U.S. patent application number 10/281043 was filed with the patent office on 2003-06-26 for method of enriching rare cells.
This patent application is currently assigned to John Hopkins University School of Medicine. Invention is credited to Lesko, Stephen A., Nelson, William G., Partin, Alan W., Ts'o, Paul O. P., Wang, Zheng-Pin.
Application Number | 20030119077 10/281043 |
Document ID | / |
Family ID | 21768614 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030119077 |
Kind Code |
A1 |
Ts'o, Paul O. P. ; et
al. |
June 26, 2003 |
Method of enriching rare cells
Abstract
The present invention provides for enriching rare cells in a
fluid comprising the rare cells and non-rare cells. Examples of
rare cells enriched include prostate cancer cells.
Inventors: |
Ts'o, Paul O. P.; (Ellicot
City, MD) ; Wang, Zheng-Pin; (Ellicott City, MD)
; Lesko, Stephen A.; (Baltimore, MD) ; Nelson,
William G.; (Towson, MD) ; Partin, Alan W.;
(Baltimore, MD) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
700 THIRTEENTH ST. NW
SUITE 300
WASHINGTON
DC
20005-3960
US
|
Assignee: |
John Hopkins University School of
Medicine
Baltimore
MD
|
Family ID: |
21768614 |
Appl. No.: |
10/281043 |
Filed: |
October 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10281043 |
Oct 25, 2002 |
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09348670 |
Jul 6, 1999 |
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09348670 |
Jul 6, 1999 |
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08832468 |
Apr 2, 1997 |
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5962237 |
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60014929 |
Apr 5, 1996 |
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Current U.S.
Class: |
435/7.21 ;
435/366; 435/7.23 |
Current CPC
Class: |
G01N 33/56966 20130101;
G01N 33/57488 20130101; G01N 33/57438 20130101; G01N 33/57434
20130101; G01N 33/57407 20130101 |
Class at
Publication: |
435/7.21 ;
435/7.23; 435/366 |
International
Class: |
G01N 033/567; G01N
033/574; C12N 005/08 |
Claims
What is claimed is:
1. A method for enriching rare cells of interest in a bodily fluid
sample comprising rare cells and non-rare cells, comprising: (a)
obtaining a bodily fluid sample comprising rare cells of interest
and non-rare cells; (b) subjecting said sample to a binding agent
that binds non-rare cells; (c) removing the bound non-rare cells
from the sample so as to provide a sample enriched with the rare
cells of interest.
2. A method as in claim 1 wherein removing the bound non-rare cells
from the sample comprises application of a magnetic field so as to
separate the bound non-rare cells.
3. A method as in claim 1 wherein said bodily fluid sample is
selected from the group consisting of blood, urine, saliva, lymph,
spinal fluid, semen, amniotic fluid, cavity fluids and tissue
extracts.
4. A method as in claim 1 wherein said bodily fluid sample is
blood.
5. A method as in claim 1 wherein said bodily fluid sample is
amniotic fluid.
6. A method as in claim 1 wherein said rare cells of interest are
cancer cells.
7. A method as in claim 1 wherein said rare cells of interest are
fetal cells.
8. A method for enriching rare cells of interest in a bodily fluid
sample comprising rare cells and non-rare cells, comprising: (a)
obtaining a bodily fluid sample comprising rare cells of interest
and non-rare cells; (b) subjecting said sample to density gradient
separation; (c) selecting at least one gradient of interest and
subjecting said gradient of interest to a binding agent that binds
non-rare cells; (d) removing the bound non-rare cells from the
gradient of interest so as to provide a sample enriched with the
rare cells of interest.
9. A method as in claim 8 wherein said binding agent has magnetic
properties, and wherein removing the bound non-rare cells from the
sample comprises application of a magnetic field so as to separate
the bound non-rare cells.
10. A method as in claim 8 wherein said bodily fluid sample is
selected from the group consisting of blood, urine, saliva, lymph,
spinal fluid, semen, amniotic fluid, cavity fluids and tissue
extracts.
11. A method as in claim 8 wherein said bodily fluid sample is
blood.
12. A method as in claim 8 wherein said bodily fluid sample is
amniotic fluid.
13. A method as in claim 8 wherein said rare cells of interest are
cancer cells.
14. A method as in claim 8 wherein said rare cells of interest are
fetal cells.
Description
[0001] This application is a Continuation of U.S. application Ser.
No. 09/348,670, filed Jul. 6, 1999, which is a Continuation of U.S.
application Ser. No. 08/832,468, filed Apr. 2, 1997, now U.S. Pat.
No. 5,962,237. This application claims the benefit of U.S.
Provisional Patent Application Serial No. 60/014,929, filed Apr. 5,
1996, which is incorporated by reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to a method of enriching rare
cells in a fluid containing a mixture of rare cells and non-rare
cells, and particularly to a method of enriching rare non-blood
cells such as cancer cells, from bodily fluids, such as blood.
BACKGROUND OF THE INVENTION
[0003] There has been a growing interest in enriching rare cells
(e.g., for subsequent isolation and characterization) over the past
several years. This may be attributed at least in part to the
recognition that rare cells, such as cancer cells, can provide
information that is helpful in the diagnosis and/or treatment of
various medical conditions.
[0004] The desire to enrich cancer cells is based in part on the
knowledge that a majority of cancer deaths occur due to the
metastasis of tumors. As such, the presence of carcinoma cells in
the peripheral blood is an indication of cancer cell spread, and
enriching such cancer cells would be of great diagnostic benefit.
This need is particularly acute in prostate cancer, wherein
approximately two-thirds of such cancers are clinically localized
at the time of diagnosis, but only about half of these prove to be
confined to the prostate at the time of surgery. Thus, nearly
one-third to one-half of cancers have spread beyond the prostate
when first identified, cancers which could be detected at any
earlier stage if accurate, highly sensitive enrichment methods were
available.
[0005] Much of the activity with respect to the early detection of
prostate cancer has centered around the usefulness of serum
prostate specific antigen (PSA). However, PSA is organ specific and
not cancer specific, and is produced by normal, benign, and
malignant prostate epithelium. As a result, the positive predictive
value for PSA as a screen for prostate cancer is generally less
than 50 percent.
[0006] Additionally, the maximal level of cancer cells in the
peripheral blood has been estimated to be two in 10.sup.7
leukocytes. Fidler, Cancer Res., 50, 6130 (1990). Thus, while
studies have suggested that prostate cancer cells circulate in the
bloodstream of men with advanced disease, it is difficult to detect
these few circulating cancer cells.
[0007] Methods for separating and detecting cancer cells have
included, for example, using immunomagnetic beads and the
polymerase chain reaction (e.g., Hardingham et al. Cancer Res, 53,
3455 (1993)), using density gradient gels (e.g., U.S. Pat. No.
4,255,256), or using density gradient centrifugation followed by
immunological separation to bind the cancer cells (e.g., Griwatz et
al., J. Immunol Methods., 183, 251-265 (1995)).
[0008] These methods have been generally unsatisfactory as they
lack the efficiency and sensitivity to separate the few cancer
cells in a blood sample. Additionally, these methods may provide
low cell recovery, since the highly fragile cancer cells can be
damaged during the separation process and/or the relatively sticky
cancer cells can become inappropriately bound during the separation
process.
[0009] For example, conventional processes utilize "positive
selection", wherein a rare cell is bound to a binding agent such as
an antibody, and the bound rare cell is separated from the non-rare
cells. Thereafter, the rare cell is separated from the antibody by
heat or other suitable means, which can damage or destroy the rare
cell, making it difficult to detect and/or culture. Additionally,
or alternatively, some processes involve concentrating cancer cells
by centrifugation. However, since some cancer cells are fragile
and/or tend to stick to surfaces onto which they come into contact,
these processes can also damage or destroy the rare cells, which is
undesirable as described above. Furthermore, some processes provide
for "fixing" the cells during the separation process, thus
rendering them unsuitable for culturing or PCR analysis.
[0010] In view of the foregoing, there exists a need for an
efficient, highly sensitive and highly reproducible method for
enriching rare cells from a population of cells. There is also a
need for a method that can minimize damage to those rare cells that
are fragile and/or sticky.
[0011] The present invention provides for ameliorating at least
some of the disadvantages of the prior art. These and other
advantages of the present invention will be apparent from the
description as set forth below.
SUMMARY OF THE INVENTION
[0012] The present invention provides a method for enriching rare
cells in a fluid comprising the rare cells (that are preferably
rare non-blood cells) and non-rare cells. An embodiment of the
method comprises (a) subjecting the fluid comprising rare cells and
non-rare cells to density gradient separation and producing a fluid
comprising an increased concentration of rare cells; (b) subjecting
the fluid having an increased concentration of rare cells to an
agent that binds non-rare cells; and (c) removing the bound
non-rare cells from the fluid so as to enrich the rare cells in the
fluid.
[0013] Another embodiment of the method comprises (a) subjecting
the fluid to density gradient separation and producing a first
fluid comprising an increased concentration of rare cells and a
second fluid comprising an increased concentration of rare cells;
(b) subjecting at least one of said first fluid and said second
fluid to an agent that binds non-rare cells; and (c) removing the
bound non-rare cells from the first and/or the second fluid so as
to enrich the rare cells in the fluid(s). Typically, after the
bound non-rare cells are removed from the first and/or the second
fluid, the rare cell-containing first fluid and second fluid are
combined.
[0014] Embodiments of the present invention also provide for
further processing the rare cells. For example, rare cells (such as
cancer cells) can be identified and/or cultured. Illustratively, in
some embodiments involving identification, specific antigens in
and/or on the cancer cells can be detected. Additionally, or
alternatively, the expression of specific nucleic acids can be
detected, and, if desired, chromosomal changes (e.g., aneuploidy)
can be detected. In one embodiment, an identification protocol
includes combination staining (involving immunocytochemistry
staining) and fluorescent in situ hybridization (FISH). Embodiments
of the invention also provide improved methods of diagnosis,
staging, and monitoring of cancer in a patient.
[0015] The present invention further provides certain nucleic acid
sequences suitable as probes for cancer cells, particularly
prostate cancer cells. The present invention further provides
compositions comprising the rare cells isolated by the various
processes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 schematically depicts a density gradient column
before (on the left) and after (on the right) centrifuging a fluid
sample (e.g., blood). Four regions are formed after centrifugation:
Plasma I, Interface I, Gradient I, and Cell Pellet I. Interface I
and Gradient I are combined to provide a first fluid including an
increased concentration of rare cells, referred to below as the
Collection I fluid. Plasma I and Pellet I can be combined and
centrifuged to form another column having four regions.
[0017] FIGS. 2A and 2B schematically depict centrifuging the
combined Plasma I and Pellet I (from FIG. 1) on a single density
gradient column (FIG. 2A) or a double density gradient column (FIG.
2B). The schematics illustrate the columns before (on the left) and
after (on the right) centrifugation. Four regions are formed after
centrifugation: Plasma II, Interface II, Gradient II, and Pellet
II. Gradient II and Interface II will be combined to provide a
second fluid including an increased concentration of rare cells,
referred to below as the Collection II fluid.
