U.S. patent application number 13/542000 was filed with the patent office on 2012-12-27 for chemo-sensitivity assays using tumor cells exhibiting persistent phenotypic characteristics.
This patent application is currently assigned to Precision Therapeutics, Inc.. Invention is credited to Stacey Brower, Anuja Chattopadhyay, Michael Gabrin, Holly Gallion, Sean McDonald, Payal Nanavati, Shara Dawn Rice.
Application Number | 20120329086 13/542000 |
Document ID | / |
Family ID | 37809652 |
Filed Date | 2012-12-27 |
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United States Patent
Application |
20120329086 |
Kind Code |
A1 |
Gabrin; Michael ; et
al. |
December 27, 2012 |
CHEMO-SENSITIVITY ASSAYS USING TUMOR CELLS EXHIBITING PERSISTENT
PHENOTYPIC CHARACTERISTICS
Abstract
The assays, methods, tools and systems discussed herein
represent an improved and unified system for monitoring the
progression of an individual patient malignancy. The assays,
methods, tools and systems discussed herein represent an improved
and unified system for monitoring and for identifying cellular and
secreted markers, for screening cells to detect phenotypic and
genotypic drift and for predicting chemotherapeutic response of
patient tumor cells to at least one therapeutic agent. The assays,
methods, tools and systems discussed herein also represent an
improved and unified system for monitoring and for screening
multiple pharmaceutical agents for efficacy and long term effect as
to a specific patient.
Inventors: |
Gabrin; Michael;
(Pittsburgh, PA) ; Brower; Stacey; (Pittsburgh,
PA) ; McDonald; Sean; (Pittsburgh, PA) ;
Gallion; Holly; (Pittsburgh, PA) ; Nanavati;
Payal; (Pittsburgh, PA) ; Rice; Shara Dawn;
(Pittsburgh, PA) ; Chattopadhyay; Anuja;
(Pittsburgh, PA) |
Assignee: |
Precision Therapeutics,
Inc.
Pittsburgh
PA
|
Family ID: |
37809652 |
Appl. No.: |
13/542000 |
Filed: |
July 5, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12622022 |
Nov 19, 2009 |
8236489 |
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13542000 |
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11785984 |
Apr 23, 2007 |
7642048 |
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12622022 |
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11514172 |
Sep 1, 2006 |
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11785984 |
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60712815 |
Sep 1, 2005 |
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60712814 |
Sep 1, 2005 |
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60735813 |
Nov 14, 2005 |
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Current U.S.
Class: |
435/32 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/136 20130101; G01N 33/57407 20130101; C12Q 2600/106
20130101 |
Class at
Publication: |
435/32 |
International
Class: |
C12Q 1/18 20060101
C12Q001/18 |
Claims
1-91. (canceled)
92. A method for predicting a cancer patient's response to a
therapeutic agent, comprising the steps of: contacting tumor cells
from a patient tumor sample with one or more therapeutic agents,
and measuring a response of the tumor cells to the one or more
therapeutic agents, wherein said one or more therapeutic agents
comprises Herceptin, an anti-EGFR antibody, or a small molecule
kinase inhibitor, to thereby predict a cancer patient's response to
the therapeutic agent.
93. The method of claim 92, wherein the cancer is an epithelial
carcinoma.
94. The method of claim 92, wherein the cancer is breast cancer,
colorectal cancer, ovarian cancer, or head and neck cancer.
95. The method of claim 92, wherein the tumor cells are from one or
more tumor explants.
96. The method of claim 95, wherein the tumor cells are released
from the one or more tumor explants, and expanded in monolayer
culture.
97. The method of claim 96, wherein a suspension of from 4,000 to
12,000 cells/mL is prepared, and inoculated into a plurality of
segregated sites for testing.
98. The method of claim 92, wherein the response of the tumor cells
is measured with an apoptosis assay.
99. The method of claim 92, wherein the response of the tumor cells
is measured by determining the number of viable cells after
treatment.
100. The method of claim 99, wherein a dose-response curve is
prepared.
101. The method of claim 92, wherein the therapeutic agent is
Herceptin.
102. The method of claim 92, wherein the therapeutic agent is an
anti-EGFR antibody.
103. The method of claim 102, wherein the therapeutic agent is
Erbitux.
104. The method of claim 92, wherein the therapeutic agent is a
small molecule kinase inhibitor.
105. The method of claim 104, wherein the therapeutic agent is
Tarceva or Iressa.
106. The method of claim 92, further comprising, selecting the
therapeutic agent(s) for treatment when the tumor cells are
responsive to the agent(s).
107. A method for predicting a cancer patient's response to a
therapeutic agent, comprising the steps of: preparing a monolayer
culture of tumor cells from a patient tumor sample, contacting the
tumor cells with one or more therapeutic agents, and measuring
viability of the tumor cells to the one or more therapeutic agents,
wherein said one or more therapeutic agents comprises Herceptin, an
anti-EGFR antibody or a small molecule kinase inhibitor, to thereby
predict a cancer patient's response to the therapeutic agent.
108. A method for predicting a cancer patient's response to a
therapeutic agent, comprising the steps of: contacting tumor cells
from a patient tumor sample with one or more therapeutic agents,
and measuring a response of the tumor cells to the one or more
therapeutic agents, wherein said one or more therapeutic agents
comprises pemetrexed, to thereby predict a cancer patient's
response to the therapeutic agent.
109. The method of claim 108, wherein the cancer is an epithelial
carcinoma or mesothelioma
110. The method of claim 108, wherein the tumor cells are from one
or more tumor explants.
111. The method of claim 110, wherein the tumor cells are released
from the one or more tumor explants, and expanded in monolayer
culture.
112. The method of claim 108, wherein a suspension of from 4,000 to
12,000 cells/mL is prepared, and inoculated into a plurality of
segregated sites for testing.
113. The method of claim 108, wherein the response of the tumor
cells is measured with an apoptosis assay.
114. The method of claim 108, wherein the response of the tumor
cells is measured by determining the number of viable cells after
treatment.
115. The method of claim 108, wherein a dose-response curve is
prepared.
116. The method of claim 108, further comprising, selecting the
therapeutic agent for treatment when the tumor cells are responsive
to the agent.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application 60/712,815, filed Sep. 1, 2005, and U.S. Provisional
Application 60/712,814, filed Sep. 1, 2005, both of which are
incorporated by reference in their entirety.
[0002] This application is related to but does not claim the
benefit of U.S. application Ser. No. 08/679,056, filed Jul. 12,
1996, now U.S. Pat. No. 5,728,541; PCT Application PCT/US97/11595,
filed Jul. 10, 1997; U.S. application Ser. No. 09/040,161, filed
Mar. 17, 1998, now U.S. Pat. No. 6,900,027; U.S. application Ser.
No. 10/205,887, filed Jul. 26, 2002, now U.S. Pat. No. 6,887,680;
U.S. application Ser. No. 11/081,827, filed Mar. 17, 2005; U.S.
application Ser. No. 09/039,957, filed Mar. 16, 1998, now U.S. Pat.
No. 6,933,129; U.S. application Ser. No. 11/073,931, filed Mar. 8,
2005; U.S. application Ser. No. 11/504,098, filed Aug. 16, 2006;
09/189,310, filed Nov. 10, 1998, now U.S. Pat. No. 6,416,967; U.S.
application Ser. No. 10/399,563, filed Oct. 18, 2001; PCT
Application PCT/US01/32540, filed Oct. 18, 2001; U.S. application
Ser. No. 10/208,480, filed Jul. 30, 2002; PCT Application
PCT/US03/23888, filed Jul. 30, 2003; U.S. Provisional Application
60/417,439, filed Oct. 10, 2002; U.S. application Ser. No.
