U.S. patent application number 09/189310 was filed with the patent office on 2001-12-13 for method for using multicellular particulates to analyze malignant or hyperproliferative tissue.
Invention is credited to KORNBLITH, PAUL L..
Application Number | 20010051353 09/189310 |
Document ID | / |
Family ID | 27365637 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010051353 |
Kind Code |
A1 |
KORNBLITH, PAUL L. |
December 13, 2001 |
METHOD FOR USING MULTICELLULAR PARTICULATES TO ANALYZE MALIGNANT OR
HYPERPROLIFERATIVE TISSUE
Abstract
A comprehensive and integrated system for monitoring
(identifying, tracking and analyzing) an individual patient's
malignancy through the duration of a malignancy as to a specific
patient is provided. The method of the present invention allows for
initial identification of a malignancy, identification of
malignancy-specific cellular or secretal markers, identification of
cellular or secreted markers indicative of complications, study of
the invasiveness and aggressiveness of the malignancy, study of the
growth rate of the malignancy, study of the effect of therapies on
the malignancy as compared to control cells of the same patient
(chemosensitivity versus toxicity) and the identification of a
therapeutic index (i.e., the ratio of chemosensitivity:toxicity),
study of tumor morphology and study of histological, cytochemical
and immunocytochemical markers.
Inventors: |
KORNBLITH, PAUL L.;
(PITTSBURGH, PA) |
Correspondence
Address: |
BARBARA E JOHNSON
WEBB ZIESENHEIM BRUENING LOGSDON ORKIN
AND HANSON
436 7TH AVENUE SUITE 700
PITTSBURGH
PA
152191818
|
Family ID: |
27365637 |
Appl. No.: |
09/189310 |
Filed: |
November 10, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09189310 |
Nov 10, 1998 |
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08679056 |
Jul 12, 1996 |
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5728541 |
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08679056 |
Jul 12, 1996 |
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09095993 |
Jun 11, 1998 |
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09095993 |
Jun 11, 1998 |
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09039957 |
Mar 16, 1998 |
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Current U.S.
Class: |
435/29 ; 435/261;
435/30; 435/32; 435/803 |
Current CPC
Class: |
G01N 33/5017 20130101;
G01N 33/5008 20130101; G01N 2800/44 20130101; G01N 33/5005
20130101; G01N 33/5091 20130101; G01N 33/5011 20130101; G01N 33/574
20130101 |
Class at
Publication: |
435/29 ; 435/30;
435/32; 435/261; 435/803 |
International
Class: |
C12Q 001/02; C12Q
001/24; C12Q 001/18 |
Claims
I claim:
1. A method for identifying and monitoring progress of an
individual patient having, a malignancy comprising the steps of: a.
collecting a specimen of a patient's cells; b. separating the
specimen into cohesive multicellular particulates; c. growing a
tissue culture monolayer from the multicellular particulates to
form a prime culture; and d. monitoring said tissue culture
monolayer over a period of time.
2. A method for identifying and monitoring progress of a malignancy
in an individual patient as claimed in claim 1 further comprising
the step of maintaining the prime culture.
3. A method for identifying and monitoring progress of a malignancy
in an individual patient as claimed in claim 1 further comprising
the steps of preparing a reference culture from the prime culture
and treating the reference culture with one or more treatments as
given to the patient.
4. A method for identifying and monitoring progress of a malignancy
in an individual patient as claimed in claim 3 further comprising
the steps of preparing a subculture of one of the prime culture and
the reference culture.
5. A method for identifying and monitoring progress of a malignancy
in an individual patient as claimed in claim 4 further comprising
the step of assaying for a malignancy-specific one of the prime
culture, the reference culture, the subculture and tissue culture
medium used to grow one of the prime culture, the reference culture
or the subculture.
6. A method for identifying and monitoring progress of a malignancy
in an individual patient as claimed in claim 5 wherein the marker
indicates one of aggressiveness and invasiveness of the
malignancy.
7. A method for identifying and monitoring progress of a malignancy
in an individual patient as claimed in claim 5 wherein the marker
is indicative of complications associated with the malignancy.
8. A method for identifying and monitoring progress of a malignancy
in an individual patient as claimed in claim 7 wherein the marker
is indicative of a thrombogenic potential.
9. A method for identifying and monitoring progress of a malignancy
in an individual patient as claimed in claim 5 wherein the marker
is identified by one of cytochemistry or immunohistochemistry.
10. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 9 wherein
the marker is selected from the group consisting of an estrogen
receptor, a progesterone receptor, an oncogene, a product of an
oncogene, a marker for multi-drug resistance and a marker for
phenotypic characterization.
11. A method for identifying and monitoring progress of a
malignancy of an individual patient as claimed in claim 5 wherein
one or more of the steps are at least partially automated.
12. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 5 wherein
the marker is characterized by a molecular biological
technique.
13. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 12 wherein
the molecular biological technique characterizes one of tumor cell
heterogeneity or specific mutations of cancer-related genes
14. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 2 further
comprising the steps of: d. inoculating cells from one of the prime
culture, the reference culture or a subculture of the prime culture
or of the reference culture into a plurality of segregated sites;
and e. treating the plurality of sites with at least one treating
means, followed by assessment of sensitivity of cells in the site
to the treating means.
15. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 14 wherein
one or more of the steps are at least partially automated.
16. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 14 further
comprising the step of phenotypically or genotypically analyzing
the cells in one or more sites for drug resistance.
17. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 14 further
comprising the steps of: f. collecting a specimen of a patient's
non-malignant cells; g. separating the non-malignant cells into
cohesive multicellular particulates; h. growing a tissue culture
monolayer from the multicellular particulates of non-malignant
cells to form a control culture; i. inoculating the control culture
in a plurality of non-segregated sites; j. treating the plurality
of segregated sites of the control culture with the same treating
means as the segregated sites of the prime culture or a subculture
thereof, followed by assessment of the sensitivity of the
segregated cells of the control culture to the treating means; and
k. comparing the sensitivity of the segregated cells of the prime
culture or a subculture thereof with the sensitivity of the
segregated cells of the control culture to the treating means.
18. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 17 wherein
the assessment of steps e and j are calculations of the percentage
or fraction of cells sensitive to the treatment and further
comprising the step of: l. creating a therapeutic index of a ratio
of one of the percentage of or the fraction of sensitive cells or
insensitive cells in the segregated cells of the control culture to
one of the percentage of or the fraction of sensitive cells or
insensitive cells in the segregated cells of the prime culture or
subculture thereof.
19. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 18 further
comprising the step of programming a computer to automatically
perform calculations to create said therapeutic index.
20. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 19 wherein
the segregated sites are in a readable plate having a plurality of
culture wells and a scanner is used to automatically scan the
segregated sites to determine the percentage or fraction of cells
sensitive to the treatment and an interface is provided between the
scanner and the computer allowing automated input of scanner data
into the computer for calculation of the therapeutic index.
21. A method for identifying and monitoring progress of a
malignancy in an individual patient as claimed in claim 18 wherein
the non-malignant cells are epithelial cells.
22. A method for treating a patient having a malignancy comprising
the steps of: a. analyzing a patient's cells prepared according to
the method of claim 1 for malignancy-associated markers; b.
determining a therapeutic regimen according to the results of the
analysis; and c. treating a patient according to the regimen.
23. A method for treating a patient having a malignancy as claimed
in claim 22 further comprising the step of treating one of cells
cultured as a subculture of the prime culture and cells of the
prime culture according to the method of claim 14.
24. A method for treating a patient having a malignancy as claimed
in claim 22 wherein the analyzing step further includes the steps
of: i. inoculating cells from one of the prime culture, the
reference culture or a subculture of the prime culture or the
reference culture into a plurality of segregated sites; ii.
treating the plurality of sites with at least one treating means,
followed by assessment of sensitivity of cells in the site to the
treating means; and iii. determining a therapeutic index for each
treating means according to the method of claim 18.
Description
RELATED APPLICATION
[0001] This is a Continuation-In-Part of U.S. application Ser. No.
08/679,056, filed Jul. 12, 1996, now U.S. Pat. No. 5,728,541,
granted Mar. 17, 1998; U.S. application Ser. No. 09/095,993, filed
Jun. 11, 1998; and U.S. application Ser. No. 09/039,957, filed Mar.
16, 1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] A system is provided for in vitro tracking of cancerous
tissue over the course of the malignancy. The system provides a
method for identifying the malignancy and for determining a
patient's prognosis. Further, the system provides for assessing a
malignancy's invasiveness, aggressiveness, growth rate, production
of extracellular markers, possible side effects and for determining
the efficacy on the malignancy of a given therapeutic regimen. The
system also allows for generation of a therapeutic index, which
serves as an indicator of a given therapy's effectiveness against
the malignancy as compared to its undesirable side effects, such as
lethality to a patient's normal cells.
Introduction
[0004] Tracking a malignancy in a patient according to prior art
methods is an inaccurate process which involves identification of
the malignancy through techniques including biopsy and subsequent
histological, biochemical, and immunochemical techniques and
regularly monitoring the malignancy's progression by invasive
(i.e., biopsy) or noninvasive (i.e., x-ray, nuclear imaging,
Magnetic Resonance Imaging (MRI) and Positron Emission Tomography
(PET)) methods. These methods are often expensive, inconvenient,
painful and usually involve hospital visits and safety risks. It
is, therefore, desirable to reduce a patient's exposure to such
methods. Furthermore, identification of a malignancy as a known
variety of malignancy is often helpful in determining a suitable
therapeutic approach and expected prognosis. However, even
individually identifiable malignancies differ from
patient-to-patient in their growth characteristics and in their
responsiveness to treatment.
[0005] Determination of the growth rate, invasiveness and
aggressiveness of a given malignancy is critical to prognosis and
to the choice of therapies. A patient with a poor prognosis might
be given a therapeutic regimen which might be more effective than
another regimen but more risky to the patient. A patient with a
better prognosis might be given a therapeutic regimen which is less
aggressive and less risky to the patient, but which might not be as
effective as often as a more dangerous therapy. Similarly, if a
malignancy produces factors or creates conditions which cause a
dangerous side effect, such as a thrombogenesis, the patient can be
treated, preferably prophylactically, for the condition.