[0018] FIG. 3 schematically depict another embodiment of the
invention, wherein a double density gradient column can be utilized
to form six regions after centrifugation. The left side of FIG. 3
illustrates the double density gradient column and fluid sample
(e.g., blood) before centrifugation, and the right side illustrates
the column after centrifugation. Six regions are formed after
centrifugation: Plasma, Interface I, Gradient I, Interface II,
Gradient II, and Pellet. Interface I and Gradient I will be
combined to form the Collection I fluid, and Interface II and
Gradient II will be combined to form the Collection II fluid. The
Collection I and II fluids each have an increased concentration of
rare cells.
[0019] FIG. 4 schematically depicts one exemplary embodiment of the
negative selection process of the present invention. The Collection
II fluid is incubated with one or more primary antibodies to the
non-rare cells, e.g., antibodies specific to white blood cell
and/or red blood cell antigens. The Collection II fluid containing
the primary antibodies is then incubated with secondary antibodies
that are bound to supports such as magnetic beads. The primary
antibodies bind to the non-rare cells, and the secondary antibodies
(that are bound to the beads) bind to the primary antibodies.
Accordingly, the removal of the beads from the fluid provides a
fluid enriched with the rare cells, referred to below as the
Collection III fluid.
[0020] FIG. 5 schematically depicts that the Collection I and III
fluids, that each include rare cells, can be combined. If desired,
the rare cells can be used for cell culturing and/or slide
preparation.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0021] The present invention provides a sensitive, economical, and
reproducible method for enriching rare cells in a fluid comprising
rare and non-rare cells. In accordance with the invention, a fluid
comprising rare and non-rare cells is subjected to density gradient
separation, before producing at least one fluid comprising an
increased concentration of rare cells. The fluid comprising an
increased concentration of rare cells is subjected to a "negative
selection process" comprising contacting the fluid with an agent
which binds the non-rare cells. The bound non-rare cells are then
separated from the fluid, providing a fluid enriched with the rare
cells. The rare cells can be further processed, e.g., to identify,
characterize, and/or culture the cells. For example, the rare cells
can be identified and characterized to detect one or more types of
cancer. Embodiments of the present invention provide for monitoring
the progress, or regression, of cancer during or after therapy, and
are particularly useful for monitoring prostate cancer in men.
[0022] In an embodiment, a fluid comprising rare and non-rare cells
is subjected to density gradient separation, before producing a
first fluid comprising an increased concentration of rare cells and
a second fluid comprising an increased concentration of rare cells.
This first fluid and/or second fluid is subjected to the negative
selection process comprising contacting the fluid(s) with an agent
which binds the non-rare cells. The bound non-rare cells are then
separated from the fluid(s), providing the fluid(s) enriched with
the rare cells.
[0023] Additionally, since embodiments of the method according to
the invention can be carried out while minimizing stress to those
rare cells that are fragile and/or sticky, the rare cells can be
recovered essentially unscathed. This is especially desirable, as
the live recovered rare cells have a variety of uses, e.g., for
studies of the whole cell and/or cell culturing. Moreover,
embodiments of the method allow different forms of rare cells in
the same sample (e.g., "light" and "heavy" rare cells) to be
processed differently, thus allowing a great proportion, if not
substantially all, of the rare cells to be recovered, while
reducing the presence of the non-rare cells in the rare
cell-enriched fluid.
[0024] An embodiment of the invention provides a method for
enriching rare cells in a fluid sample comprising rare cells and
non-rare cells, comprising (a) obtaining the sample comprising rare
cells and non-rare cells; (b) subjecting the fluid sample to
density gradient separation and producing a fluid having an
increased concentration of rare cells; (c) subjecting the fluid
having an increased concentration of rare cells to a binding agent
that binds non-rare cells; (d) removing the bound non-rare cells
from the fluid to provide a fluid enriched with rare cells.
Preferably, the rare cells are cancer cells. In some embodiments,
the non-rare cells comprise blood cells, i.e., white blood cells
(leukocytes) and/or red blood cells (erythrocytes).
[0025] In one embodiment of the invention, a method for enriching
rare non-blood cells in a fluid sample comprising rare non-blood
cells and non-rare cells, wherein the ratio of the rare non-blood
cells to the non-rare cells is at least about 1:100,000, comprises
(a) obtaining the fluid sample comprising rare non-blood cells and
non-rare cells; (b) subjecting the fluid sample to density gradient
separation and producing a first fluid (I) comprising an increased
concentration of rare non-blood cells, and a second fluid (II)
comprising an increased concentration of rare non-blood cells; (c)
subjecting at least one of said first fluid (I) and said second
fluid (II) to a binding agent that binds non-rare cells; (d)
removing the bound non-rare cells from the first fluid (I) and/or
the second fluid (II) to provide a first fluid (Ia) enriched with
rare non-blood cells and/or a second fluid (IIa) enriched with rare
non-blood cells.
[0026] In another embodiment, a method for enriching rare non-blood
cells in a fluid sample comprising rare non-blood cells and
non-rare cells, wherein the ratio of the rare non-blood cells to
the non-rare cells is at least about 1:100,000, comprises (a)
obtaining the fluid sample comprising rare non-blood cells and
non-rare cells; (b) subjecting the fluid sample to density gradient
separation and producing a fluid comprising an increased
concentration of rare non-blood cells; (c) subjecting the fluid
comprising an increased concentration of rare non-blood cells to a
binding agent that binds non-rare cells; (d) removing the bound
non-rare cells from the fluid to provide a fluid enriched with rare
non-blood cells.
[0027] Another embodiment according to the invention provides a
method for enriching cancer cells in a blood sample comprising (a)
obtaining the blood sample comprising cancer cells; (b) subjecting
the blood sample to density gradient separation and producing a
first fluid comprising an increased concentration of cancer cells
of a first density, and a second fluid comprising an increased
concentration of cancer cells of a second density, wherein the
second density is greater than the first density; (c) subjecting
said second fluid to a binding agent that binds white blood cells
and/or red blood cells; (d) removing the bound white and/or red
blood cells from the second fluid to provide a second fluid
enriched with the greater density cancer cells. In some
embodiments, the second fluid comprising an increased concentration
of cancer cells of a second density is subjected to a binding agent
that binds white blood cells and red blood cells, and the bound
blood cells, i.e., the white and red blood cells, are removed from
the fluid. In one preferred embodiment, the cancer cells having
different densities are prostate cancer cells.
[0028] Any fluid containing rare and non-rare cells can be
processed according to the invention. Embodiments of the invention
are suitable for enriching rare cells in a fluid wherein the ratio
of rare cells to non-rare cells in the fluid is at least about
1:10,000, and are especially suitable for enriching rare cells in a
fluid wherein the ratio of rare cells to non-rare cells in the
fluid is at least about 1:100,000. In accordance with the
invention, the concentration of rare cells can be increased by at
least about 10-fold, preferably, increased by at least about
100-fold, and in some embodiments, increased by at least about
500-fold, as compared to the ratio of rare cells to non-rare cells
in the original sample.
[0029] The present invention, particularly for some of those
embodiments wherein the rare cells to be enriched are cancer cells,
is capable of providing relatively high levels of cancer cell
recovery from fluids. For example, recoveries as high as 70%, or
more, based on the number of cancer cells in a blood sample have
been observed. In addition, some embodiments provide sufficiently
highly sensitivity to allow one to detect at least 1.5 cancer cells
per milliliter of blood (e.g., from a 20 ml blood sample).
[0030] The method of the present invention is surprising and
unexpected in that it can provide the foregoing advantages while
utilizing "negative selection", i.e., binding the non-rare cells, a
procedure that is precisely the opposite of conventional processes,
that utilize "positive selection", i.e., binding the rare
cells.
[0031] Examples of fluids that can be processed in accordance with
the invention include bodily fluids, e.g., blood, urine, saliva,
lymph, spinal fluid, semen, amniotic fluid, cavity fluids, and
tissue extracts.
[0032] The rare cells that can be enriched in accordance with the
invention include a variety of cells of therapeutic or diagnostic
interest, including but not limited to, cancer cells. For example,
the rare cells in the fluid can be cancer cells, and the non-rare
cells can be non-cancer cells. In one embodiment, the rare cells
are rare non-blood cells, such as, for example, prostate cancer
cells.
[0033] The cancer cells, of course, can comprise a cell from any
one of a number of different cancers including, but not limited to,
those of epithelial origin. The term cancer should be further
understood to encompass localized cancer (e.g., localized in
tumors), as well as non-localized cancer. In particular, carcinomas
of the bladder, brain, breast, colon, kidney, liver, lung, ovary,
pancreas, prostrate, rectum, and stomach are included, as are
tumors in the form of a sarcoma (e.g., a fibrosarcoma or
rhabdosarcoma), a hematopoietic tumor of lymphoid or myeloid
lineage, or another tumor, including, but not limited to, a
melanoma, teratocarcinoma, neuroblastoma, or glioma.
[0034] In accordance with the invention, fluids comprising rare and
non-rare cells are subjected to density gradient separation before
carrying out negative selection. This is advantageous, particularly
for those embodiments wherein the rare cells are cancer cells,
since in general (using a bodily fluid such as blood for example),
the density of most cancer cells is less than other circulating
blood cells, such as nucleated white blood cells, due to the fact
that such cancer cells are much larger, and thus lighter per unit
mass, than the other blood cells. This being said, however, some
cancer cells are heterogenous in nature, and certain kinds of
cancer cells can have densities that are similar to that of
nucleated white blood cells. Accordingly, as will be described in
more detail below, some embodiments of the invention include
carrying out density gradient separation at least twice, and/or
using one or more multiple density gradient columns (i.e., columns
having two or more density gradients) to further improve the
efficiency of the enrichment process.
[0035] Density Gradient Separation
[0036] Generally, density gradient separation processes involve
preparing one or more layers of gradient media, wherein the density
or densities of the gradient media should be higher than the
density of the rare cells to be separated. Typically, the fluid
comprising rare cells and non-rare cells is placed onto the upper
layer of the gradient medium (or uppermost gradient medium), the
media and the fluid are centrifuged until the components of the
fluid separate from one another according to their individual
component densities.
[0037] For example, using FIG. 1 for reference, and using a bodily
fluid such as blood as an illustrative fluid comprising rare cells
and non-rare cells, the contents of the centrifuge tube can appear
after centrifugation as follows: a plasma layer (Plasma I), an
interface layer (Interface I), a density gradient layer (Gradient
I), and a cell pellet (Pellet I) which resides at the bottom of the
tube. The interface layer is flanked by the plasma layer on one
side, and the density gradient layer on the other.
[0038] Rare cells that exist in both relatively light and heavy
forms (e.g., some cancer cells such as prostate cancer cells), will
be present in the interface layer, the adjacent density gradient
layer, and in the cell pellet. Typically, the lighter cancer cells
will be located in the interface layer and in the gradient layer,
while the relatively heavier cancer cells will be located in the
cell pellet along with the white and red blood cells.
[0039] In accordance with embodiments of the invention, one can
prepare a first fluid suspension comprising an increased
concentration of the "lighter" rare cells, and a second fluid
suspension comprising an increased concentration of the "heavier"
rare cells. This can be advantageous, since the rare cells having
different characteristics can be processed differently according to
the invention to improve rare cell recovery and reduce the presence
of non-rare cells, while minimizing stress to the more fragile rare
cells. Illustratively, the suspension comprising an increased
concentration of heavier rare cells can be exposed to an agent that
binds the non-rare cells, and the bound non-rare cells can be
removed. However, the suspension comprising an increased
concentration of the lighter rare cells (that may be larger, more
fragile and/or sticky) need not be exposed to the binding
agent.
