consisting of: titanium, nickel, chromium,
and a combination thereof.
19. The array of claim 17, wherein the second bonding layer is
selected from the group consisting of: gold, silver, copper,
palladium, platinum, nickel, alloys of each of these, and a
combination thereof.
20. The array of claim 17, wherein the adhesive layer is selected
from the group consisting of: a silane, a compound including an
ethylene oxide group, a compound including a acrylamide group, a
compound including an amine group, a compound including a
hydrophobic group, and a combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part application of
and claims priority to U.S. patent application entitled "CELL-BASED
ARRAYS, METHODS OF MAKING, AND METHODS OF USING," having Ser. No.
13/375,051 and filed on Nov. 2, 2011, which claims priority to U.S.
provisional patent application of the same title having Ser. No.
61/409,223, filed on Nov. 2, 2010, both of which are entirely
incorporated herein by reference. This application also claims
priority to U.S. provisional patent application entitled
"CELL-BASED ARRAYS, METHODS OF MAKING, AND METHODS OF USING,"
having Ser. No. 61/826,139 and filed on May 22, 2013, which is
entirely incorporated by reference herein.
BACKGROUND
[0003] It is becoming increasingly evident in cancer treatment that
simultaneously targeting multiple critical pathways, using
combinations of chemotherapeutic drugs, can enhance
outcome.sup.1-5. Currently, oncologists lack the tools necessary to
predict the success of various combination treatments from one
patient to the next. Sensitivity to different classes of
chemotherapeutics is highly variable, due in part to intratumor
heterogeneity.sup.1. Recent findings attribute this heterogeneity
to a rare population of cancer stem cells (CSCs) which are now
being targeted for therapy. A barrier to this approach is the
limitation of having very few available cells on which to test drug
combinations.sup.6, 7.
[0004] Colon cancer is the third most common cause of cancer and
cancer death in the United States. Colon cancer stem cells (CCSC's)
have only recently been recognized as a potential cause of colon
cancer with several markers identified. As such, this cell
population has also been targeted for future therapeutics, but the
rarity of CCSC's makes it difficult to screen potential agents.
SUMMARY
[0005] Embodiments of the present disclosure provide for arrays,
systems, and methods for the analyzing cells, methods of making
arrays, and the like.
[0006] An embodiment of the array, among others, includes: a
non-fouling layer disposed in a first area of the array, where
cells do not substantially adhere to the non-fouling layer; and a
plurality of cell binding sites, each being disposed in a cell
binding site area of the array distinct from the non-fouling layer,
where the cell binding sites include a cell adhesion layer and a
timed-release polymer layer, where each timed-release polymer layer
corresponding to a cell binding site includes one or more types of
an agent, where one or more types of target cells adhere to the
cell adhesion layer, and where the timed-release polymer has the
characteristic of releasing the agent to the cell or cells adhered
to the cell binding site. The timed-release polymer layer of at
least one cell binding site includes at least one type of agent
different from at least one type of agent in the timed-release
polymer layer of at least one other cell binding site or has a
different concentration of agent than the concentration of agent in
the timed-release polymer layer of at least one other cell binding
site
[0007] An embodiment of the array, among others, includes: a first
substrate having a first area and a plurality of cell binding site
areas, wherein the first area of the array includes: a first
bonding layer disposed on the first area of the first substrate; a
second bonding layer disposed on the first bonding layer; a
non-fouling layer disposed on the second bonding layer, wherein
cells do not adhere to the non-fouling layer; and wherein the cell
binding site areas are different areas of the first substrate,
wherein the cell binding site areas include: an adhesive layer
disposed on each of the cell binding site areas of the first
substrate; a timed-release polymer layer disposed on the adhesive
layer; and a cell adhesion layer disposed on the timed-release
polymer layer. In this embodiment, the timed-release polymer layer
of at least one cell binding site includes at least one type of
agent different from at least one type of agent in the
timed-release polymer layer of at least one other cell binding site
or has a different concentration of agent than the concentration of
agent in the timed-release polymer layer of at least one other cell
binding site
[0008] Other structures, arrays, methods, features, and advantages
of the present disclosure will be, or become, apparent to one with
skill in the art upon examination of the following drawings and
detailed description. It is intended that all such additional
structures, methods, features, and advantages be included within
this description, be within the scope of the present disclosure,
and be protected by the accompanying claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Many aspects of the disclosed devices and methods can be
better understood with reference to the following drawings. The
components in the drawings are not necessarily to scale, emphasis
instead being placed upon clearly illustrating the relevant
principles. Moreover, in the drawings, like reference numerals
designate corresponding parts throughout the several views.
[0010] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0011] FIG. 1.1 illustrates a cross-section of an embodiment of the
present disclosure.
[0012] FIG. 1.2 illustrates a cross-section through the a-a plane
of the embodiment shown in FIG. 1.1.
[0013] FIG. 1.3A illustrates a schematic of an embodiment of the
present disclosure, while FIG. 1.3B illustrates a representative
microarray having more than 1000 spots.
[0014] FIG. 2.1 illustrates a phase micrograph of array seeded with
murine colon cancer stem cells.
[0015] FIGS. 2.2A to 2.2C illustrate micrographs of CCSC's attached
to PLGA island imaged using (a) Phase, (a,b) DAPI, and (a,c) FITC,
respectively.
[0016] FIG. 2.3 is a graph that illustrates a release profile for
PLGA films loaded with coumarin, 355 nm/460 nm.
[0017] FIG. 3.1 illustrates human epithelial cells (HCE-T corneal
epithelial cells) that are shown seeded on a PLGA film array
(over-spotted with adhesion molecules collagen and
fibronectin).
[0018] FIG. 3.2 illustrates the release over time of coumarin
(fluorescent dye) from a chip printed with 169 coumarin-loaded PLGA
films.
[0019] FIG. 4.1 illustrates a phase contrast/fluorescence overlay
micrograph displaying SW480 cells attached to isolated ethylene
vinyl acetate (EVA) islands. Cells were seeded onto a
small-molecule eluting array and stained with Hoechst 34580.
[0020] FIG. 4.2 illustrates a mosaic of SW480 cells seeded onto
small-molecule eluting array and stained with Hoechst 34580.
[0021] FIG. 4.3 illustrates a micrograph of one column in 7.times.7
small-molecule-eluting cellular array separated by a fluorescent
channel. Arrays were seeded with SW480 cells and stained for BrdU
incorporation (green), indicating proliferating cells, and nuclear
counter-stain (blue).
[0022] FIG. 4.4 illustrates a release profile from arrays printed
with 20% (w/w) coumarin-loaded EVA. The red curve represents arrays
printed with single spots of coumarin-loaded EVA, while the green
curve represents a blank EVA film printed over the coumarin-loaded
EVA films to delay dye release.
[0023] FIGS. 5.1A to 5.1C illustrate HCT116 cells that are nuclear
stained with Hoechst over a time of 5 h, 52 h, and 68 h, without
any azide present.
[0024] FIGS. 5.2A to 5.2C illustrate HCT116 cells that are nuclear
stained with Hoechst over a time of 5 h, 52 h, and 68 h, with 37.5
mM azide present.
[0025] FIGS. 5.3A to 5.3C illustrate HCT116 cells that are nuclear
stained with Hoechst over a time of 5 h, 52 h, and 68 h, with 75 mM
azide present.
[0026] FIGS. 5.4A to 5.4C illustrate HCT116 cells that are nuclear
stained with Hoechst over a time of 5 h, 52 h, and 68 h, with 75 mM
azide present.
[0027] FIGS. 6A-6C illustrate an embodiment of drug-eluting
cellular microarrays of the present disclosure. FIG. 6A is a
schematic illustration of printing of glass coverslips to form the
arrays. FIG. 6B shows a schematic of a single spot highlighting the
substrate architecture, the chemistry of the non-fouling PEG
coating, and the drug eluting polymer with cells attached (not to
scale). FIG. 6C is a fluorescence microscopy mosaic image of a
10.times.11 microarray seeded with HCT116 colon carcinoma cells
illustrating fidelity of cell adhesion to isolated islands of
drug-eluting polymer films. Also shown is a detail of a single drug
eluting island demonstrating adherent cells (nuclear staining is
highlighted/outlined in lighter shading). Scale bar=200 .mu.m.
[0028] FIGS. 7A-7L illustrate cumulative drug release from array
spots and HCT116 cell responses to drug-loaded microarrays. FIG. 7A
is a graph illustrating nutlin-3a release profile from microarray
revealed a burst release of approximately 8 h followed by a steady
release rate over five days. Release profiles show
means.+-.standard deviations of three replicates, and data is
modeled using exponential decay. FIG. 7B is a graph illustrating
that the percent of non-proliferative HCT116 cells on nutlin-3a
loaded microarray increases with increasing drug loading
concentration. (Proliferation was quantified via BrdU incorporation
and data is normalized to unloaded control. Significant differences
were determined by ANOVA, {F(4,138)=19.068, p<0.05}, followed by
Tukey's post-hoc analysis.) The images in FIG. 7C illustrates
representative fluorescence micrographs of non-proliferating cells
on a 25 .mu.M nutlin-3a-loaded polymer island (low BrdU staining).
FIG. 7D is an image illustrating representative fluorescence
micrographs of an unloaded control island with highly proliferative
cells (high BrdU staining). FIG. 7E is a graph illustrating the
camptothecin release profile from microarray revealing a burst
release of approximately 24 h followed by a steady release rate
over five days. (Release profiles show means.+-.standard deviations
of three replicates, and data is modeled using exponential decay.)
FIG. 7F is a graph illustrating that the percent of apoptotic cells
on camptothecin loaded microarray increases with increasing drug
loading concentrations. (Apoptosis was quantified by annexin V
staining and significant differences were determined by ANOVA,
{F(4,479)=52.778, p<0.05}, followed by Tukey's post-hoc
analysis.) FIG. 7G shows representative fluorescence micrographs
displaying high levels of cells undergoing apoptosis on a 10 .mu.M
camptothecin-loaded polymer island (high annexin V staining). The
images of FIG. 7H illustrate representative fluorescence
micrographs of an unloaded control island with low levels of
apoptotic cells (low annexin V staining). FIG. 7I is a schematic
illustration of a single factor dosing array layout with increasing
drug loading concentrations. The schematic of FIG. 7J illustrates a
randomized single factor array with loading concentrations
configured in randomized fashion. The graph of FIG. 7K illustrates
statistical comparison of cell apoptosis between the array
configurations of FIGS. 7I and 7J and indicates results are
independent of array configuration (n=3). This indicates that there
is negligible cellular cross-talk or drug interaction between
neighboring islands. FIG. 7 is a schematic illustration of a
randomized two-factor dosing array used in combinatorial
microarrays. Different patterns represent the 16 different
combinations of two drugs (four concentrations per drug). (*:
p<0.05 compared to all other conditions, #: p<0.05 compared
to control). Scale bar=200 .mu.M.