[0006] Current methodologies for determining growth rate,
invasiveness, aggressiveness or which track the progression of a
malignancy include biopsy and short-term culture, which can include
drawing of blood or other bodily fluids, or semi- or non-invasive
techniques such as x-ray and nuclear imaging. At any given time, a
patient could be subject to multiple procedures, depending upon
when the information is needed by the physician. Each procedure
requires the presence of the patient and usually creates risk or
pain. These procedures also can increase the stress level of the
patient, which often is an exacerbating factor in cancer and
associated prognoses. It is therefore, desirable to reduce the
frequency of such procedures.
[0007] Identification of an effective therapeutic regimen is
critically important to a patient. Often, once the malignancy is
identified, a therapy is chosen based upon prior research on that
type of malignancy and is not tailored to the sensitivities of the
malignancy of a given patient. Often secondary therapies are needed
because a first choice was ineffective. Valuable treatment time can
be lost and a patient's life can be threatened.
[0008] All active agents including chemotherapeutic active agents
are subjected to rigorous testing as to efficacy and safety prior
to approval for medical use in the United States. Methods of
assessing efficacy have included elaborate investigations of large
populations in double blind studies as to a given treatment method
and/or active agent, with concomitant statistical interpretation of
the resulting data, but these conclusions are inevitably
generalized as to patient populations taken as a whole. In many
pharmaceutical disciplines and particularly in the area of
chemotherapy, however, the results of individual patient therapy
may not comport with generalized data--to the detriment of the
individual patient. The need has been long recognized for a method
of assessing the therapeutic potential of active agents, including
but not limited to chemotherapeutic agents, for their efficacy as
to a given individual patient, prior to the treatment of that
patient. This need also applies to assessing the therapeutic
potential as to radiation therapies, combined radiation/drug
therapies and cellular immunotherapies.
[0009] Prior art assays already exist which expose malignant tissue
of various types to a plurality of active agents, for the purpose
of assessing the best choice for therapeutic administration. For
example, in Kruczynski, A., et al., "Evidence of a direct
relationship between the increase in the in vitro passage number of
human non-small-cell-lung cancer primocultures and their
chemosensitivity," Anticancer Research, vol. 13, no. 2, pp. 507-513
(1993), chemosensitivity of non-small-cell-lung cancers was
investigated in in vivo grafts, in in vitro primocultures and in
commercially available long-term cancer cell lines. The increase in
chemosensitivity was documented and correlated with morphological
changes in the cells in question. Sometimes animal model malignant
cells and/or established cell cultures are tested with prospective
therapy agents, see for example Arnold, J.T., "Evaluation of
chemopreventive agents in different mechanistic classes using a rat
tracheal epithelial cell culture transformation assay," Cancer
Res., vol. 55, no. 3, pp. 537-543 (1995).
[0010] In vitro prior art techniques present the further
shortcoming that assayed cells do not necessarily express the
cellular markers they would express in vivo. This is regrettable
because the determination of expression of certain secreted or
cellular markers, secreted factors or tumor antigens or lack
thereof can be useful for both identification and therapeutic
purposes. For instance, members of the fibrinolytic system such as
urokinase-type plasminogen activator (u-PA) and plasminogen
activator inhibitors type 1 (PAI-1) are up-regulated in malignant
brain tumors. See, e.g., Jasti S. Rao, et al., "The Fibrinolytic
System in Human Brain Tumors: Association with Pathophysiological
Conditions of Malignant Brain Tumors," Advances in Neuro-Oncology
II, Kornblith PL, Walker MD (eds) Futura (1997). Other secreted
factors such as .alpha.-fetoprotein, carcinoembryonic antigen and
transforming growth factors .alpha. and .beta. have been found to
be indicative of various cancers and/or cancer progression (see
also, Singhal et al., "Elevated Plasma Osteopontin in Metastatic
Breast Cancer Associated with Increased Tumor Burden and Decreased
Survival," Clinical Cancer Research, vol. 3, 605-611, (April 1997);
Kohno et al., "Comparative Studies of CAM 123-6 and
Carcinoembryonic Antigen for the Serological Detection of Pulmonary
Adenocarcinoma," Cancer Detection and Prevention, 21(2): 124-128
(1997)). These examples are but a few of the many factors that may
be used to identify diseased cells.
[0011] Cellular markers also include metastatic markers, indicative
of metastatic potential, i.e., invasiveness and aggressiveness,
which is relevant to the progression of a given malignancy and to a
patient's prognosis. First, markers indicating the invasiveness of
a given malignancy indicate the ability of the malignancy to
infiltrate and to destroy adjacent tissue. As an example, for
epithelial malignancies, invasiveness markers are indicative of the
ability of the malignancy to infiltrate beneath the epithelial
basement membrane. Invasiveness markers can include the presence of
proteolytic enzymes or angiogenic factors. A second category of
metastatic marker indicates growth conditions of the malignancy.
For instance, a malignancy could require for instance a
prostate-specific factor for growth. Invasiveness and
aggressiveness factors are often present in serum or in tissue
culture media.
[0012] Relevant to a patient's prognosis and, incidentally, to the
identification of a malignancy is the presence of markers, cellular
or secreted, which lead to complications beyond those involved with
uncontrolled growth and invasion by a malignancy. For instance,
secretion by the malignancy of thrombogenic substances by the
malignancy can result in blood clotting, resulting in
thrombophlebitis or other thrombotic events such as pulmonary
thrombosis. Identification of a thrombotic potential indicates
treatment (preferably prophylactically) with thrombolytic
substances.
[0013] When a specific patient's cells are used in in vitro assays
in typical prior art processes the cells are harvested (biopsied)
and trypsinized (connective tissue digested with the enzyme
trypsin) to yield a cell suspension purportedly suitable for
conversion to the desired tissue culture form. The in vitro tissue
culture cell collections which result from these techniques are
generally plagued by their inability accurately to imitate the
chemosensitivity or therapeutic sensitivity of the original tumor
or other cell biopsy. These collections often do not express
cellular markers in the same manner that they would in vivo. A need
thus remains for a technique of tissue culture preparation which
provides cell cultures, allowing identification of a malignancy,
accurate tracking of the malignancy's progress in a patient and
therapy screening, in which, after simple preparation, the cell
cultures react in a manner equivalent to their in vivo reactivity.
The culture method would enable drug or chemotherapeutic agent,
radiation therapy and/or cellular immunotherapy screening as to a
particular patient for whom such screening is indicated.
[0014] A need also remains for a technique of tissue culture
preparation which provides cell cultures for screening for
expressed markers or factors where the cultured cells express the
markers or factors in a manner indicative of their in vivo
expression of the same. A further need also remains for a tissue
culture preparation which allows for morphological study of the
cells. Lastly, a need remains for a tissue culture system in which
progression of an individual malignancy can be studied as
indicative of the in vivo progression of the malignancy.
SUMMARY OF THE INVENTION
[0015] A comprehensive and integrated unified system for monitoring
(i.e., identifying, tracking and analyzing) an individual patient's
malignancy through the duration of a malignancy as to a specific
patient is provided. The method of the present invention allows for
initial identification of a malignancy, identification of
malignancy-specific cellular or secreted markers, identification of
cellular or secreted markers indicative of complications, study of
the invasiveness and aggressiveness of the malignancy, study of the
growth rate of the malignancy, study of the effect of therapies on
the malignancy as compared to control cells of the same patient
(chemosensitivity versus toxicity) and the identification of a
therapeutic index (i.e., the ratio of chemosensitivity:toxicity),
study of tumor morphology and study of histological and
cytochemical markers.
[0016] The method of the present invention includes the steps of
collecting a tissue sample or specimen of a patient's cells and
separating the specimen into cohesive multicellular particulates
(explants) of the tissue sample, rather than enzymatically digested
cell suspensions or preparations. The cells are then grown as a
tissue culture monolayer from the multicellular particulates to
form a prime culture. A specimen can be taken from a patient at any
relevant site, including but not limited to tissue, ascites or
effusion fluid. Samples may also be taken from body fluid or
exudates, as is appropriate. A tissue culture monolayer, designated
as the prime culture, can be grown in any method known in the art
for growing such a monolayer, for instance in tissue culture plates
or flasks. If the malignant cells originate from solid tissue,
however, the tissue must be subdivided into small pieces from which
a tissue culture monolayer is then grown out.
[0017] Once a prime culture is established from a patient's
malignancy, the prime culture can be maintained without any
treatments beside normal feedings and passage techniques, as
indicative of the growth of the malignancy absent treatment.
However, subcultures of the prime culture are prepared so that the
prime culture is preferably left untreated, and the cells of the
prime culture are not affected by any testing. However, either the
prime culture or a subculture thereof can be propagated as a
reference culture. The reference culture is a culture which is
treated with therapies reflective of a patient's actual treatments.
For instance, if a patient is treated with a chemotherapeutic
agent, the reference culture is treated with the same agent in the
same concentration. The reference culture can be monitored
genotypically or phenotypically to reflect actual progress of the
malignancy in the patient. Treatment of the reference culture need
not be limited to anticancer therapies, but can reflect all of a
patient's treatments. For instance, thrombolytic or
anti-thrombogenic treatments can be applied to the reference
culture to reflect a patient's treatment. Subcultures of either the
prime culture or the reference culture can be used for further
analysis. Preferably, since the reference culture is indicative of
the current state of a malignancy at a given time, subcultures of
the reference culture are analyzed further. At various points in
the passage of the control culture and the reference culture,
aliquots of cells from those cultures can be stored cryogenically
or otherwise.
[0018] The tissue sample technique of the present invention is also
useful in assaying expression and/or secretion of various markers,
factors or antigens present on or produced by the cultured cells.
These assays can be used for diagnostic purposes for monitoring the
applicability of certain candidate therapeutic or chemotherapeutic
agents or for monitoring the progress of treatment of the cancer
with those agents.
[0019] A method for identifying and monitoring progress of a
malignancy in an individual patient is provided including the steps
of inoculating cells from either the prime culture, the reference
culture or a subculture of the prime culture or of the reference
culture into a plurality of segregated sites; treating the
plurality of sites with at least one treating means or therapy,
followed by assessment of sensitivity of cells in the site to the
treating means; collecting a specimen of a patient's non-malignant
cells; separating the non-malignant cells into cohesive
multicellular particles; growing a tissue culture monolayer from
the multicellular particulates of non-malignant cells to form a
control culture; inoculating the control culture in a plurality of
non-segregated sites; treating the plurality of segregated sites of
the control culture with the same treating means as the segregated
sites of the prime culture or a subculture thereof, followed by
assessment of the sensitivity of the segregated cells of the
control culture to the treating means; and comparing the
sensitivity of the segregated cells of the prime culture or a
subculture thereof with the sensitivity of the segregated cells of
the control culture to the treating means. The assessments
described above are calculations of the percentage or fraction of
cells sensitive, or insensitive, to the treatment and the method
further includes the step of creating a therapeutic index of a
ratio of one of the percentage of or the fraction of sensitive
cells or insensitive cells in the segregated cells of the control
culture to one of the percentage of or the fraction of sensitive
cells or insensitive cells in the segregated cells of the prime
culture or subculture thereof.