[0040] For example, again using FIG. 1 for reference, a first fluid
suspension comprising an increased concentration of lighter rare
cells can be prepared by removing the interface layer (Interface I)
and, preferably, the portion of the density gradient layer
(Gradient I) adjoining the interface layer, and placing the
interface layer and the gradient layer in another tube. Care should
be exercised in removing the gradient layer so as not to disturb
the cell pellet (Pellet I). Typically, about two-thirds of the
adjoining gradient layer is removed.
[0041] Thereafter, the cells in the new tube are gently washed with
a suitable diluent, such as phosphate buffered saline (PBS), and
are then lightly centrifuged (e.g., centrifuged at a force of about
200.times.g). The cell pellet that results from this processing is
then suspended in a solution to form a first fluid comprising an
increased concentration of rare cells, illustrated as the
"Collection I" fluid in FIGS. 1 and 5. Suitable solutions for use
in forming the Collection I fluid include, for example, an albumin
solution, such as a 1 wt. % bovine serum albumin solution. The
resulting cell suspension (the Collection I fluid), in which the
relatively light cancer cells predominate, can be used for a
variety of purposes, e.g., cell identification, and/or culturing,
as will be discussed in greater detail herein. If desired, this
fluid can be subjected to a negative selection process to bind
non-rare cells contained in the fluid, and the bound cells can be
removed to produce a fluid enriched with rare cells.
[0042] In the case of rare cells such as cancer cells that are
relatively heavy, or which comprise relatively light and heavy
cells, a second fluid suspension comprising an increased
concentration of rare cells (i.e., the relatively heavy cancer
cells) can be prepared. For example, using FIGS. 1, 2A, and 2B for
reference, the plasma layer (Plasma I in FIG. 1) and the cell
pellet (Pellet I in FIG. 1), which were not used when the
relatively light cells were enriched, are removed and combined in a
new tube, as illustrated on the left in FIGS. 2A and 2B. This
combination of the plasma and the pellet includes the relatively
heavy cancer cells as well as red and white blood cells.
Subsequently, this combination is subjected to a density gradient
separation process. In some embodiments, prior to subjecting the
combination to this separation process, the density of the
combination is adjusted to correspond to approximate that of the
original fluid sample. For example, in those embodiments wherein
the original sample comprises blood, the density of the combination
can be adjusted by adding plasma.
[0043] As a result of the separation process, the contents of the
centrifuge tube can, as before, appear as four layers. For example,
the right sides of FIGS. 2A and 2B illustrate a plasma layer
(Plasma II); an interface layer (Interface II) containing the
cancer cells as well as some white blood cells and red blood cells,
a density gradient layer (Gradient II), and a cell pellet at the
bottom (Pellet II).
[0044] The interface layer (Interface II) and, preferably, the
portion of the density gradient layer (Gradient II) adjoining the
interface layer, are removed and placed into a new tube.
Thereafter, the cells in the new tube are gently washed with a
suitable diluent, such as phosphate buffered saline (PBS), and,
typically, are then lightly centrifuged. The cell pellet that
results from this processing is then suspended in a solution to
form a second fluid comprising an increased concentration of rare
cells (as well as some white blood cells and red blood cells),
illustrated as the "Collection II" fluid in the right side of FIGS.
2A, 2B, and in the left side in FIG. 4. Suitable solutions for use
in forming the Collection II fluid include an albumin solution,
such as a 1 wt. % bovine serum albumin solution. The resulting cell
suspension (the Collection II fluid) is typically subjected to a
negative selection process as will be described in more detail
below in the section entitled "negative selection".
[0045] In an alternative embodiment, for example, as illustrated in
FIG. 3, a multiple density gradient column can be utilized to
provide a plurality of interface and gradient layers, and the
appropriate layers can be combined and processed to provide one or
more fluids having an increased concentration of rare cells.
[0046] For example, an embodiment of the gradient column as
illustrated in FIG. 3 can be utilized to provide the Collection I
fluid and the Collection II fluid, wherein each fluid has an
increased concentration of rare cells. Illustratively, a bodily
fluid such as blood can be placed on the upper layer of the
gradient column, wherein the upper gradient density layer (Gradient
I) has a density less than that of the lower layer (Gradient II).
After centrifugation, the contents of the tube can appear as
follows: a plasma layer (Plasma), a first interface layer
(Interface I), a first density gradient layer (Gradient I), a
second interface layer (Gradient II), a second density gradient
layer (Gradient II), and a cell pellet (Pellet) that resides at the
bottom of the tube.
[0047] Typically, some of the lighter rare cells will be located in
the Interface I and Gradient I layers, while some of the heavier
rare cells will be located in the Interface II and Gradient II
layers. In one embodiment (again using FIG. 3 for reference), the
Interface I and Gradient I layers are combined to provide the
Collection I fluid, and the Interface II and Gradient II layers are
combined to provide the Collection II fluid. If desired, diluents
can be used and/or suspensions can be formed as described above.
The Collection I fluid and/or the Collection II fluid can be
subjected to a negative selection process to bind the non-rare
cells contained in the fluid(s), and the bound cells can be removed
to produce fluid(s) enriched with rare cells.
[0048] A variety of density gradient media and protocols for
carrying out density gradient separation are suitable for carrying
out the invention. Thus, single and/or multiple density columns can
be used, and any suitable combination of media densities can be
employed. Of course, density gradient separation according to the
invention can also be carried out using continuous and/or
discontinuous gradients. Different media and protocols can be
utilized depending on the fluid to be processed and the cells of
interest. Density gradient separation can be carried out any number
of times to provide one or more fluids having an increased
concentration of rare cells. The gradient medium or media can also
include one or more additives, e.g., to provide a desired density,
or viscosity. Alternatively, or additionally, the additive(s) can
provide for, for example, clumping and/or aggregating of non-rare
cells during the density separation process.
[0049] In some embodiments, e.g., for separating relatively dense
rare cells, such as, for example, dense prostate cancer cells,
FICOLL 400.TM. is a preferred medium. The medium is generally used
in combination with a compound, in solution, of relatively high
density and relatively low viscosity, for example sodium metrizoate
and sodium diatrizoate.
[0050] By way of example, and in some embodiments wherein the fluid
comprises blood, density gradients containing cell aggregating or
clumping agents such as methylcellulose, ISOPAQUE.TM., dextran, and
FICOLL.TM. can be used. Bhat, N. M. J. Immuno Meth., 158, 277-280
(1993). ISOPAQUE.TM. is a sodium
N-methyl-3,5,-diacetamino-2,4,6-triiodobenzoate. FICOLL.TM.
(Accurate Chemical and Scientific Corporation, Westbury N.Y.) is a
synthetic high polymer made by the copolymerization of sucrose and
epichlorohydrin. These agents cause erythrocyte clumping, and thus
can be utilized to separate leukocytes from red blood cells.
[0051] PERCOLL.TM. (available from Pharmacia) density gradients are
also suitable for the purposes of the present invention.
PERCOLL.TM. is a colloidal polyvinyl pyrrolidone coated silica
having a pH of 8.9.+-.0.3 at 20.degree. C., a density of
1.13.+-.0.005 g/mL, and a viscosity of 10.+-.5 cps at 20.degree.
C.
[0052] The following section describes using PERCOLL.TM. to provide
a density gradient medium of any suitable density. It should be
clear that other media and preparation protocols are also suitable,
and can be readily determined by one of ordinary skill in the art.
A stock solution of PERCOLL.TM. is prepared by combining the
following ingredients: 90 mL of PERCOLL.TM., 9 mL of
10.times.Hank's Balanced Salt Solution (HBSS without calcium,
magnesium, and phenol red), 1 mL of HEPES buffer (pH of 7.3), and
0.4 mL of 1 M HCl. The resulting solution has a pH of 7.4. Media
having various illustrative densities can be obtained as follows. A
medium having a density of 1.070 g/mL can be obtained by mixing 24
volumes of the PERCOLL.TM. stock solution and 20 volumes of
1.times.HBSS. A medium having a density of 1.079 g/mL can be
obtained by mixing 27 volumes of the PERCOLL.TM. stock solution and
15.9 volumes of 1.times.HBSS. A medium having a density of 1.088
g/mL can be obtained by mixing 23 volumes of the PERCOLL.TM. stock
solution and 10 volumes of 1.times.HBSS.
[0053] It may be advantageous to dilute the fluid comprising rare
cells and non-rare cells with a suitable diluent prior to placing
it on the density gradient column, particularly for those
embodiments wherein the fluid comprises blood. Any suitable diluent
known to those of ordinary skill in the art can be employed.
Examples of such diluents include buffers, e.g., physiological
buffers such as Tris buffer, phosphate buffer, citrate buffer, and
phosphate buffered saline (PBS), and salt solutions, e.g.,
commercially available balanced salt solutions such as Hanks
balanced salt solution (HBSS), Earl's balanced salt solution, Gey's
balanced salt solution,and the like. PBS is a preferred diluent for
diluting blood.
[0054] The fluid can be diluted with the aforesaid diluent to any
desired ratio. Typically, however, in those embodiments wherein the
fluid is a blood sample, it is diluted in a volume ratio of from
about 0.1 to about 10 (blood:diluent), advantageously in a volume
ratio of from about 0.5 to about 5 (blood:diluent), and preferably
in a volume ratio of from about 1 to about 2 (blood:diluent).
[0055] After the fluid sample is placed on the column, the column
and the sample are centrifuged. Centrifugation will typically be
performed in any suitable centrifuge, and at a suitable force and
for a suitable length of time, so that the lighter rare cells are
separated from the heavier non-rare cells and other material. In
some embodiments, the force of centrifuging should generally range
from a force of from about 300.times.g to about 600.times.g,
preferably, from about 350.times.g to about 450.times.g. Of course,
in other embodiments, the centrifuge can operate at a higher force,
or a lower force, than described above.
[0056] Centrifugation can be carried out to any suitable length of
time. In some embodiments, centrifugation is carried out for about
1 minute to about 60 minutes, advantageously for about 10 minutes
to about 50 minutes, and preferably for about 20 minutes to about
40 minutes. In the case where blood is the fluid being processed,
the centrifuging is preferably carried out for a period of about 30
minutes at a force of about 400.times.g.
[0057] Although those skilled in the art will be able to determine
the appropriate densities, in the specific case of enriching
prostate cancer cells in blood, the gradient medium (gel) should
have a density of no less than about 1.06 g/mL, more preferably no
less than about 1.068 g/mL. In one embodiment involving the
enrichment of prostate cancer cells, and utilizing a double density
gradient, the double gradient should include layers having a
density ranging from about 1.06 g/ml to about 1.10 g/ml, with about
1.077 g/ml to about 1.083 g/ml being preferred.
