Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Title: A pre-clinical method for monitoring serial changes in circulating
breast
cancer cells in mice.
Inventor: Gerald V. Doyle
Cross-Reference to Related Applications
This is a non-provisional application which claims priority to U.S.
Provisional Applications 61/001,418, filed November 01, 2007. The
aforementioned application is incorporated in full by reference herein.
Background
Field of the Invention
The invention relates generally to cancer monitoring and assessing
disease progression in metastatic cancer patients, based on the presence of
morphologically intact circulating cancer cells (CTC) in blood. More
specifically, methods, reagents and apparatus are described for assessing
circulating cancer cells in animal models.
Background Art
Non-hematogenous epithelial tumor cells were first identified in the blood
of a breast cancer patient over 150 years ago. Since then, CTC's have been
shown to be a critical link between primary cancer, a disease stage at which
cure is possible, and metastatic disease, which continues to be the leading
cause of death for most malignancies. Clinical studies have shown that CTC's
are a powerful prognostic and predictive biomarker in metastatic breast
cancer,
and similar findings have been reported in prostate cancer and colorectal
cancer. From this data, CTC's have been shown to be representative of the
underlying biology driving metastatic cancer and suggest that further cellular
and molecular analyses of these cells can reveal new insights into molecular
regulation of metastasis and response to therapy.
Research on the role of CTC in metastasis and expansion of their use as
a biomarker in pharmacokinetic and pharmacodynamic studies has been
limited to the clinical phase of drug development. It is generally accepted
that
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most cancer patients are not killed by their primary tumor, but they succumb
instead to metastases: multiple widespread tumor colonies established by
malignant cells that detach themselves from the original tumor and travel
through the body, often to distant sites. The most successful therapeutic
strategy in cancer is early detection and surgical removal of the tumor while
still
organ confined. Early detection of cancer has proven feasible for some
cancers, particularly where appropriate diagnostic tests exist such as PAP
smears in cervical cancer, mammography in breast cancer, and serum prostate
specific antigen (PSA) in prostate cancer. However, many cancers detected at
early stages have established micrometastases prior to surgical resection.
Thus, early and accurate determination of the cancer's malignant potential is
important for selection of proper therapy.
If a primary tumor is detected early enough, it can often be eliminated by
surgery, radiation, or chemotherapy or some combination of those treatments.
Unfortunately, the metastatic colonies are difficult to detect and eliminate
and it
is often impossible to treat all of them successfully. Therefore, metastasis
can
be considered the conclusive event in the natural progression of cancer.
Moreover, the ability to metastasize is a property that uniquely characterizes
a
malignant tumor.
Based on the complexity of cancer and cancer metastasis and the
frustration in treating cancer patients over the years, many attempts have
been
made to develop diagnostic tests to guide treatment and monitor the effects of
such treatment on metastasis or relapse.
One of the first attempts to develop a useful test for diagnostic oncology
was the formulation of an immunoassay for carcinoembryonic antigen (CEA).
This antigen appears on fetal cells and reappears on tumor cells in certain
cancers. Extensive efforts have been made to evaluate the usefulness of
testing for CEA as well as many other "tumor" antigens, such as prostate
specific antigen (PSA), CA 15.3, CA 125, prostate-specific membrane antigen
(PSMA), CA 27.29, p27 found in either tissue samples or blood as soluble
cellular debris.
Additional tests used to predict tumor progression in cancer patients
have focused upon correlating enzymatic indices like telomerase activity in
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biopsy-harvested tumor samples with an indication of an unfavorable or
favorable prognosis (US 5,693,474; US 5,639,613). Assessing enzyme activity
in this type of analysis can involve time-consuming laboratory procedures such
as gel electrophoresis and Western blot analysis. Also, there are variations
in
the signal to noise and sensitivity in sample analysis based on the origin of
the
tumor. Despite these shortcomings, specific soluble tumor markers in blood
can provide a rapid and efficient approach for developing a therapeutic
strategy
early in treatment. For example, detection of serum HER-2/neu and serum CA
15-3 in patients with metastatic breast cancer have been shown to be
prognostic factors for metastatic breast cancer (Ali S.M., Leitzel K.,
Chinchilli
V.M., Engle L., Demers L., Harvey H.A., Carney W., Allard J.W. and Lipton A.,
Relationship of Serum HER-2/neu and Serum CA 15-3 in Patients with
Metastatic Breast Cancer, Clinical Chemistry, 48(8):1314-1320 (2002)).