10/336,659 filed Jan. 2, 2003; PCT Application PCT/US03/32285,
filed Oct. 10, 2003; U.S. Provisional Application 60/735,813 filed
Nov. 14, 2005; and U.S. Provisional Application 60/819,631, filed
Jul. 11, 2006, all of which are herein incorporated by reference in
their entirety.
FIELD OF INVENTION
[0003] The present invention relates to methods of preparing a
tumor cell sample for use in an assay before substantial phenotypic
drift of the tumor cell population occurs. In one embodiment of the
invention, a cell culture monolayer is formed from a tissue explant
treated with collagenase and DNase. In another embodiment, the cell
culture is formed from a tissue explant that has been mechanically
agitated. The methods of the invention can be used in conjunction
with chemosensitivity and chemoresistance assays.
BACKGROUND
[0004] In spite of the progress made against cancer, it is still
the second-leading cause of death in America after cardiovascular
diseases. One of the major hurdles in the battle against cancer is
that chemotherapy agent selections for any individual patient are
not truly personalized. While "cancers" share many characteristics
in common, each particular cancer has its own specific
characteristics. Genetics and environmental factors have a complex
interplay in severity and prognosis of treatment.
[0005] It has been recognized that when patient cells are removed
from their in situ locations in tissues and cultured in vitro, the
cells are subject to phenotypic and genotypic drift, i.e., they
begin to lose some of the morphological features (and components)
of some characteristic of their tissue or organ of origin,
sometimes as a result of changes in expression of a gene, or
expression of mutated gene. As a result, simply excising cells from
normal and tumor tissues and culturing them in vitro is not
satisfactory, since adaptation to culture conditions causes
repression of components that are expressed in tumor tissue or in
normal tissue and may also cause expression of components that are
not normally present in tumor or normal tissue.
[0006] Currently, chemotherapy choices are based primarily on a
combination of the average population response, as published in
peer reviewed journal articles, and the treating physician's
professional experience. In treating cancer patients with highly
toxic chemotherapy, oncologists are faced with the challenge of
selecting a therapy regimen for a particular patient with
prospective indicators as to what drug might actually work best for
that specific patient.
[0007] Culture condition variations, selective overgrowth of some
cells in the population, and genetic variation of in vitro cultured
cells may result in inaccurate and unreliable prospective
information regarding therapeutic treatments. Physicians need a
reliable method of obtaining prospective information to assist in
personalizing the therapy based on a patient's in vitro tumor
behavior.
SUMMARY OF THE INVENTION
[0008] The present invention discloses methods of preparing a tumor
cell sample comprising agitating a tumor explant to substantially
release tumor cells from the tumor explant, culturing a cell
culture monolayer from the released cells and forming a cell
suspension from the monolayer before substantial phenotypic drift
occurs. In one embodiment, the cell suspension is about 4,000 to
12,000 cells/ml. In one embodiment, the cell suspension is about
4,000 to 9,000 cells/ml. In another embodiment, the cell suspension
is about 7,000 to 9,000 cells/ml.
[0009] The tumor explant can optionally be treated with collagenase
and DNase prior to culturing of a cell culture monolayer. For
instance, the Inventors of the present invention have found that
ovarian and colorectal tumor explants culture favorably when
treated with a Collagenase II and DNase cocktail. In one
embodiment, the tumor explant is treated with a cocktail comprising
about 0.010% to about 0.60% Collagenase II and about 0.0007% to
about 0.005% DNase. In another embodiment, the tumor explant is
treated with a cocktail comprising about 0.25% Collagenase II and
about 0.001% DNase. In yet another embodiment, the tumor explant is
treated with a cocktail comprising about 0.025% Collagenase II and
about 0.001% DNase.
[0010] Cells from the cell suspension can be inoculated into at
least one segregated site. The segregated site can comprise about
100 to 10,000 cells. Each segregated site can comprise, for
instance, about 100 to 5,000 cells, about 100 to 2,500 cells, about
100 to 1,000 cells, about 200 to 1,000 cells or about 200 to 500
cells.
[0011] Cells from the cell suspension or at a segregated site can
be contacted with one or more pharmaceutical agents such as one or
more chemotherapeutic drugs or biological agents. In one
embodiment, cells are incubated in one or more segregated sites
prior to be contacted with a pharmaceutical agent. For instance,
cells can be incubated about 4 to about 30 hours prior to contact
with an agent. Cells can also optionally be analyzed, for instance,
counted, prior to contact with a pharmaceutical agent. In one
embodiment, cells are counted after incubatation for about 24 hours
prior to contact with an agent.
[0012] In one embodiment, cells are kept in contact with one or
more pharmaceutical agents for 25 to 200 hours. The time a
pharmaceutical agent is kept in contact with a cell population can
vary based on factors, including, but not limited to, the identity
of the pharmaceutical agent. At the end of the period of contact,
cells can be counted. In one embodiment, a dose response curve is
generated. In another embodiment, a Cytotoxicity Index or
normalized Cytotoxicity Index is calculated.
[0013] The assays, methods, tools and systems included in the
invention disclosed herein address the challenge presented by
patients who will undergo initial chemotherapy, have typically
failed earlier chemotherapy, and/or have built up drug resistance
through multiple lines or courses of chemotherapy, i.e., the most
resistant cancer has survived, and become chemoresistant. The
assays, methods, tools and systems included in the invention
disclosed herein provide prospective information that will assist
the oncologist in personalizing the therapy based on the
individual's in vitro tumor behavior.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1. Representative population distribution achieved
during dose setting for carboplatin on ovarian tumor. Specimens are
processed and cultured as described in the text. Wells of the
microtiter plate are treated with decreasing concentrations from
dose 10 to dose 1. Dose 0 is an untreated control well. Fraction
cells surviving is determined by averaging three replicate wells at
each dose divided by the average of the control well replicates.
Each specimen is indicated by a different color line. Lines are
non-linear curve fits of raw data.
[0015] FIG. 2. Representative population distribution achieved
during dose setting for Carboplatin on Breast tumor. Specimens are
processed and cultured as described in the text. Wells of the
microtiter plate are treated with decreasing concentrations from
dose 10 to dose 1. Dose 0 is an untreated control well. Fraction
cells surviving is determined by averaging three replicate wells at
each dose divided by the average of the control well replicates.
Each specimen is indicated by a different color line. Lines are
non-linear curve fits of raw data.
[0016] FIG. 3. Representative population distribution achieved
during dose setting for Carboplatin on Colon tumor. Specimens are
processed and cultured as described in the text. Wells of the
microtiter plate are treated with decreasing concentrations from
dose 10 to dose 1. Dose 0 is an untreated control well. Fraction
cells surviving is determined by averaging three replicate wells at
each dose divided by the average of the control well replicates.
Each specimen is indicated by a different color line. Lines are
non-linear curve fits of raw data.
[0017] FIG. 4. Representative results for treatment of tumor
derived cells with combination treatment of taxol and carboplatin.
Specimens are processed and cultured as described in the text.
Wells of the microtiter plate are treated with decreasing
concentrations from dose 10 to dose 1. Dose 0 is an untreated
control well. Fraction cells surviving is determined by averaging
three replicate wells at each dose divided by the average of the
control well replicates. Each specimen is indicated by a different
color line. Lines are non-linear curve fits of raw data.