[0029] FIG. 8A is a graph illustrating the release kinetics of
coumarin-loaded EVA films and showing that overspotting of unloaded
EVA over coumarin-loaded EVA mitigates bolus release. (*:
p<0.05). The bar graph in FIG. 8B illustrates cell counts of
HCT116 on azide loaded film after attachment. (Microarrays were
fixed with 4% paraformaldehyde and stained with Hoechst 34580
nuclear dye 1 h after seeding to quantify initial cell density. No
statistical difference was found by ANOVA (p=0.490)).
[0030] FIGS. 9A-9B are graphs illustrating that HCT116 cell numbers
exhibit dose dependent responses to drug loading concentration.
Cell numbers decreased with increasing loading concentrations of
azide after 24 h (FIG. 9A) and nutlin-3a after 72 h (FIG. 9B). (*:
p<0.05 compared to all other concentrations).
[0031] FIGS. 10A-I illustrate proliferation and dose-response
curves from combinatorial microarrays of HCT116 cells. FIG. 10A is
a three-dimensional graph illustrating that increasing
concentrations of combination treatments increased the overall
antiproliferative activity. Following 24 h incubation with both
nutlin-3a and camptothecin, proliferation of HCT116 cells
significantly decreased. A significant primary effect on
proliferation relative to nutlin-3a, {F(3,619)=18.253, p<0.01},
and camptothecin, {F(3,619)=25.056, p<0.01} was revealed by
two-way ANOVA. Additionally, a sub-additive effect was observed
from combination treatments. The graphs in FIGS. 10B-10E illustrate
dose response curves of fixed camptothecin concentrations with
variable nutlin-3a concentration. The addition of camptothecin
increased the sensitivity to nutlin-3a by over 5-fold (19.6 for 50
.mu.M CPT compared to 3.0 for 0 .mu.M CPT). The graphs in FIGS.
10E-10I illustrate dose response curves of fixed nutlin-3a
concentrations with variable camptothecin concentration. The
presence of nutlin-3a increased the sensitivity to camptothecin by
over 16-fold (78.1 for 125 .mu.M nutlin compared to 4.83 for 0
.mu.M nutlin). Proliferation data were transformed to
non-proliferation data by subtracting the former from 100%. (*:
p<0.05 compared to 0 drug) (Bars atop columns represent
SEM).
[0032] FIGS. 11A-11I illustrate apoptosis and dose-response curves
from combinatorial microarrays of HCT116 cells. FIG. 11A is a
three-dimensional graph illustrating that Nutlin-3a and
camptothecin had varying effects on inducing apoptosis of HCT116
cells. A significant antagonistic effect on apoptosis was observed
from combination treatments as revealed by two-way ANOVA,
{F(9,342)=3.371, p<0.05}. FIGS. 11B-11E illustrate dose response
curves of fixed camptothecin concentrations with variable nutlin-3a
concentration. The graphs of FIGS. 11C-11E illustrate that
increasing the nutlin-3a concentration conferred protection from
the apoptotic response to camptothecin. FIGS. 11F-11I are graphs
showing a dose response curve of fixed nutin-3a concentrations with
variable camptothecin concentration. The graphs of FIGS. 11G-11I
illustrate that addition of nutlin-3a attenuated the apoptotic
response to camptothecin. Sensitivity could not be statistically
compared when evaluating apoptosis as the hyperbolic curve fit does
not apply to the linear dose response curves. (*: p<0.05
compared to 0 .mu.M) (Bars atop columns represent SEM).
[0033] FIGS. 12A-12D are a series of graphs illustrating apoptosis
and proliferation dose-response curves from HCT116 cells incubated
with soluble drugs. FIG. 12A illustrates the percent of apoptotic
HCT116 cells incubated 24 h with soluble nutlin-3a. FIG. 12B shows
the percent of apoptotic HCT116 cells incubated 24 h with soluble
camptothecin. Significant differences were determined by ANOVA,
{F(5,282)=19.694, p<0.05}, followed by Tukey's post-hoc
analysis. FIG. 12C illustrates the percent of non-proliferating
HCT116 cells incubated 24 h with soluble nutlin-3a. Significant
differences were determined by ANOVA, {F(4,226)=12.643, p<0.05},
followed by Tukey's post-hoc analysis. FIG. 12D shows the percent
of non-proliferating HCT116 cells incubated 24 h with soluble
camptothecin. Significant differences were determined by ANOVA,
{F(5,252)=102.485, p<0.05}, followed by Tukey's post-hoc
analysis. (*: p<0.05 compared to all other conditions,
#:p<0.05 from all conditions marked *).
[0034] FIGS. 13A-13B are bar graphs illustrating ALDH and MUC2
expression in patient-derived CCSCs. FIG. 13A shows that ALDH (a
marker for CCSCs) expression was unchanged in cells from CA1 when
cultured as spheres or monolayers, whereas cells from CA2 showed
markedly decreased expression of ALDH when cultured as monolayers
compared to spheroids. FIG. 13B shows that MUC2 (a marker for
goblet cell lineage) expression decreased slightly in cells from
CA1 when cultured as monolayers compared to spheroids. By contrast,
cells from CA2 showed higher MUC2 expression in monolayers versus
spheroid culture.
[0035] FIGS. 14A-14R illustrate patient-derived CCSC responses to
drug-loaded microarrays. FIG. 14A is a three dimensional graph
illustrating proliferation response of CA1 cells on drug-eluting
cellular microarrays. FIGS. 14B-141 are graphs showing dose
responses of CA1 cells exposed to ranges of one drug in combination
with a fixed amount of a second drug. Proliferation values were
transformed to non-proliferation by subtracting values from 100%.
FIG. 14J is another three dimensional graph illustrating
proliferation response of CA2 cells on drug-eluting cellular
microarrays. FIGS. 14K-14R illustrate dose responses of CA2 cells
exposed to ranges of one drug in combination with a fixed amount of
a second drug. Proliferation values were transformed to
non-proliferation outcomes by subtracting values from 100%. (*:
p<0.05 from 0 .mu.M). (Bars atop columns represent SEM).
DETAILED DESCRIPTION
[0036] Before the present disclosure is described in greater
detail, it is to be understood that this disclosure is not limited
to particular embodiments described, as such may, of course, vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present disclosure
will be limited only by the appended claims.
[0037] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
(unless the context clearly dictates otherwise), between the upper
and lower limit of that range, and any other stated or intervening
value in that stated range, is encompassed within the disclosure.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the disclosure, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the disclosure.
[0038] 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 disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present disclosure, the preferred methods and materials are now
described.
[0039] Publications and patents cited in this specification are
incorporated by reference where indicated and are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present disclosure is not entitled to antedate such publication by
virtue of prior disclosure. Further, the dates of publication
provided could be different from the actual publication dates that
may need to be independently confirmed.
[0040] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present disclosure. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0041] Embodiments of the present disclosure will employ, unless
otherwise indicated, techniques of chemistry, synthetic organic
chemistry, biochemistry, biology, molecular biology, and the like,
which are within the skill of the art. Such techniques are
explained fully in the literature.
[0042] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to perform the methods and use the compositions
and compounds disclosed and claimed herein. Efforts have been made
to ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.), but some errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C., and pressure is at or near
atmospheric. Standard temperature and pressure are defined as
20.degree. C. and 1 atmosphere.
[0043] Before the embodiments of the present disclosure are
described in detail, it is to be understood that, unless otherwise
indicated, the present disclosure is not limited to particular
materials, reagents, reaction materials, manufacturing processes,
or the like, as such can vary. It is also to be understood that the
terminology used herein is for purposes of describing particular
embodiments only, and is not intended to be limiting. It is also
possible in the present disclosure that steps can be executed in
different sequence where this is logically possible.
[0044] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a support" includes a plurality of
supports. In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings unless a contrary intention is
apparent.
DEFINITIONS
[0045] In describing and claiming the disclosed subject matter, the
following terminology will be used in accordance with the
definitions set forth below.
[0046] In accordance with the present disclosure there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory
Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and
II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait
ed. 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J.
Higgins eds. (1985)); "Transcription and Translation" (B. D. Hames
& S. J. Higgins eds. (1984)); "Animal Cell Culture" (R. I.
Freshney, ed. (1986)); "Immobilized Cells and Enzymes" (IRL Press,
(1986)); B. Perbal, "A Practical Guide To Molecular Cloning"
(1984), each of which is incorporated herein by reference.
[0047] Use of the term "affinity" can include biological
interactions and/or chemical interactions between or among a
material (e.g., a compound or bio-molecule (e.g., polypeptide or
polynucleotide)) and a cell. The biological interactions can
include, but are not limited to, bonding or hybridization among one
or more biological functional groups of the compound or cell. The
chemical interaction can include, but is not limited to, bonding
among one or more functional groups (e.g., organic and/or inorganic
functional groups) located on the compound of cells.
[0048] The term "array" encompasses the term "microarray" and
refers to an ordered array presented for binding to polynucleotides
and the like.
[0049] An "array" includes any two-dimensional or substantially
two-dimensional (as well as a three-dimensional) arrangement of
addressable regions including nucleic acids (e.g., particularly
polynucleotides or synthetic mimetics thereof) and the like. Where
the arrays are arrays of polynucleotides, the polynucleotides may
be adsorbed, physisorbed, chemisorbed, and/or covalently attached
to the arrays at any point or points along the nucleic acid
chain.
[0050] A substrate may carry one, two, four or more arrays disposed
on a front surface of the substrate. Depending upon the use, any or
all of the arrays may be the same or different from one another and
each may contain multiple spots or features. A typical array may
contain one or more, including more than two, more than ten, more
than one hundred, more than one thousand, more ten thousand
features, or even more than one hundred thousand features, in an
area of less than about 20 cm.sup.2 or even less than about 10
cm.sup.2 (e.g., less than about 5 cm.sup.2, including less than
about 1 cm.sup.2 or less than about 1 mm.sup.2 (e.g., about 100
.mu.m.sup.2, or even smaller)). For example, features may have
widths (that is, diameter, for a round spot) in the range from
about 10 .mu.m to 1.0 cm. Non-round features may have area ranges
equivalent to that of circular features with the foregoing width
(diameter) ranges.
[0051] An array "package" may be the array plus a substrate on
which the array is deposited, although the package may include
other features. It will also be appreciated that throughout the
present application, that words such as "top," "upper," and "lower"
are used in a relative sense only.
[0052] An array, such as those described herein, is "addressable"
when it has multiple regions of different moieties (e.g., cell
binding sites) such that a region at a particular predetermined
location (i.e., an "address") on the array can detect a particular
outcome for a particular cell type and/or agent, interaction. Array
features are typically, but need not be, separated by intervening
spaces.