[0020] Lastly, a method for treating a patient having a malignancy
is provided, including the steps of: analyzing a patient's cells
prepared according to the above-described methods for
malignancy-associated markers; determining a therapeutic regimen
according to the results of the analysis; and treating a patient
according to the regimen. The method can further include the step
of treating one of either cells cultured as a subculture of the
prime culture or cells of the prime culture according to the
regimen as representative of the patient's malignancy. Lastly, the
method further includes determining a therapeutic index for each
treating means as described above.
[0021] When applicable, cultures can be grown in a readable
(scannable) plate and to determine percent confluence of the cells
or any other parameter which can be determined in such a manner.
The scanner can be operably linked with a computer or CPU to
automatically input data into the computer or CPU. The computer or
CPU can be programmed to automatically calculate a therapeutic
index (or other relevant indices) based upon the data provided by
the scanner. Alternatively, the data can be entered manually into
the programmed computer or CPU to calculate the index.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A, 1B and 1C are graphs of the growth rates of three
independent cell cultures.
[0023] FIGS. 2A-2F through 5A-5F are graphs depicting the results
of short-term and long-term chemotherapy assays. FIGS. 2A-2F and
3A-3F show short-term and long-term assays for a first patient.
FIGS. 4A-4F and 5A-5F show short-term and long-term assays for a
second patient.
[0024] FIGS. 6 and 7 show two radiation dose versus surviving
fraction curves for two glioblastoma cell lines. Cells were
irradiated in microtiter plates and assayed four days
post-irradiation.
[0025] FIGS. 8A-8C are graphs of survival rates of cell cultures
treated with radiation (FIG. 8A) or with radiation and Taxol (FIGS.
8B and 8C).
[0026] FIGS. 9A and 9B are graphs showing data from a series of
experiments where target cells from two tumor types were exposed to
Activated Natural Killer (ANK) cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] The present invention is an improved and unified system for
monitoring the progression of an individual malignancy and for
identifying cellular and secreted markers, markers indicative of
certain side effects of the malignancy and for screening multiple
candidate therapeutic or chemotherapeutic agents for efficacy and
long term effect as to a specific patient. In the method, a tissue
sample from the patient is harvested, cultured and separately
exposed to a plurality of treatments and/or therapeutic agents for
the purpose of objectively identifying the best treatment for the
cultured cells obtained from the patient. The culture techniques of
the present invention also result in a monolayer of cells that
express cellular markers, secreted factors and tumor antigens in a
manner representative of their expression in vivo. Specific method
innovations such as tissue sample preparation techniques render
this method practically as well as theoretically useful. One
particularly important tissue sample preparation technique is the
initial preparation of cohesive multicellular particulates of the
tissue sample, rather than enzymatically dissociated cell
suspensions or preparations, for initial tissue culture monolayer
preparation. With respect to the culturing of malignant cells, for
example, it is believed (without any intention of being bound by
the theory) that by maintaining the malignant cells within a
multicellular particulate of the originating tissue, growth of the
malignant cells themselves is facilitated versus the overgrowth of
fibroblasts or other cells which tends to occur when suspended
tumor cells are grown in culture. Practical monolayers of cells may
thus be formed to enable meaningful screening of a plurality of
treatments and/or agents as well as meaningful identification of
cellular markers. In the drug assays, growth of cells is monitored
to ascertain the time to initiate the assay and to determine the
growth rate of the cultured cells; sequence and timing of drug
addition is also monitored and optimized. By subjecting uniform
samples of cells to a wide variety of active agents (and
concentrations thereof), the most efficacious agent can be
determined. For assays concerning cancer treatment, a two-stage
evaluation is contemplated in which both acute cytotoxic and longer
term inhibitory effects of a given anti-cancer agent are
investigated.
[0028] With regard to the identification of expressed cellular
markers, secreted factors or tumor antigens, with the initial
culturing of the multicellular particulates it is believed (without
any intention of being bound by the theory) that because the cells
are grown under conditions closer to those found in vivo, the cells
express their cellular markers, secreted factors and tumor antigens
in a manner more closely resembling their expression in vivo. By
assaying the culture media obtained from growing a monolayer
according to the inventive method or by histochemically and/or
immunohistochemically assaying the cells grown under such
conditions, a more accurate profile of the cellular markers or
factors is obtained.
[0029] Thus, a comprehensive and integrated system for identifying,
tracking and analyzing an individual patient's malignancy through
the duration of the malignancy and thereafter is provided. The
method of the present invention allows for initial identification
of a malignancy, identification of malignancy-specific cellular or
secreted markers, identification of cellular or secreted markers
indicative of complications, study of the invasiveness and
aggressiveness of the malignancy, study of the growth rate of the
malignancy, study of the effect of therapies on the malignancy as
compared to control cells of the same patient (chemosensitivity
and/or radiosensitivity versus toxicity) and the identification of
a therapeutic index (i.e., the ratio of chemosensitivity:toxicity),
study of tumor morphology and study of histological, cytochemical
and immunocytochemical markers.
[0030] The method of the present invention includes the steps of
collecting a tissue sample or specimen of a patient's cells and
separating the specimen into cohesive multicellular particulates
(explants) of the tissue sample, rather than enzymatically digested
cell suspensions or preparations. The cells are then grown as a
tissue culture monolayer from the multicellular particulates to
form a prime culture. A specimen can be taken from a patient at any
relevant site, including but not limited to tissue, ascites or
effusion fluid. Samples may also be taken from body fluid or
exudates, as is appropriate. A tissue culture monolayer can be
grown in any method known in the art for growing such a monolayer,
for instance in tissue culture plates or flasks.
[0031] Once a prime culture is established from a patient's
malignancy, the prime culture can be maintained without any
treatments beside normal feedings and passage techniques, as
indicative of the growth of the malignancy absent treatment with a
therapeutic regimen. Subcultures of the prime culture are prepared
so that the cells of the prime culture are not affected by any
subsequent testing or treatments. Although prime culture is
preferably left untreated, either the prime culture or a subculture
thereof can be propagated as a reference culture. The reference
culture is a culture which is treated with therapies reflective of
a patient's actual treatment regimen. For instance, if a patient is
treated with a chemotherapeutic agent, the reference culture is
treated with the same agent in the same concentration. The
reference culture can be monitored genotypically or phenotypically
to reflect actual progress of the malignancy in the patient.
Treatment of the reference culture need not be limited to
anticancer therapies, but can reflect all of a patient's
treatments. For instance, thrombolytic or anti-thrombogenic
treatments, can be applied to the reference culture to reflect a
patient's treatment. Subcultures of either the prime culture or the
reference culture can be used for further analysis. Preferably,
since the reference culture is indicative of the current state in a
patient of a malignancy, subcultures of the reference culture are
analyzed. At various points in the passage of the control culture
and the reference culture, aliquots of cells from those cultures
can be stored cryogenically, or otherwise.
[0032] An important further aspect of the present invention is to
provide a system for screening specific tissue samples from
individual patients for expressed cellular markers, secreted
factors or antigens, including tumor antigens, characteristic of
the tissue sample. A tissue sample from a patient is harvested and
grown in a monolayer culture as described above. Culture medium in
which the cultures or subcultures thereof are assayed for the
presence or absence of certain factors, such as secreted tumor
antigens such as PAI-1, u-PA, cancer associated serum antigen
(CASA) or carcinoembryonic antigen (CEA). These factors may be
detected through use of standard assays such as radioimmunoassay
(RIA) or enzyme-linked immunosorbent assay (ELISA), although other
assays known to those skilled in the art may be used to detect
and/or to quantify the soluble factors. The cell cultures grown in
this manner may also be assayed histochemically and or
immunohistochemically for identification or quantification of
cellular or membrane-bound markers. Examples of such markers
include, without limitation, CEA, tissue polypeptide specific
antigen, (TPS) and mucin antigens, such as CA 15-3, CA 549, CA
27.29 and MCA. By screening tissue samples in this manner, for
production of such factors, markers or antigens, the cultured cells
may be further identified, aiding the physician in treatment
strategies and as a prognosis indicator. Furthermore, by combining
the use of the culture technique with assaying for such markers,
factors and antigens, a treatment strategy for a disease state may
be optimized and treatment progression may be monitored.
[0033] One important aspect of analyzing tissue culture medium is
that complications of a malignancy can be predicted. For instance,
one common complication is thrombogenesis. A propensity towards
blood clot formation can be detected in tissue culture medium by
identifying thrombogenic or procoagulant factors such as, without
limitation, the Lewis Y antigen (Ley), HLA-DR and other tumor
procoagulants, such as cancer procoagulant (CP) and tissue factor
(TF). By identifying production of thrombogenic factors, a
physician can prescribe drug and/or exercise regimens, as
appropriate, to prevent life and/or limb-threatening clotting.
[0034] Cells and/or tissue culture media from any of the prime
culture, the reference culture or subcultures thereof can be
analyzed for tumor aggressiveness and invasiveness markers.
Presence of these markers or absence thereof is highly relevant to
a patient's prognosis. Furthermore, the effect of a given therapy
on any of these markers can be analyzed. For instance, a tumor may
produce angiogenic factors, such as, without limitation, vascular
endothelial growth factor (VEGF), which would lead a doctor to give
a patient a less favorable prognosis. Other markers can include,
without limitation, factors which allow cancer cells to affix to
organs other than those from which the cancer cells derive, for
instance, beta 3 integrin, which participates in the ability of
melanoma cells to adhere to blood vessel walls. However, the
effectiveness of therapies can be assessed if the presence of the
angiogenic marker is analyzed in segregated sites according to the
method of the present invention. A physician can suppress a
malignancy by preventing expression of factors or markers which
cause the malignancy's aggressiveness or invasiveness.