Increased HER-2/neu results in decreased response to hormone therapy, and
is a significant prognostic factor in predicting responses to hormone receptor-
positive metastatic breast cancer. Thus in malignancies where the HER-2/neu
oncogene product is associated, methods have been described to monitor
therapy or assess risks based on elevated levels (US 5,876,712). However in
both cases, the base levels during remission, or even in healthy normals, are
relatively high and may overlap with concentrations found in patients, thus
requiring multiple testing and monitoring to establish patient-dependent
baseline and cut-off levels.
In prostate cancer, PSA levels in serum have proven to be useful in
early detection. When used with a follow-up physical examination and biopsy,
the PSA test has improved detection of prostate cancer at an early stage when
it is best treated.
However, PSA or the related PSMA testing leaves much to be desired.
For example, elevated levels of PSA weakly correlate with disease stage and
appear not to be a reliable indicator of the metastatic potential of the
tumor.
This may be due in part to the fact that PSA is a component of normal prostate
tissue and benign prostatic hyperplasia (BHP) tissue. Moreover, approximately
30% of patients with alleged localized prostate cancer and corresponding low
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serum PSA concentrations, may have metastatic disease (Moreno et al.,
Cancer Research, 52:6110 (1992)).
Genetic markers:
One approach for determining the presence of malignant prostate tumor
cells has been to test for the expression of messenger RNA from PSA in blood.
This is being done through the laborious procedure of isolating all of the
mRNA
from the blood sample and performing reverse transcriptase PCR. No
significant correlation has been described between the presence of shed tumor
cells in blood and the ability to identify which patients would benefit from
more
vigorous treatment (Gomella LG., J of Urology, 158:326-337 (1997)).
Additionally, false positives are often observed using this technique. There
is
an added drawback, which is that there is a finite and practical limit to the
sensitivity of this technique based on the sample size. Typically, the test is
performed on 105 to 106 cells separated from interfering red blood cells,
corresponding to a practical lower limit of sensitivity of one tumor cell/0.1
ml of
blood (about 10 tumor cells in one ml of blood) before a signal is detected.
Higher sensitivity has been suggested by detecting hK2 RNA in tumor cells
isolated from blood (US 6,479,263; US 6,235,486).
Qualitative RT-PCR based studies with blood-based nucleotide markers
has been used to indicate that the potential for disease-free survival for
patients
with positive CEA mRNA in pre-operative blood is worse than that of patients
negative for CEA mRNA (Hardingham J.E., Hewett P.J., Sage R.E., Finch J.L.,
Nuttal J.D., Kotasel D. and Dovrovic A., Molecular detection of blood-borne
epithelial cells in colorectal cancer patients and in patients with benign
bowel
disease, Int. J. Cancer 89:8-13 (2000): Taniguchi T., Makino M., Suzuki K.,
Kaibara N., Prognostic significance of reverse transcriptase-polymerase chain
reaction measurement of carcinoembryonic antigen mRNA levels in tumor
drainage blood and peripheral blood of patients with colorectal carcinoma,
Cancer 89:970-976 (2000)). The prognostic value of this endpoint is
dependent upon CEA mRNA levels, which are also induced in healthy
individuals by G-CSF, cytokines, steroids, or environmental factors. Hence,
the
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CEA mRNA marker lacks specificity and is clearly not unique to circulating
colorectal cancer cells.
The aforementioned studies, while seemingly prognostic under the
experimental conditions, do not provide for consistent data with a long follow-
up
period or at a satisfactory specificity. Accordingly, these efforts have
proven to
be somewhat futile as the appearance of mRNA for antigens in blood have not
been generally predictive for most cancers and are often detected when there
is little hope for the patient.