[0053] A "scan region" refers to a contiguous (preferably,
rectangular) area in which the array features of interest (cell
binding sites), as defined above, are found or detected.
[0054] An "array layout" refers to one or more characteristics of
the features, such as feature positioning on the substrate, one or
more feature dimensions, and an indication of a moiety at a given
location.
Discussion
[0055] In accordance with the purpose(s) of the present disclosure,
as embodied and broadly described herein, embodiments of the
present disclosure, in one aspect, relate to arrays, systems, and
methods for the analyzing cells, methods of making arrays, and the
like. In particular, embodiments of the present disclosure include
an agent (e.g., drug(s)) delivering cell-based array (e.g.,
microarrays) that can be used to analyze the timed-release delivery
of the agent to cells such as rare cells (e.g., cancer cells, stem
cells, precancerous cells, and the like), any patient-derived
cells, or other rare or low population cells.
[0056] Analyzing chemosensitivity on an established panel of cancer
cell lines is the conventional method to screening
chemotherapeutics.sup.8. An emerging strategy in cancer treatment
involves performing in vitro chemosensitivity testing of tumor
biopsies as a predictive procedure to individualize chemotherapy
treatments.sup.9. Benefits to date have been limited due to
apparent poor correlations between in vitro sensitivity and in vivo
responses. Traditional chemotherapeutic drugs are designed to
target the rapidly dividing cells of the bulk tumor in vivo, or
transformed cell lines derived from the bulk, in vitro. However,
tumors include multiple phenotypes, due in part to the presence of
CSCs.sup.10 6, 7. In the CSC model, these tumor-initiating cells
perpetually self-renew and give rise to tumor heterogeneity,
metastasis, and disease recurrence.sup.11, 12. Recent
identification of unique cell surface markers that enrich
colon-cell isolates for CSCs have led to techniques for isolating
enriched CCSC populations from patient tumor samples.sup.13-18.
After transplantation of a single CCSC enriched for high Wnt
signaling activity, tumors have been generated that recapitulate
the diverse phenotypic heterogeneity of the original tumor.sup.17.
Thus, isolating and identifying CCSCs from an individual cancer
patient and determining their sensitivity to chemotherapeutic drugs
in vitro is possible and could potentiate personalized treatment of
cancer.sup.18, 19.
[0057] While promising, cell source limitations make targeting CSCs
for treatment problematic. CSCs with the greatest tumor-initiating
and metastatic potential are exceptionally rare (.about.1% of tumor
cells), making them difficult to isolate. Moreover, the time
required for gold standard methods of CSC isolation and propagation
makes it impractical to develop individualized therapy using
traditional screening methods which require large quantities of
cells. The methods and systems embodied in the present disclosure
address these challenges with a device and methods capable of
facilitating personalized chemosensitivity screening.
[0058] Embodiments of the present disclosure can permit multiple
different biological or pharmaceutical agents and combinations
thereof to be tested on rare cell populations. Cell binding sites
that include a thin film(s) or layer(s) of timed-released polymer,
loaded with an agent(s) of interest, are microarrayed onto a
substrate, where the substrate includes a non-fouling layer or
background around the cell binding sites that resists or prevents
cell adhesion to the non-fouling layer on areas excluding the cell
binding sites. Each cell binding site is able to provide a unique
agent or combination of agents to be released. In addition, a
thousand spots or more may be arrayed onto a single substrate
(e.g., standard glass slide).
[0059] An embodiment of the array can be implemented with the
seeding of a rare cell population of interest onto the array,
requiring relatively less cells than alternative systems such as
microwell plate and/or microfluidics systems. Non-adherent cells
can be removed providing isolated islands of adherent cells
disposed on the cell binding sites in close contact with the
timed-release polymer that includes the agent. Outcome parameters
of cellular response are able to be determined through
immunostaining or use of a contrast agent including, but not
limited to, proliferation, apoptosis, and differentiation using
defined agonists/antagonists, in multiple concentrations of one or
more combinations of agents, in the assay. Multiple conditions
(e.g., one or more agents in the timed-release polymer, different
agents at different cell binding sties, different concentrations of
agents at different cell binding sites, and the like) can be
evaluated simultaneously using simple common laboratory protocols,
with a limited cell number, and without the use of expensive
automated microfluidics machines with application toward
personalized medicine (i.e., focused screening of drug interactions
with rare cell populations from patients, for example diagnostics
for cancer stem cells). Embodiments of this array would lower
expenses since fewer reagents/cells would be required, thereby
increasing throughput and productivity. These increases would
result in more rapid diagnostic capacity.
[0060] In an embodiment shown in FIG. 1.1, the array 2 includes a
non-fouling layer 4 disposed in a first area of the array 2. The
array 2 also includes a plurality of cell binding sites 6. Each of
the cell binding sites 6 is disposed in a cell binding site area of
the array 2 that is distinct from the non-fouling layer 4. In other
words, the non-fouling layer and the cell binding sites areas are
separate and distinct areas. Each of the cell binding sites 6 can
have an area of about 20 .mu.m.sup.2 to 5 mm.sup.2, where each cell
binding site 6 does not have to have the same area. In an
embodiment, the area of each ceiling binding site 6 can be
polygonal, circular, semicircular, a combination thereof, or
amorphous. Each area of the cell binding site 6 can have the same
shape, a combination of shapes, or different shapes. The cell
binding sites 6 can be positioned a distance (e.g., about 10 .mu.m
to 2 mm or more) from one another so that cross-talk or other
interference is substantially reduced or is not exhibited. An array
2 can include a few (e.g., 2, 3, 4, 5, 6, 10, 20, 50, 100, and the
like) cell binding sites 6 to a 1000 or more cell binding sites 6
in an area of about 100 .mu.m.sup.2 to 1,800 mm.sup.2 or more for
larger array substrates.
[0061] Each of the cell binding sites 6 includes a cell adhesion
layer 28 and a timed-release polymer layer(s) 26 (additional
details provided below). Each timed-release polymer layer 26
corresponding to a cell binding site 6 includes one or more types
of an agent (e.g., drug, biological, or other agent that can be
tested as to its affect on the cell). One or more types of target
cells can adhere to the adhesion layer 28. Cells that are not
adhered to the cell binding sites 6 can be removed, so only cell
binding sites 6 have target cells adhered thereto since the
non-fouling layer 4 eliminates or substantially (e.g., about 80%,
about 90%, about 95%, about 99%, or about 99.9% or more, in
particular about 95% or more) eliminates non-target cells adhering
to it.
[0062] In an embodiment, the interaction between the cell and the
cell binding sites can include electrostatic interactions, van der
Waals interactions, hydrogen bonding, hydrophobic interactions, or
a combination thereof. In an embodiment, the interaction between
the cell and the cell binding sites can also be bound through
specific biological binding, covalent binding, and/or entrapment in
a gel (e.g., PEG hydrogel, fibrin gel, collagen gel, etc.).
[0063] Once the non-adhered cells are removed, the adhered target
cells can be exposed over time (e.g., hours to days to weeks) to
the agent released from the timed-release polymer layer 26. The
effect of the agent on the cells can be studied and analyzed as a
function of time. In addition, the effect of the agent on the cells
can be studied and analyzed as a function of agent type,
combinations of agents, concentrations of agent(s), and the
like.
[0064] In an embodiment, the target cell of interest may not adhere
to a known compound or bio-molecule, but may adhere to another cell
type (e.g., fibroblasts, epithelial cells). One way to analyze the
target cell is to first adhere a cell that adheres to the target
cell to the cell binding site 6. Then the un-adhered cells can be
removed, and the target cell of interest can be introduced to the
array so that the target cell of interest adheres to the cell on
the cell binding site 6. In other words, the cell binding site 6
has a first cell type bonded to the adhesion surface layer 28 and
the target cell of interest is adhered to this cell. In another
embodiment, the two cells are adhered to one another prior to
introduction to the array, and then the cell that adheres to the
cell adhesion layer becomes disposed on the cell binding site. In
embodiments the target cell is a cancer stem cell (CSC) or other
rare cell type, and in embodiments the CSC is a colorectal cancer
stem-like cell (CCSC).
[0065] FIG. 1.2 is a cross-section of an array illustrating one
cell binding site (a-a plane shown in FIG. 1.1). FIG. 1.3A
illustrates a schematic of an embodiment of the present disclosure,
while FIG. 1.3B illustrates a representative microarray having more
than 1000 spots.
[0066] As shown in FIG. 1.2 the array includes a substrate 12
having a first area and a cell binding site area. The first area
and the cell binding site areas are different and distinct areas of
the first substrate 12. A first bonding layer 14 is disposed on the
first area of the first substrate 12. A second bonding layer 16 is
disposed on the first bonding layer 14. In an embodiment, one could
combine the first bonding layer 14 and the second bonding layer 16
into a single bonding layer. In an embodiment, the non-fouling
layer 18 and 22 can be formed of two layers and they are disposed
on the second bonding layer 16. In an embodiment, the non-fouling
layer 18 and 22 can be attached directly to the substrate 12. An
adhesive layer 24 is disposed on each of the cell binding site
areas of the first substrate 12. The timed-release polymer layer 26
is disposed on the adhesive layer 24. The cell adhesion layer 28 is
disposed on the timed-release polymer layer 26.
[0067] The substrate 12 enables imaging live cells and fixed cells,
e.g., via brightfield or fluorescence microscopy. In an embodiment,
the substrate 12 can be a rigid and optically transparent
substrate. In an embodiment, the substrate 12 can be glass (e.g.
mica, Pyrex.RTM., and the like); PET, polycarbonate, styrene, and
other amorphous polymers; silicon wafer; quartz; and the like. In
an embodiment, the substrate 12 can have a thickness of about 0.05
mm to 10 mm. The area of the substrate 12 can vary depending on the
desired number of cell binding sites, the distance between the cell
binding sites, the size of the cell binding sites, and the like. In
an embodiment the area is about 10 mm.sup.2 to 1,800 mm.sup.2.
[0068] The first bonding layer 14 provides a bonding construct for
the substrate 12 and the second bonding layer 16. In an embodiment,
the first bonding layer 14 can be titanium, nickel, chromium, and
the like. The first bonding layer 14 can have a thickness of about
1 nm to 500 nm.
[0069] The second bonding layer 16 provides a surface for
alkanethiols to form bonds for formation of self-assembled
monolayers. In an embodiment, the second bonding layer 16 can be
gold, silver, copper, palladium, platinum, nickel, and alloys of
any of these. The second bonding layer 16 can have a thickness of
about 1 nm to 500 nm.