[0035] An important application of the present invention is the
screening of chemotherapeutic agents and other anti-neoplastic
therapies in tissue culture preparations of malignant cells from
the patients from whom malignant samples are biopsied. Related
anti-cancer therapies which also can be screened using the
inventive system include radiation therapy and agents which enhance
the cytotoxicity of radiation, as well as immunotherapeutic
anti-cancer agents. Screening processes for treatments or
therapeutic agents for nonmalignant syndromes are also embraced
within this invention and include without limitation agents which
combat hyper-proliferative syndromes, such as psoriasis, or wound
healing agents. Nor is the present efficacy assay limited only to
the screening of active agents which speed up (healing) or slow
down (anti-cancer, anti-hyper-proliferative) cell growth because
agents intended to enhance or to subdue intracellular biochemical
functions may be tested in the present tissue culture system also.
For example, the formation or blocking of enzymes,
neurotransmitters and other biochemicals may be screened with the
present assay methods prior to treatment of the patient.
[0036] When a patient is to be treated for the presence of tumor,
in the preferred embodiment of the present invention a tumor biopsy
of >100 mg of non-necrotic, non-contaminated tissue is harvested
from the patient by any suitable biopsy or surgical procedure known
in the art. Biopsy sample preparation generally proceeds as follows
under a Laminar Flow Hood which should be turned on at least 20
minutes before use. Reagent grade ethanol is used to wipe down the
surface of the hood prior to beginning the sample preparation. The
tumor is then removed, under sterile conditions, from the shipping
container and is minced with sterile scissors. If the specimen
arrives already minced, the individual tumor pieces should be
divided into four groups. Using sterile forceps, each undivided
tissue quarter is then placed in 3 ml sterile growth medium
(Standard F-10 medium containing 17% calf serum and a standard
amount of Penicillin and Streptomycin) and systematically minced by
using two sterile scalpels in a scissor-like motion, or
mechanically equivalent manual or automated opposing incisor
blades. This cross-cutting motion is important because the
technique creates smooth cut edges on the resulting tumor
multicellular particulates. Preferably but not necessarily, the
tumor particulates each measure 1 mm.sup.3. After each tumor
quarter has been minced, the particles are plated in culture flasks
using sterile pasteur pipettes (9 explants per T-25 or 20
particulates per T-75 flask). Each flask is then labeled with the
patient's code, the date of explanation and any other
distinguishing data. The explants should be evenly distributed
across the bottom surface of the flask, with initial inverted
incubation in a 37.degree. C. incubator for 5-10 minutes, followed
by addition of about 5-10 ml sterile growth medium and further
incubation in the normal, non-inverted position. Flasks are placed
in a 35.degree. C., non-CO.sub.2 incubator. Flasks should be
checked daily for growth and contamination. Over a period of a few
weeks, with weekly removal and replacement of 5 ml of growth
medium, the explants will foster growth of cells into a monolayer.
With respect to the culturing of malignant cells, it is believed
(without any intention of being bound by the theory) that by
maintaining the malignant cells within a multicellular particulate
of the originating tissue, growth of the malignant cells themselves
is facilitated versus the overgrowth of fibroblasts (or other
unwanted normal cells) which tends to occur when suspended tumor
cells are grown in culture.
[0037] The use of the above procedure to form a cell monolayer
culture maximizes the growth of malignant cells from the tissue
sample, and thus optimizes ensuing tissue culture assay of
chemotherapeutic action of various agents to be tested. Enhanced
growth of actual malignant cells is only one aspect of the present
invention; however, another important feature is the growth rate
monitoring system used to oversee growth of the monolayer once
formed. Once a primary culture and its derived secondary monolayer
tissue culture has been initiated, the growth of the cells is
monitored to ascertain the time to initiate the chemotherapy assay
and to determine the growth rate of the cultured cells.
[0038] Monitoring of the growth of cells is conducted by counting
the cells in the monolayer on a periodic basis, without killing or
staining the cells and without removing any cells from the culture
flask. The counting may be done visually or by automated methods,
either with or without the use of estimating techniques known in
the art (counting in a representative area of a grid multiplied by
number of grid areas, for example). Data from periodic counting is
then used to determine growth rates which may or may not be
considered parallel to growth rates of the same cells in vivo in
the patient. If growth rate cycles can be documented, for example,
then dosing of certain active agents can be customized for the
patient. The same growth rate can be used to evaluate radiation
treatment periodicity, as well. It should be noted that with the
growth rate determinations conducted while the monolayers grow in
their flasks, the present method requires no hemocytometry, flow
cytometry or use of microscope slides and staining, with all their
concomitant labor and cost.
[0039] Protocols for monolayer growth rate generally use a
phase-contrast inverted microscope to examine culture flasks
incubated in a 37.degree. C. (5% CO.sub.2) incubator. When the
flask is placed under the phase-contrast inverted microscope, ten
fields (areas on a grid inherent to the flask) are examined using
the 10.times. objective, with the proviso that the ten fields
should be non-contiguous, or significantly removed from one
another, so that the ten fields are a representative sampling of
the whole flask. Percentage cell occupancy for each field examined
is noted, and averaging of these percentages then provides an
estimate of overall percent confluency in the cell culture. When
patient samples have been divided between two or among three or
more flasks, an average cell count for the total patient sample
should be calculated. The calculated average percent confluency
should be entered into a process log to enable compilation of
data--and plotting of growth curves--over time. Monolayer cultures
may be photographed to document cell morphology and culture growth
patterns. The applicable formula is: 1 Percent confluency =
estimate of the area occupied by cells total area in an observed
field.
[0040] As an example, therefore, if the estimate of area occupied
by the cells is 30% and the total area of the field is 100%,
percent confluency is 30/100, or 30.
[0041] Adaptation of the above protocol for non-tumor cells is
straightforward and generally constitutes an equivalent
procedure.
[0042] Active agent and/or radiation therapy screening using the
cultured cells proceeds with subcultures of the prime culture or,
preferably, of the reference culture. The screening can be carried
out in an incubation flask, but generally proceeds using plates
such as microtiter plates. In a chemotherapy/radiotherapy assay, it
is desirable to grow a control culture of a patient's cells in a
culture parallel to the reference or prime culture. The control
culture can be grown from skin cells, as an easy source of
non-malignant cells, from the same organ from which the malignant
cells are derived, or from other sources, so long as the cells are
typical of non-malignant cells of the patient.
[0043] The performance of the chemosensitivity/radiosensitivity
assay used for screening purposes depends on the ability to deliver
a reproducible cell number to each row in a plate and/or a series
of plates, as well as the ability to achieve an even distribution
of cells throughout a given well. The following procedure assures
that cells are reproducibly transferred from flask to microtiter
plates, and cells are evenly distributed across the surface of each
well.
[0044] The first step in preparing the microtiter plates is, of
course, preparing and monitoring the monolayer as described above.
The following protocol is exemplary and susceptible of variation as
will be apparent to one skilled in the art. Cells are removed from
the culture flask and a cell pellet is prepared by centrifugation.
The cell pellet derived from the monolayer is then suspended in 5
ml of the growth medium and mixed in a conical tube with a vortex
for 6 to 10 seconds. The tube is then rocked back and forth 10
times. A 36 .mu.l droplet from the center of the conical tube is
pipetted onto one well of a 96 well plate. A fresh pipette is then
used to pipette a 36 .mu.l aliquot of trypan blue solution, which
is added to the same well, and the two droplets are mixed with
repeated pipette aspiration. The resulting admixture is then
divided between two hemocytometer chambers for examination using a
standard light microscope. Cells are counted in two out of four
hemocytometer quadrants, under 10.times. magnification. Only those
cells which have not taken up the trypan blue dye are counted. This
process is repeated for the second counting chamber. An average
cell count per chamber is thus determined. Using means known in the
art, the quadrant count values are checked, logged, multiplied by
10.sup.4 to give cells/ml, and the total amount of fluid (growth
medium) necessary to suspend remaining cell aliquots is calculated
accordingly.
[0045] After the desired concentration of cells in medium has been
determined, additional cell aliquots from the monolayer are
suspended in growth medium via vortex and rocking and loaded into a
Terasaki dispenser known in the art. Aliquots of the prepared cell
suspension are delivered into the microtiter plates using Terasaki
dispenser techniques known in the art. A plurality of plates may be
prepared from a single cell suspension as needed. Plates are then
wrapped in sterile wet cotton gauze and incubated in an incubator
box by means known in the art.
[0046] After the microtiter plates have been prepared, exposure of
the cells therein to active agent and/or radiation is conducted
according to the following exemplary protocol. During this portion
of the inventive assay, the appropriate amount of specific active
agent is transferred into the microtiter plates prepared as
described above. A general protocol, which may be adapted, follows.
Each microtiter plate is unwrapped from its wet cotton gauze sponge
and microscopically examined for cell adhesion. Control solution is
dispensed into delineated rows of wells within the grid in the
microtiter plate, and appropriate aliquots of active agent to be
tested are added to the remaining wells in the remaining rows.
Ordinarily, sequentially increasing concentrations of the active
agent or higher doses of radiation being tested are administered
into progressively higher numbered rows in the plate. The plates
are then rewrapped in their gauze and incubated in an incubator box
at 37.degree. C. under 5% CO.sub.2. After a predefined exposure
time, the plates are unwrapped, blotted with sterile gauze to
remove the agent, washed with Hank's Balance Salt Solution, flooded
with growth medium, and replaced in the incubator in an incubator
box for a predefined time period, after which the plates may be
fixed and stained for evaluation.
[0047] Fixing and staining may be conducted according to a number
of suitable procedures; the following is representative. After
removal of the plates from the incubator box, culture medium is
poured off and the plates are flooded with Hank's Balance Salt
Solution. After repeated flooding (with agitation each time) the
plates are then flooded with reagent grade ethanol for 2-5 minutes.
The ethanol is then poured off. Staining is accomplished with
approximately 5 ml of Giemsa Stain per plate, although volume is
not critical and flooding is the goal. Giemsa stain should be left
in place 5 min. .+-.30 seconds as timing influences staining
intensity. The Giemsa stain is then poured off and the plates are
dipped three times in cold tap water in a beaker. The plates are
then inverted, shaken vigorously, and air dried overnight (with
plate lids off) on a rack on a laboratory bench. Cells per well are
then counted manually or by automated and/or computerized means, to
derive data regarding chemosensitivity of cells at various
concentrations of exposure. One particularly useful computer
operating environment for counting cells is the commercially
available OPTIMATE compiler, which is designed to permit an optical
counting function well suited to computerized cell counting
procedures and subsequent calculations.
[0048] The above procedures do not change appreciably when cell
growth promoters are assayed rather than cell arresting agents such
as chemotherapeutic or radiotherapeutic agents. The present assay
allows cell death or cell growth to be monitored with equal ease.