[0058] Embodiment of the method of the present invention encompass
the enrichment of many types of cancer cells that can circulate in
a fluid such as blood. For example, as described below, cells from
the classical Hepatoma G.sub.2 cell lines were cultured and put
into normal human blood in known numbers. These samples were
centrifuged in various density gradients. The density gradient
having a density of 1.068 g/mL was found to be the gradient layer
from which 80% of added hepatoma cells were recovered. The hepatoma
cells also were found to be very sticky and fragile. It is believed
that these hepatoma cells have a similar density to the prostate
cancer cells of the LNCaP line.
[0059] Negative Selection
[0060] As noted earlier, the negative selection process comprises
subjecting a fluid comprising rare cells and non-rare cells to an
agent that binds non-rare cells, and removing the bound non-rare
cells from the fluid. The removal of the bound non-rare cells
provides a rare cell-enriched fluid.
[0061] For example, the Collection II fluid as described in any of
the embodiments above can comprise the heavier rare cells, as well
as some non-rare cells that may have similar densities (e.g., some
white blood cells and red blood cells). Accordingly, the Collection
II fluid can be subjected to an agent that binds these non-rare
cells. The removal of the bound non-rare cells provides a rare
cell-enriched fluid, represented as the "Collection III" fluid in
FIG. 5.
[0062] In accordance with embodiments of the invention, the agent
that binds to the non-rare cells typically comprises one or more
antibodies, preferably monoclonal antibodies, that specifically
bind to the non-rare cells. A variety of antibodies are suitable
for carrying out the invention, and they can be derived from any
suitable source. For example, in some embodiments, e.g., wherein
the fluid comprising rare cells and non-rare cells includes blood
cells, suitable binding agents include antibodies that specifically
bind to one or more normal white blood cell surface antigens and/or
red blood cell surface antigens. Alternatively, or additionally,
the binding agent can comprise, for example, anti-human antibodies,
e.g., that specifically bind to human normal white blood cells
and/or human red blood cells.
[0063] The negative selection process encompasses both "direct" and
"indirect" protocols. For example, one example of a direct negative
selection process includes utilizing an antibody bound to a support
wherein the antibody binds to a non-rare cell. An example of an
indirect negative selection process includes using a "primary"
antibody to bind to the non-rare cell, and a "secondary" antibody
(that is bound to a support) to bind to the "primary" antibody.
Preferably, the primary and secondary antibodies are from different
species of animals. A variety of primary and secondary antibodies
are suitable, and are commercially available.
[0064] The use of a support (e.g., a particle such as a bead, more
preferably a microbead) is desirable, since it allows the
antibody-non-rare cell combination or the secondary
antibody-primary antibody-non-rare cell combination to be more
readily removed from the fluid. Microbeads, which are well-known in
the art, can be made of any suitable material, including plastic
and magnetic materials, with magnetic microbeads being preferred,
and superparamagnetic microbeads being even more preferred. A
variety of suitable supports, particularly particles such as
microbeads (with or without antibodies bound thereto) are
commercially available. Any separation method and system known to
those of ordinary skill in the art that bis capable of removing the
support (e.g., the particles) from the fluid can be utilized.
[0065] In one embodiment of a direct negative selection process, a
fluid comprising an increased concentration of rare cells (e.g.,
the Collection II fluid), is contacted with a mixture of anti-human
antibodies bound to support particles. The fluid thus produced is
then incubated at a suitable temperature and for a suitable period
of time so as to effect substantially complete binding of the
antibodies to the non-rare cells. While the temperature and time of
incubation will vary, the incubation is preferably carried out at a
subambient temperature, and more preferably at about 4.degree. C.,
for a period of from about 5 minutes to 60 minutes, and preferably
for a period of from about 10 minutes to about 50 minutes.
[0066] During the incubation, the antibody/support particles and
the cells are preferably gently mixed, e.g., by using a suitable
mixing or shaking device. The support particles/antibodies, that
now have non-rare cells bound to the antibodies, are separated from
the fluid as is known in the art. Illustratively, in some
embodiments wherein the support particles are paramagnetic
microbeads, the separation can be carried out using a magnetic
particle concentrator. Suitable concentrators are commercially
available, e.g., from Dynal, Inc. (Lake Success, N.Y.).
[0067] In an embodiment of an indirect negative selection process,
a fluid comprising an increased concentration of rare cells (e.g.,
the Collection II fluid), is contacted with a mixture of primary
antibodies, e.g., anti-human antibodies. These primary antibodies
are not bound to supports. The resulting mixture can then incubated
as described above for the direct method. Thereafter, the fluid is
contacted with secondary antibodies which are bound to support
particles. These secondary antibodies are selected so as to be
specific to the primary antibodies. The support particles/secondary
antibodies, that now also have primary antibodies/non-rare cells
bound thereto, can be separated from the fluid as described above
with respect to the direct negative selection process, e.g., by
using a magnetic particle concentrator.
[0068] As noted above, in some embodiments, the use of
superparamagnetic particles is preferred. Exemplary
superparamagnetic microbeads have a magnetic susceptibility of from
about 10.sup.-9 cgs units to about 10.sup.-7 cgs units, and
preferably from about 8.times.10.sup.-9 cgs units to about
10.sup.-7 cgs units. One embodiment involving the use of a magnetic
particle concentrator to separate the paramagnetic (or
superparamagnetic) particles from the fluid can be described as
follows. When the fluid is placed within the magnetic field
generated by magnetic particle concentrator, the paramagnetic
particles are attracted to and held close to the wall of the tube
in proximity to the magnet of the magnetic particle concentrator,
providing for the separation of the non-rare cells (bound to the
paramagnetic particles) from the rare cells (that are unbound). The
rare cells enriched according to this embodiment are substantially
completely free of contamination by non-rare cells. In the case of
the separation of cancer cells from blood, it was found that the
cancer cells could be almost completely separated from nucleated
white blood cells. This is significant, because nucleated white
blood cells, if present, can interfere with cell identification,
particularly when polymerase chain reaction (PCR) methods are
used.
[0069] For some of the embodiments wherein the fluid comprising
rare cells and non-rare cells is blood, it may be desirable to use
antibodies that bind to white blood cells (leukocytes) and/or red
blood cells (erythrocytes). Examples of suitable leukocyte
antibodies include the human and anti-human leukocyte CD
antibodies, e.g., CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b,
CD11c, CD14, CD15, CD16, CD19, CD20, CD28, CD36, CD42a, CD43, CD44,
CD45, CD45R, CD45RA, CD45RB, CD45RO, CD57, and CD61 antibodies, and
the like. Antibodies targeted to human CD45, CD3, CD19, CD14, and
CD36 are preferred. For example, when an CD45 specific antibody is
used, it recognizes the CD45 leukocyte common antigen (LCA) family
which is comprised of at least four isoforms of membrane
glycoproteins (220, 205, 190, 180 kD) present on cells of the
leukocyte lineage. Of course, human and anti-human red blood cell
antibodies can also be included.
[0070] By way of example of a direct negative separation
embodiment, the rare cell-containing fluid can be contacted with a
mixture of anti-human CD45, anti-human CD19, anti-human CD14, and
anti-human CD3 antibodies (e.g., a mixture of mouse anti-human CD45
IgG, mouse anti-human CD19 IgG, mouse anti-human CD14 IgG, and
mouse anti-human CD3 IgG antibodies). Optionally, a suitable
anti-human red blood cell antibody (e.g., glycophorin A) can also
be included in the antibody mixture. In one embodiment, the
antibodies are bound to magnetic particles before exposing the
mixture to the rare cell-containing fluid, and the particles are
removed from the fluid using a magnetic particle concentrator as
described above.
[0071] By way of example of an indirect negative separation
embodiment, where a mouse anti-human CD45 IgG antibody is used as
the primary antibody, the secondary antibody would be anti-mouse
IgG antibody. The secondary antibodies can be bound to particles
before use, and removed from the rare cell-containing fluid, as
described above.
[0072] In accordance with an embodiment of the invention, a kit for
the enrichment of cancer cells from blood is provided, comprising
at least first and second gradient density media, wherein the first
gradient density medium has a density of at least about 1.067 g/mL,
and the second gradient density medium has a density of at least
about 1.077 g/mL, wherein the kit further comprises support
particles, and at least one antibody capable of binding to a cell
surface antigen of a cell that is more dense than the cancer cell.
In a more preferred embodiment, the first gradient density medium
has a density of about 1.068 g/mL to about 1.077 g/mL, and the
second medium has a density of from about 1.077 g/mL to about 1.085
g/mL.
[0073] In other embodiments of kits according to the invention, the
kit can include one or more nucleic acid probes (described below in
the section entitled "Further Processing of the Enriched Rare
Cells") and/or one or more antibodies. If desired, such kits can
also include one or more gradient density media.
[0074] Further Processing of the Enriched Rare Cells
[0075] As noted earlier, a fluid comprising rare cells and non-rare
cells can be processed to provide a plurality of fluids, each
having an increased concentration of rare cells. One or more of the
fluids having an increased concentration of rare cells can be
subjected to a binding agent to bind the non-rare cells, to provide
one or more rare cell-enriched fluids. If desired, the fluids can
be combined. For example, two rare cell-enriched fluids can be
combined, or a rare cell-enriched fluid can be combined with a
fluid that has an increased concentration of rare cells, but was
not subjected to a binding agent.
[0076] Embodiments of the invention provide one or more rare blood
cell enriched fluids that are suitable for a variety of
purposes.
[0077] Embodiments of the method according to the present invention
also provide for processing or using the enriched rare cells, e.g.,
to identify, characterize, and/or culture the rare cells.
Additionally, the method provides for diagnosing cancer,
particularly prostate cancer in men, and also allows monitoring the
progress, or regression, of cancer, particularly during or after
therapy.
[0078] The present invention further provides a method of
identifying cancer cells in a patient's blood comprising enriching
the cancer cells from the patient's blood by any of the methods set
forth above, and identifying the cells using any suitable protocol
and system.
[0079] Embodiments of the present invention also provide for
preparing a therapeutic product, including, but not limited to, a
vaccine.
[0080] The cells can be prepared (e.g., for identification,
characterization, and/or culturing) by any suitable procedure.
Typically, an embodiment of the method for identifying and/or
characterizing the rare cells includes preparing a suspension
including the enriched cells, transferring the suspension of cells
to a microscope slide (e.g., to prepare a smear), and examining the
smear using alight microscope. In the direct smear procedure, it is
preferred to avoid packing the cancer cells down through a
centrifugal force or redistributing these packed cells by
mechanical means.
[0081] On the other hand, if the cells are gently sedimented or
centrifuged down, and after the supernatant is carefully removed,
the loosely sedimented cells can be resuspended by a small volume
of liquid (about1-10 .mu.l), and then be directly transferred onto
a slide (e.g., for identification) or onto a growth medium (e.g.,
for culturing). One protocol for transferring the cells onto a
slide includes resuspending the sedimented cells in BSA solution,
and cytospinning the cells onto the slide, e.g., by using a
commercially available Megafunnel.TM. large volume sample chamber
(Shandon).
[0082] If desired, the sedimented cells can be fixed by addition of
a fixative (such as ethanol), thus rendering the cells more
damage-resistant. This can be advantageous, as the fixed cells can
be readily transferred to the slide. However, since the cells are
fixed, they cannot be cultured or used for PCR studies.