[0070] In an embodiment, the first and second bonding layer could
be a single layer that achieves both the functions of the first
bonding layer 14 and the second bonding layer 16.
[0071] The non-fouling layer (or surface) functions to resist,
prevent, or substantially prevent cell attachment in the area that
the non-fouling surface is disposed. In an embodiment, the
non-fouling layer can be made up of two layers, 18 and 22. In
another embodiment, the non-fouling layer can be made of a single
layer or multiple layers that achieve the same function as the
first layer 18 and the second layer 22.
[0072] The first layer 18 functions to attach to the second bonding
layer 16. In an embodiment, the first layer 18 can be made of
self-assembled monolayer of methyl-terminated
alkanethiol--treatment to promote adsorption of pluronic;
hydrophobic polymers (e.g., polyethylene, polyethylene
terephthalate, siloxanes); non-polar peptides/amino acids (e.g.,
alanine, leucine, valine, isoleucine); micro/nano textures; and the
like. The first layer 18 can have a thickness of about 1 nm to 100
nm.
[0073] The second layer 22 is attached to the first layer 18 and
can resist attachment by cells. In an embodiment, the second layer
22 can be made of glycol-based polyethylene; a neutral polymer
(e.g., poly(2-hydroxyethyl methacrylate, polyacrylamide,
poly(N-vinyl-2-pyrolidone, and poly(N-isopropyl acrylamide) (below
31.degree. C.))); an anionic polymer; a phosphoryl choline polymer;
gas discharge-deposited coatings (especially from PEG-like
monomers); self-assembled n-alkyl molecules with oligo-PEG head
groups; self-assembled n-alkyl molecules with other polar head
groups; passivating proteins (e.g., albumin and casein);
polysaccharides (e.g., hyaluronic acid); liposaccharide;
phospholipid mono/bilayers (e.g., phosphorylcholine); glycoproteins
(e.g., mucin), and the like. The second layer 22 can have a
thickness of about 1 nm to 20 .mu.m.
[0074] The adhesive layer 24 provides a surface for the
timed-release polymer layer 26 and attaches to the substrate 12. In
an embodiment, the adhesive layer 24 can be a silane; chemical
groups forming covalent bonds to polymer such as: ethylene oxide,
acrylamide, other crosslinking schemes; chemical groups promoting
non-specific interactions (electrostatic, hydrophobic, van der
Waals) such as amine groups (e.g., the amine-terminated silane
depicted, polylysine, polyethyleneimmine) or hydrophobic groups
(e.g., methyl-terminated silane) or micro/nanotextures; and the
like. The adhesive layer 24 can have a thickness of about 1 nm to
500 nm.
[0075] The timed-release polymer layer 26 functions to delivers
drugs (or agents) and in some instances can promote cell adhesion.
In an embodiment, the timed-release polymer layer 26 can be a
poly(lactic-co-glycolic acid); polycaprolactone; polyglycolide;
polylactic acid; poly(vinylpyridine); chitosan; alginate; and the
like. The timed-release polymer layer 26 can have a thickness of
about 10 nm to 5 .mu.m. In an embodiment, the timed-release polymer
layer 26 can include a plurality of layers each having a thickness
of about 10 nm to 5 .mu.m. In an embodiment, the additional layers
can function to increase the time that the agent is delivered. In
addition to or in the alternative to the timed-release polymer
layer 26, the drugs (or agents) can be bound and/or tethered to the
one or more layers of the cell binding site to achieve the same
function of the timed-release polymer layer. In an embodiment, the
cellular uptake can process through a process such as
phagocytosis.
[0076] The agents can be used to test, study, analyze, and the
like, outcomes of the interaction of the agent with the cell. The
concentration of the agents can be varied between or among the
timed-release polymer layers of the array. Advantages to the arrays
and methods of the present disclosure include the ability to test
various different agents (e.g., drugs), agent concentrations, and
combinations on a single array without having to use a large amount
of target cells. In embodiments various agents can be combined in
different combinations in the timed-release polymer layers of each
cell binding site, such that multiple cell binding sites have
different agents present in combination with other agents in
various concentrations. In embodiments, the timed-release polymer
layer for each cell binding site is prepared to have one or more
drugs in one or more different concentrations or combinations of
concentrations. For instance, the timed-release polymer layer of at
least one cell binding site can include at least one type of agent
different from at least one type of agent in the timed-release
polymer layer of at least one other cell binding site. Similarly,
the timed-release polymer layer of at least one cell binding site
can include a first concentration of one type of agent different
from a concentration of that type of agent in the timed-release
polymer layer of at least one other cell binding site. In
embodiments, different cell binding sites include both different
agents and different concentrations or combinations of agents that
other cell binding sites. In embodiments, multiple cell binding
sites have one or more different types of agent in the
timed-release polymer layer than the types of agent in the
timed-release polymer layers of other cell binding sites.
Similarly, multiple cell binding sites have one or more different
types of agents or combinations of agents present in different
concentrations than the types and combinations of agents in the
timed-release polymer layers of other cell binding sites. Thus, one
or more or a plurality of agents can be present in each cell
binding site in different concentrations and combinations with
other agents to test different combinations of agents in different
amounts on the target cells. In embodiments, the array includes at
least a first and second type of agent, where the first agent is
present in at least two different concentrations in the
timed-release polymer layers of at least two different cell binding
sites and the second agent is present in at least two different
concentrations in at least two different cell binding sites, and
where the first agent and second agent are combined in different
concentrations in at least two different cell binding sites. The
agents can include drugs, compounds, bio-molecules, and the like.
In embodiments, one or more agents can be selected from nutlin-3a,
camptothecin, and combinations of those agents.
[0077] The cell adhesion layer 28 functions to promote adhesion of
the cells to the cell binding site by capturing the cells so that
the agent can be delivered to the cells. The cell adhesion layer 28
has an affinity for one or more types of cells such as tumor
initiating cells, stem cells, inflammatory/immune cells,
hematologic cellular components, neural cells, micro-environmental
cellular elements, and the like. In an embodiment, the cell
adhesion layer 28 can promote adhesion of a specific cell type(s)
or can be a material that promotes non-specific binding (e.g.,
positively-charged treatments such as polylysine,
polyethyleneimmine). In an embodiment the cell adhesion layer 28
can include: fibronectin (e.g., endothelia); polylysine (e.g.,
epithelia); collagen (e.g., epithelia); vitronectin (e.g.,
fibroblasts); intercellular adhesion molecules (ICAM-1,2,3,4,5);
immunoglobulin superfamily Cell Adhesion Molecules (IgSF CAMs)
(e.g., dSynCAMs Synaptic Cell Adhesion Molecules (e.g., epithelia),
NCAMs Neural Cell Adhesion Molecules (e.g., neural cells), ICAM-1
Intercellular Cell Adhesion Molecule (e.g., leukocytes), VCAM-1
Vascular Cell Adhesion Molecule (e.g., leukocytes), PECAM-1
Platelet-endothelial Cell Adhesion Molecule (e.g., platelets), L1,
integrin (e.g., leukocytes); cadherin (e.g., epithelia); and the
like. In an embodiment, the cell adhesion layer 28 can have a
thickness of about 0.2 nm to 2 .mu.m.
[0078] As described above, methods of the present disclosure can
include separating cells (e.g., rare cells) from other cells using
an array of the present disclosure. Subsequently, the captured
cells can be exposed to an agent. In addition, embodiments of the
present disclosure include systems using an array of the present
disclosure to capture and analyze cells, where the system includes
the array and equipment to introduce, remove, etc., reagents and
the like.
EXAMPLES
[0079] Now having described the embodiments of the disclosure, in
general, the examples describe some additional embodiments. While
embodiments of the present disclosure are described in connection
with the example and the corresponding text and figures, there is
no intent to limit embodiments of the disclosure to these
descriptions. On the contrary, the intent is to cover all
alternatives, modifications, and equivalents included within the
spirit and scope of embodiments of the present disclosure.
Example 1
[0080] As noted above, CCSC's are rare and therefore it is
difficult to screen potential agents. In this example we provide an
array that can be used to analyze CCSC's (e.g., FIG. 1.3A) using a
limited number of cells. This technique utilizes arrays of spotted
PLGA films loaded with signaling pathway inhibitors. Signaling
pathways govern self-renewal and as such have been identified as a
target for therapy. The selected factors include KAAD-Cyclopamine,
a sonic hedgehog antagonist, DKK-1, a WNT inhibitor, Compound E, a
notch pathway antagonist, and rapamycin, an mTOR inhibitor. Various
concentrations were used and factors were printed in randomized
arrays in order to factor out potential cross-talk between arrayed
spots. Colon stem cells, both cancerous and precancerous, were
isolated using ALDH and CD44 expression and identified by cytokine
array analyses. Cells were seeded onto colon stem cell inhibitor
microarrays, cell attachment was assessed through DAPI staining and
proliferation quantified by immunostaining for BrdU incorporation.
This example shows that a microarray platform has been developed
that allows for a systematic investigation of the role of signaling
pathway inhibitors on the response of CCSC's isolated from murine
colon crypts, requiring limited cell numbers.
[0081] FIG. 2.1 illustrates a phase micrograph of array seeded with
murine colon cancer stem cells. FIGS. 2.2 illustrate micrographs of
CCSC's attached to PLGA island imaged using (a) Phase, (a,b) DAPI,
and (a,c) FITC. FIG. 2.3 is a graph that illustrates a release
profile for PLGA films loaded with coumarin, 355 nm/460 nm.
[0082] The array can be fabricated using an oxygen plasma cleaned
coverslips were printed with silane in specific array formats.
Printed coverslips were coated with 175 .ANG. of titanium followed
by 250 .ANG. of gold. Gold coated coverslips were sonicated to
expose silane islands. The coverslips were then incubated with
methyl-terminated alkanethiol followed by 10% pluronic F-127 to
create a nonfouling background. Appropriate drug concentrations
were loaded into 10% poly(D,L lactide-co-glycolide) (PLGA)
dissolved in polycarbonate. The following drugs were used:
Rapamycin--mTOR inhibitor; DKK1--WNT inhibitor; KAAD--Sonic
hedgehog inhibitor; Compound E--Notch pathway antagonist; and
Wortmannin--P13K. Cell adhesion molecules are then over spotted
onto PLGA film islands.
[0083] The method of isolating colon cancer stem cell is described
below. Colon cancer xenografts were dissociated and colon stem
cells, both cancerous and precancerous, were isolated using ALDH,
CD44, and ESA expression and identified by cytokine array analyses.
Cells are seeded onto array, allowed to attach specifically to
islands, and pulsed with BrdU. After 24 h, samples were fixed and
stained. Fluorescence and phase contrast micrographs were then
taken.