In any case, optimization of use of the present system will involve
the comparative testing of a variety of candidate active agents for
selection of the best candidate for patient treatment based upon
the in vitro results. One particularly advantageous embodiment of
the above described invention comprises a two-stage assay for
cytotoxicity followed by evaluation of longer-term inhibitory
effect. Chemotherapeutic agents may thus be evaluated separately
for both their direct chemotherapeutic effect as well as for their
longer duration efficacy.
[0049] As discussed in brief, above, in parallel with growth of the
prime or reference culture, a control culture can be grown. The
control culture is a culture of normal cells taken from the same
patient from whom the prime culture is collected. The control
culture can derive from an epithelial cell sample or can be
collected from the same organ as the prime culture so long as the
control culture contains no malignant cells. More than one control
culture can be maintained. For instance, cultures of both normal
skin cells and normal cells of an organ from which the malignancy
is derived can be maintained. The value of maintaining a control
culture is many fold. Primarily, the control culture serves as a
negative control (or positive control, depending upon the marker to
be analyzed) in the various analyses to be carried out on the prime
culture, the reference culture or subcultures thereof.
[0050] A second value of the control culture is an indicator of
toxicity, the toxicity or undesirable effects of a given therapy
upon normal cells. For instance, in the segregated analysis of
chemotherapeutic agents described above, concomitant analysis of
the same agents on segregated sites of the control culture would
yield an indication of cytotoxicity of the agent with regard to
malignant cells versus the toxicity of the agent to control cells.
A therapeutic index can be calculated based on the ratio of
cytotoxicity to malignant cells to toxicity. Cytotoxicity and
toxicity can be quantified as a percentage or fraction of cells
killed by a given therapy, or as a percentage or fraction of cells
surviving a given therapy. A therapeutic index is a ratio of these
percentages or fractions and is reflective of the desirability of a
given treatment in a patient. An optimal treatment would be
maximally cytotoxic (or even cytostatic) to the malignant cells and
minimally toxic to a patient's normal cells.
[0051] Other indices may be generated, depending upon the desired
effect of a therapy. For instance, if a desired therapy is designed
to up-regulate a malignancy-specific antigen to promote destruction
of the malignancy by a patient's immune system, an index could be
generated to discern a treatment which reflects maximal
up-regulation of the antigen in the malignant cells and minimal or
negative up-regulation in a patient's normal cells. A similar index
can be calculated based upon down-regulation of a desired marker
(i.e., an angiogenic factor) which can be assayed as either a
secreted or a cellular marker and reflects maximal down-regulation
of the marker with minimal toxicity or other undesirable effects on
the control culture.
[0052] Often the diseased cells express a cellular marker that is
indicative of a certain disease state or lack thereof. However, one
aspect of the culture techniques of the present invention is that
the cultured diseased cells do not necessarily have to be the cells
expressing the factor to be assayed. One question that inevitably
arises when considering whether a serum marker is indicative of a
particular cancer cell is, which cells produce the marker, the cell
or the tissue in which the cancer cells grow? See e.g. Singhal et
al., p 610. By co-culturing the cancerous tissue within a
multicellular particulate of its originating tissue, the cells
(both the diseased cells or the surrounding cells) are better able
to retain their production of characteristic markers.
[0053] Identification of one or more active agents or
chemotherapeutic agents is peripheral to the present invention,
which is intended for the efficacy screening of any or all of them
as to a given patient. Literally any active agent may be screened
according to the present invention; listing exemplary active agents
is thus omitted here.
[0054] One important focus of the present invention thus includes
the simplicity of the present system--cohesive multicellular
particulates of the patient tissue to be tested are used to form
cell monolayers; growth of those monolayers is monitored for
accurate prediction of correlating growth of the same cells in
vivo; and differing concentrations of a number of active agents may
be tested for the purpose of determining not only the most
appropriate agent but the most appropriate concentration of that
agent for actual patient exposure (according to the calculated cell
growth rates). It is also important to note, in the context of the
invention, that the present system allows in vitro tests to be
conducted in suspensions of tissue culture monolayers grown in
nutrient medium under fast conditions (a matter of weeks), rather
than with single cell progeny produced by dilution cloning over
long periods of time. In some cases, the present invention is a two
stage assay for both cytotoxicity and the longer-term growth
inhibitory.
[0055] It is additionally possible to increase the value of the
assay with the use of staining compositions and protocols designed
to characterize the malignant cells thus grown. In other words, the
tissue preparation and cell culturing technique itself offers a
first assurance that the cells grown out of the tumor are really
the malignant tumor cells and not fibroblasts or other nonmalignant
cells of no diagnostic value. As a separate confirmation, the
present staining compositions and protocols offer a second,
independent assurance that the cells subject to diagnostic or
prognostic assay are in fact malignant cells in culture. One
important characterization has to do with the nature of the
malignant cells as epithelial, which is in turn an indicator of the
carcinoma type of malignancy. Other characterizations of malignant
cells are intended to fall within the scope of the present
invention as well, although the characterization of the cells as
epithelial or not is of primary importance.
[0056] The technique is practiced as follows. The same cell
culturing and well distribution process is used as in the
cytotoxicity assay described above, but rather than exposing the
cells to chemotherapeutic or other agents, the cells are instead
fixed and stained. With the stain or stain cocktail described
below, the epithelial cells are identified by their intermediate
filaments and/or specific membrane antigens by means of a
monoclonal antibody immunoperoxidase technique. The fixative used
can be any fixative which does not alter the cellular molecular
markers of interest. The fixed, stained cells are then counted. If
the specimen is positive for epithelial cells, the process is
complete. If the specimen is negative for epithelial cells, an
independent fixing and staining process is subsequently completed,
with fresh cells from identical wells, using Vimentin as a stain to
confirm the non-epithelial nature of the cells.
[0057] The importance of having a stain or stain cocktail (i.e.,
antibody cocktail), as well as an overall protocol, for identifying
epithelial cells in biopsies of malignant tumors is as follows. In
the basic cytotoxicity assay, the tissue culture technique is
designed to grow out the cells of the tumor of origin and in fact
consistently does so. Despite such reliable predictability,
however, the fact that the cells of the tumor of origin did in fact
grow out, and not fibroblasts or other cells, must be confirmed
with independent proof before the cells can be used with complete
assurance in the appropriate patient assay(s). The present
technology provides a means to obtain this confirmation, which in
turn furthers the interests of good laboratory and medical
practice.
[0058] As a general consideration, the staining compounds or
compositions of interest for use in the present technology are
those which bind with cellular molecular markers unique either to
epithelial or to non-epithelial cells. A further aspect of the
invention therefore inheres in the following two aspects: the
improvement of the cytotoxicity assay by adding the epithelial
staining protocol with any known epithelial stain; and the further
improvement wherein specially designed stain cocktails maximize the
likelihood that the presence of any known intermediate filament or
specific membrane antigen, characteristic of epithelial cells, will
be identified if present.
[0059] Many carcinomas are positive for any one of the intermediate
filaments or specific membrane antigens characteristic of
epithelial cells; virtually all if not all carcinomas are positive
for one of a number of such intermediate filaments or specific
membrane antigens. For example, "epithelial membrane antigen" (EMA)
glycoproteins are known in the art and can be bound with various
antiepithelial membrane antigen antibodies including monoclonal
antibodies. Cytokeratin is another important epithelial cell marker
and binding reagents including monoclonal antibodies are available
which are specific to cytokeratin. While antisera can be raised in
vivo against markers such as EMA glycoproteins and cytokeratin, as
a practical matter commercially available polyclonal or monoclonal
antibodies are used in the following protocols, with monoclonal
antibodies being preferred.
[0060] Binding of the epithelial marker is revealed with associated
staining procedures and reactions which give a visual indication
that the marker binding took place. Those skilled in the art
already appreciate various techniques already available--in the
general field of "immunocytochemistry" --to reveal antibody-antigen
reactions. One known way to accomplish this visualization when
antibody binding reagents are used is with the "labeled
streptavidin procedure". In this procedure, after the specimen is
exposed to antibodies specific to the target antigen, a secondary
"link" antibody is added. The secondary biotinylated "link"
antibody consists of anti-mouse and anti-rabbit antibodies which
bind universally to most primary monoclonal or polyclonal
antibodies. The "link" will also connect to the tertiary reagent
(peroxidase-labeled streptavidin) through chemical bonding between
the biotin on the secondary reagent and the streptavidin on the
streptavidin/peroxidase conjugate. Staining is completed by
incubating the specimen and primary, secondary and tertiary agents
in the presence of a chromogen, so that the peroxidase and the
chromogen form a visible precipitate. Alternatively, a
fluorescein-based detection system can be used to visualize the
primary antibody, or a third alternative known in the art as the
digoxigenin-conjugated detection system may be used.
[0061] Of the various epithelial markers, three have received the
most widespread attention in the literature: EMA glycoproteins,
cytokeratin, and carcinoembryonic antigen. In the context of this
invention, the first two are the most important because literally
any epithelial cell will have at least either one EMA glycoprotein
on the surface thereof or a cytokeratin intermediate filament
present. Therefore, the present invention resides not only in
binding and staining for an epithelial marker on the surfaces of
the specimen cells, but in simultaneously assaying for either or
both of EMA glycoprotein(s) and cytokeratin. The cocktails of the
present invention therefore contain binding reagents for both EMA
glycoproteins and cytokeratin and, importantly, are selected to
include the most generally applicable binding reagents in
combination so that the cocktail has the broadest binding scope
possible. The cocktails identified in Examples 1 and 2, for
example, represent a combination of two general binding reagents
(containing a total of three monoclonal antibodies) for
cytokeratin, admixed with a general binding reagent for EMA
glycoprotein. The dual benefit of this admixture of general binding
agents is that the incidence of false negatives for epithelial
cells is minimized, and the visible staining reactions are
generally stronger when the combined binding reagents are used in
lieu of a single binding reagent.