DETAILED DESCRIPTION
[0018] The following embodiments and aspects thereof are described
and illustrated in conjunction with assays, methods, tools and
systems included in the invention and are meant to be exemplary and
illustrative, not limiting in scope. In various embodiments, one or
more of the herein-described problems have been reduced or
eliminated, while other embodiments are directed to improvements of
the assays, methods, tools and systems described herein.
[0019] The invention includes a method of preparing a tumor cell
sample comprising agitating a tumor explant to substantially
release tumor cells from the tumor explant; culturing the released
cells to produce a cell culture monolayer; and, forming a cell
suspension from the monolayer cells before substantial phenotypic
drift of the tumor cell population occurs. In one embodiment, the
cell suspension is about 4,000 to 12,000 cells/ml. In another
embodiment, the cell suspension is 4,000 to 9,000 cells/ml or 7,000
to 9,000 cells/ml. In another embodiment, the method further
comprises inoculating cells from the cell suspension into at least
one segregated site. In one aspect of the method, each segregated
site comprises about 10.sup.2 to 10.sup.4 cells after the
inoculating. In another aspect of the method, each segregated site
comprises about 10.sup.2 to 10.sup.3 cells after the inoculating.
In a different aspect of the method, each segregated site comprises
about 200 to about 1000 cells. In yet another aspect, each
segregated site comprises about 200 to about 500 cells.
[0020] Embodiments of the methods of the invention further comprise
contacting the cells with at least one pharmaceutical agent. In one
aspect of the method, the cells are cultured for about 4 to about
30 hours prior to contact with an agent. In another aspect, the
method further comprises at least one combination treatment. In one
aspect of the method, each combination treatment contacts the cells
for about 25 to about 200 hours. In one aspect, each combination
treatment comprises at least two agents. In another aspect, each
combination treatment comprises a serial dilution series of 3-20
dose levels for each agent. The method further comprises adjusting
the dose level of each agent to obtain from 0% up to and including
maximal cell killing. In one aspect, each agent is initially used
at a dose level below to above the range determined to be in the
extracellular fluid surrounding a tumor in vivo. In another aspect,
a dose response curve is generated for each agent. In one aspect of
the method, cell viability is maintained for about 25 to about 200
hours.
[0021] In one embodiment of the method, media and nonadherent cells
are removed at the end of about 25 to about 200 hours. In another
aspect, the media and nonadherent cells are analyzed at the end of
about 25-200 hours. In a different aspect, the adherent cells are
analyzed at the end of about 25-200 hours. The adherent cells can
be analyzed at any time or at any step in the procedures disclosed
herein. In a variation of the method, the method is repeated at
least once using cells which had been frozen after being grown in
monolayers from explants.
[0022] The method also includes an automated cell imaging system
which takes images of the cells using one or more of visible light,
UV light and fluorescent light at predetermined intervals before,
simultaneously with, or beginning immediately after, contact with
each treatment. In a different embodiment, the cells are imaged
after about 25 to 200 hours of contact with each treatment. In
another embodiment, the cells are imaged once or multiple times,
prior to or during contact with each treatment. Alternatively, UV
or fluorescent light is used to take images to count cells.
Visible, UV or fluorescent imaging can occur at multiple times
prior to drug exposure, at predetermined intervals during drug
exposure and at the end of the assay.
[0023] In a different embodiment, the method further comprises
quantifying the number of viable or non-viable cells. In yet
another aspect, the method comprises analyzing the genotypic or
phenotypic state of the adherent cells after 25 to 200 hours. In
one aspect of the method, the quantifying is by one or more of
visible light, UV light and fluorescent light. In one embodiment of
the method, the percent of cell confluency is determined.
[0024] In another embodiment, at least one pharmaceutical agent is
a targeting agent. In a specific embodiment, the targeting agent
targets a marker. In a more specific embodiment, the marker is
selected from the group consisting of: markers of mesenchymal
cells, epithelial cells, tumor markers and tissue specific markers.
In another aspect, the marker is, but not limited to, one or more
of: vimentin, desmin, S100, fibronectin and collagen, cell adhesion
molecules and cytokeratins, tumor markers including but not limited
to total levels and mutations in p53, cyclins, ras, src, growth
factor receptors, hormone receptors, molecules involved in signal
transduction and tissue specific markers including but not limited
to CA125, PSA, PSM, milk proteins, surfactants and homeobox nuclear
proteins.
[0025] Certain methods of the invention also further comprise
assaying the cells of the cell suspension for the expression of at
least one gene. In one aspect of the method, at least one gene is
selected from the group consisting of ABCB1; ABCC1; ABCC2; ABCG2;
ABL1; ACLY; ADH1A; ADPRT; ADSS; AKAP2; AKT1; AKT2; ALDH1A1; ALDH4;
ANK3; ANXA8; AP2B1; APAF-1; APH-1A; API5; APOE; ATF5; ATP7B; B4-2;
BAD; BAG1; BAK1; BARX2; BAX; BBC3; BCL2; BCL2L1; BCL2L2; BNIP3;
BRCA1; BRCA2; BRF2; BTF3; BUB1; BUB3; C8orf2; CASP2; CBR1; CCNL2;
CCNB1; CCNE2; CD44; CD68; CDA; CDC45L; CDK9; CEACAM6; CEGP1; CENPA;
CES1; CFFM4; CFLAR; COL1A1; COL4A2; COX17; CPR2; CREM; CSNK2B;
CTSL2; CUL1; CYP1B1; CYP2A6; CYP2B6; CYP2C8; CYP2C9; CYP2C19;
CYP2D6; CYP3A4; CYP3A5; CYR61; DC13; DCK; DCTD; DD96; DDB1; DIA4;
DLC1; DNAJD1; DPYD; DPYS; ECGF1; ECT2; EFEMP1; EGR1; EMP-1; EPB42;
EPRS; ER; ERBB2; ERCC1; ERCC2; ERCC4; ERG; ESM1; EXT1; FAAH; FCGRT;
FDXR; FGF18; FGFR2; FLJ10948; FLJ11190; FLJ11196; FLJ13855;
FLJ14299; FLJ20323; FLJ20585; FLNA; FLT1; FN 1; GADD34; GADD153;
GBX2; GJB1; GNAZ; GMPS; GRB7; GSR; GSTM1; GSTM3; GSTP1; GTF2H3;
HBOA; HCFC1; HEC; HER2; HLA-C; HMG1; HN1; HSPC134; IGFBP5; IL4R;
ISGF3G; ITGA5; Ki67; KIAA0175; KIAA0281; KIAA0303; KIAA1041;
KIAA1067; KIAA1442; KIP2; KIT; KLK4; KNTC2; KPNA2; KRT13; L2DTL;
LAMB1; LCHN; LDHA; LOC51061; LOX; MAD2L1; MAP2K4; MAP4; MAPT; MCM2;
MCM6; MGMT; MGST1; MLH1; MMP9; MMP11; MP1; MPO; MSH2; MSN; MUC1;
MYBL2; MYC; NDP; NFAT5; NFATC3; NFKB1; NME1; NME2; NMT1; NMU; NPM
1; NR1I2; ORC6L; ORM1/2; OXCT; p21/WAF; PAPPA; PB1; PCDHB2; PCSK7;
PECI; PGK1; PGR; PK428; PLD3; POLA2; POLB; POLE; POLH; POR; PP591;
PPP2R1A; PRC1; PRKDC; PRPSAP1; PSME 1; PTK2; PTPRC; RAB6B;
RAB11FIP1; RALGDS; RFC4; RNF2; RPL27; RRM1; RRM2; RTKN; SCARA3;
SCUBE2; SEC61A1; SERF1A; SIAH2; SLC2A3; SLC7A10; SLC28A1; SLC28A2;
SLC29A1; SLC29A2; SLC35B1; SM20; SOD1; SPARC; STK15; STOML1; SURF4;
SURVIVIN; TBPL1; TCEB3; TDP1; TFRC; TGFB3; TIMP1; TIMP3; TLOC1;
TNC; TNF; TNFSF6; TOP1; TOP2A; TP53; TRAG3; TUBB/TUBA2; TWIST; TXN;
TYMS; UBE2M; UBCH10; UBPH; UCH37; UMP-CMPK; UMPS; UP; UPB1; USP22;
WISP1; XIAP; XIST; XPA; XPB and XRCC1.