Example 2
[0084] Human epithelial cells (HCE-T corneal epithelial
cells--representative of an adherent cell type that could be
tested) are shown seeded on a PLGA film array (over-spotted with
adhesion molecules collagen and fibronectin) (FIG. 3.1). Seeding
density was 1 million cells in 3 ml serum-free media, seeding time
was approximately 10 minutes, followed by washing to remove
loosely-adherent cells from the non-fouling PEG background.
[0085] Shown is the release over time of coumarin (fluorescent
dye--representative of a small hydrophobic molecule such as many
drugs) from a chip printed with 169 coumarin-loaded PLGA films
(FIG. 3.2). Characteristics of interest are a burst release within
the first 24 hr followed by a more linear release over the next 5
d. These release characteristics are in line with other
configurations of PLGA-loaded delivery vehicles (e.g.,
microparticles, wafers) and are amenable to the cellular array
device.
Example 3
[0086] FIG. 4.1 illustrates a phase contrast/fluorescence overlay
micrograph displaying SW480 cells attached to isolated ethylene
vinyl acetate (EVA) islands in a similar manner to methods
described for FIG. 3.1 however with a different cell line and
different drug-releasing polymer. This is a close-up image of an
array similar to that shown in FIG. 4.2. SW480 cells are a human
colorectal adenocarcinoma line which are epithelial like in
morphology and used as an in vitro model for colorectal cancer.
Cells were seeded onto small-molecule eluting array and stained
with nuclear stain (blue).
[0087] FIG. 4.2 illustrates a whole 11.times.13 array of SW480
cells seeded onto small-molecule eluting array and stained with
nuclear stain (blue). This figure illustrates high specificity of
cell adhesion onto small-molecule releasing islands with little
off-spot adhesion across the entire array.
[0088] FIG. 4.3 illustrates one column in an array similar to that
in FIG. 4.2. This array was seeded with SW480 cells and incubated
for 4 days. At 80 hours post seeding, the array was pulsed with
BrdU for 16 hours and later stained for BrdU incorporation (green),
indicating proliferating cells, and nuclear counter-stain (blue).
This figure displays the spots separated by fluorescent channel and
a final merged image.
[0089] FIG. 4.4 illustrates a release profile from arrays printed
with 20% (w/w) coumarin-loaded EVA. The methods were similar to
those from FIG. 2.3 with exception to the over-spotted samples
illustrated by the green curve. The red curve represents arrays
printed with single spots of coumarin-loaded EVA, while the green
curve represents an extra layer of polymer over-spotted to delay
release of the loaded factor.
Example 4
[0090] The following figures (FIGS. 5.1 to 5.4) are micrographs
taken from drug-eluting cellular microarrays, manufactured as
described herein. The images are taken from individual cellular
islands from the same array under various drug-loading conditions
over a period of three days. HCT116 cells are nuclear stained with
Hoechst for easy visibility. All polymer formulations have 5% ELVAX
in cyclohexanol (w/w) loaded with 8.5% water phase (loaded with
drug) and 8.5% polyvinyl alcohol (to form a stable emulsion). The
water phase for this experiment was loaded with azide at various
concentrations in addition to a control with no drug loaded. Azide
is a useful probe reagent, mutagen, and preservative. Azide
inhibits cytochrome oxidase by binding irreversibly to the heme
cofactor in a process similar to the action of carbon monoxide. As
such, it is expected to induce necrosis in cells at physiologically
relevant doses. Our array demonstrates a dose-dependent response to
azide as shown in FIGS. 5.1 to 5.4. In the absence of azide (Blank
ELVAX), the attached cells appear viable after 68 hour incubation.
However, in the presence of azide, cell death is evidenced by the
decrease in cell density, which is intensified at the higher
concentrations.
Example 5
Colon Cancer Stem Cell and Combinatorial Drug Interaction Screening
Using Drug-Eluting Microarrays
[0091] Modern cancer treatments seek to simultaneously target
multiple critical pathways with combinations of chemotherapeutic
drugs.sup.1-5. Intratumor heterogeneity gives rise to varying
sensitivity among patients to different classes of
chemotherapeutics making it difficult to predict the success of
various combination treatments from one patient to the next.sup.1.
This heterogeneity is attributed to a rare population of cancer
stem cells (CSCs). Testing possibly new therapies targeted for
these CSC's is complicated by the limitation of having very few
available cells on which to test drug combinations.sup.6, 7. The
present example sought to resolve this challenge with the
fabrication of a miniaturized microarray platform to which a
minimal quantity of cells can adhere and be exposed to unique
treatment conditions. Using this method, colorectal cancer
stem-like cells (CCSCs) isolated from two different patients
exhibited unique responses to drug combinations when cultured on
the microarray, highlighting its potential utility as a prognostic
tool for identifying effective, personalized chemotherapeutic
regimens.
[0092] This platform includes hundreds of drug-loaded polymer
islands acting as drug depots that are surrounded by a non-fouling
background, thus creating isolated culture environments capable of
screening a large number of unique drug combinations on small
numbers of cells. The embodiment of the array can screen up to
4,845 unique conditions in the footprint of a standard microtiter
plate. Statistically significant results can be obtained by testing
approximately one-eighth the amount of cells as a typical 96 well
plate experiment. Moreover, the readout of the device is obtained
using fluorescence microscopy, allowing for easy translation to
laboratory settings. Whereas state-of-the-art industrial
pharmaceutical capabilities can surpass this reduction in cell
requirements, such facilities are typically unavailable for use by
clinicians or core pathology labs. This small, easily manufactured
device can be utilized to screen libraries of small molecules on
rare cells, establishing a new class of microarray, the
drug-eluting cellular microarray.
Materials and Methods:
Polymer Formulation
[0093] Poly(ethylene-co-vinyl acetate) (EVA) (Sigma) (40% vinyl
acetate by weight) was first washed to remove butylhydroxytoluene
(BHT) according to a protocol adapted from Langer et al..sup.29
(reference 29 is hereby incorporated by reference herein). Briefly,
polymer pellets were washed ten times each, first in water and then
in ethanol at 37.degree. C. with stirring. After each successive
wash, the absorbance of the ethanol was analyzed
spectrophotometrically at 230 nm to quantify the relative level of
BHT until its absorbance was less than twenty times the original
reading. Following washing, pellets were dried in a desiccator at
room temperature. Polymer pellets were then dissolved in
cyclohexanol (Acros, Morris Plains, N.J.) at a 5% w/w
concentration.
[0094] To embed molecules into the polymer matrix, they were first
dissolved in an appropriate solvent, depending on their respective
hydrophobicity. A stock solution of azide (99%, Acros), a
hydrophilic molecule, was dissolved in dH.sub.2O whereas Nutlin-3a
(EMD Chemicals, Gibbstown, N.J.) and camptothecin (Sigma), both
hydrophobic molecules, were dissolved in DMSO. Dissolved drugs were
then added to 5% EVA at a 1:10 ratio of solvent:EVA. Prior to
printing, polymer mixtures were vortexed for 30 s and then
homogenized for 1 min before being loaded onto the source plate of
the robotic mini-arrayer.
Array Fabrication
[0095] Glass coverslips were cleaned in an oxygen plasma etcher
(Terra Universal, Fullerton, Calif.). Arrays of isolated spots of
(3-Aminopropyl) trimethoxysilane (NH2-terminated silane)
(Sigma-Aldrich, St. Louis, Mo.) were robotically printed on clean
coverslips using a Calligrapher Miniarrayer printer (Bio-Rad,
Hercules, Calif.) with 1500 .mu.m center to center distances and a
pin diameter of 400 .mu.m. The silane printed coverslips were then
coated with 175 .ANG. of titanium (Ti; 99.995% pure) and 225 .ANG.
of gold (Au; 99.999% pure) (Williams Advanced Materials, Buffalo,
N.Y.). Following coating, gold-coated arrays were sonicated to
remove gold from the amine spots, exposing NH2-terminated silane
islands. The coverslips were incubated with 0.1 M,
methyl-terminated alkanethiol (CH.sub.3(CH.sub.2).sub.11SH) (Sigma)
for 30 min. Substrates were incubated in 10% Pluronic.RTM. F-127
(BASF Corporation, USA) for 3 h and 1% heat-denatured BSA for 30
min to create a non-fouling surface around the adhesive amine
islands. Three layers of the drug-loaded ethylene vinyl acetate
(EVA) polymers were printed over the amine islands and placed in a
desiccator between each successive layer. Poly-d-lysine (0.1%) was
over-spotted onto the EVA films to promote cell attachment. (The
EVA film was fabricated using water-oil emulsion to promote uniform
film thickness during drying, and had a mottled appearance) The
arrays were placed in 35 mm petri dishes containing PBS with 2%
penicillin and 2% streptomycin for 15 minutes to rehydrate the
non-fouling PEG background and as a non-caustic sterilization step.
Schematic diagrams of the arrays are illustrated in FIGS. 6A and
6B.
Drug Release and Loading Efficiency
[0096] To test the effect of over-spotting on release of drugs, 5%
EVA in cyclohexanol was loaded with 20% (w/w)
7-Diethylamino-4-methycoumarin (Acros Organics, Morris Plains,
N.J.) and printed onto glass coverslips in an arrayed fashion as
described above. Following printing, arrays were placed in 35 mm
petri dishes with 3 ml PBS, and 20 .mu.l samples were taken at
defined intervals and analyzed on a Wallac 1420 Multilabel Counter
(PerkinElmer, Waltham, Mass.). For over-spotted samples, unloaded
EVA was immediately printed over the dye-loaded islands before
being incubated in PBS. Sampling and analysis thereafter were
identical.
[0097] To determine the loading efficiency of factors, polymer
formulations were made as described above. Films derived from 100
.mu.l EVA loaded with factors were then made on glass coverslips
and allowed to dry overnight under vacuum. Films were removed from
the glass substrates and weighed. Films were washed briefly with
PBS before being placed in 1 ml toluene to dissolve the polymer.
Samples were then analyzed using a Nanodrop-ND-1000
spectrophotometer. Standard curves were generated for each factor,
and loading efficiency was calculated as the percent of drug
embedded in the polymer compared to the theoretical drug
concentration.
[0098] For drug release studies, polymer formulations were again
made as described above. Glass coverslips were weighed prior to
printing. Arrays consisting of 900 drug-loaded polymer islands were
manufactured and allowed to dry overnight. Samples were weighed and
placed in 3 ml of PBS containing 0.1% Tween-80 at 37.degree. C.
with gentle agitation. Samples were taken at the specified times
and analyzed.
Human Subjects
[0099] Tissues from colon cancer patients were retrieved under
pathologic supervision with Institutional Review Board approvals at
the University of Michigan and the University of Florida as
previously described.sup.32 (reference 32 is hereby incorporated by
reference herein with respect to obtaining tissue samples from
colon cancer patients).