[0062] Although the binding reagents and other reagents identified
in the Examples are the preferred reagents for use in the context
of the invention, the invention is intended to encompass
epithelial-specific binding and staining reagents generally. These
include, without limitation: Boehringer-Mannheim AE1
anti-cytokeratin antibody; Boehringer-Mannheim AE3 anti-cytokeratin
antibody; Boehringer-Mannheim AE1/AE3 anti-cytokeratin antibody
(AE1 and AE3 in admixture); Becton-Dickinson CAM 5.2 antibody, DAKO
EMA antibody, Biomeda's Anti-Cytokeratin Cocktail CK22, Biomeda's
Anti-Cytokeratin Cocktail CK23, Biomeda's Anti-Pan-Cytokeratin
CK56, Biomeda's polyclonal goat or rabbit anti-cytokeratin
antisera, ScyTek Laboratories' anti-EMA antigen antibody clone E29,
and many others. Those skilled in the art and in possession of the
guidance provided herein can readily determine alternative,
equivalent binding and staining reagents and cocktails, to
accomplish the disclosed result. These binding agents and cocktails
may be used in combination with any known visualization system,
such as the streptavidin, fluorescein- and digoxigenin-conjugated
systems identified above.
[0063] As a control, Vimentin antibody is used as a binding
alternative either in conjunction with binding and staining of the
test cells, or subsequently thereto. In the context of this
invention, Vimentin can be considered a binding reagent which is
specific to non-epithelial cells of mesenchymal origin.
[0064] In a further aspect of the present invention, immunological
markers may be monitored in applications requiring up- or down-
regulation of such markers (i.e., Major histocompatibility complex
molecules). This aspect of the present invention can be especially
useful in transplantation applications where, for instance, through
chemical or biological means rejection of transplanted cells is
sought to be avoided by down-regulation of the various
transplantation antigens present on the cells to be transplanted.
The present invention would be especially useful in monitoring such
immunoregulation.
[0065] Lastly, cell morphology can be assayed by culturing cells
of, i.e., the prime culture or the reference culture, removing the
cells from the surface upon which they grow, centrifuging cells
into a loose pellet and growing the cell pellet over a defined time
period. By growing cells in this manner, it is possible to view the
cohesive morphology of cells in a cluster resembling a tumor.
EXAMPLE 1
Radiation Therapy
[0066] Separate 50 mg samples from residual tissue from specimens
of three human glioblastomas and one human ovarian carcinoma were
minced in medium with sterile scissors to a particle size of
roughly 1 mm.sup.3 and with a particle size distribution between
about 0.25 and about 1.5 mm.sup.3. The medium was Standard F-10
medium containing 17% calf serum and a standard amount of
Penicillin and Streptomycin. Each 50 mg sample was minced and was
divided into four groups of particulates and each of 16 groups was
charged to a separate labeled culture flask containing the
above-described medium. Visual confirmation was made that the
particulates were evenly distributed along the bottom of each flask
and the flasks were placed in a 35.degree. C., non-CO.sub.2
incubator. Flasks were checked daily for growth and contamination.
Over a period of a few weeks, with weekly removal and replacement
of 5 ml of growth medium, the particulates grew into
monolayers.
[0067] Enough cells were then removed from the monolayers grown in
the flasks for centrifugation into standard size cell pellets for
each of the 16 flasks. Each cell pellet was then suspended in 5 ml
of the above-described medium and was mixed in a conical tube with
a vortex for 6 to 10 seconds, followed by manual rocking back and
forth 10 times. A 36 ml droplet from the center of each tube was
then pipetted into one well of a 96-well microtiter plate together
with an equal amount of trypan blue, plus stirring. The resulting
admixture was then divided between two hemocytometer quadrants for
examination using a standard light microscope. Cells were counted
in two out of four hemocytometer quadrants, under 10.times.
magnification--only those cells which had not taken up the trypan
blue dye were counted. This process was repeated for the second
counting chamber. An average cell count per chamber was calculated
and by means known in the art the optimum concentration of cells in
the medium was determined.
[0068] Accommodating the above calculations, additional cell
aliquots from the 16 monolayers were separately suspended in growth
medium via vortex and rocking and were loaded into a Terasaki
dispenser adapted to a 60-well plate. Aliquots of the prepared cell
suspension were delivered into the microtiter plates using Terasaki
dispenser techniques known in the art. Cells were plated into
60-well microtiter plates at a concentration of 100 cells per
well.
[0069] Twenty-four (24) hours later, the cells were irradiated
using a Siemens Stabilipan X-ray machine at 250 kVp, 15 mA with a
dose rate of 75 rad/minute. For each radiation dose from 1Gy to
6Gy, cell number per well was monitored as a function of time
through five days post-irradiation.
[0070] Cell number relative to controls was determined and survival
curves were fit to the data. The rate of decrease in survival as a
function of time was proportional to dose. A differential radiation
response among the four cell lines was observed.
EXAMPLE 2
Immuno Therapy
[0071] Separate 50 mg samples from residual tissue from specimens
of a human brain tumor, renal carcinoma, and breast carcinoma were
minced in medium with sterile scissors to a particle size of
roughly 1 mm.sup.3 and with a particle size distribution between
about 0.25 and about 1.5 mm.sup.3. The medium was Standard F-10
medium containing 17% calf serum and a standard amount of
Penicillin and Streptomycin. Each 50 mg sample was minced and was
divided into four groups of particulates and each of 12 groups was
charged to a separate labeled culture flask containing the
above-described medium. Visual confirmation was made that the
particulates were evenly distributed along the bottom of each flask
and the flasks were placed in a 35.degree. C., non-CO.sub.2
incubator. Flasks were checked daily for growth and contamination.
Over a period of a few weeks, with weekly removal and replacement
of 5 ml of growth medium, the particulates grew into
monolayers.
[0072] Enough cells were then removed from the monolayers grown in
the flasks for centrifugation into standard size cell pellets for
each of the twelve flasks. Each cell pellet was then suspended in 5
ml of the above-described medium and was mixed in a conical tube
with a vortex for 6 to 10 seconds, followed by manual rocking back
and forth 10 times. A 36 ml droplet from the center of each tube
was then pipetted into one well a 96-well microtiter plate together
with an equal amount of trypan blue, plus stirring. The resulting
admixture was then divided between two hemocytometer quadrants for
examination using a standard light microscope. Cells were counted
in two out of four hemocytometer quadrants, under 10.times.
magnification--only those cells which had not taken up the trypan
blue dye were counted. This process was repeated for the second
counting chamber. An average cell count per chamber was calculated
and by means known in the art the optimum concentration of cells in
the medium was determined.
[0073] Accommodating the above calculations, additional cell
aliquots from the 12 monolayers were separately suspended in growth
medium via vortex and rocking and were loaded into a Terasaki
dispenser adapted to a 60-well plate. Aliquots of the prepared cell
suspension were delivered into the microtiter plates using Terasaki
dispenser techniques known in the art. Cells were plated into
60-well microtiter plates at a concentration of 100 cells per
well.
[0074] Twenty-four (24) hours post-plating, Activated Natural
Killer (ANK) cells were delivered into a row of six wells by means
of a micropipette. In each microtiter plate three rows of six wells
each served as controls. The effector (ANK cells):target cell
(tumor cells) ratio varied from 2.5:1 to 20:1. The ANK cells were
exposed to the target cells for four hours. Subsequently, the wells
were washed with Hanks Balanced Salt Solution and the number of ANK
cells remaining in the wells was observed with a phase contrast
microscope. This process was repeated until no ANK cells remained
in the wells (usually 3 washes). Following removal of the ANK
cells, the tumor cells were incubated in the wells for another 24
hours.
[0075] Cell number relative to control was determined. For the
three tumor types increasing the effector: target cell ratio from
2.5:1 to 20:1 resulted in an increase in the number of tumor cells
killed by the ANK cells.
EXAMPLE 3
Gene Therapy/Antisense Oligonucleotides
[0076] A 50 mg sample from a residual human mesothelioma was minced
in medium with sterile scissors to a particle size of roughly 1
mm.sup.3 and with a particle size distribution between about 0.25
and about 1.5 mm.sup.3. The medium was Standard F-10 medium
containing 17% calf serum and a standard amount of Penicillin and
Streptomycin. The 50 mg sample was minced and was divided into four
groups of particulates and each of four groups was charged to a
separate labeled culture flask containing the above-described
medium. Visual confirmation was made that the particulates were
evenly distributed along the bottom of each flask and the flasks
were placed in a 35.degree. C., non-CO.sub.2 incubator. Flasks were
checked daily for growth and contamination. Over a period of a few
weeks, with weekly removal and replacement of 5 ml of growth
medium, the particulates grew into monolayers.
[0077] Enough cells were then removed from the monolayers grown in
the flasks for centrifugation into standard size cell pellets for
each of the four flasks. Each cell pellet was then suspended in 5
ml of the above-described medium and was mixed in a conical tube
with a vortex for 6 to 10 seconds, followed by manual rocking back
and forth 10 times. A 36 ml droplet from the center of each tube
was then pipetted into one well of a 96-well microtiter plate
together with an equal amount of trypan blue, plus stirring. The
resulting admixture was then divided between two hemocytometer
quadrants for examination using a standard light microscope. Cells
were counted in two out of four hemocytometer quadrants, under
10.times. magnification--only those cells which had not taken up
the trypan blue dye were counted. This process was repeated for the
second counting chamber. An average cell count per chamber was
calculated and by means known in the art the optimum concentration
of cells in the medium was determined.
[0078] Accommodating the above calculations, additional cell
aliquots from the 4 monolayers were separately suspended in growth
medium via vortex and rocking and were loaded into a Terasaki
dispenser adapted to a 60-well plate. Aliquots of the prepared cell
suspension were delivered into the microtiter plates using Terasaki
dispenser techniques known in the art. Cells were plated into
60-well microtiter plates at a concentration of 100 cells per
well.
[0079] Twenty-four (24) hours post-plating, antisense
oligonucleotide for the urokinase-type plasminogen activator
receptor (uPAR) was delivered to wells in the microtiter plate.
Proteolysis of plasminogen to plasmin by urokinase-type plasminogen
activator has been implicated in the processes of tumor cell
proliferation and invasion. The concentrations of the uPAR
antisense oligonucleotide were 1, 10 and 100 micromolar. uPAR sense
and missense oligonucleotides at the concentrations of 1, 10 and
100 micromolar served as controls. The tumor cells were exposed to
the oligonucleotides for 24 hours and then the agents were removed.
The cells were allowed to incubate for another 72 hours so that
inhibition of cell proliferation could be observed.
[0080] Cell number relative to control was then determined.
Antisense oligonucleotides to uPAR suppressed the proliferative
activity of the tumor cells in a concentration dependent
manner.