[0026] The methods of the invention also further comprise assaying
the cells of the cell suspension for at least one SNP from at least
one gene. In one aspect, the at least one gene is selected from the
group consisting of ABCB1; ABCC1; ABCC2; ABCG2; ABL1; ACLY; ADH1A;
ADPRT; ADSS; AKAP2; AKT1; AKT2; ALDH1A1; ALDH4; ANK3; ANXA8; AP2B1;
APAF-1; APH-1A; API5; APOE; ATF5; ATP7B; B4-2; BAD; BAG1; BAK1;
BARX2; BAX; BBC3; BCL2; BCL2L1; BCL2L2; BNIP3; BRCA1; BRCA2; BRF2;
BTF3; BUB1; BUB3; C8orf2; CASP2; CBR1; CCNL2; CCNB1; CCNE2; CD44;
CD68; CDA; CDC45L; CDK9; CEACAM6; CEGP1; CENPA; CES1; CFFM4; CFLAR;
COL1A1; COL4A2; COX17; CPR2; CREM; CSNK2B; CTSL2; CUL1; CYP1B1;
CYP2A6; CYP2B6; CYP2C8; CYP2C9; CYP2C19; CYP2D6; CYP3A4; CYP3A5;
CYR61; DC13; DCK; DCTD; DD96; DDB1; DIA4; DLC1; DNAJD1; DPYD; DPYS;
ECGF1; ECT2; EFEMP1; EGR1; EMP-1; EPB42; EPRS; ER; ERBB2; ERCC1;
ERCC2; ERCC4; ERG; ESM1; EXT1; FAAH; FCGRT; FDXR; FGF18; FGFR2;
FLJ10948; FLJ11190; FLJ11196; FLJ13855; FLJ14299; FLJ20323;
FLJ20585; FLNA; FLT1; FN 1; GADD34; GADD153; GBX2; GJB1; GNAZ;
GMPS; GRB7; GSR; GSTM1; GSTM3; GSTP1; GTF2H3; HBOA; HCFC1; HEC;
HER2; HLA-C; HMG1; HN1; HSPC134; IGFBP5; IL4R; ISGF3G; ITGA5; Ki67;
KIAA0175; KIAA0281; KIAA0303; KIAA1041; KIAA1067; KIAA1442; KIP2;
KIT; KLK4; KNTC2; KPNA2; KRT13; L2DTL; LAMB1; LCHN; LDHA; LOC51061;
LOX; MAD2L1; MAP2K4; MAP4; MAPT; MCM2; MCM6; MGMT; MGST1; MLH1;
MMP9; MMP11; MP1; MPO; MSH2; MSN; MUC1; MYBL2; MYC; NDP; NFAT5;
NFATC3; NFKB1; NME1; NME2; NMT1; NMU; NPM 1; NR1I2; ORC6L; ORM1/2;
OXCT; p21/WAF; PAPPA; PB1; PCDHB2; PCSK7; PECI; PGK1; PGR; PK428;
PLD3; POLA2; POLB; POLE; POLH; POR; PP591; PPP2R1A; PRC1; PRKDC;
PRPSAP1; PSME 1; PTK2; PTPRC; RAB6B; RAB11FIP1; RALGDS; RFC4; RNF2;
RPL27; RRM1; RRM2; RTKN; SCARA3; SCUBE2; SEC61A1; SERF1A; SIAH2;
SLC2A3; SLC7A10; SLC28A1; SLC28A2; SLC29A1; SLC29A2; SLC35B1; SM20;
SOD1; SPARC; STK15; STOML1; SURF4; SURVIVIN; TBPL1; TCEB3; TDP1;
TFRC; TGFB3; TIMP1; TIMP3; TLOC1; TNC; TNF; TNFSF6; TOP1; TOP2A;
TP53; TRAG3; TUBB/TUBA2; TWIST; TXN; TYMS; UBE2M; UBCH10; UBPH;
UCH37; UMP-CMPK; UMPS; UP; UPB1; USP22; WISP1; XIAP; XIST; XPA; XPB
and XRCC1.
[0027] Definitions
[0028] As is generally the case in biotechnology and chemistry, the
description of the present methods has required the use of a number
of terms of art. Although it is not practical to do so
exhaustively, definitions for some of these terms are provided here
for ease of reference. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which the methods
described herein belong. Definitions for other terms also appear
elsewhere herein. However, the definitions provided here and
elsewhere herein should always be considered in determining the
intended scope and meaning of the defined terms. Other than in the
operating examples or where otherwise indicated, all numbers or
expressions referring to quantities of ingredients, reaction
conditions, etcetera, used in the specification and claims are to
be understood as modified in all instances by the term "about."
[0029] As used herein, the term "cancer" refers to a class of
diseases of humans (and animals) characterized by uncontrolled
cellular growth. As used herein, "cancer" is used interchangeably
with the terms "tumor," "malignancy," "hyperproliferation" and
"neoplasm(s)." The term "cancer cell(s)" is interchangeable with
the terms "tumor cell(s)," "malignant cell(s)," "hyperproliferative
cell(s)," and "neoplastic cell(s)" unless otherwise explicitly
indicated. Similarly, the terms "hyperproliferative,"
"hyperplastic," "malignant" and "neoplastic" are used
interchangeably, and refer to those cells in an abnormal state or
condition characterized by rapid proliferation. Collectively, these
terms are meant to include all types of hyperproliferative growth,
hyperplastic growth, neoplastic growth, cancerous growths or
oncogenic processes, metastatic tissues or malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness.
[0030] As used herein, the term "candidate therapeutic agent"
refers to an agent administered to a particular cell population
causing a desired chemotherapeutic response.
[0031] As used herein, the term "cell culture" refers to cultures
derived from dispersed cells taken from the original tissue or from
a primary culture. It is not intended that the present invention be
limited to cell cultures from any particular species, as the
present invention finds use with any type of animal cell. See, for
example, U.S. Pat. No. 6,528,309.
[0032] As used herein, the term "chemoresistant" refers to tumor
cells (and interchangeable terms discussed, above) which show
little or no significant detectable response to an agent used in
chemotherapy.
[0033] As used herein, the term "chemosensitive" refers to tumor
cells (and interchangeable terms discussed, above) which show a
detectable response to an agent used in chemotherapy.
[0034] As used herein, the terms "chemotherapeutic agent,"
"cytotoxic agent," "anticancer agent" and "antitumor agent" are
used interchangeably and refer to agents that have the property of
inhibiting the growth or proliferation (e.g., a cytostatic agent),
or inducing the killing, of tumor cells (and interchangeable terms
as discussed above). The chemotherapeutic agent inhibits or
reverses the development or progression of a cancer, such as for
example, solid tumor, or a soft tissue tumor. See, for example,
U.S. Pat. No. 6,599,912.