Cell Lines and Seeding
[0100] HCT116 (p53+/+, ATCC, Manassas, Va.) human colon cancer
cells were maintained in McCoy's 5a Medium supplemented with 10%
fetal bovine serum (Thermo Scientific, Waltham, Mass.), 1%
penicillin G and 1% streptomycin (Thermo Scientific). The cells
were cultured at 37.degree. C. in a humidified incubator containing
5% CO.sub.2. Following microarray fabrication, 100,000 HCT116 cells
were seeded over each array in 3 ml serum-free media and allowed to
incubate on a rocking plate at room temperature until cell
attachment to the EVA islands occurred, with minimal attachment to
background, typically 10-15 min. Microarrays were gently washed in
PBS, placed in a 35 mm petri dish with complete media, and placed
in an incubator for 24-72 h. ALDH.sup.high spheres were generated
from tumor cells obtained from patients with colon cancer. Isolated
cells were cultured in serum-free media as previously
described.sup.14 (reference 14 is hereby incorporated by reference
herein with respect to methods for culturing cells in serum-free
media). Using these cultures, adherent cell growth was established
with 0.1% gelatin (Millipore) coatings on tissue culture plates
(TPP, Switzerland) and the cells were serially propagated. For CA1
and CA2 cells, 25,000 cells were tested per array. Cells were
seeded in PBS with 0.1% gelatin and treated identically to the
microarrays with HCT116.
[0101] For p53 mutational analysis, genomic DNA was isolated using
a DNeasy Tissue kit (Qiagen GmbH, Hilden, Germany); exons 4-9 were
amplified with a Taq polymerase Master Mix (Promega, Madison, Wis.)
using a Touchdown PCR program (45 cycles; 60.degree. C. to
50.degree. C.; 0.5.degree. C. decrease per cycle) and previously
described primers.sup.33 (reference 33 is hereby incorporated
herein with respect to the primers). The resulting PCR products
were fractionated by agarose gel electrophoresis; excised and
isolated using a QIAquick extraction kit (Qiagen GmbH, Hilden,
Germany) and sequenced using an ABI 3130xl Genetic Analyzer
(Applied Biosystems, Carlsbad, Calif.). Sequences were analyzed
using Sequencher v. 5.0 (Gene Codes, Ann Arbor, Mich.).
[0102] To establish microarray fidelity, microarrays were
manufactured as described above. HCT116 cells were then seeded over
microarrays and incubated for 24 or 72 h. Microarrays were fixed in
4% paraformaldehyde and stained with Hoechst 34580 dye.
Staining and Image Analysis
[0103] Camptothecin-loaded microarrays were stained with Annexin V
(BD Pharminigen), fixed in 4% paraformaldehyde, and placed in PBS
with Hoechst dye 34580 (Invitrogen, USA) for 30 min. Azide-loaded
microarrays were fixed with 4% paraformaldehyde and incubated with
PBS containing Hoechst 34580 dye. Finally, nutlin-3a-loaded
microarrays were fixed with 4% paraformaldehyde, stained with BrdU
(BD Bioscience, San Jose, Calif.), and incubated in PBS containing
Hoechst 34580 dye. All arrays were mounted with Fluoro-Gel
(Electron Microscope Sciences, Hatfield, Pa.) and imaged using an
Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany).
Analysis was performed with Axiovision (Carl Zeiss, Oberkochen,
Germany) by quantifying, in each drug-eluting island, the area of
fluorescence and reported as relative fluorescence intensity
(RFI).
Crosstalk
[0104] To evaluate the potential influence of neighbouring
drug-eluting islands on the array, multiple arrays were printed in
randomized configurations. Data were then analysed using student's
t-test to see if pairs with significant crosstalk existed between
the same groups (i.e., the outcome changed when the pairs were
arranged differently on the array).
Calculating Comparison to Microtiter Plates
[0105] Dimensions for a typical microtiter plate were obtained from
Corning.RTM.. The length.times.width is 127.8 mm.times.85.6 mm.
Based on island spacing of 1.5 mm, 85 islands (i.e., (127.8)/(1.5))
can fit along the length axis, and 57 (i.e., (85.6)/(1.5)) islands
can fit along the width of a traditional plate. Thus (85.times.57)
yields 4845 total islands that fit within the footprint of a
standard microtiter plate. For calculating the comparison between
the amount of cells required for a traditional screen using a 96
well plate 10,000 cells per well was assumed as a typical seeding
density. Performing experiments in triplicate with 16 unique drug
combinations therefore requires 480,000 cells total (i.e.
(10,0000)*(16)*(3)).
Statistical Analyses
[0106] Statistical analyses were performed using either a one-way
ANOVA or a two-way ANOVA, using Systat (Version 12, Systat
Software, Inc., San Jose, Calif.). Post-hoc pair-wise comparisons
were made using Tukey's Honestly-Significant-Difference, with
p10.05 being significant. Curve-fitting of drug-release and
dose-response curves were performed using SigmaPlot (Version 10,
Systat Software, Inc., San Jose, Calif.).
[0107] Proliferation values were normalized to control (0 .mu.M
drug). Values for concentration-response curves were transformed to
non-proliferation by subtracting the normalized value from 100%.
Curve fitting analysis was performed to obtain E.sub.max and
D.sub.50 values using the equation
E=E.sub.0+(E.sub.max.times.D)/(D+D.sub.50) where E is the effect
(either non-proliferation or apoptosis), E.sub.0 is the initial
value, E.sub.max is the maximum effect, D is the dose, and D.sub.50
is the dose at which a 50% maximum effect (E.sub.max) is
observed.sup.34. Drug sensitivity values were obtained by taking
the inverse of the D.sub.50 and multiplying by 100. Those values
marked with "#" indicate that the r.sup.2 value of the curve-fit
was below 0.65. N/A values are present where negative parameters
were obtained.
Results & Discussion
[0108] The present example describes development of a drug-eluting
cellular microarray to screen libraries of small molecules for
their effects on populations of rare cells. Arrays with PEG-based
non-fouling backgrounds and amine-terminated silane adhesion
islands were manufactured as reported.sup.20 (Reference 20 is
hereby incorporated by reference herein with respect to
manufacturing of the described PEG-based arrays. (FIG. 6a).
Micropatterning of NH2-terminated silane onto the glass substrate
provided 400 .mu.m diameter islands. PEG was back-filled around the
silane islands to resist cell attachment off-spot (FIG. 6b). These
silane islands were then over-spotted with oil/water emulsions of
ethylene vinyl acetate (EVA) loaded with drugs of interest or
unloaded (control). EVA is a biocompatible polymer commonly used in
drug delivery applications, and when formulated as an oil/water
emulsion both hydrophilic and hydrophobic molecules can be
loaded.sup.30, 31. Dry polymer films were over-spotted with
poly-d-lysine to facilitate cell adhesion.
[0109] Site-specific attachment of cells to polymer islands with
minimal cell adhesion to the background was achieved (FIG. 6c).
Fidelity of fabrication and cell attachment was quantified at 24 h
incubation using the following criteria (results in parentheses):
(1) lysine printing misalignment with polymer islands (<1.3%);
(2) proportion of islands with adherent cells (>95%); (3)
islands with <65% cell coverage (<11%); (4) proportion of
cells on islands (vs. background) (>94%).
[0110] Loading efficiency of small molecules from EVA films on the
microarrays was quantified (Table 1). Release kinetics from
microarrayed drug-eluting EVA films demonstrated an initial burst
during the first 24 h followed by a steady rate of release over
four days (FIGS. 7A and 7E). Drug release can be delayed by
over-spotting unloaded EVA films onto drug-loaded films, creating a
diffusion barrier (FIG. 8A). Cell number per island at 1 h was
unaffected by drug loading concentration (FIG. 8B).
TABLE-US-00001 TABLE 1 Loading efficiencies of small molecules in
microarrayed EVA films. Compound Loading efficiency (%) Azide 73
+/- 8 Camptothecin 92 +/- 3 Nutlin-3a 81 +/- 7
Feasibility of eliciting dose-dependent responses to hydrophilic
drugs was demonstrated (FIG. 9A). Having thus demonstrated
feasibility, two classes of clinically relevant drugs were
investigated. Nutlin-3a is a hydrophobic drug that inhibits human
double minute 2 (HDM2) and is being investigated clinically in
combination with numerous therapeutic agents.sup.21. Nutlin-3a
binding to HDM2 disrupts turnover of p53, increasing p53 protein
levels and inducing cells to enter into either a state of cell
cycle arrest, or apoptosis at higher concentrations.sup.22, 23.
HCT116 cells were cultured on nutlin-3a loaded microarrays for 24 h
and proliferation was quantified. With increasing concentrations of
nutlin-3a, the ratio of non-proliferating cells expanded (FIGS.
7B-7D). Correspondingly, cell numbers were diminished following 72
h incubation (FIG. 9B). Hence, the HCT116 cell line evidenced
dose-dependent cell cycle arrest when cultured on nutlin-3a loaded
microarrays. Camptothecin, a hydrophobic topoisomerase inhibitor
that induces apoptosis is also of interest as various analogues are
used in chemotherapy.sup.24, 25. As expected, the proportion of
HCT116 cells undergoing apoptosis was greater with increasing
concentrations of camptothecin after 72 h incubation on the
microarray (FIGS. 7F-H).
[0111] Seminal cell-based microarray studies previously
demonstrated that experimental design can control for undesirable
interactions between islands through island spacing, randomized
configurations and robust statistical analysis.sup.26. Prior work
on a different microarray configuration indicated 1.5 mm spacing
between islands was sufficient to isolate cell populations from
agents released from neighboring islands.sup.27. To corroborate
with the setup of this example, and determine whether paracrine
signaling or leaching of drugs from adjacent polymer islands
occurred using this 1.5 mm island spacing, camptothecin loaded
arrays were analyzed in a variety of configurations (FIGS. 7I and
7J). No significant differences were noted between the
configurations, indicating the negligible interaction with cells or
drugs from neighbouring islands (FIG. 7K).
[0112] Chemotherapy for colorectal cancer is often a combination of
two drugs. Dual-drug microarrays were developed to investigate
possible interaction effects of nutlin-3a and camptothecin on the
HCT116 cell line. Ranges of loading concentrations for the two
drugs were combinatorially encapsulated and spotted in randomized
microarray configurations (FIG. 7L). Results demonstrate
feasibility of the drug-eluting microarray approach to identify
combined effects of drugs on proliferation (FIGS. 10A-10I, Table
2), and apoptosis (FIGS. 11A-11I, Table 3) using the HCT116 cell
line. Following 24 h incubation with both nutlin-3a and
camptothecin, proliferation of HCT116 cells significantly decreased
(FIG. 10A). A significant primary effect on proliferation relative
to nutlin-3a, {F(3,619)=18.253, p<0.01}, and camptothecin,
{F(3,619)=25.056, p<0.01} was revealed by two-way ANOVA.