EXAMPLE 4
Combination Chemotherapy
[0081] Separate 50 mg samples from residual tissue from specimens
from four human ovarian tumors were minced in medium with sterile
scissors to a particle size of roughly 1 mm.sup.3 and with a
particle size distribution between about 0.25 and about 1.5
mm.sup.3. The medium was Standard F-10 medium containing 17% calf
serum and a standard amount of Penicillin and Streptomycin. Each 50
mg sample was minced and was divided into 4 groups of particulates
and each of 16 groups was charged to a separate labeled culture
flask containing the above-described medium. Visual confirmation
was made that the particulates were evenly distributed along the
bottom of each flask and the flasks were placed in a 35.degree. C.,
non-CO.sub.2 incubator. Flasks were checked daily for growth and
contamination. Over a period of a few weeks, with weekly removal
and replacement of 5 ml of growth medium, the particulates grew
into monolayers.
[0082] Enough cells were then removed from the monolayers grown in
the flasks for centrifugation into standard size cell pellets for
each of the 16 flasks. Each cell pellet was then suspended in 5 ml
of the above-described medium and was mixed in a conical tube with
a vortex for 6 to 10 seconds, followed by manual rocking back and
forth 10 times. A 36 ml droplet from the center of each tube was
then pipetted into one well of a 96-well microtiter plate together
with an equal amount of trypan blue, plus stirring. The resulting
admixture was then divided between two hemocytometer quadrants for
examination using a standard light microscope. Cells were counted
in two out of four hemocytometer quadrants, under 10.times.
magnification--only those cells which had not taken up the trypan
blue dye were counted. This process was repeated for the second
counting chamber. An average cell count per chamber was calculated
and by means known in the art the optimum concentration of cells in
the medium was determined.
[0083] Accommodating the above calculations, additional cell
aliquots from the 16 monolayers were separately suspended in growth
medium via vortex and rocking and were loaded into a Terasaki
dispenser adapted to a 60-well plate. Aliquots of the prepared cell
suspension were delivered into the microtiter plates using Terasaki
dispenser techniques known in the art. Cells were plated into
60-well microtiter plates at a concentration of 100 cells per
well.
[0084] Twenty-four (24) hours post-plating, the chemotherapeutic
agent Taxol was applied to the wells in the microtiter plates. The
first three treatment rows in the plates (Rows 2, 3, and 4) were
designed to have escalating Taxol doses (1.0, 5.0, and 25 .mu.M)
with a fixed carboplatin dose (200 .mu.M). The last three treatment
rows in the plates (Rows 6, 7, and 9) were designed to have a fixed
Taxol dose (5 .mu.M) with an escalating carboplatin dose (50, 200,
and 1000 .mu.M). Rows 5 and 9 served as a control. The Taxol
exposure time was two hours. Twenty-four hours later, the cells in
the wells were exposed to carboplatin for two hours. The tumor
cells in the wells were then incubated for another 48 hours.
[0085] Cell number relative to control was determined. For the
cells from the four tumor specimens a dose response relationship
was observed for both the escalating Taxol/fixed carboplatin and
fixed Taxol/escalating carboplatin treatment schema.
EXAMPLE 5
Hormonal Therapy
[0086] Separate 50 mg samples from residual tissue from specimens
from four human breast tumors were minced in medium with sterile
scissors to a particle size of roughly 1 mm.sup.3 and with a
particle size distribution between about 0.25 and about 1.5
mm.sup.3. The medium was Standard F-10 medium containing 17% calf
serum and a standard amount of Penicillin and Streptomycin. Each 50
mg sample was minced and was divided into four groups of
particulates and each of 16 groups was charged to a separate
labeled culture flask containing the above-described medium. Visual
confirmation was made that the particulates were evenly distributed
along the bottom of each flask and the flasks were placed in a
35.degree. C., non-CO.sub.2 incubator. Flasks were checked daily
for growth and contamination. Over a period of a few weeks, with
weekly removal and replacement of 5 ml of growth medium, the
particulates grew into monolayers.
[0087] Enough cells were then removed from the monolayers grown in
the flasks for centrifugation into standard size cell pellets for
each of the 16 flasks. Each cell pellet was then suspended in 5 ml
of the above-described medium and was mixed in a conical tube with
a vortex for 6 to 10 seconds, followed by manual rocking back and
forth 10 times. A 36 ml droplet from the center of each tube was
then pipetted into one well of a 96-well microtiter plate together
with an equal amount of trypan blue, plus stirring. The resulting
admixture was then divided between two hemocytometer quadrants for
examination using a standard light microscope. Cells were counted
in two out of four hemocytometer quadrants, under 10.times.
magnification--only those cells which had not taken up the trypan
blue dye were counted. This process was repeated for the second
counting chamber. An average cell count per chamber was calculated
and by means known in the art the optimum concentration of cells in
the medium was determined.
[0088] Accommodating the above calculations, additional cell
aliquots from the 16 monolayers were separately suspended in growth
medium via vortex and rocking and were loaded into a Terasaki
dispenser adapted to a 60-well plate. Aliquots of the prepared cell
suspension were delivered into the microtiter plates using Terasaki
dispenser techniques known in the art. Cells were plated into
60-well microtiter plates at a concentration of 100 cells per
well.
[0089] Twenty-four (24) hours post-plating, the antiestrogenic
compound Tamoxifen was delivered to wells in the microtiter plates.
A stock solution of Tamoxifen was initially prepared by dissolving
1.5 mg of Tamoxifen powder in 1 ml of absolute ethanol and then
adding 9 ml of growth medium. This stock solution was then used to
make Tamoxifen solutions in the concentration range of 10 nM to 20
.mu.M. Six doses of Tamoxifen were used for cells from each of the
four breast tumor specimens. An ethanol solution at a concentration
equivalent to that at the highest Tamoxifen concentration served as
a control. The tumor cells were exposed to Tamoxifen for 24 hours
and then the agent was removed. The cells were allowed to incubate
for another 72 hours so that inhibition of cell proliferation could
be observed.
[0090] Cell number relative to control was then determined. There
was no effect observed when the ethanol-only control wells were
compared to the growth medium-only control wells. The cells of two
of the four breast specimens tested showed an inhibition of cell
proliferation by Tamoxifen exposure. These responses occurred in
the mid to high Tamoxifen concentration ranges.
EXAMPLE 6
Differentiating Agent Therapy ("Biological Response
Modification")
[0091] Separate 50 mg samples from residual tissue from specimens
from four human breast tumors were minced in medium with sterile
scissors to a particle size of roughly 1 mm.sup.3 and with a
particle size distribution between about 0.25 and about 1.5
mm.sup.3. The medium was Standard F-10 medium containing 17% calf
serum and a standard amount of Penicillin and Streptomycin. Each 50
mg sample was minced and was divided into four groups of
particulates and each of 16 groups was charged to a separate
labeled culture flask containing the above-described medium. Visual
confirmation was made that the particulates were evenly distributed
along the bottom of each flask and the flasks were placed in a
35.degree. C., non-CO.sub.2 incubator. Flasks were checked daily
for growth and contamination. Over a period of a few weeks, with
weekly removal and replacement of 5 ml of growth medium, the
particulates grew into monolayers.
[0092] Enough cells were then removed from the monolayers grown in
the flasks for centrifugation into standard size cell pellets for
each of the 16 flasks. Each cell pellet was then suspended in 5 ml
of the above-described medium and was mixed in a conical tube with
a vortex for 6 to 10 seconds, followed by manual rocking back and
forth 10 times. A 36 ml droplet from the center of each tube was
then pipetted into one well of a 96-well microtiter plate together
with an equal amount of trypan blue, plus stirring. The resulting
admixture was then divided between two hemocytometer quadrants for
examination using a standard light microscope. Cells were counted
in two out of four hemocytometer quadrants, under 10.times.
magnification--only those cells which had not taken up the trypan
blue dye were counted. This process was repeated for the second
counting chamber. An average cell count per chamber was calculated
and by means known in the art the optimum concentration of cells in
the medium was determined.
[0093] Accommodating the above calculations, additional cell
aliquots from the 16 monolayers were separately suspended in growth
medium via vortex and rocking and were loaded into a Terasaki
dispenser adapted to a 60-well plate. Aliquots of the prepared cell
suspension were delivered into the microtiter plates using Terasaki
dispenser techniques known in the art. Cells were plated into
60-well microtiter plates at a concentration of 100 cells per
well.
[0094] Twenty-four (24) hours post-plating the differentiating
agent retinoic acid was delivered to wells in the microtiter
plates. A stock solution of retinoic acid was initially prepared by
dissolving retinoic acid powder in 1 ml of dimethyl sulfoxide
(DMSO) and then adding 9 ml of growth medium. This stock solution
was then used to make retinoic acid solutions in the concentration
range of 0.1 to 1.0 mM. Six doses of retinoic acid were used for
cells from each of the four breast tumor specimens. A DMSO solution
at a concentration equivalent to that at the highest retinoic acid
concentration served as a control. The tumor cells were exposed to
retinoic acid for 24 hours and then the agent was removed. The
cells were allowed to incubate for another 72 hours so that
inhibition of cell proliferation could be observed.
[0095] Cell number relative to control was then determined. There
was no effect observed when the DMSO-only control wells were
compared to the growth medium-only control wells. The cells of
three of the four breast specimens tested showed an inhibition of
cell proliferation by retinoic acid exposure. These responses
occurred in the mid to high retinoic acid concentration ranges.
EXAMPLE 7
Combined Modality Therapy Drug/Radiation
[0096] Separate 50 mg samples from residual tissue from specimens
from two human brain tumors and two human ovarian tumors were
minced in medium with sterile scissors to a particle size of
roughly 1 mm.sup.3 and with a particle size distribution between
about 0.25 and about 1.5 mm.sup.3. The medium was Standard F-10
medium containing 17% calf serum and a standard amount of
Penicillin and Streptomycin. Each 50 mg sample was minced and was
divided into four groups of particulates and each of 16 groups was
charged to a separate labeled culture flask containing the
above-described medium. Visual confirmation was made that the
particulates were evenly distributed along the bottom of each flask
and the flasks were placed in a 35.degree. C., non-CO.sub.2
incubator. Flasks were checked daily for growth and contamination.
Over a period of a few weeks, with weekly removal and replacement
of 5 ml of growth medium, the particulates grew into
monolayers.