[0035] As used herein, the terms "chemotherapeutic response" and
"chemoresponse" are used interchangeably. A chemoresponse refers to
the response obtained upon administration of a pharmaceutical
agent. The desired chemoresponse may be a genotypic response, such
as, for example, a change in expression of one or more genes, for
example. The desired chemoresponse may also be a phenotypic
response, such as, for example, the slowing of, or regression of,
the growth of tumor cells. Upon identification of a
chemotherapeutic agent giving a desired chemoresponse in the assays
or methods disclosed herein, the agent is then administered to the
patient in vivo. In one embodiment of the methods disclosed herein,
the tumor cell population is a chemoresistant cell population and
the desired chemoresponse is slowing of, death or regression of the
chemoresistant cells.
[0036] As used herein, the term "chemotherapy" refers to
administration of at least one chemotherapeutic agent to patients
having a cancer.
[0037] As used herein, the term "combination treatment" refers to a
treatment of the cells with at least two pharmaceutical agents. The
pharmaceutical agents which are used either at the same time, or
separately, or sequentially, according to the methods disclosed
herein, do not represent a mere aggregate of known agents, but a
new combination with the surprising valuable property that modifies
the chemoresistance and/or chemosensitivity of the tumor cells and
allows a new effective treatment (partial or complete response) for
cancer.
[0038] As used herein, the term "contacting" refers to the
interaction of the tumor cells and at least one pharmaceutical
agent.
[0039] As used herein, the term "Cytotoxicity Index" (CI) is the
ratio of the number of treated cells to number of control cells
(e.g., untreated cells) after treatment with an agent. A
"normalized Cytotoxicity Index" is a CI that has been corrected to
take into account variations in the assay such as variations in the
starting number of cells.
[0040] As used herein, the term "effective amount" of a compound
refers to a sufficient amount of the drug or agent which provides
the desired effect.
[0041] As used herein, the term "empiric chemotherapy" refers to
selecting chemotherapy based on outcomes reported in the literature
for groups of patients with a particular type of tumor.
[0042] As used herein, the term "epithelial cell marker" refers to
a marker expressed by epithelial cells. As used herein, the term
"malignant epithelial cell marker" refers to a marker expressed by
malignant epithelial cells. Many are known in the art and
optionally intended for use as part of, or in conjunction with the
assays, methods, tools and systems as included in the invention
disclosed herein. Epithelial cell markers, malignant or
nonmalignant, may be used as markers for aid in determining whether
phenotypic drift and/or genotypic drift has occurred in the
cultured tumor cell population at any point during the cell culture
period.
[0043] As used herein, the term "gene" refers to any segment of DNA
associated with a biological function. Thus, genes include, but are
not limited to, coding sequences and/or the regulatory sequences
required for their expression. Genes can also include non-expressed
DNA segments that, for example, form recognition sequences for
other proteins. Genes can be obtained from a variety of sources,
including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters.
[0044] As used herein, the term "marker" refers to any genotypic or
phenotypic characteristic of a cell or cell population that, alone
or in combination with other marker(s), can be used to identify the
particular cell type. Markers can be, without limitation,
genotypic, such as an insertion, deletion or substitution, or
phenotypic, such as the presence of high levels of a receptor or a
secreted peptide. A marker may be a molecular predictor of
response. For example, a molecular predictor of response such as
EGR1, a gene involved in cell proliferation, may also be useful as
a marker in identifying a particular cell type. The marker can be
any molecule detectable on the surface of tumor cells, in tumor
cells or both. Thus, the marker is any one or more of a protein, a
lipid, a carbohydrate, a nucleic acid and any combination thereof
(for example, a glycoprotein). The marker may or may not be
expressed in tumor cells and therefore is typically evaluated prior
to initiating chemotherapy.
[0045] As used herein, the term "maximal cell killing" refers to
the cytotoxic index associated with the greatest amount of cell
kill for a given chemotherapeutic agent that is maintained over at
least 2 of the highest doses tested (Doses 9 and 10). Most agents
do not kill all (100%) of the tumor cell population. Cytotoxic
chemotherapeutic agents exert fractional cell kill whereby a
constant fraction, and not number, of the live tumor cells are
killed such that 100% tumor cell kill is only asymptotically
approached but rarely achieved. Cytostatic chemotherapeutic agents
halt the proliferation of tumor cells but are ineffective at
killing tumor cells so that 100% of the tumor cells are not
killed.
[0046] As used herein, the term "molecular predictor of response"
refers to at least one gene in a pathway such as, for example,
chemotherapeutic drug metabolism (such as, for example, CYP3A4,
CYP3A5, CYP2D6, CYP2C8 and CYP2C9), drug transport (such as, for
example, ABCB1, ABCC1, ABCC2 and ABCG2), cell apoptosis (such as,
for example, BCL2, BAD, BAX and BAK1), cell proliferation (such as,
for example, EGR1, CYR61, p21/WAF and TP53) and DNA repair pathways
(such as, for example, ERCC1, ERCC2, MLH1 and MSH2). A molecular
predictor of response is predictive of whether the patient is
likely to respond favorably to a chemotherapeutic regimen
comprising a given agent or given combination therapy or whether
long term survival of the patient following termination of
chemotherapy or other treatment is likely.
[0047] As used herein, the term "neoadjuvant treatment" refers to
administration of chemotherapy prior to surgical intervention or
resection. Neoadjuvant treatment may be used optionally in
conjunction with any of the assays, methods, tool or systems
disclosed herein. For example, the patient may receive neoadjuvant
treatment before a tumor biopsy is obtained from the patient.
[0048] As used herein, the terms "nucleic acid" or "polynucleotide"
refer to deoxyribonucleotides or ribonucleotides and polymers
thereof in either single- or double-stranded form. Unless
specifically limited, the terms encompass nucleic acids containing
analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions) and complementary sequences as well
as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J.
Biol. Chem. 260:2605-2608; Cassol et al., 1992; and Rossolini et
al., 1994, Mol. Cell. Probes 8:91-98). The term nucleic acid is
used interchangeably with gene, cDNA, and mRNA encoded by a
gene.
[0049] As used herein the terms "optional" or "optionally" means
that the subsequently described circumstance may or may not occur,
so that the description includes instances where the circumstance
occurs and instances where it does not.
[0050] As used herein, the term "pharmaceutical agent" includes,
without limitation, biologically active molecules, enzymes,
proteins, lipids, carbohydrates, glycoproteins, glycolipids,
nucleic acids such as DNA and/or RNA and fragments of DNA and/or
RNA, antisense nucleic acids, siRNA molecules, antibodies, small
molecules and inorganic pharmaceutical molecules. As used herein,
"small molecules" are those molecules having a molecular weight of
about 2000 Daltons or less. The term "pharmaceutical agent" is used
interchangeably with the terms "agent," "drug," "compound,"
"therapeutic," "chemotherapeutic," and "biological agent"
herein.
[0051] The term "pharmaceutical agent" encompasses not only the
specified molecular entity but also its pharmaceutically
acceptable, pharmacologically active analogs, including, but not
limited to, salts, esters, amides, prodrugs, conjugates, active
metabolites and other such derivatives, analogs and structurally,
biologically and functionally related compounds. The agents or
derivatives thereof disclosed herein are in a pharmaceutically
acceptable carrier when necessary, i.e., when required by a method
or assay. More than one agent can be simultaneously used at a time.
For example, combination treatment may comprise two or three or
four or more pharmaceutical agents used together.