Additionally, a sub-additive effect was observed from combination
treatments. Camptothecin increased sensitivity to nutlin-3a and
vice versa (FIGS. 10B-I). By contrast, the Emax values were
unaffected by combination drug treatments, as values obtained from
single drug regimens were already at maximum levels. This indicates
that in HCT116 cells, a maximum plateau effect is present when
evaluating proliferation in the presence of nutlin-3a and
camptothecin, though significantly lower concentrations are able to
obtain a given effect when these drugs are used in concert.
Nutlin-3a showed no effect on the proportion of HCT116 cells that
underwent apoptosis when administered alone (6.0% for 0 .mu.M vs
5.6% for 125 .mu.M, p>0.05) (FIGS. 11A, 11B). A greater
percentage of cells underwent apoptosis after exposure to
camptothecin. (FIG. 11F). Increasing the nutlin-3a concentration
conferred protection from the apoptotic response to camptothecin,
particularly evident at higher fixed concentrations of camptothecin
(FIGS. 11C-11E), and addition of nutlin-3a attenuated the apoptotic
response to camptothecin.sup.35, 36, resulting in a 65% decrease in
the E.sub.max of the camptothecin concentration response curve in
the presence of 125 .mu.M nutlin-3a compared to 0 .mu.M nutlin-3a
(FIGS. 11F-11I).
[0113] Hyperbolic curve fits were generated for the dose responses
from a first drug in the presence of a fixed amount of a second
drug for each combination (Tables 2 and 3, below). The curves were
modeled using the following equation
E=Eo+EmaxC/(C+D50)
where E.sub.max is the maximum biological response obtainable,
(1/D.sub.50) is the sensitivity (where an increasing value
indicates a lower necessary dose to approach E.sub.max), and C is
the concentration.
TABLE-US-00002 TABLE 2 E.sub.max and D.sub.50 values generated from
combinatorial microarrays of HCT116 cells. HCT116 Proliferation
Hyperbolic Fit Parameters of Nutlin Dose Hyperbolic Fit Parameters
of Camptothecin Response Upon Addition of Camptothecin Dose
Response Upon Addition of Nutlin 2.sup.nd Drug Conc (.mu.M)
E.sub.max 1 D50 .times. 100 ##EQU00001## Source Data 2.sup.nd Drug
Conc (.mu.M) E.sub.max 1 D50 .times. 100 ##EQU00002## Source Data
CPT 0 108 +/- 110 3.00 +/- 0.49* Sup FIG. 4b NUT 0 86.4 +/- 39 4.83
+/- 0.54* Sup FIG. 4f 1 96.1 +/- 25 1.78 +/- 1.7 Sup FIG. 4c 1 88.8
+/- 20 5.65 +/- 5.2 Sup FIG. 4g 10 86.5 +/- 19 2.97 +/- 3.7 Sup
FIG. 4d 25 86.3 +/- 22 12.1 +/- 18 Sup FIG. 4h 50 81.4 +/- 1.9 19.6
+/- 12 Sup FIG. 4e 125 79.1 +/- 3.7 78.1 +/- 26* Sup FIG. 4i
TABLE-US-00003 TABLE 3 E.sub.max and D.sub.50 values generated from
combinatorial microarrays of HCT116 cells undergoing apoptosis.
HCT116 Apoptosis Hyperbolic Fit Parameters of Nutlin Dose
Hyperbolic Fit Parameters of Camptothecin Response Upon Addition of
Camptothecin Dose Response Upon Addition of Nutlin 2.sup.nd Drug
Conc (.mu.M) E.sub.max 1 D50 .times. 100 ##EQU00003## Source Data
2.sup.nd Drug Conc (.mu.M) E.sub.max 1 D50 .times. 100 ##EQU00004##
Source Data CPT 0 95.2 +/- 9.2.sup.# N/A Sup FIG. 5b NUT 0 21.6 +/-
3.0* 2.53 +/- 1.1 Sup FIG. 5f 1 112 +/- 23.sup.# N/A Sup FIG. 5c 1
12.8 +/- 2.5 83.3 +/- 76 Sup FIG. 5g 10 79.3 +/- 9.7.sup. 2.48 +/-
0.83 Sup FIG. 5d 25 9.31 +/- 2.5 15.9 +/- 2.8 Sup FIG. 5h 50 131
+/- 38 .sup. 143 +/- 98 Sup FIG. 5e 125 7.46 +/- 2.5* N/A Sup FIG.
5i
[0114] Results were corroborated using soluble drugs in 96-well
plates (FIGS. 12A-D). Results showed no effect of nutlin-3a on
inducing apoptosis of HCT116 cells (FIG. 12A), similar to results
shown on microarray. Camptothecin displayed a dose-dependent effect
on inducing apoptosis of HCT116 cells (FIG. 12B). Nutlin-3a and
Camptothecin both showed a dose-dependent effect on reducing
proliferation of HCT116 cells (FIGS. 12C-12D).
[0115] CCSCs have recently been linked to tumor initiation,
potentiation, and as the genesis for metastatic deposits.sup.11.
The cancer stem cell hypothesis states that these rare cells,
constituting <10% of the tumor mass, are responsible for both
the heterogeneity and the hierarchy within the tumor.sup.7, 10. As
these cells are challenging to isolate and target therapeutically,
they were selected as targets to determine the feasibility of the
microarray to delineate the potency of pathway-specific
chemotherapeutic agents. CCSCs were thus isolated and enriched for
aldehyde dehydrogenase (ALDH) from patients with colon cancer and
propagated as spheroid cultures as recently described.sup.14, 15,
17 (References 14, 15, and 17 are hereby incorporated by reference
herein with respect to the propagation of CCSCs as spheroid
cultures). Two such patient-derived populations of CCSCs, labeled
herein as CA1 and CA2, were investigated. In contrast to HCT116
cells, which express wild type p53, both CA1 and CA2 have a single
base pair transition substitution at amino acid 273 of the DNA
binding domain (arginine to histidine). For compatibility with
drug-eluting microarrays, adherent cell growth was established and
serially propagated. Phenotypes were compared to those maintained
as spheroid cultures with regards to expression of ALDH and mucin 2
(MUC2, which delineates differentiation along the goblet cell
lineage). While ALDH expression was maintained in CA1 cells, in CA2
cells, the proportion of cells expressing ALDH declined from 75% in
cells cultured in spheroid form to 16% of cells in the adherent
form. Correspondingly, MUC2 expression increased from 9% (spheroid)
to 15% (adherent) (FIGS. 13A-B).
[0116] Different trends were observed for CCSCs from each patient
when exposed to camptothecin and nutlin-3a combinations on the
microarrays (FIGS. 14A and 14J). Cells from both patients exhibited
decreasing proliferation with increasing nutlin-3a or camptothecin
exposure (FIGS. 14A, 14B, 14F, 14J, 14K, and 14O). For CA1 cells
(FIGS. 14B-14I) sub-additive effects were observed from combination
treatments. Significant effects on proliferation due to nutlin-3a,
{F(3,233)=5.762, p<0.01}, and camptothecin, {F(3,233)=16.884,
p<0.01} were found by ANOVA. Trends were evident, indicating
increased antiproliferative activity with combination treatments.
For CA2 cells (FIGS. 14K-14R), significant primary effect on
proliferation due to nutlin-3a was revealed {nutlin-3a,
F(3,329)=2.854, p=0.037}, though not to camptothecin
{F(3,329)=0.508, p=0.677} by ANOVA. Additionally, an antagonistic
effect was observed from combination treatments {F(9,329)=2.382,
p=0.013}, where increasing both drugs reversed drug-induced
non-proliferation compared to high doses of individual drugs.
Negative slopes at high doses indicate an antagonistic interaction
(FIGS. 14N, 14R). The concentration response curves indicated
significant differences between patients (FIGS. 14B-14I and
14K-14R). To assess these differences, maximum response (E.sub.max)
and drug sensitivity values (1/D.sub.50) were obtained by
hyperbolic fit to the concentration response curves (Tables 4 and
5).
TABLE-US-00004 TABLE 4 E.sub.max and D.sub.50 values generated from
combinatorial microarrays of CA1 CCSCs. CA1 Hyperbolic Fit
Parameters of Nutlin Dose Hyperbolic Fit Parameters of Camptothecin
Response Upon Addition of Camptothecin Dose Response Upon Addition
of Nutlin 2.sup.nd Drug Conc (.mu.M) E.sub.max 1 D50 .times. 100
##EQU00005## Source Data 2.sup.nd Drug Conc (.mu.M) E.sub.max 1 D50
.times. 100 ##EQU00006## Source Data CPT 0 38.0 +/- 12* 28.6 +/-
4.7* FIG. 5b NUT 0 61.9 +/- 25 10.8 +/- 16 FIG. 5f 1 34.7 +/- 5.9
N/A FIG. 5c 1 105 +/- 160 1.97 +/- 7.7 FIG. 5g 10 50.9 +/- 0.15
50.0 +/- 1.3* FIG. 5d 25 109 +/- 4.1 3.18 +/- 0.40 FIG. 5h 50 81.7
+/- 3.1* 55.6 +/- 180 FIG. 5e 125 221 +/- 400 0.62 +/- 1.8 FIG.
5i
TABLE-US-00005 TABLE 5 E.sub.max and D.sub.50 values generated from
combinatorial microarrays of CA2 CCSCs. CA2 Hyperbolic Fit
Parameters of Nutlin Dose Hyperbolic Fit Parameters of Camptothecin
Response Upon Addition of Camptothecin Dose Response Upon Addition
of Nutlin 2.sup.nd Drug Conc (.mu.M) E.sub.max 1 D50 .times. 100
##EQU00007## Source Data 2.sup.nd Drug Conc (.mu.M) E.sub.max 1 D50
.times. 100 ##EQU00008## Source Data CPT 0 73.3 +/- 9.3* 171 +/- 49
.sup. FIG. 6b NUT 0 46.6 +/- 0.57 133 +/- 5.3* .sup. FIG. 6f 1 43.8
+/- 11 189 +/- 328 .sup. FIG. 6c 1 52.3 +/- 49 1.60 +/- 7.9* .sup.