In spite of this, real-time reverse transcriptase-polymerase chain
reaction (RT-PCR) has been the only procedure reported to correlate the
quantitative detection of circulating tumor cells with patient prognosis. Real-
time RT-PCR has been used for quantifying CEA mRNA in peripheral blood of
colorectal cancer patients (Ito S., Nakanishi H., Hirai T., Kato T., Kodera
Y.,
Feng Z., Kasai Y., Ito K., Akiyama S., Nakao A., and Tatematsu M.,
Quantitative detection of CEA expressing free tumor cells in the peripheral
blood of colorectal cancer patients during surgery with real-time RT-PCR on a
Light Cycler, Cancer Letters, 183:195-203 (2002)). These results suggest that
tumor cells were shed into the bloodstream (possibly during surgical
procedures or from micro metastases already existing at the time of the
operation), and resulted in poor patient outcomes in patients with colorectal
cancer. The sensitivity of this assay provided a reproducibly detectable range
similar in sensitivity to conventional RT-PCR. As mentioned, these detection
ranges are based on unreliable conversions of amplified product to the number
of tumor cells. The extrapolated cell count may include damaged CTC
incapable of metastatic proliferation. Further, PCR-based assays are limited
by
possible sample contamination, along with an inability to quantify tumor
cells.
Most importantly, methods based on PCR, flowcytometry, cytoplasmic
enzymes and circulating tumor antigens cannot provide essential morphological
information confirming the structural integrity underlying metastatic
potential of
the presumed CTC and thus constitute functionally less reliable surrogate
assays than the highly sensitive imaging methods embodied, in part, in this
invention.
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Detection of intact tumor cells in blood provides a direct link to recurrent
metastatic disease in cancer patients who have undergone resection of their
primary tumor. Unfortunately, the same spreading of malignant cells continues
to be missed by conventional tumor staging procedures. Recent studies have
shown that the presence of a single carcinoma cell in the bone marrow of
cancer patients is an independent prognostic factor for metastatic relapse
(Diel
IJ, Kaufman M, Goerner R, Costa SD, Kaul S, Bastert G. Detection of tumor
cells in bone marrow of patients with primary breast cancer: a prognostic
factor
for distant metastasis. J Clin Oncol, 10:1534-1539, 1992). But these invasive
techniques are deemed undesirable or unacceptable for routine or multiple
clinical assays compared to detection of disseminated epithelial tumor cells
in
blood.
An alternative approach incorporates immunomagnetic separation
technology and provides greater sensitivity and specificity in the unequivocal
detection of intact circulating cancer cells. This simple and sensitive
diagnostic
tool, as described (US6,365,362; US6,551,843; US6,623,982; US6,620,627;
US6,645,731; WO 02/077604; W003/065042; and WO 03/019141) is used in
the present invention to provide a preclinical animal model to enumerate
CTC's.
The assay depends upon the acquisition of a preserved blood sample
from a patient. The blood sample from a cancer patient (WO 03/018757) is
incubated with magnetic beads, coated with antibodies directed against an
epithelial cell surface antigen as for example EpCAM. After labeling with anti-
EpCAM-coated magnetic nanoparticles, the magnetically labeled cells are then
isolated using a magnetic separator. The immunomagnetically enriched
fraction is further processed for downstream immunocytochemical analysis or
image cytometry, for example, in the CeIlTracks System (Veridex LLC, NJ).
The magnetic fraction can also be used for downstream immunocytochemical
analysis, RT-PCR, PCR, FISH, flowcytometry, or other types of image
cytometry.
The CeIlTracks System utilizes immunomagnetic selection and
separation to highly enrich and concentrate any epithelial cells present in
whole
blood samples. The captured cells are detectably labeled with a leukocyte
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specific marker and with one or more tumor cell specific fluorescent
monoclonal
antibodies to allow identification and enumeration of the captured CTC's as
well
as unequivocal instrumental or visual differentiation from contaminating non-
target cells. This assay allows tumor cell detection even in the early stages
of
low tumor mass. The embodiment of the present invention is not limited to the
CeIlTracks System, but includes any isolation and imaging protocol of
comparable sensitivity and specificity.