[0052] As used herein, the term "pharmaceutically acceptable"
refers to a material that is not biologically or otherwise
undesirable, i.e., the material may be incorporated into a
pharmaceutical composition without causing any undesirable
biological effects or interacting in a deleterious manner with any
of the other components of the composition in which it is
contained. When the term "pharmaceutically acceptable" is used to
refer to a pharmaceutical carrier or excipient, it is implied that
the carrier or excipient has met the required standards of
toxicological and manufacturing testing or that it is included on
the Inactive Ingredient Guide prepared by the U.S. Food and Drug
administration.
[0053] As used herein, the term "phenotypic drift" refers to
phenotypic plasticity, which is a phenomenon in which a given
genotype may develop different states for a character or group of
characters in different environments, the phenotypic variability
produced by a given genotype under the range of environmental
conditions common to the natural habitat of the species or under
the standard culture or experimental conditions. (A Dictionary of
Genetics, 5th edition, King et al., Oxford University Press, NY
Oxford 1997). Phenotypic drift may also include changes in a cell
character or a group of characters due to a genetic change.
"Substantial phenotypic drift" refers to a detectable change in the
character or nature of one or more biochemical markers, functional
markers or physical markers characteristic of a tumor cell; and/or
to a change in one or more molecular predictors of response. The
change is detected using any detection method known in the art
including morphometry by cell shape changes and the epithelial
markers discussed herein. Changes in fibroblast markers, malignant
markers, proliferation markers can also be detected using detection
methods known in the art.
[0054] As used herein, the term "predicting the chemoresponse"
refers to any method of analyzing the response of tumor cells
contacted with at least one pharmaceutical agent. Methods for
evaluating the molecular chemoresponse include expression assays
(microarrays, PCR-based technology) disclosed herein and other
assays known to those of skill in the art. In one embodiment of the
invention, evaluating the chemoresponse comprises performing an
analysis of the expression of one or more molecular predictors of
response. In another embodiment of the invention, evaluating the
chemoresponse comprises counting the cells before and after
treatment with an agent and calculating a Cytotoxicity Index
(CI).
[0055] As used herein, the term "primary culture" refers to a
culture that has been developed from a patient's tumor cells and
before the first subculture. Thus, a primary culture represents the
first in vitro growth of cells. It is not intended that the present
methods be limited to primary cultures from any particular species,
as the present invention finds use with any type of animal cell.
See, for example, U.S. Pat. No. 6,528,309. It is not intended that
the present invention be limited to primary cultures but may
include first or subsequent subcultures.
[0056] As used herein, the term "SNP" (single nucleotide
polymorphism) refers to nucleotide sequence variations that occur
when a single nucleotide (A, T, C or G) in the genome sequence is
altered. SNPs can occur in both coding (gene) and noncoding regions
of the genome. Many SNPs have no effect on cell function while
other SNPs predispose people to cancer or a disease or influence
their response to a drug or are linked to a locus that predisposes
a person to cancer or a disease or influences their response to a
drug. Such linkages can be determined by any commonly available
means such as those procedures that produce linkage disequilibrium
maps for a given SNP or a group of SNPs.
[0057] As used herein, "staining" refers to any number of processes
known to those in the field that are used to allow visualization
and/or improve visualization of cell component(s) and/or
feature(s). Many such processes are publicly available and known to
those of skill in the art and may optionally be used in or in
conjunction with the assays, methods, tools and systems disclosed
herein. Many stains or other molecules allowing visualization of
the cells and/or cell features are known in the art and may
optionally used in or in conjunction with the assays, methods,
tools and systems disclosed herein.
[0058] As used herein, the term "substantially release tumor cells"
refers to the number of tumor cells released from the explant upon
sudden agitation or motion of the explant. This releases a
significant number of viable and representative tumor cells from
the explant as compared to the number released if the explant is
not subjected to sudden agitation or motion. This process may be
facilitated with the use of chemicals or enzymes designed to
enhance the release of tumor cells from tissue segments.
[0059] As used herein, the term "targeting agent" refers to an
agent designed to target a marker expressed in tumor and nontumor
cells, on tumor and nontumor cells or both in and on tumor and
nontumor cells. Targeting agents useful in the practice of the
methods disclosed herein include antibodies, cell surface ligands,
nucleic acids, etcetera. Targeting agents are also useful for
tracking changes in markers during culture and for determining the
occurrence of phenotypic and/or genotypic drift. Many targeting
agents are known to those of skill in the art and are optionally
used in, or in conjunction with, the assays, methods, systems and
tools described herein. The targeting agent may itself be
detectable by any method known in the art, such as for example, by
radioactive labeling, linkage to fluorescent molecules or linkage
to other molecules which are detectable.
[0060] I. General Overview
[0061] In one embodiment of the methods disclosed herein, 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 effective treatments for
the cultured cells obtained from the patient. The culture
techniques of the present methods 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.
The culture techniques of the present methods also allow for
monitoring of tumor antigen expression or other tumor markers in
order to detect possible phenotypic and/or genotypic drift by the
cultured tumor cells in order to ascertain whether in vitro tumor
antigen expression is correlative of, or similar to, in vivo tumor
antigen expression. Specific methods disclosed herein, such as
tissue sample preparation techniques, render this method
practically as well as theoretically useful. See, for example, U.S.
Pat. Nos. 5,728,541; 6,887,680 and 6,416,967.
[0062] The ChemoFx Assay.RTM. disclosed herein may include the use
of a predictive algorithm of patient response to chemotherapy by
evaluating tumor factors via the response of patient-derived tumor
cells to various chemotherapeutic agents in vitro. Furthermore, the
incorporation of host factors (in the form of genomic or phenotypic
markers) into the algorithm further enhances its predictive
ability. The invention includes a method of predicting
chemotherapeutic response of patient tumor cells to at least one
therapeutic agent comprising assaying expression levels of at least
one gene selected from the group consisting of genes involved in
chemotherapeutic drug metabolism, in drug transport, in cell
apoptosis, in cell proliferation, and in DNA repair, in the patient
tumor cells. The invention also includes a method of predicting
chemotherapeutic response of patient tumor cells to at least one
therapeutic agent comprising detecting at least one SNP from at
least one gene selected from the group consisting of genes involved
in chemotherapeutic drug metabolism, in drug transport, in cell
apoptosis, in cell proliferation, and in DNA repair, in the patient
tumor cells.
[0063] II. ChemoFx.RTM. Assays: Version 1 and Version 2
[0064] The proprietary ChemoFx.RTM. Assays disclosed herein involve
the isolation, short-term growth, and drug dosage treatment of
epithelial cells derived from solid tumors. At the time of surgical
"debulking," or biopsy (e.g., vacuum-assisted and core biopsy) or
fine needle aspiration of a tumor site, pieces of solid tumor are
obtained by the surgeon, radiologist, or pathologist and placed in
tissue culture media. The tumor is minced into small pieces and
placed with cell culture media (Lifetech, Gibco BRL) into small
flasks or other appropriately sized culture dishes for cell
outgrowth. Over time, cells move out of the tumor pieces and form a
monolayer on the bottom of the vessel. Once enough cells have
migrated out of the ex vivo explant pieces, they are then
trypsinized and reseeded into microtiter plates for either
ChemoFx.RTM. Assay (versions 1 and 2 described below) or for
immunohistochemistry (IHC) analysis.
[0065] A. Version 1
[0066] In Version 1 of the ChemoFx.RTM. Assay, cultured cells are
seeded into 60 well microtiter plates at a density of about 100-500
cells per well and allowed to attach and grow for about 24 hours.