[0083] If desired, a machine collection procedure for preparing
cells for identification can avoid exposing the cells to the
stresses of centrifugation. For example, the few cancer cells in
solution can be advantageously collected from the suspension and
deposited on a membrane while creating gentle suction. The liquid
will pass through the pores of the membrane, and the cells will be
collected on the membrane. These cells are then transferred to the
slide by putting the cell-containing surface of the membrane onto
the slide.
[0084] A variety of techniques are suitable for identifying and/or
characterizing the rare cells. Additionally, embodiments of the
invention can include identifying and/or characterizing a plurality
of types of rare cells, e.g., different cancer cell types, in a
single sample. Suitable techniques include, for example,
immunocytochemical staining with monoclonal antibodies, nucleic
acid hybridization (including in situ hybridization) and polymerase
chain reaction (PCR) studies. The technique can include utilizing a
"cocktail" of antibodies and/or probes.
[0085] Illustratively, in some embodiments wherein the rare cells
are cells of epithelial origin, e.g., prostate cancer cells, they
can be identified by immunocytochemically staining them with
monoclonal antibodies that specifically bind to, for example, PSA
(prostate specific antigen), PSMA (prostate specific membrane
antigen), PSAP (prostate specific acid phosphatase), cytokeratin
protein, or albumin. Although PSA is widely used for identification
of prostatic cells' activities, there are certain prostate cells
which secrete little or no PSA. Therefore, in some embodiments it
may be desirable to alternatively, or additionally, use antibodies
that specifically bind to PSMA.
[0086] Of course, rare cells can also be identified and/or
characterized using nucleic acid hybridization protocols. For
example, in some embodiments wherein the rare cell is a liver
cancer cell, suitable nucleic acid probes include oligomeric probes
that specifically bind to serum albumin mRNA and
.alpha.-fetoprotein mRNA, for example. Alternatively, in some
embodiments wherein the rare cell is a prostate cancer cell,
suitable probes include those specific for, for example, PSA, PSMA,
chromosome 7, chromosome 8, and/or chromosome 18. As noted above,
there are certain prostate cells which secrete little or no PSA.
Thus, probing for PSA may be less sensitive than probing for
PSMA.
[0087] Illustrative probes that are specific for mRNA encoding PSA,
for mRNA encoding PSMA, and for the centromeric regions of
chromosomes 7, 8, and/or 18, are described in more detail below.
These probes are particularly suitable for in situ
hybridization.
[0088] Representative probes that are specific for PSMA (prostate
specific membrane antigen) mRNA include:
[0089] SEQ. ID. No. 1: TGGCTGTGCG CTGGGGCGCT GGTGCTGGCG GGTGGCTTCT
TTCTCCTCGG CTTCCTCTTC GGGTGGTTTA TA,
[0090] SEQ. ID. No. 2: AGTGTCTATG AAACATATGA GTTGGTGGAA AAGTTTTATG
ATCCAATGTT, and
[0091] SEQ ID. No. 6: GTGTTTGAGC TAGCCAATTC CATAGTGCTC CCTTTTGATT
GTCGAGATTA.
[0092] Representative probes that are specific for PSA (prostate
specific antigen) mRNA include:
[0093] SEQ ID. No. 3: GGTCCTCACA GCTGCCCACT GCATCAGGAA CAAAAGCGTG
ATCTTGCTGG GTCGGCACAG,
[0094] SEQ ID. No. 4: CGCTGGACAG GGGGCAAAAG CACCTGCTCG GGTGATTCTG
GGGGCCCACT TGTCTGTAAT,
[0095] SEQ ID. No. 7: TCTTCCTCAC CCTGTCCGTG ACGTGGATTG GTGCTGCACC
CCTCATCCTG TCTCGGATTG, and
[0096] SEQ ID. No. 8: CAGGCTGGGG CAGCATTGAA CCAGAGGAGT TCTTGACCCC
AAAGAAACTT CAGTGTGTGG.
[0097] Representative probes that are specific for the repetitive
sequences in centromeric regions of chromosomes 7, 8 and 18
include:
[0098] SEQ ID. No. 5: GCTGTGGCAT TTTCAGGTGG AGATTTCAAG CGATTTGAGG
ACAATTGCAG (chromosome 7).
[0099] The probes for the centromeres can be used to determine the
number of chromosomes in the cells, e.g., to determine aneuploidy.
For example the probes for the centromere of chromosome 7 can be
used to count the number of chromosome 7's in the cell. The normal
cell should be diploid, and thus exhibit two stained probe "dots".
Deviation from the diploid state (i.e., 1, 3, 4 or a greater number
of chromosome 7's) would indicate aneuploidy or an abnormal number
of chromosomes which is a very strong indication of a
cancerous/neoplastic state.
[0100] A suitable probe for the chromosome 8 centromere can be
obtained commercially, for example, from Vysis, Inc. (Downers
Grove, Ill).
[0101] A suitable probe for the chromosome 18 centromere is SEQ ID.
No. 9: GTACTCACAC TAAGAGAATT GAACCACCGT. Meyne et al. in Methods in
Molecular Biology, 33: In Situ Hybridization Protocols, Choo, H. K.
(ed.), 63-74 (1994). This sequence can be converted to a longer
sequence. For example, it can be converted to SEQ. ID No. 10:
ATGTGTGTAC TCACACTAAG AGAATTGAAC CACCGTTTTG AA. Although a sequence
length of about 20 to about 60 nucleotides can be used, a preferred
length is 42.
[0102] Of course, rare cells can also be identified by polymerase
chain reaction (PCR) techniques. Any PCR technique and suitable
probe(s) known to those of ordinary skill in the art can be
employed.
[0103] The present invention further provides method of identifying
cancer cells in a patient's blood comprising enriching the cancer
cells from the patient's blood by any of the methods set forth
above, and subjecting the cells to in situ hybridization, including
Fluorescent In Situ Hybridization (FISH). Suitable probes include
those described above. Additionally, exemplary in situ
hybridization protocols band probes used therein can be found in,
for example, Meyne et al. in Methods in Molecular Biology, Vol. 33,
as referenced above.
[0104] Illustratively, the FISH probes can be synthesized with
deoxyribose nucleotidyl units, or 2'-O-methylribosyl nucleotidyl
units, or the nonionic analogs consisting of methyphosphonate
backbone or phosphorothiolate nucleotidyl backbone. Suitable probes
include oligodeoxyribonucleotide probes, and preferably those
labelled with a fluorescent residue. Any suitable fluorescent
residue can be employed. Thus, fluorescent dyes such as fluorescein
(green), cy3 (red), cy5 (far red), cy7 (infrared), and Texas red
can be the labels. Dual and triple in situ hybridization also can
be carried out by using a combination of mRNA and centromere probes
(differentially labeled) under the conditions described above.
After a high stringency wash, the nuclei of the cells can be
counterstained with a fluorescent DNA stain such as DAPI (diamidino
phenylindole) or PI (propidium iodide). The stained cells can be
analyzed for specific mRNA and aneuploidy using any suitable
fluorescence microscope. Suitable systems and protocols including
the use of a fluorescent microscope include those described in, for
example, Callahan, et al. Cytometry 13, 453-461 (1992), and Lesko
et al. Exp. Cell Res 219, 499-506 (1995).
[0105] One alternate procedure for assaying aneuploidy in cancer
cell nuclei is to first conduct specific immunocytochemical
staining (for example, by staining PSA, PSMA, PSAP, or albumin) and
crosslinking the antibodies and antigens, followed by in situ
hybridization with fluorescently labelled centromere probes.
[0106] One procedure for detecting epithelioid cancer cells
includes specific immunocytochemical staining (e.g., by
cytokeratonal protein antibodies that specifically bind to
cytokeratin protein expressed by the cells), then postfixation
and/or crosslinking the antibodies and antigens, followed by in
situ hybridization for the detection of specific mRNA and
chromosome aneuploidy.
[0107] In order to carry out FISH, the slides should be cleaned
prior to placing the cells on them, by immersing in a dilute
hydrochloric acid solution, e.g., O.1N HCl, at room temperature for
about 20 minutes for denaturation of any DNA and RNA residues. The
HCl solution should contain 0.1% Triton X100 surfactant. The slides
are then rinsed with PBS.
[0108] The rare cells, e.g., cancer cells, can be loaded on the
slides by any suitable procedure as previously discussed. The
slides are then dehydrated by immersing sequentially in 75%
ethanol, 85% ethanol, and 95% ethanol, for a period of about 2
minutes for each ethanol immersion.
[0109] An exemplary FISH cocktail includes 200 ng of each PSMA and
PSA probes as well as 250 ng of chromosome 7 centromere probe, 10
.mu.l of in situ hybridization buffer (25% formamide 4.times.SSC
for oligomere probes, and 50% formamide and 1.times.SSC for
commercial probes) are added to the slide. The cells are then
covered with a cover slip, and the edges of the slide are
surrounded by rubber cement and sealed. The slide should be kept in
the oven (e.g., at 80.degree. C. for about 5 minutes) for
denaturation and then incubated, e.g., at 42.degree. C. for about 3
hours. The cells are then washed, e.g., with 1.times.SSC,
65.degree. C. for about 10 minutes. The cells are subsequently
stained by a suitable dye, such as, for example, diamidino
phenylindole (DAPI), and then examined under a suitable
microscope.
[0110] Embodiments of the present invention further include
culturing rare cells. For example, rare cells such as cancer cells
can be enriched as described above, and subsequently placed in
contact with a suitable growth medium. Typically, in order to carry
out culturing the cells, the cells are loaded onto a sterile
membrane filter.
[0111] Any suitable membrane filter known to those of ordinary
skill in the art can be employed. Examples of suitable membranes
include microporous membranes. The membranes can have any suitable
pore size, preferably a pore size of from about 0.2 .mu.m to about
15 .mu.m, and more preferably 15 .mu.m. Examples of suitable
microporous membranes include nylon 6, nylon 46, nylon 66, and
nitrocellulose membranes. Suitable membranes are commercially
available.
[0112] The cells are not "fixed" prior to loading onto the
membrane. The membrane with the cells loaded onto it is then
typically placed in a collagen coated petri dish containing a
growth medium with the cells being in contact with the collagen
coated surface. Any suitable growth medium known to those of
ordinary skill in the art can be employed. An example of a growth
medium is PFMR-4A supplemented with 1% serum and additional factors
(Peehl, J. of Tissue Culture Methods, 9, 53-60 (1985)). Examples of
other suitable growth media include RPMI 1640, Coon's F12,
Dulbecco's Modified Eagle Medium, McCoy's Medium, and the like.
[0113] The cell growth can be monitored by a suitable method known
to those of ordinary skill in the art. For example, prostate cancer
cell growth can be monitored by analyzing for PSA secretion into
the culture medium with an Enzyme Linked Immunosorbent Assay
(ELISA), and liver cell growth can be monitored with an ELISA by
assaying the secretion of albumin or .alpha.-fetoprotein.
[0114] The cultured cells have a variety of additional uses. For
example, the cells can be used to provide a therapeutic product,
including, but not limited to, a vaccine.