Currently available preclinical protocols have not demonstrated a
consistently reliable means for repetitively monitoring CTC's in assessing
metastatic breast cancer (MBC) progression. The development of a reliable
mouse model to assess diagnostic and therapeutic advancements in cancer
research would provide a means to further research development in these
areas. Thus, there is a clear need for accurate detection of cancer cells with
metastatic potential, not only in MBC but in metastatic cancers in general.
Moreover, this need is accentuated by the need to select the most effective
therapy for a given patient.
The inability to repetitively monitor CTC's in the small blood volumes
available in pre-clinical animal models of breast and other cancers has
restricted their use to analysis of samples obtained from terminal blood
draws.
As a consequence, the study of temporal changes in CTC's during tumor
progression and therapy in a living animal model, such as in mice, as not been
established. However, using this technology to serially assay CTC's in mice
would permit integration of CTC's assessments into pre-clinical as well as
clinical studies. Further characterization of specific molecular markers on
these
cells would permit early development of "companion" diagnostic assays for
targeted therapies, which would accelerate translation of new assay protocols
into clinical trials in patients and ultimately into clinical practice.
Summary of the Invention
The present invention provides a method and means for preclinical
modeling of cancer metastasis in xenograft mice, incorporating clinical
analysis
tools such as the CeIlTracks System, and is based upon the absolute
number, change, or combinations of both of circulating epithelial cells in
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patients with metastatic cancer. The system immunomagnetically concentrates
epithelial cells, fluorescently labels the cells, identifies and quantifies
CTC's for
positive enumeration in zenograft tumor models of human breast cancer.
Brief Description of the Drawings
Figure 1: CellTracks fluorescent analysis profile used to confirm objects
captured as human tumor cells. Check marks signify a positive tumor cell
based on the composite image. Composite images are derived from the
positive selection for Epithelial Cell Marker (EC-PE) and for the nuclear dye
(NADYE). A negative selection is also needed for the leukocyte marker (L-
APC) and for control (CNTL).
Figure 2: Quantification of human breast cancer cells in mouse blood
samples. MDA-MB-231 human breast cancer cells without or with stable
transduction of GFP were added to 100 l blood samples from mice without
tumor xenografts. Samples were fixed, and epithelial cells were enriched by
immunomagnetic bead isolation using an antibody to epithelial cell adhesion
molecule. Recovered cells then were stained with an antibody to cytokeratin
(8, 18, and 19) to identify epithelial cells and distinguish them from
leukocytes
stained with CD45. Nucleated cells were identified by staining with the
fluorescent nucleic acid dye 4,2-diamidino-2-phenylindoledihydrochloride
(DAPI). GFP on cancer cells was detected in the FITC channel.
Representative images of recovered breast cancer cells are shown.
Figure 3: Quantification of human breast cancer cells in mouse blood samples.
Terminal blood samples from mice bearing xenografts of MDA-MB-231 human
breast cancer cells were obtained by cardiac puncture and analyzed for CTC.
Numbers of CTC are plotted versus tumor volumes measured by calipers.
Figure 4: Serial analysis of CTC in mice. Mice were implanted with orthotopic
tumor xenografts of SUM-159 (A) or SKBR-3 (B) human breast cancer cells,
and CTC in approximately 100 l blood samples were measured by cardiac
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puncture at approximately weekly intervals until mice were euthanized because
of tumor burden. CTC data were normalized to 100 l volume and plotted
against tumor volume for individual. Mean numbers of CTC were significantly
greater on day 30 as compared with prior days (p < 0.05).
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Detailed Description of the Invention
While any effective mechanism for isolating, enriching, and analyzing
CTC's in blood is appropriate, one method for collecting circulating tumor
cells
combines immunomagnetic enrichment technology, immunofluorescent
labeling technology with an appropriate analytical platform after initial
blood
draw. The associated test has been shown to have the sensitivity and
specificity to detect these rare cells in a sample of whole blood and to
investigate their role in the clinical course of the disease in malignant
tumors of
epithelial origin. From a sample of whole blood, rare cells are detected with
a
sensitivity and specificity to allow them to be collected and used in modeling
disease progression in an animal model.