After about 24 hours in culture the cells are then exposed for
about 2 hours to a battery of chemotherapeutic agents. At the end
of the incubation with the chemotherapeutic agents, the plates are
washed to remove non-adherent cells. The remaining cells are fixed
with 95% ethanol and stained with the DNA intercalating blue
fluorescent dye, DAPI, or 6-diamidino 2-phylindole dihydrochloride
(Molecular Probes, Eugene, Oreg., USA) or equivalent. The surviving
cells are then counted using an operator-controlled,
computer-assisted image analysis system (Zeiss Axiovision,
Thornwood, N.Y., USA). A cytotoxic index is then calculated using
methods known in the art. The data are presented graphically as the
cytotoxic index (CI). A dose-response curve is then generated for
each drug or drug combination evaluated.
[0067] B. Version 2
[0068] For the Version 2 ChemoFx.RTM. Assay, proprietary software,
named Resource Allocator, is utilized to generate logical scripts
that direct the activity of a liquid handling machine. The
procedure, however, may be carried out using any liquid handling
machine with appropriate software, known in the art. This software
employs the ideology behind the assay, a plating cell suspension of
about 4,000 to 12,000 cells/ml and 1-10 replicates per dose for
each of a multiple dose drug treatments, to calculate the number of
cells necessary to accommodate testing of all requested drugs. In
one embodiment, the assay comprises about 8,000 cells/ml and 3
replicates per dose for each of 10 dose drug treatments. After
those calculations are complete, Resource Allocator will determine
the quantity of disposable pipette tips, 8 row deep-well basins and
384 well microplates necessary for cell plating as well as the
location of those consumables on the stage of the liquid handler.
Finally, Resource Allocator will determine the specific location of
cells in an 8 row deep-well basin prior to plating, and the
specific location of cells in a 384 well microplate after plating.
This information is provided in a printable format for easy
interpretation of results. Using the information provided by
Resource Allocator, a cell suspension is prepared at a
concentration of about 4,000 to 12,000 cells/ml and delivered to a
reservoir basin on the stage of the liquid handling machine. The
machine then seeds about 200 to 400 cells in about 30 to 50 .mu.l
of medium into the wells of a 384 well microplate in replicates of
about 1-10, after which the cells are allowed to adhere to the
plate and grow for about 24 hours at 37.degree. C. In one
embodiment, the cell suspension is prepared at a concentration of
about 8,000 cells/ml, and the liquid handling machine seeds about
320 cells in about 40 .mu.l of medium into the wells of a
microplate in replicates of 3.
[0069] After all cell suspensions have been delivered to the
appropriate 384 well microplate, Resource Allocator is initiated
again to calculate the number of drugs, and volume of each, that
are needed to accommodate treatment of all cells plated. The
software uses a volume of about 30-50 .mu.l per replicate for each
dose of a drug treatment and the number of unique cell lines
needing that particular treatment to calculate the total volume of
drug required. For instance, in one embodiment, the software uses a
volume of about 40 .mu.l per replicate for each dose. After
determining the necessary volume of each drug, the software
calculates the number of disposable pipette tips, 96 well deep-well
plates, and medium basins necessary for drug preparation. Resource
Allocator will then determine into which 96 well deep-well plate
each drug will go, the specific location in a 384 well microplate
the treatment will be delivered, and the stage location for all of
the consumables. For ease of interpretation, Resource Allocator
provides these results in a printable format.
[0070] Following the approximately 4-28 hour incubation of the cell
plates, the liquid handling machine prepares ten doses of each
drug, in the appropriate growth medium, via serial dilutions in a
96 well deep-well microplate. When the drugs are ready, the liquid
handling machine dispenses 30-50 .mu.l of a drug (at 2.times. the
final testing concentration) into the appropriate wells of the deep
well plate. After treatment, the drugs can be left on the cells for
an incubation of about 25-200 hours thus necessitating their
preparation in growth medium. In one embodiment of the invention,
the drugs are left on the cells for an incubation of 48-96 hours.
During this period, cell viability is maintained with a standard
incubator. During imaging of the cells, their viability is
maintained with a device named the BioBox and visible light images
are taken at predetermined intervals using proprietary software
named Plate Scanner. The BioBox is a humidified incubator
environment on the stage of a microscope. While the procedure uses
the BioBox, other equipment known in the art may be used in
practice. Temperature and gas composition are maintained at
37.degree. C. and 5% CO.sub.2 with air balance, respectively. It
serves the purpose of providing an environment suitable for cell
growth, while maintaining limited exposure to ambient air, which
reduces potential contamination of the plates. Plate Scanner
automates the acquisition of images from each well that has
received cells in a microtiter plate. Plate Scanner provides the
ability to choose which wavelengths of light to use as well as the
ability to decide exposure duration for each wavelength of light
chosen. In addition, the software uses focal stack imaging to
determine the physical geometry of each plate in order to optimize
image quality. The software automatically alters the light (either
visible, UV or fluorescent) to capture the necessary image and
stores the image on a hard drive. While the procedure uses Plate
Scanner, other equipment and software known in the art may be used
in practice.
[0071] At the end of the 25-200 hour incubation period, the liquid
handling machine is used to remove the media and any non-adherent
cells. Then, the remaining cells are fixed for at least 20 minutes
in 95% ethanol followed by the DNA intercalating blue fluorescent
dye, DAPI. Following fixation and staining, the automated
microscope is used to take visible and UV images of the stained
cells in every well. Afterwards, the number of cells per well in
both visible and UV light is quantified using proprietary software
named Cell Counter.
[0072] Cell Counter scans through each unique image and ascertains
the cell locations by measuring the peak pixel intensity and
aggregating pixels that are significantly above the background
signal. The software provides various filters, such as minimum
pixel intensity threshold, which allow better distinction of cells
from background noise. While the procedure uses Cell Counter, any
cell counting machine known in the art may be used in the practice
of the methods of the inventions disclosed herein.
[0073] A complete dose response curve is generated for each drug
evaluated. An Image analysis system is used in analysis of the
cells. Here, cells grown in plates are imaged using equipment and
methods known to those of ordinary skill in the art.
[0074] Modification of ChemoFx.RTM. assays, disclosed herein, are
within the ordinary skill in the art. Inclusion of other assays,
methods, procedures, tools, materials, drugs, systems, compounds
and equipment (such as for example, liquid handling machines and
the operating software) known in the art is intended to be an
option in the practice of the assays, methods, tools and systems
included in the invention disclosed herein.
[0075] In the agent 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 agent
addition is also monitored and optimized. By subjecting uniform
samples of cells to a wide variety of pharmaceutical agents (and
concentrations thereof), the most efficacious agent or combination
of agents can be determined.
[0076] For assays concerning cancer treatment, a two-stage
evaluation may be carried out in which both acute cytotoxic and
longer term inhibitory effects of a given anti-cancer agent (or
combination of agents) are investigated. 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 duration of the
malignancy is intended to cover both the initial cell culture and
determination, using one or more of the assays or methods disclosed
herein, of agents as well as the culture of chemoresistant cells
and determination, using one or more of the assays or methods
disclosed herein, of agents effective to affect the progress of the
malignancy.
[0077] The commercial potential of the assays, methods, tools and
systems disclosed herein is considerable for many reasons, but most
notably because it minimizes the number of valuable patient cells
necessary to generate dose response information, the system
optionally uses a nearly automated system for data accrual that
requires very little user intervention and data generated from the
assays disclosed herein can be used with a software package to
generate patient dose response information.