[0115] The present invention further provides a method of
diagnosing cancer, particularly prostate cancer in men, the method
comprising enriching and identifying the cancer cells from the
blood of the patient as described above
[0116] The present invention further provides an improved method of
staging cancer in human beings, particularly a method of staging
prostatic cancer in men. For example, in one embodiment, the blood
of a suspected cancer patient is processed as described above to
enrich the prostate cancer cells (if present). An enhanced reverse
transcriptase (RT) polymerase chain reaction (PCR) assay utilizing
oligonucleotide primers is then carried out. Since the present
inventive method is highly efficient in enriching cells and
embodiments are capable of detecting 1 cancer cell in 6 million
cells, the method of the present invention is significantly more
sensitive than the methods reported in the literature, which are
said to be capable of detecting one PSA-producing cell in 100,000
lymphocytes (Katz et al., Urology, 43, 765-775 (1994)) and 1 in 1
million cells (Israeli et al., Cancer Research, 54, 6306-6310
(1994)). In addition, the present method identifies
PSA-synthesizing cancer cells, as well as non-PSA-synthesizing
cancer cells, such as PSMA-synthesizing cells.
[0117] The present invention further provides a method of
monitoring the progress, or regression, of cancer during or after
therapy, and finds particular use with respect to prostate cancer
in men. The method comprises taking repeated blood samples over
time and enriching, isolating, and subsequently identifying the
cancer cells (if present) from the blood of a patient suspected of
having cancer as described above. Embodiment of the present
invention, in view of their enhanced sensitivity, are particularly
useful in monitoring the efficacy of various cancer treatments by
isolating and detecting cancer cells in the patient's blood
stream.
[0118] The following examples further illustrate the present
invention but, of course, should not be construed as in any way
limiting its scope. In all of the following examples, the enriched
cells are identified using an automated Zeiss Axiovert 35
epifluorescent microscope equipped with a cooled charge coupled
device (CCD) camera and filter cubes which will allow additional
differential detection of fluorescein cy3, cy5, and cy7 fluorescent
signals. The camera has a computer controlled shutter. The computer
also controls the movement of the slide stage of the
microscope.
[0119] The microscope is put in automated mode and multiple
wavelength exposures are taken. The images are downloaded to the
computer via an A to D converter. The computer processes and
records the images in digital form.
EXAMPLE 1
[0120] This example illustrates the density measurements of
prostatic cancer cells and hepatoma cells, e.g., to improve the
efficiency in selecting suitable density gradient media for the
practice of the present invention. The density of the cancer cells
is measured in a density gradient column by determining the percent
recovery of the cancer cells at the interface of the culture medium
and the gradient medium.
[0121] A stock solution of PERCOLL.TM. (Sigma Chemical Co., St.
Louis, Mo.) is prepared by adding 9 parts of PERCOLL.TM. to 1 part
(V/V) of 1.5 M NaCl solution. The osmolality of the PERCOLL.TM.
solution is adjusted with physiological saline. Final adjustment to
the required osmolality can be made by adding distilled water or
salts. The density of the stock PERCOLL.TM. solution can be
calculated from the following formulas: 1 Vx = Vo ( Po - P1 ) ( P1
- P10 ) P1 = VoPo + VxP10 Vx + Vo
[0122] wherein
[0123] Vx=Volume of diluting medium (ml)
[0124] Vo=Volume of PERCOLL.TM. (ml)
[0125] Po=density of PERCOLL.TM. (1.130.+-.0.005 g/ml)
[0126] P10=density of 1.5M NaCl=1.058 g/ml of 2.5 M sucrose=1.316
g/ml
[0127] P1=density of stock solution produced (g/ml)
[0128] Thus, for stock PERCOLL.TM. in saline P1=1.123 g/ml, and for
stock PERCOLL.TM. in sucrose P1=1.149 g/ml.
[0129] Solutions of stock PERCOLL.TM. can be diluted to lower
densities by diluting with 0.15M saline(density=1.008 g/ml) for
cell isolation. The following formula can be used to calculate the
volumes required to obtain a solution of the desired density. 2 Vy
= Vi ( P1 - P ) ( P - Py )
[0130] wherein
[0131] Vy=Volume of diluting medium (ml)
[0132] Vi=Volume of stock PERCOLL.TM. (ml)
[0133] P1=density of stock solution (g/ml)
[0134] Py=density of diluting medium (g/ml)
[0135] P density of diluted solution produced (g/ml)
1TABLE 1 The preparation of several densities of PERCOLL .TM.
solution for the measurement of cancer cell densities. Stock
Solution 0.15 M NaCl Solution density CP1 = 1.123 (g/m) (ml) (g/ml)
70 28 1.090 60 40 1.077 50 47 1.067 40 56 1.056 30 69 1.043 20 80
1.031
[0136] The density of the cancer cells is measured as follows:
[0137] The cancer cell lines used were obtained from commercial
suppliers (e.g., the American Type Culture Collection). The LNCaP,
TSU, and Hepatoma G.sub.2 cell lines were cultured in RPMI 1640
with 10% FBS and 5% CO.sub.2 at 37.degree. C. Cells in 5 ml of
culture medium were layered on 5 ml of single PERCOLL.TM. solution
with a known density and centrifuged at 400.times.g for 20 minutes
at room temperature. The interface and the PERCOLL.TM. solution
above any visible pellet were collected. The number of cells in
this suspension were counted and used for the calculation of
recovery. The data are presented in Table 2.
2TABLE 2 Density Measurements of Cancer Cells Percoll density
Recovery (%) (g/ml) LNCaP TSU Hepatoma G.sub.2 1.031 10.0 10.0 10.0
1.043 20.5 15.0 17.0 1.056 25.0 20.5 24.0 1.067 76.0 84.5 85.0
1.077 76.5 98.5 98.0 1.090 76.0 98.5 98.0
[0138] As can be seen in Table 2, 76% to 85% of the cancer cells
are recovered using a gradient with a density of 1.067 g/ml. With
Hepatoma G.sub.2 and TSU cells, an additional 13% to 14% cell
recovery could be obtained using gradients with a density of 1.077
g/ml. No additional recovery of cells was found with LNCaP cells at
the higher density.
EXAMPLE 2
[0139] This example illustrates a method of separation of prostatic
cancer cells using a single density gradient column.
[0140] Twenty ml of fresh blood was taken in two tubes. The blood
was diluted 1:2 with phosphate buffered saline (PBS). Thirty ml of
the diluted blood containing 2.3.times.10.sup.5 LNCaP cells
(prostate cancer cells) were layered on 15 ml of a PERCOLL.TM.
gradient with a density of 1.068 g/ml (Gradient I in FIG. 1). The
gradient column was centrifuged at 400.times.g for twenty minutes
at room temperature.
[0141] The cells at the interface between the blood plasma and the
PERCOLL.TM. medium were carefully removed to a new tube. Forty ml
of PBS was added into the new tube and mixed. The PBS diluted cells
were centrifuged at 250.times.g for five minutes. The resulting
pellet was suspended in 50 .mu.l of 0.1% bovine serum albumin (BSA)
solution.
[0142] The cell suspension thus prepared was smeared on slides as
spots, each with 10 .mu.l of the suspension. The slides were
allowed to air-dry for two hours. The cells were fixed with 95%
ethanol for fifteen minutes, and then with modified Carnoy's
fixative for ten minutes. The slide was stored in 75% ethanol at
40.degree. C. until used.
[0143] The above experiment was repeated nine more times, each time
with a fresh blood sample. The average recovery of the prostate
cancer cells in the ten experiments was 76-86%.
EXAMPLE 3
[0144] This example illustrates the method of separation of
prostatic cancer cells using a higher single density gradient
column. (See FIGS. 1 and 2A).
[0145] The pellet from the b 1.068 g/ml density gradient (Gradient
I in FIG. 1) of Example 2 was resuspended in the plasma fraction
from Example 2, and layered on a higher density gradient column
containing 10 ml of FICOLL.TM. medium having a density of 1.083
g/ml (Gradient II in FIG. 2A).The density gradient column was
centrifuged at 400.times.g for twenty minutes at room
temperature.
[0146] The cells at the interface between the blood plasma and the
medium having a density of 1.083 g/ml were carefully removed with a
cell transfer pipette and placed in a new tube. Forty ml of PBS was
added to the interface cells and mixed. The PBS diluted cells were
then centrifuged at 250.times.g. The resulting pellet was suspended
in 0.5 ml of 0.1% by weight BSA solution. The white blood cells
were counted using a light microscope.
[0147] Thirty .mu.l of mouse anti-human CD45, CD19, CD3, CD14
monoclonal antibody (Sigma Chemical Co.), respectively, and 10
.mu.l of glycophorin A monoclonal antibody (Dako, Inc.) were added
to the cell suspension and the tube was incubated on ice for thirty
minutes. The cell suspension was spin down and the supernatant was
aspirated. The cell pellet was resuspended with 8.times.10.sup.7
magnetic beads coated with anti-mouse IgG antibody (Dynal, Inc.) in
2 ml of PBS-BSA. The cells and beads were incubated at 4.degree. C.
for 30 minutes while rotating the tube at 10 rpm/minute. The
cell-monoclonal antibody-mouse IgG-magnetic bead complexes were
removed using a magnetic particle concentrator. The remaining cells
were collected on a slide. The slide was prepared and the cells
were fixed as described above in Example 2.
EXAMPLE 4
[0148] This example illustrates the efficiency of the procedure
described in Example 2 for isolating prostate cancer cells from
blood.
[0149] Blood samples were subjected to the procedure set forth in
Example 1, except that the centrifugation was for twenty minutes
instead of thirty minutes.
[0150] The total number of cells, the number of cells at the
interface, the number of cells at the bottom of the gradient were
measured, and the number of cells lost was determined. The data are
set forth in Table 3.
3TABLE 3 The Efficacy of Isolation of Prostatic Cancer Cells Using
a Single Density Gradient Prostatic Cancer Cell Lines LNCaP P100
TSU wt Total Cell Counts 2.3 .times. 10.sup.5 100 .times. 10.sup.7
Counts of Cells at Interface (% 1.75 .times. 10.sup.5 8.45 .times.
10.sup.6 Recovery) (76%) (84.5%) Counts of Cell at Bottom Few cells
1.50 .times. 10.sup.5 (higher density cells %) (.about.1.0%) (15%)
Counts of Cells Lost* 5.5 .times. 10.sup.4 5.00 .times. 10.sup.4 (%
loss) (23%) (0.5%) *Includes cells that were stuck on the tube wall
and that were broken during the separation and wash.
EXAMPLE 5
[0151] This example illustrates the efficiency of the procedure
illustrated in Example 3 for isolating prostate cancer cells from
blood.
[0152] Blood samples were subjected to the procedure set forth in
Example 2, except that the centrifugation was for twenty
minutes.
[0153] The total number of cells, the number of cells at the
interface and the number of cells at the bottom of the gradient
were measured, and the number of cells lost was determined. The
data are set forth in Table 4.
4TABLE 4 The Efficacy of Isolation of Prostatic Cancer Cells Using
a Secondary Density (1.083 g/ml) Gradient Prostatic Cancer Cell
Lines LNCaP P100 TSU wt Total Cell Counts* 2.3 .times. 10.sup.3
1.50 .times. 10.sup.4 Counts of Cells at Interface (% 1.87 .times.