Circulating tumor cells (CTC's) have been shown to exist in the blood in
detectable amounts. This created a tool to investigate the significance of
cells
of epithelial origin in the peripheral circulation of cancer patients (Racila
E.,
Euhus D., Weiss A.J., Rao C., McConnell J., Terstappen L.W.M.M. and Uhr
J.W., Detection and characterization of carcinoma cells in the blood, Proc.
NatI.
Acad. Sci. USA, 95:4589-4594 (1998)). This study demonstrated that these
blood-borne cells might have a significant role in the pathophysiology of
cancer.
Having a detection sensitivity of 1 epithelial cell per 5 ml of patient blood,
the
assay incorporated immunomagnetic sample enrichment and fluorescent
monoclonal antibody staining followed by flowcytometry for a rapid and
sensitive analysis of a sample.
The CellSearchTM System (Veridex LLC, NJ) previously has been used
to isolate and enumerate circulating epithelial tumor cells from human blood
samples 2. This is an automated system that enriches for epithelial cells
using
antibodies to epithelial-cell adhesion molecule coupled to magnetic beads.
Isolated cells then are stained with the fluorescent nucleic acid dye 4,2-
diamidino-2-phenylindole dihydrochloride (DAPI) to identify nucleated cells.
Recovered cells subsequently are stained with fluorescently labeled
monoclonal antibodies to CD45 (APC channel) and cytokeratin 8, 18, 19 (PE
channel) to distinguish epithelial cells from leukocytes. Nucleated epithelial
cells then are quantified as circulating tumor cells. There is an additional
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fluorescence channel for FITC that is not part of the standard CellSearchTM
assay and may be used for further characterization of tumor cells.
As shown in Example 1, the assay was further configured to an image
cytometric analysis such that the immunomagnetically enriched sample is
analyzed by the CeIlTracks System. This is a fluorescence-based
microscope image analysis system, which in contrast with flowcytometric
analysis permits the visualization of events and the assessment of morphologic
features to further identify objects.
Example 1
Enumeration of circulating cytokeratin positive cells
The CeIlTracks System refers to an automated fluorescence
microscopic system for automated enumeration of isolated cells from blood.
The system contains an integrated computer controlled fluorescence
microscope and automated stage with a magnetic yoke assembly that will hold
a disposable sample cartridge. The magnetic yoke is designed to enable
ferrofluid-labeled candidate tumor cells within the sample chamber to be
magnetically localized to the upper viewing surface of the sample cartridge
for
microscopic viewing. Software presents suspect cancer cells, labeled with
antibodies to cytokeratin and having epithelial origin, to the operator for
final
selection..
While isolation of tumor cells for the CeIlTracks System can be
accomplished by any means known in the art, one embodiment uses
immunomagentic enrichment for isolating tumor cells from a biological sample.
Epithelial cell-specific magnetic particles are added and incubated for 20
minutes. After magnetic separation, the cells bound to the immunomagnetic-
linked antibodies are magnetically held at the wall of the tube. Unbound
sample is then aspirated and an isotonic solution is added to resuspend the
sample. A nucleic acid dye, monoclonal antibodies to cytokeratin (a marker of
epithelial cells) and CD 45 (a broad-spectrum leukocyte marker) are incubated
with the sample. After magnetic separation, the unbound fraction is again
aspirated and the bound and labeled cells are resuspended in 0.2 ml of an
isotonic solution. The sample is suspended in a cell presentation chamber and
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placed in a magnetic device whose field orients the magnetically labeled cells
for fluorescence microscopic examination in the CeIlTracks System. Cells are
identified automatically in the CeIlTracks System and candidate circulating
tumor cells presented to the operator for checklist enumeration. An
enumeration checklist consists of predetermined morphologic criteria
constituting a complete cell.