FIG. 6g 10 57.0 +/- 39 0.95 +/- 4.6.sup.# FIG. 6d 25 43.9 +/- 1.9
N/A FIG. 6h 50 34.1 +/- 9.3* 303 +/- 260 .sup. FIG. 6e 125 N/A 0.00
+/- 1.01.sup.# FIG. 6i
[0117] In CA1 cells, the E.sub.max of the nutlin-3a concentration
response curve was increased by 115% when 50 .mu.M camptothecin was
present as compared to nutlin-3a alone (81.7 vs 38.0) (Table 4,
FIGS. 14B, 14E). The sensitivity also increased by 75% when 10
.mu.M camptothecin was present compared to nutlin-3a alone (50.0 vs
28.6) (Table 4, FIGS. 14B, 14D). By contrast, in CA2 cells the
E.sub.max of the concentration response curve to nutlin-3a
decreased by 53% when 50 .mu.M camptothecin was present as compared
to nutlin-3a alone, but not significantly (p=0.097) (FIGS. 14K and
14N). No statistical differences were found on the effects to
sensitivity between the drugs in CA2 cells. The sensitivity of the
CCSCs to nutlin-3a was unexpected due to the p53 mutation
identified in these cells. However, responsiveness could
potentially be explained by a nutlin-3a-induced increase in p73
expression, which can promote genes required for cell cycle arrest,
senescence, and apoptosis as previously shown.sup.28.
Alternatively, the p53 mutation in the CCSCs could be silent.
[0118] These results demonstrate that CA1 and CA2 cells differ
greatly in their responses to combinations of drugs. Cells from
patient CA1 exhibited an improved reduction in proliferation. In
particular, introduction of camptothecin significantly increased
the anti-proliferative activity of nutlin-3a in this patient's
CCSCs. In contrast, responses from CA2 cells were less pronounced
to drug combinations, with treatments interacting antagonistically.
CA2 cells exhibited significant responses to either nutlin-3a or
camptothecin treatment alone, but combination treatments muted
anti-proliferative effects. Therefore, in contrast to CA1, CA2
cells may respond to topoisomerase I inhibitors or p53 activating
agents alone or possibly with other classes of agents, to achieve
an increased reduction in proliferation.
[0119] The present example demonstrates creation of a novel
platform capable of performing chemosensitivity screens on
patient-derived CCSCs using limited cell numbers. The results
presented here (i) indicate that there can be considerable
variability in responses to drugs by CCSCs from different patients,
and (ii) suggest that chemosensitivity screening on patient-derived
CCSCs can lead to valuable information regarding chemotherapy
decisions. Although efficacy of drugs against CCSCs was
demonstrated, the approach could be adopted for any CSC or rare
cellular subpopulation where cell numbers are limiting, and
identifying responsiveness to drug combinations is paramount. This
platform can facilitate personalized medicine approaches centered
on the eradication of CCSCs.
[0120] It should be noted that ratios, concentrations, amounts, and
other numerical data may be expressed herein in a range format. It
is to be understood that such a range format is used for
convenience and brevity, and thus, should be interpreted in a
flexible manner to include not only the numerical values explicitly
recited as the limits of the range, but also to include all the
individual numerical values or sub-ranges encompassed within that
range as if each numerical value and sub-range is explicitly
recited. To illustrate, a concentration range of "about 0.1% to
about 5%" should be interpreted to include not only the explicitly
recited concentration of about 0.1 wt % to about 5 wt %, but also
include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and
the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the
indicated range. In an embodiment, the term "about" can include
traditional rounding according to significant figures of the
numerical value. In addition, the phrase "about `x` to `y`"
includes "about `x` to about `y`".
[0121] It should be emphasized that the above-described embodiments
of the present disclosure are merely possible examples of
implementations, and are merely set forth for a clear understanding
of the principles of this disclosure. Many variations and
modifications may be made to the above-described embodiment(s) of
the disclosure without departing substantially from the spirit and
principles of the disclosure. All such modifications and variations
are intended to be included herein within the scope of this
disclosure and protected by the following claims.
REFERENCES
[0122] 1. Lee, M. J. et al. Sequential application of anticancer
drugs enhances cell death by rewiring apoptotic signaling networks.
Cell 149, 780-794 (2012). [0123] 2. Lake, R. A. & Robinson, B.
W. Immunotherapy and chemotherapy--a practical partnership. Nat Rev
Cancer 5, 397-405 (2005). [0124] 3. DeVita, V. T., Young, R. C.
& Canellos, G. P. Combination versus single agent chemotherapy:
a review of the basis for selection of drug treatment of cancer.
Cancer 35, 98-110 (1975). [0125] 4. DeVita, V. T. & Chu, E. A
history of cancer chemotherapy. Cancer Res 68, 8643-8653 (2008).
[0126] 5. Lage, H. An overview of cancer multidrug resistance: a
still unsolved problem. Cell Mol Life Sci 65, 3145-3167 (2008).
[0127] 6. Maitland, M. L., DiRienzo, A. & Ratain, M. J.
Interpreting disparate responses to cancer therapy: the role of
human population genetics. J Clin Oncol 24, 2151-2157 (2006).
[0128] 7. Magee, J. A., Piskounova, E. & Morrison, S. J. Cancer
stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 21,
283-296 (2012). [0129] 8. Shoemaker, R. H. et al. Development of
human tumor cell line panels for use in disease-oriented drug
screening. Prog Clin Biol Res 276, 265-286 (1988). [0130] 9.
Ugurel, S. et al. In vitro drug sensitivity predicts response and
survival after individualized sensitivity-directed chemotherapy in
metastatic melanoma: a multicenter phase II trial of the
Dermatologic Cooperative Oncology Group. Clin Cancer Res 12,
5454-5463 (2006). [0131] 10. Shackleton, M., Quintana, E., Fearon,
E. R. & Morrison, S. J. Heterogeneity in cancer: cancer stem
cells versus clonal evolution. Cell 138, 822-829 (2009). [0132] 11.
Reya, T., Morrison, S. J., Clarke, M. F. & Weissman, I. L. Stem
cells, cancer, and cancer stem cells. Nature 414, 105-111 (2001).
[0133] 12. O'Brien, C. A., Kreso, A. & Jamieson, C. H. Cancer
stem cells and self-renewal. Clin Cancer Res 16, 3113-3120 (2010).
[0134] 13. Ricci-Vitiani, L. et al. Identification and expansion of
human colon-cancer-initiating cells. Nature 445, 111-115 (2007).
[0135] 14. Shenoy, A., Butterworth, E. & Huang, E. H. ALDH as a
marker for enriching tumorigenic human colonic stem cells. Methods
Mol Biol 916, 373-385 (2012). [0136] 15. Huang, E. H. et al.
Aldehyde dehydrogenase 1 is a marker for normal and malignant human
colonic stem cells (SC) and tracks SC overpopulation during colon
tumorigenesis. Cancer Res 69, 3382-3389 (2009). [0137] 16. Boman,
B. M. & Huang, E. Human colon cancer stem cells: a new paradigm
in gastrointestinal oncology. J Clin Oncol 26, 2828-2838 (2008).
[0138] 17. Shenoy, A. K. et al. Transition from colitis to cancer:
high wnt activity sustains the tumor-initiating potential of colon
cancer stem cell precursors. Cancer Res 72, 5091-5100 (2012).
[0139] 18. Huang, E. H. & Wicha, M. S. Colon cancer stem cells:
implications for prevention and therapy. Trends Mol Med 14, 503-509
(2008). [0140] 19. Zhao, C. et al. Hedgehog signalling is essential
for maintenance of cancer stem cells in myeloid leukaemia. Nature
458, 776-779 (2009). [0141] 20. Acharya, A. P., Clare-Salzler, M.
J. & Keselowsky, B. G. A high-throughput microparticle
microarray platform for dendritic cell-targeting vaccines.
Biomaterials 30, 4168-4177 (2009). [0142] 21. Hoe, K. K., Verma, C.
S. & Lane, D. P. Drugging the p53 pathway: understanding the
route to clinical efficacy. Nat Rev Drug Discov 13, 217-236 (2014).
[0143] 22. Vassilev, L. T. et al. In vivo activation of the p53
pathway by small-molecule antagonists of MDM2. Science 303, 844-848
(2004). [0144] 23. Vassilev, L. T. p53 Activation by small
molecules: application in oncology. J Med Chem 48, 4491-4499
(2005). [0145] 24. Goldwasser, F., Bae, I., Valenti, M., Torres, K.
& Pommier, Y. Topoisomerase I-related parameters and
camptothecin activity in the colon carcinoma cell lines from the
National Cancer Institute anticancer screen. Cancer Res 55,
2116-2121 (1995). [0146] 25. Motwani, M. et al. Augmentation of
apoptosis and tumor regression by flavopiridol in the presence of
CPT-11 in Hct116 colon cancer monolayers and xenografts. Clin
Cancer Res 7, 4209-4219 (2001). [0147] 26. Soen, Y., Mori, A.,
Palmer, T. D. & Brown, P. O. Exploring the regulation of human
neural precursor cell differentiation using arrays of signaling
microenvironments. Mol Syst Biol 2, 37 (2006). [0148] 27. Bailey,
S. N., Sabatini, D. M. & Stockwell, B. R. Microarrays of small
molecules embedded in biodegradable polymers for use in mammalian
cell-based screens. Proc Natl Acad Sci USA 101, 16144-16149 (2004).
[0149] 28. Lau, L. M., Nugent, J. K., Zhao, X. & Irwin, M. S.
HDM2 antagonist Nutlin-3 disrupts p73-HDM2 binding and enhances p73
function. Oncogene 27, 997-1003 (2008). [0150] 29. Langer, R. et
al. Controlled release and magnetically modulated systems for
macromolecular drugs. Ann N Y Acad Sci 446, 1-13 (1985). [0151] 30.
Langer, R., Brem, H. & Tapper, D. Biocompatibility of polymeric
delivery systems for macromolecules. J Biomed Mater Res 15, 267-277
(1981). [0152] 31. Sefton, M. V., Brown, L. R. & Langer, R. S.
Ethylene-vinyl acetate copolymer microspheres for controlled
release of macromolecules. J Pharm Sci 73, 1859-1861 (1984). [0153]
32. Carpentino, J. E. et al. Aldehyde dehydrogenase-expressing
colon stem cells contribute to tumorigenesis in the transition from
colitis to cancer. Cancer Res 69, 8208-8215 (2009). [0154] 33.
Sanchez, J. A., Dejulius, K. L., Bronner, M., Church, J. M. &
Kalady, M. F. Relative role of methylator and tumor suppressor
pathways in ulcerative colitis-associated colon cancer. Inflamm
Bowel Dis 17, 1966-1970 (2011). [0155] 34. Tallarida, R. (2000).
[0156] 35. Gupta, M. et al. Inactivation of p53 increases the
cytotoxicity of camptothecin in human colon HCT116 and breast MCF-7
cancer cells. Clin Cancer Res 3, 1653-1660 (1997). [0157] 36.
Kranz, D. & Dobbelstein, M. Nongenotoxic p53 activation
protects cells against S-phase-specific chemotherapy. Cancer Res
66, 10274-10280 (2006).
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