10.sup.3 1.45 .times. 10.sup.4 Recovery) (81.3%) (96.6%) Counts of
Cell at Bottom No detection No detection (higher density cells %)
(.about.0%) (.about.0%) Counts of Cells Lost** 4.5 .times. 10.sup.2
5.00 .times. 10.sup.2 (% loss) (.about.18.7%) (.about.3.4%) *The
cell suspension was the collection from the tube bottom of single
gradient (1.068 g/ml) separation. **Includes cells that were stuck
on the tube wall and that were broken during the separation and
wash.
EXAMPLE 6
[0154] This example illustrates a method of culturing cancer cells.
LNCaP cells were counted and added into normal adult blood. The
methods illustrated in Examples 2 and 3 were used for isolation of
LNCaP cells in the artificial blood. The LNCaP cells isolated from
the artificial blood were cultured in the growth medium RPMI 1640
supplemented with 10% serum and additional factor. The culture
medium was changed every three days. The cultured cells showed
positive immunostaining PSA and PSAP, and the number of cells in
each flask increased with time in culture.
EXAMPLE 7
[0155] This example illustrates the identification of LNCaP cells
or prostatic cancer cells from patients blood by fluorescent in
situ hybridization (FISH) with PSA-mRNA, PSMA-mRNA and chromosome
centromere probes.
[0156] Oligonucleotide probes specific for PSA-mRNA, PSMA-mRNA and
the centromeres of chromosomes 7 and 18 were synthesized and
conjugated with fluorescent dyes such as fluorescein, cy3 and cy5.
The probe for chromosome centromere 8 was from a commercial source
(Vysis).
[0157] The cancer cells isolated, fixed and stored by the method
described in Examples 1 and 2 were pretreated in the solution of
0.1 M HCI-0.1% Triton X-100 for thirty minutes at room temperature,
and dehydrated in series grades of ethanol at 75%, 85% and 95% for
two minutes in each grade. The samples were air dried.
[0158] The FISH "Cocktail" comprises FISH buffer which mainly
includes 25% Formamide and 4.times.SS (for oligomer probes) or 50%
Formamide and 1.times.SSC (for commercial probes), 20 .mu.g/ml
PSA-mRNA probe and PSMA-mRNA probe, and 25 .mu.g/ml chromosome 7, 8
and 18 centromere probes. Ten .mu.l of FISH "Cocktail" were added
on to each slide, under a coverslip. The samples were denatured at
80.degree. C. for ten minutes and incubated at 42.degree. C. for
two hours. The slides were washed in 1.times.SSC at 70.degree. for
ten minutes. Ten .mu.l of antifade mounting medium containing 0.2
.mu.Ag/ml diamidino phenylindole (DAPI) were used for
counterstaining. After counterstaining, the samples were examined
under a fluorescent microscope. The chromosome 7 centromeres (which
are multiploid) exhibited green stain, and the nucleus was stained
inblue with DAPI.
[0159] The chromosome 8 centromeres (which are tetraploid)
exhibited yellow stain, and the nucleus was stained in blue with
DAPI.
[0160] The chromosomal centromeres were counted and the data are
set forth in Table 5.
5TABLE 5 Aneuploidy of Chromosome 7 and 8 in the Nucleus of LNCaP
Cell from Culture and from Cancer Cells Isolated from Cancer
Patient's Blood Chromosomal Centromere 7 Chromosomal Centromere 8
Patient Patient Cell No. LNCaP cancer cell LNCaP cancer cell 1 14 8
14 8 2 4 8 4 8 3 7 4 7 4 4 4 4 4 4 5 4 4 3 3 6 4 8 4 8 7 4 8 4 8 8
5 4 3 3 9 8 3 7 4 10 2 4 3 4
EXAMPLE 8
[0161] This example illustrates the identification of LNCaP cells
or prostatic cancer cells from patient's blood by
immunocytochemistry stain, as well as by chromosomal centromere 7
and 8 detection. The cancer cells enriched, isolated, fixed and
stored as described in Examples 2 and 3 were stained by
immunocytochemistry with primary antibodies against PSA and PAP,
and secondary antibodies conjugated by fluorescent dyes. After
immunocytochemistry staining, the samples were treated by 1%
paraformaldehyde for 10 minutes at room temperature. The
paraformaldehyde treatment provides for post-fixation of the cancer
cells before fluorescent in situ hybridization (FISH) as well as
crosslinking the antibodies and antigens and making the complex
more stable during the FISH procedures. The slides pre-treated by
the solution of 0.1 M HCI 0.1% Triton X-100 for twenty minutes at
room temperature and then dehydrated by 75%, 85% and 95% ethanol
for two minutes in each grade. The slides were air dried.
[0162] The FISH cocktail fluid was prepared for chromosomal
centromere 7 and 8 stain. The "Cocktail" comprised FISH buffer
which contained 50% formamide and 2.times.SSC, and chromosomal
centromere 7 and 8 DNA probes conjugated by fluorescent dyes. The
cells, covered by 10 .mu.l FISH "Cocktail" and a coverslip, were
denatured at 80.degree. C. for five minutes, and incubated at
42.degree. C. for two to three hours. The slides were washed in
1.times.SSC for ten minutes at 60.degree. C. Ten .mu.l of an
antifade mounting medium containing of 0.2 .mu.g/ml DAPI were used
for counterstaining. The samples were examined under a fluorescent
microscope.
[0163] LNCaP cells immunofluorescently stained for Prostate
Specific Antigen (PSA) exhibited green stain that represented the
immunoreaction of PSA antibody in the cytoplasm.
[0164] LNCaP cells showed the immunofluorescent stain for the cell
nucleus, as exhibited by the blue stain (DAPI).
[0165] LNCaP cells stained by immunocytochemistry with Prostatic
Specific Acidic Phosphatase (PSAP) antibody showed the stain in the
cytoplasm, while the blue stain showed the nucleus stained with
DAPI.
[0166] Prostatic cells from the blood of prostatic cancer patients
were stained in the cytoplasm (green) by immunochemistry with PSA
antibodies, and then stained by FISH with chromosome centromere 7
(blue) and chromosome centromere 8 (red) probes in the nucleus.
EXAMPLE 9
[0167] This example illustrates the efficiency of enriching and
isolating cancer cells by the method described in Examples 2 and
3.
[0168] Blood samples were reconstituted with LNCaP cells at varying
ratios of white blood cells (WBC) to LNCaPcells. The cell recovery
data set forth in Tables 6 and 7 confirm that the cancer cells are
recoverable at high recovery percentages.
6TABLE 6 Recovery of LNCaP Cells from Reconstituted Blood Recovery
WBC:LNCaP WBC LNCaP of LNCaP, Counts % 500:1 2.76 .times. 10.sup.7
6.5 .times. 10.sup.4 5.5 .times. 10.sup.4 84.6 1000:1 2.76 .times.
10.sup.7 3.2 .times. 10.sup.4 2.7 .times. 10.sup.4 84.6 10000:1
2.76 .times. 10.sup.7 3.2 .times. 10.sup.3 2.8 .times. 10.sup.3
86.2 0:20000 0 2.3 .times. 10.sup.5 1.8 .times. 10.sup.5 78.3
(control)
[0169]
7TABLE 7 Recovery of prostatic cancer cells (LNCaP) in the
reconstituted blood Quantity Cell Sample No. of Blood WBC Counts
LNCaP Counts Recovery 51-9B3329 9.0 ml 1.57 .times. 10.sup.8
.about.100 85 85% 51-5B3320 9.0 ml 1.47 .times. 10.sup.8 .about.100
80 80% 51-0B3340 9.0 ml 1.78 .times. 10.sup.8 .about.100 97 97%
51-0B3337 9.0 ml 1.70 .times. 10.sup.8 .about.100 95 95% 51-0B3314
9.0 ml 6.64 .times. 10.sup.7 .about.100 82 82% 51-7B3333 9.0 ml
1.26 .times. 10.sup.8 .about.100 94 94% 51-0B3323 9.0 ml 1.09
.times. 10.sup.8 .about.100 90 90% 51-6B3339 9.0 ml 7.84 .times.
10.sup.7 .about.100 96 96% 51-2B3330 9.0 ml 2.32 .times. 10.sup.8
.about.100 75 75% 51-8B3310 9.0 ml 1.59 .times. 10.sup.8 .about.100
90 90% N = 10 9.0 ml 1.42 .times. 10.sup.8 .about.100 88.4
88.4%
[0170] The LNCaP cells isolated from the reconstituted blood had
less than 1% of the original WBC concentration. The components of
contamination were in the following order:
monocytes>lymphocytes>eosinophils. The LNCaP cells isolated
from the reconstituted blood have been found to grow in RPMI 1640
culture medium. The WBC contamination can be lowered by changing
the culture medium after 3 days.
EXAMPLE 10
[0171] This example illustrates the identification of LNCaP cells
or prostatic cancer cells from patients' blood by the combination
of immunocytochemistry stain with cytokeratin monoclonal antibody,
FISH with chromosomal centromere 7 and 18 probes, and PSMA mRNA
probe.
[0172] The sample is fixed with 100% acetone at room temperature
for two to three minutes. The slides are air-dried, and stored at
room temperature in a slide box. The slides are incubated in 0.1M
Tris washing buffer at room temperature for ten minutes, and the
liquid is removed from the surface of the slides. Twenty five .mu.l
of FITC conjugated Anti-Cytokeratin (CAM5.2) monoclonal antibody
(Becton-Dickinson, San Jose, Calif.; Cat. 347653) (1:2 dilution) is
added onto the slides. The slides are incubated with a coverslip in
a humid box at 37.degree. C. for one hour. The slides are uncovered
and washed in the 0.1M Tris washing buffer at room temperature for
ten minutes. The slides are then air-dried in a dark area.
[0173] The 1% paraformaldehyde with 0.1 M MgC12 is prepared and
pre-cooled on ice. 1.0 ml of the 1% paraformaldehyde is dropped on
the sample area. The sample is fixed at room temperature for 2-5
minutes. The fixative is removed from the surface of the slide, and
the slide is air-dried at room temperature.
[0174] The FISH mixture (per sample) is prepared according to the
following:
8 FISH buffer (Oncor) 9.0 .mu.l Cy3-Chromosomal centromere probe 18
28 ng 0.5 .mu.l Cy3 conjugated PSMA-mRNA probe 25 ng 0.5 .mu.l
Cy5-PSA-mRNA probe 25 ng 0.5 .mu.l Cy5-Chromosomal centromere probe
7 28 ng 0.5 .mu.l Cy5-Chromosomal centromere probe 8 28 ng 0.5
.mu.l
[0175] The FISH mixture is added onto the sample area. The
coverslip is added and sealed with rubber cement. The sample is
denatured at 85.degree. C. for seven minutes, and the slide is
incubated at 42.degree. C. for two hours. The slide is washed in
1.times.SSC at 60.degree. C. for five minutes, and the sample is
air-dried at room temperature. The sample is counterstained with
DAPI, and the slide is examined under a fluorescent microscope.
[0176] The results are summarized in Table 8.