Cytokeratin positive cells are isolated by immunomagnetic enrichment
using a 7.5 ml sample of whole blood from humans. Epithelial cell-specific
immunomagnetic fluid is added and incubated for 20 minutes. After magnetic
separation for 20 minutes, the cells bound to the immunomagnetic-linked
antibodies are magnetically held at the wall of the tube. Unbound sample is
then aspirated and an isotonic solution is added to resuspend the sample. A
nucleic acid dye, monoclonal antibodies to cytokeratin (a marker of epithelial
cells) and CD 45 (a broad-spectrum leukocyte marker) are incubated with the
sample for 15 minutes. After magnetic separation, the unbound fraction is
again aspirated and the bound and labeled cells are resuspended in 0.2 ml of
an isotonic solution. The sample is suspended in a cell presentation chamber
and placed in a magnetic device whose field orients the magnetically labeled
cells for fluorescence microscopic examination in the CeIlTracks System.
Cells are identified automatically in the CeIlTracks System; control cells
are
enumerated by the system, whereas the candidate circulating tumor cells are
presented to the operator for enumeration using a checklist as shown in Figure
1.
Example 2
in vitro Recovery of Human Epithelial Cells
To accomplish this, 500 MDA-MB-231 breast cancer cells were spiked
into 100 l blood samples collected from mice without tumors. Since the
clinical version of the assay requires blood be drawn into a proprietary
vacuum
tube, such as the CeIlSave tube, containing both an anticoagulant and a
preservative, a proportionately reduced amount of CeIlSave solution was added
to the specimens. The spiked specimens were then prepared, the CTC
quantified and the percent recovery calculated. As a positive control,
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additional samples using MDA-MB-231 cells stably transduced with GFP were
prepared. Fluorescence from GFP was detected in an open channel (FITC) of
the system to confirm that all cells quantified as epithelial cells
corresponded
with 231 -GFP cells added to mouse blood. As a negative control, mouse blood
samples without cancer cells were collected, processed in an identical manner
and analyzed. Of the 500 cells added to mouse blood (n = 4 samples), 482-
526 cells per specimen were recovered, which is within the range of the
dilution
error for spike-in experiments at this concentration (Figure 2). For samples
using 231 -GFP cells, all cells identified as epithelial cells also expressed
GFP,
verifying that these were human breast cancer cells and not contaminating
murine epithelial cells. No epithelial cells were recovered from normal mouse
blood, confirming the specificity of the assay.
Example 3
Recovery of CTC from Xenografts in Mice
The preferred method to serially monitor CTC's in mouse models of
human breast cancer incorporates the use of the CeIlTracks System. As
previously discussed, the system uses immunomagnetic isolation of epithelial
cells from blood and immunofluorescent staining to further differentiate
epithelial cancer cells from leukocytes. Because the CeIlTracks system was
originally developed to process 7.5 to 30 ml human blood samples, it is
necessary that human epithelial breast cancer cells could be reliably
recovered
from small volumes of mouse blood using this assay (see Example 2).
The system was used to identify CTC's that spontaneously intravasate
into the circulation from orthotopic tumor xenografts of MDA-MB-231 cells. 0.7
to 1 ml blood samples were collected from each mouse by puncture of the left
ventricle when animals were euthanized for tumor burden at 10 weeks. Total
numbers of CTC's ranged from approximately 100 to 1000 cells per ml of blood
(Figure 3). No CTC's were recovered from blood samples collected from mice
without tumor xenografts (data not shown). The number of CTC's did not
correlate with size of the primary tumor. These data suggest that numbers of
CTC's reflect the underlying biology of various primary tumors, which is
consistent with previous studies showing that MDA-MB-231 cells contain
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subpopulations with differing metastatic potential. Using the same method,
CTC's were also detectable in mice with tumor xenografts of MCF-7, MCF-7
cells stably transfected with fibroblast growth factor (FGF), SUM-159, and
SKBR-3 cell-lines.
While the system was successful in detecting CTC's using cardiac
puncture to collect blood, this procedure is invasive compared to other sites
of
blood sampling in mice. One aspect of the present invention is to repetitively
draw blood samples for analysis of CTC's, blood samples from the lateral tail
vein and retro-orbital venous plexus and thereby avoid the invasive nature of
cardiac puncture. In mice with or without orthotopic MDA-MB-231 tumor
xenografts were compared to direct cardiac sampling. No epithelial cells were
detected in any of the lateral tail vein samples, independent of the presence
of
a tumor xenograft. One possible explanation for the failure to detect CTC's in
tumor-bearing mice was the small volume of blood (<_ 25 l) that could be
collected from the lateral tail vein. Although larger volumes of blood (50 -
75
l) could be obtained from the retro-orbital venous plexus, 3 of 3 blood
samples
from this site contained epithelial cells (5-500 cells) in mice without
tumors.