[0097] Enough cells were then removed from the monolayers grown in
the flasks for centrifugation into standard size cell pellets for
each of the 16 flasks. Each cell pellet was then suspended in 5 ml
of the above-described medium and was mixed in a conical tube with
a vortex for 6 to 10 seconds, followed by manual rocking back and
forth 10 times. A 36 ml droplet from the center of each tube was
then pipetted into one well of a 96-well microtiter plate together
with an equal amount of trypan blue, plus stirring. The resulting
admixture was then divided between two hemocytometer quadrants for
examination using a standard light microscope. Cells were counted
in two out of four hemocytometer quadrants, under 10.times.
magnification--only those cells which had not taken up the trypan
blue dye were counted. This process was repeated for the second
counting chamber. An average cell count per chamber was calculated
and by means known in the art the optimum concentration of cells in
the medium was determined.
[0098] Accommodating the above calculations, additional cell
aliquots from the 16 monolayers were separately suspended in growth
medium via vortex and rocking and were loaded into a Terasaki
dispenser adapted to a 60-well plate. Aliquots of the prepared cell
suspension were delivered into the microtiter plates using Terasaki
dispenser techniques known in the art. Cells were plated into
60-well microtiter plates at a concentration of 100 cells per
well.
[0099] Twenty-four (24) hours post-plating, cells in the microtiter
plate wells were exposed to the chemotherapeutic agent Taxol. One
set of plates was designed to have escalating Taxol doses with
(0.5-25.0 .mu.M) with a fixed radiation dose (2Gy). A second set of
plates was designed to have a fixed Taxol dose (5 .mu.M) with an
escalating radiation dose (1Gy-6Gy). The cells in the plates were
irradiated using a Siemans Stabilipan X-ray machine operating at
250 kVp, 15 mA with a dose rate of 75 rad/minute.
[0100] For each of the two treatment schema, cell number per well
was monitored as a function of time through 5 days post-treatment.
Cell number relative to controls was determined and survival curves
were fit. A differential response among the cells from the four
tumor specimens was observed. Both additive and synergistic cell
killing was noted.
EXAMPLE 8
Initiation of a Prime Culture
[0101] A tumor biopsy of approximately 100 mg of non-necrotic,
non-contaminated tissue was harvested from the patient by surgical
biopsy and transferred to the laboratory in a standard shipping
container. Biopsy sample preparation proceeded as follows. Reagent
grade ethanol was used to wipe down the surface of a Laminar flow
hood. The tumor was then removed, under sterile conditions, from
its shipping container, and cut into quarters with a sterile
scalpel. Using sterile forceps, each undivided tissue quarter was
then placed in 3 ml sterile growth medium (Standard F-10 medium
containing 17% calf serum and a standard amount of Penicillin and
Streptomycin) and was systematically minced by using two sterile
scalpels in a scissor-like motion. The tumor particulates each
measured about 1 mm.sup.3. After each tumor quarter was minced, the
particles were plated in culture flasks using sterile pasteur
pipettes (9 explants per T-25 or 20 particulates per T-75 flask).
Each flask was then labeled with the patient's code, the date of
explanation and any other distinguishing data. The explants were
evenly distributed across the bottom surface of the flask, with
initial inverted incubation in a 37.degree. C. incubator for 5-10
minutes, followed by addition of about 5-10 ml sterile growth
medium and further incubation in the normal, non-inverted position.
Flasks were placed in a 35.degree. C., non-CO.sub.2 incubator.
Flasks were checked daily for growth and contamination. Over a
period of a few weeks, with weekly removal and replacement of 5 ml
of growth medium, the explants grew out into a monolayer.
EXAMPLE 9
Unified Tracking System
[0102] a. Growth Rate
[0103] Following initiation of prime cell culture of a tumor
specimen, the growth rate of the cells was determined until the
chemosensitivity assay was performed. During this time period the
growth was monitored by observing the percent of confluency of the
cells in a flask. These data provide information valuable as a
correlation to possible growth of the tumor in the patient as well
as for the interpretation of the results of the chemosensitivity
assay.
[0104] Three examples of growth rate data are shown in FIGS. 1A-1C.
The percent of confluency of the cultured cells is plotted as a
function of time after the initial seeding of the tissue
specimen.
[0105] Slow Growth Rate (FIG. 1A): 25% confluent after 19 days
[0106] Moderate Growth Rate (FIG. 1B): 60% confluent after 21
days
[0107] Fast Growth Rate (FIG. 1C): 90% confluent after 11 days
[0108] b. Immunohistochemical Staining for Cell Characterization,
etc.
[0109] Many tumor specimens will contain a mixture of cancer and
normal cells. Although in many cases tumor cells will readily grow
in tissue culture, while the normal cells will not, it is important
to be able to distinguish the two cell types. Using
immunoperoxidase techniques to stain cells for various intermediate
filaments, the differences between normal (fibroblast-like) cells
and cells from epithelial tumors were characterized. These
techniques can also be used to identify other tumor cell
characteristics which may have prognostic value.
[0110] An initial attempt at cultured cell characterization has
been to use known epithelial tumor cell lines and a known
fibroblast cell line. The epithelial tumor cell lines all have
stained positively for a mixture ("cocktail") of epithelial
intermediate filament antibodies, (not every line, however, has
stained positively for the three antibodies within the mixture
[AE1/AE3; Cam 5.2; EMA]). Some of the epithelial tumor cells in
culture also stained mildly positive for an antibody against an
intermediate filament characteristic of fibroblasts (vimentin).
When staining for fibroblast intermediate filament (vimentin) in
cell culture, all fibroblast cells were positive. Some focal
staining by epithelial tumor cells for vimentin was also
present.
1 epithelial cocktail vimentin epithelial tumor cells ++ +
fibroblasts - ++
[0111] Testing of intermediate filaments with antibodies for
epithelial cells and vimentin appears to be a method of
distinguishing certain characteristics of tumor and normal
cells.
[0112] c. Response to Chemotherapy
[0113] The tissue culture chemosensitivity assay has been refined
to make it more sensitive for the detection of damage produced by a
variety of chemotherapeutic agents. The initial alteration was to
allow a 24-hour time period between plating of cells in microtiter
wells and the exposure to drugs. This time interval permits cells
to be in an active state of proliferation, where they are more
sensitive to cell cycle active agents. The second change was to
initiate a long-term assay (growth inhibition assay) over a period
of about 72 hours. The short-term assay is conducted 24-72 hours
after the therapeutic agent is added. The longer time between drug
exposure and assay allows for the detection of cell damage which
occurs over a protracted period and requires several cell division
cycles before it becomes apparent. "CI" is a measure of the
relative survival rates of a given cell culture. It is calculated
by according to the formula: 2 CI = ( 1 - No. of cells in treated
wells) No. of cells in control wells
[0114] The data for a short-term assay and a long-term assay
performed on two sets of patient cultured cells are presented in
FIGS. 2A-2F through 5A-5F. The long-term assay (FIGS. 3A-3F and
5A-5F) may both accentuate a positive result obtained from the
short-term assay (FIGS. 2A-2F and 4A-4F) and reveal an effect not
observed during the short-term assay. The long-term assay is now
incorporated into the tissue culture chemosensitivity on a routine
basis.
[0115] d. Response to Radiation Therapy
[0116] The use of the microtiter well assay to analyze the direct
effect of radiation therapy on tumor cells in culture has resulted
in a rapid evaluation method for the determination of inherent
cellular radiation response. As an example, two radiation
dose-response curves generated from the microtiter well assay are
presented in FIGS. 6 and 7. The cells from the tumor specimen in
FIG. 6 are more resistant than those of the specimen in FIG. 7. The
more resistant tumor has been previously irradiated.
[0117] The microtiter well assay is ideally suited for examination
of the interaction of chemotherapeutic agents and radiation. Issues
such as the differential sensitivity of drug/radiation combinations
and the timing of drug/radiation combinations may be directly
addressed with this system. An illustration of chemotherapeutic
agent enhancement of radiation response is presented in FIGS.
8A-8C.
2 FIG. 8A: Radiation-only at 2 Gy and 4 Gy FIG. 8B: Taxol 8.5 ng/ml
+ 2 Gy and 4 Gy FIG. 8C: Taxol 42.5 ng/ml + 2 Gy and 4 Gy
[0118] e. Response to Cellular Immunotherapy
[0119] Activated lymphocytes are being used as a treatment for some
types of cancer. These Activated Natural Killers (ANK) cells have
been shown to mediate highly efficient cell killing for some tumor
types. The microtiter well assay can be utilized to make a rapid
assessment of ANK-induced tumor target cell killing. An
illustration of two such interactions is presented in FIGS. 9A and
9B.
[0120] In FIGS. 9A and 9B, the target cells were from a melanoma
and a renal carcinoma, respectively. The target cells were exposed
to the ANK cells for 4 hours and then the assay was performed. The
effector:target cell ratio varied from 1:20 to 1:2.5. The data show
increasing cell killing as a function of increasing effector:
target ratio.
[0121] f. Use of Tissue Culture Medium for Determination of Factors
with Possible Prognostic/Biological Significance
[0122] A number of substances secreted by tumor cells such as Tumor
Associated Antigens and Plasminogen Activators and Inhibitors are
believed to regulate a variety of processes involved in the
progression of malignant disease. Many of these factors are
produced by tumor cells growing in tissue culture and are secreted
into the growth medium. The measurement of these factors in the
medium from cell cultures of tumor specimens may prove to be of
predictive value in the assessment of the biological behavior of
individual cancers.
[0123] Preliminary work in this area has been on the detection of
plasminogen activator inhibitor in the growth medium of
glioblastoma cell lines. Plasminogen activator inhibitor expression
has been shown to be increased in malignant brain tumors in
patients. Medium from glioblastoma cell lines showed an increase in
plasminogen activator inhibitor when compared to the medium
alone.
[0124] Any or all of the steps of the unified assays and culturing
techniques of the present invention may be automated. Indices can
be automatically calculated by a computer which is programmed
appropriately. Data can be input into the computer either manually
or automatically, into a spreadsheet or database program, or the
like. The spreadsheet or database program can be programed to
reduce the data to the indices described above, or to any other
relevant form, i.e., graphical or figurative representations of the
data.
[0125] In one example, the cells to be assayed are grown on
microtiter plates and assayed for their sensitivity to a
chemotherapeutic agent according to the above-described protocols.
The microtiter plates are read on an optical scanner and data from
the scanner is automatically exported to a computer for calculation
of a therapeutic index. Other types of scanners may be utilized
depending upon the assay. For instance, a scanner for reading RIA
data would be provided if the assay is an RIA assay.
[0126] Although the present invention has been described with
respect to specific materials and methods above, the invention is
only to be considered limited insofar as is set forth in the
accompanying claims.
* * * * *