9TABLE 8 Detection of five prostatic cancer cell lines with
cytokeratin immunocytochemistry staining and fluorescent in situ
hybridization (FISH) of prostatic specific membrane antigen (PSMA)
mRNA probe and chromosomal centromere 8 and 18 probes as well as
DAPI nucleus staining (Percentage of positive staining). Cancer
cell Immunocyto- PSMA- line chemistry mRNA Chromosome 8 & 18
DAPI LNCaP 100% 100% aneuploid (95-100%) 100% TSU-PRI 100% 100%
aneuploid (95-100%) 100% DUMS 100% 100% aneuploid (95-100%) 100%
PC-3 100% 100% aneuploid (95-100%) 100% PPC-1 (--) (--) aneuploid
(95-100%) 100%
[0177] The prostatic cancer cell is stained with Cy3 conjugated
Prostatic Specific Membrane Antigen (PSMA) mRNA probe in the
cytoplasm. The cell shows four greenish chromosomal centromere 7
signals in the nucleus stained by human 7 chromosomal centromere
probe conjugated by fluorescein. The nucleus is stained with blue
DAPI.
[0178] Another prostatic cancer cell has greenish cytokeratin stain
in the cytoplasm and chromosome 8 autopolyploidy signals (four red
spots) in the nucleus stained by DAPI. A white blood cell exhibits
a blue nucleus with two chromosomal 8 signals (the normal
chromosomal number).
EXAMPLE 11
[0179] This example illustrates the successful isolation and
identification of prostate cancer cells from the blood of advanced
prostate cancer patients by using the procedure in Example 2.
[0180] The patient number, the volume of blood samples collected,
and the number of prostate cancer cells isolated, are set forth in
Table 9.
10TABLE 9 Collection of Prostatic Cancer Cells from the Blood of
advanced Cancer Patients Patient No. Prostatic Cancer Cells (N =
13) Blood Quantity (ml) (counts) PC253 10 200 PC254 20 140 PC255 27
260 PC256 9 6 PC257 27 90 PC258 15 40 PC259 27 No detection PC260
22 Fail to spin PC261* 15 No detection PC262* 15 No detection PC263
7.5 No detection PC264 16 No detection PC265 9 20 *Control sample
from normal adult blood
EXAMPLE 12
[0181] This example illustrates the successful isolation and
identification (see the procedure in example 10) of prostate cancer
cells from the blood of advanced prostate cancer patients.
[0182] In experiment I, the parallel study, the same sample was
aliquoted into two equal parts and processed by same procedure. The
patient number, the volume of blood sample collected, and the
number of prostate cancer cells isolated, are set forth in table
10. The conclusion of the parallel study is that the isolation
procedure is reproducible.
[0183] In experiment II, the storage study, the same sample was
aliquoted into two equal parts and processed by same procedure. The
samples in the Group A were processed within 24 hours and the
samples in the Group B were processed after 72 hours storage at
4.degree. C. The patient number, the volume of blood sample
collected, and the number of prostate cancer cells isolated, are
set forth in table 11. The conclusion from the storage study is
that cancer cells were generally not preserved after 72 hours
storage. In the one case wherein the cells were preserved after 72
hours storage, the cancer cells are smaller in size (approximately
the size of monocytes), but with very typical and very intensive
cytokeratonal system staining in the cytoplasm.
11TABLE 10 Detection of Prostatic Cancer cells From Blood of
Advanced Cancer Patients Experiment I: PSA Quant. Bld. Series No.
Age (U/L) (ml) Total No. #1 58 19.9 20 3 4 7 #2 58 81.1 18 2 3 5 #3
72 76.0 18 5 4 9 #4 66 44.9 15 2 2 4 #5 69 <0.1 20 1 2 3 #6 ?
18.6 18 2 2 4 #7 63 388.2 20 2 3 5 #8 78 212.6 20 6 4 10 #9 74
379.1 20 1 5 16 1 cluster 2 cluster #10 67 61.7 20 2 1 3 #11 71
182.6 20 1 1 2 #12 42 4.4 20 0 0 0 #13 53 85.7 20 8 7 15 #14 59
37.7 20 0 0 0 #15 59 6.1 20 0 0 0 #16 58 337.3 15 5 5 10 #17 67
14.69 20 0 1 1 #18 81 17.5 20 11 12 23 #19 72 9.7 20 0 0 0 #20 81
61 20 1 2 3 #21 54 43 20 3 4 7 #22 61 <0.1 18 0 0 0 #23 69 1.15
20 0 0 0 #24 61 44.3 18 0 0 0 #25 61 25.1 16 1 1 2 *The same sample
was aliquoted into two equal parts and processed by same
procedure.
[0184]
12TABLE 11 Detection of Prostatic Cancer Cells From Blood of
Advanced Cancer Patients Experiment II: PSA Quant. Bld. Cancer
cells Series No. Age (U/L) (ml) Group A Group B Total No. #18 81
100 16 12 12 24** #26 58 44 20 0 0 0 #27 72 7 15 0 0 0 #1 58 52.6
20 2 0 2 #2 68.3 72 20 3 0 3 #7 63 381.5 20 2 0 2 #13 53 26.2 20 4
0 4 #16 58 23.2 20 0 0 0 #25 61 24.1 20 2 0 2 #27 58 14.6 20 1 0 1
**The cancer cells are smaller in size (approximately the size of
monocytes), but with very typical and very intensive staining
cytokeratonal system in the cytoplasm.
EXAMPLE 13
[0185] This example illustrates a method of separation of prostatic
cancer cells using a single density gradient column.
[0186] Blood samples were subjected to the procedure as set forth
in Example 2, except that the centrifugation of the radient column
with a density of 1.068 g/ml was carried out at 400.times.g for 30
minutes at room temperature, rather than for 20 minutes. The slides
were prepared as described in Example 2.
[0187] The experiment was repeated nine more times, each time with
a fresh blood sample. The average recovery of the prostate cancer
cells in the ten experiments is 70-80%.
EXAMPLE 14
[0188] This example illustrates a method of separation of prostatic
cancer cells using a double density gradient column.
[0189] The pellet from the density gradient of Example 13 is
resuspended in the plasma fraction from Example 13, and layered on
a double density gradient column containing 10 ml of FICOLL.TM.
medium having a density of 1.083 g/ml and 5 ml of FICOLL.TM. medium
having a density of 1.077 g/ml. The suspension is layered onto the
column so that it is in contact with the medium of lower density,
which in turn is in contact with the higher density medium. Thus,
using the left side of FIG. 2B for reference, Gradient III has a
density of 1.077 g/ml, and Gradient II has a density of 1.083 g/ml.
The density gradient column is centrifuged at 400.times.g for 30
minutes at room temperature.
[0190] Using the right side of FIG. 2B for reference, the cells at
the interface (Interface II between the blood plasma and the
Gradient II and III) were carefully removed with a cell transfer
pipette and placed in a new tube. Forty ml of PBS was also added to
the new tube and mixed. The PBS diluted cells were then centrifuged
at 250.times.g. The resulting pellet was suspended in 2 ml of 0.1
wt. % BSA solution. The white blood cells were counted using a
light microscope.
[0191] CD45-Dynalbeads were added to the above cell suspension
(about 3 beads per 1 WBC were used). The cell suspension was
incubated with the Dynalbeads for 30 minutes at 4.degree. C. with
gentle shaking. The tube containing the cells and the beads was
placed in a magnetic particle concentrator (Dynal Corp.) and the
cells in suspension were pipetted into a new tube leaving the beads
and attached blood cells held in place in the tube by the magnetic
particle concentrator.
[0192] The cell suspension was centrifuged at 250.times.g. The
pellet obtained was resuspended in 30 .mu.l of 1 wt. % BSA
solution. The cell suspension thus prepared was smeared on slides
as spots, each with 10 .mu.l of the suspension. The slides were
allowed to air dry for two hours. The cells were fixed with 95%
ethanol for 10-15 minutes, and then with modified Carnoy's
fixative.
EXAMPLE 15
[0193] This example illustrates the efficiency of the procedure
illustrated in Example 13 for isolating prostate cancer cells from
blood.
[0194] Blood samples are subjected to the procedure set forth in
Example 14, except that centrifugation of the double density
gradient column is performed for 20 minutes instead of 30
minutes.
[0195] The total number of cells, the number of cells at the
interface, the number of cells at the bottom, and the number of
cells lost are measured. The data are set forth in Table 12.
13TABLE 12 Isolation of prostatic cancer cells using a double
density gradient. Prostatic Cancer Cell Lines LNCaP P100 TSU wt %
cells at interface* .about.1% 14.5% % cells at the bottom* 0 .sup.
.about.1% % cells lost 0 0 *This column contains cancer cells that
"leaked" from the 1.068 g/ml gradient column.
EXAMPLE 16
[0196] This example illustrates the enrichment of prostatic cancer
cells using a double density gradient column.
[0197] Twenty ml of fresh blood is taken into two tubes. The blood
is diluted 1:2 PBS. Thirty ml of the diluted blood is layered on
top of a double density gradient column which has an upper layer of
10 ml of 1.068 g/ml Histopaque.TM. (Sigma Chemical Co.) and a lower
layer of 10 ml of 1.083 g/ml Histopaque.TM. as illustrated in the
left side of FIG. 3. The column is centrifuged at 400.times.g for
30 minutes at room temperature to form 6 layers as shown in the
right side of FIG. 3.
[0198] After centrifugation, Interface I and Gradient I are
carefully removed and placed in another tube, and Interface II and
Gradient II are also carefully removed and placed in yet another
tube.
[0199] Forty ml of PBS is added to the tube containing the
Interface I and Gradient I, and mixed. The PBS diluted cells are
centrifuged at 250.times.g for 5 minutes. The resultant pellet is
suspended in 50 .mu.l of 1% BSA solution to form a cell suspension
that is smeared on a slide as a spot. The slide is air dried for at
least two hours. The cells are fixed with 95% ethanol for 15
minutes, and then with modified Carnoy's fixative for 10 minutes.
The slide is stored in 75% ethanol at 4.degree. C. until used.
[0200] As noted above, Interface II and Gradient II are also
carefully removed to a new tube. Forty ml of PBS is added to the
new tube and mixed. The PBS diluted cells are centrifuged at
250.times.g for 5 minutes. The resultant pellet is suspended in 2
ml of 1% BSA solution. CD45-Dynalbeads are added to the cell
suspension (about 3-10 beads per white blood cell (leukocyte)), and
the suspension was incubated with the Dynalbeads for 30 minutes at
4.degree. C. with gentle rotation.
[0201] The tube containing the cells and beads is placed on a Dynal
magnetic concentrator. The concentrator is operated to attract the
beads and attached blood cells to the wall of the tube.
[0202] The suspension containing the cancer cells is pipetted into
another tube. Slides are prepared as described above.
EXAMPLE 17
[0203] This example illustrates the efficiency of the procedure
described in example 16 for isolating prostatic cancer cells from
blood. The procedure described in Example 16 is repeated nine more
times, each time with a fresh blood sample containing LNCaP cells.
The average recovery of the prostatic cancer cells from Interface I
and Gradient I is about 70-80%. The average recovery of the
prostatic cancer cells from Interface II and Gradient II is about
5-10%.
[0204] All of the nucleic acid sequences listed in this application
are set forth in the 5'-3' configuration.
[0205] All of the references cited herein including patents and
publications are hereby incorporated in their entireties by
reference.
[0206] While the invention has been described and disclosed herein
in connection with certain preferred embodiments and procedures, it
is not intended to limit the invention to those specific
embodiments. Rather it is intended to cover all such alternative
embodiments and modifications as fall within the spirit and scope
of the invention.
Sequence CWU 1
1
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