These contaminating cells were normal murine epithelial cells dislodged by the
microcapillary tube during blood collection. Thus sampling via the retro-
orbital
route would make it impossible to reliably identify CTC in tumor-bearing mice.
By comparison, there were no CTC's in blood samples obtained by cardiac
puncture in mice without tumor xenografts, but CTC's could be detected in
blood obtained via left ventricle cardiac puncture in mice with MDA-MB-231
xenografts.
Example 4
Temporal analysis of CTC's in Mice
After validating the assay and route of blood collection, the feasibility of
detecting temporal changes in CTC's was investigated using mice implanted
with orthotopic tumor xenografts of SUM-159 (n = 3) or SKBR-3 (n = 4) cells.
75 to 100 l blood samples were collected approximately once per week for 1
month until mice were euthanized because of tumor burden. MDA-MB-231 and
SKBR-3 human breast cancer cells were cultured in DMEM with 10% fetal
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bovine serum, 1% L-glutamine, and 0.1% penicillin/streptomycin. SUM-159
cells were cultured in Ham's F12 medium (Invitrogen) supplemented with 5%
fetal bovine serum (FBS), 5 g/ml insulin, 1 g/ml hydrocortisone, and 0.1 %
penicillin/streptomycin. Cells were maintained at 37 C in a 5% CO2 incubator.
For selected experiments, MDA-MB-231 cells were transduced with the
lentiviral vector pSico to establish cells that stably express GFP. Efficiency
of
transduction was 100%, as determined by phase-contrast and fluorescence
microscopy.
In producing tumor xenografts in mice, 5 to 6 week old female Ncr nude
(Taconic) or SCID (Jackson) mice were used. Human breast tumor xenografts
from cell lines, 1 x 106 cells were injected orthotopically into bilateral
inguinal
mammary fat pads of mice by methods know in the art. For tumor xenografts
with clinical isolates of human breast cancer cells, mice were implanted with
1-
5 x 105 cells in the fourth inguinal mammary fat pad. Mice implanted with
clinical breast cancer isolates also received a subcutaneous pellet of 60-day
sustained releasel7-J3-estradiol (Innovative Research of America). Volumes of
tumors were quantified as the product of caliper measurements in two
dimensions and calculated by the equation: width (mm) X width (mm) X length
(mm) X 0.52. For serial studies of CTC, blood samples were collected from the
left ventricle at approximately weekly intervals as shown in the figure
legend.
Assay results show low levels of CTC's (0 - 7 cells) in earlier samples
(days 8-23) (Figure 4), with numbers of CTC's increasing significantly on day
in 6 of 7 mice (26 - 55 cells) (p < 0.05), corresponding to an increase in
tumor volume. These studies establish that the assay can be used
25 successfully for serial studies of CTC's in mouse models of breast cancer.
For all CTC's measured in mice implanted with xenografts, primary
breast cancer cells were obtained from patient biopsy specimens. Blood
samples (200 pL - 800 pL) were collected via cardiac puncture at the time
animals were euthanized because of tumor burden. Breast cancer cells from 6
30 different patients formed tumors in mice, and all of these tumors produced
CTC's. Numbers of CTC's ranged from 4 - 805 cells per ml of blood with a
mean value of 118 cells 67 (n = 6). Notably, none of these animals had overt
or histologically detectable metastases (data not shown), suggesting that the
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majority of CTC's produced by primary clinical specimens may not be capable
of forming metastases in either mice or in humans. These data show that
xenografts of clinical breast cancer isolates can produce CTC's in mice and
therefore provide a model system for investigating properties and
subpopulations of human breast cancer cells involved in metastasis.
While certain of the preferred embodiments of the present invention
have been described and specifically exemplified above, it is not intended
that
the invention be limited to such embodiments. Various modification may be
made thereto without departing from the spirit of the present invention, the
full
scope of the improvements are delineated in the following claims.
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