[0078] "Cancer" as used herein, includes, without limitation,
ACTH-producing tumors, acute lymphocytic leukemia, acute
nonlymphocytic leukemia, cancer of the adrenal cortex, bladder
cancer, brain cancer, breast cancer, cervix cancer, chronic
lymphocytic leukemia, chrome myelocytic leukemia, colorectal
cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal
cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia,
head and neck cancer, Hodgkin's lymphoma, kidney cancer, liver
cancer, malignant peritoneal effusion, malignant pleural effusion,
melanoma, mesothelioma, multiple myeloma, neuroblastoma,
non-Hodgkin's lymphoma, osteosarcoma, penis cancer, prostate
cancer, retinoblastoma, soft-tissue sarcoma, squamous cell
carcinomas, stomach cancer, testicular cancer, thyroid cancer,
trophoblastic neoplasms, vaginal cancer, cancer of the vulva,
Wilm's tumor and malignancies. Also included in the term "tumor" or
"tumor cell(s)" are solid tumor cells, a soft-tissue tumor cell, a
metastatic tumor cell, a leukemic tumor cell, and a lymphoid tumor
cell. The cancer may be a fibrosarcoma, myosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangio-endotheliosarcoma,
synovioma, mesothelioma, leiomyosarcoma or rhabdomyosarcoma,
epithelial carcinoma, glioma, astrocytoma, medullobastoma,
craniopharyngioma, ependymoma, pinealoma, hemangio-blastoma,
acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neurobastoma, retinoblastoma, leukemia, lymphoma, or Kaposi
sarcoma. See, for example, U.S. Pat. No. 6,884,907.
[0079] III. Cell Culture Methods
[0080] When a patient is to be treated for the presence of tumor,
in the preferred embodiment of the present methods, a tumor biopsy
of about 15 mg or more of non-necrotic, non-contaminated tissue is
harvested from the patient by any suitable biopsy or surgical
procedure known in the art. For instance, a tumor biopsy of about
15 mg, about 20 mg, about 25 mg, about 30 mg or about 35 mg or more
can be used. In one embodiment of the invention, the biopsy sample
is about or greater than 25 mg. In another embodiment, the biopsy
sample is at least about 15 to about 35 mg. Tumor sample processing
generally proceeds as follows under a Laminar Flow Hood. 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 using sterile
forceps and placed in a sterile petri dish where it is
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,
but not necessary, because the technique creates smooth cut edges
on the resulting tumor multicellular particulates. In one
embodiment, the tumor particulates measure 0.25 mm.sup.3 to 1.5
mm.sup.3. For instance, the tumor particulates can measure about
0.25 mm.sup.3, 0.30 mm.sup.3, 0.40 mm.sup.3, 0.50 mm.sup.3, 0.60
mm.sup.3, 0.70 mm.sup.3, 0.75 mm.sup.3, 0.80 mm.sup.3, 0.90
mm.sup.3, 1 mm.sup.3, 1.1 mm.sup.3, 1.2 mm.sup.3, 1.25 mm.sup.3,
1.30 mm.sup.3, 1.40 mm.sup.3, or 1.50 mm.sup.3. Preferably but not
necessarily, the tumor particulates each measure approximately 1
mm.sup.3.
[0081] In one embodiment, the particles are then agitated to
substantially release tumor cells from the tumor explant particles.
Such agitation includes any mechanical means that enable the
enhanced plating of tumor cells and includes, but is not limited
to, shaking, swirling, or rapidly disturbing the explant particles.
These procedures may be done by hand by, for instance, sharply
hitting the container against a solid object or by the use of
mechanical agitation. For instance, a standard vortex mixer may be
used. This agitation step typically increases the number of
adherent tumor cells by at least about 5%, 10%, 20% 30%, 40%, 50%,
60%, 70%, 80%, 90%, 100%, 200%, 300% (and including any percentage
in between) or more compared to non-agitated replicate samples
after about 12-48 hours or more of incubation. Chemicals or enzymes
may be employed to facilitate the release of tumor cells from the
tumor explant. Enzymatic agitation with enzymes which may include,
but are not limited to, collagenase, DNase or dispase, is also
included as an optional step in the practice of the procedures
disclosed herein.
[0082] After each tumor has been minced to particles about 1
mm.sup.3 or less, the particles are plated in culture flasks using
sterile pasteur or serological pipettes (approximately 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 explantation 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 37.degree. C., 5% CO.sub.2 incubator. Flasks should be
checked daily for growth and contamination. Over a period of
approximately a few days to a few weeks, with weekly removal and
replacement of about 5 ml of growth medium, the explants will
foster growth of cells into a monolayer.
[0083] In another embodiment, after mincing and transferring the
particles to one or more labeled flasks, the tumor explants are
exposed to a cocktail containing Collagenase II and DNase. In one
aspect of the invention, the cocktail contains 0.25% Collagenase II
and 0.001% DNase. In another aspect of the invention, the cocktail
contains about 0.025% Collagenase II and 0.001% DNase.
[0084] The amount of collagenase and DNase can be varied to achieve
the desired beneficial outcome(s) as herein described, for
instance, enzymes concentrations can include about 0.010%
collagenase to about 0.60% collagenase and about 0.0007% DNase to
about 0.005% DNase. The amount of Collagenase II required by the
methods of the present invention is the amount necessary to reduce
the size of a tumor explant when used in conjunction with DNase or
the amount required to provide the advantageous results herein
discussed. For instance, the Collagenase II and DNase solution to
which the tumor explants are exposed can contain about 0.010% or
less Collagenase II, about 0.025% or less Collagenase II, about
0.050% or less Collagenase II, about 0.075% or less Collagenase II,
0.10% or less Collagenase II, about 0.15% or less Collagenase II,
about 0.20% or less Collagenase II, about 0.25% or less Collagenase
II, about 0.30% Collagenase II, about 0.35% Collagenase II, about
0.40% Collagenase II, about 0.45% Collagenase II, about 0.50%
Collagenase II or about 0.60% or more Collagenase II (or about
0.15% to 0.6% final concentration). In one embodiment, the
Collagenase II and DNase solution contains less than about 0.30%
Collagenase II, less than about 35% Collagenase II, less than about
40% Collagenase II, less than about 45% Collagenase II or less than
about 50% Collagenase II. For instance, about 0.25% Collagenase and
0.001% DNase can be used to process ovarian tumor tissue samples
and about 0.025% Collagenase and 0.001% DNase can be used to
process colorectal tumor tissue samples. The terms "cocktail,"
"solution," and "composition" are used interchangeably herein when
referring to the use of a collagenase and DNase solution. As used
herein, "collagenase" and "Collagenase II" are used
interchangeably.
[0085] The amount of DNase required by the methods of the present
invention is the amount necessary to reduce the size of a tumor
explant when used in conjunction with Collagenase II or the amount
required to provide the advantageous results described herein. For
instance, the Collagenase II and DNase solution can contain about
0.0007% or less DNase, about 0.0008% DNase, about 0.0009% DNase,
about 0.001% DNase, about 0.002% DNase, about 0.003% DNase, about
0.004% DNase or about 0.005% or more DNase (or about 0.0007% to
0.005% final concentration).
[0086] The Collagenase II and DNase solution can comprise
Collagenase II and DNase diluted in cell culture media. In one
embodiment of the invention, Collagenase II and DNase are diluted
in Hank's Balanced Salt Solution (HBSS) media with or without
Ca.sup.2+ and Mg.sup.2+. A skilled artisan would appreciate that
various types of tissue culture media can be used to dilute
Collagenase II and DNase. For instance, a cell culture media such
as HBSS which is not a growth medium can be used. Conversely,
growth medium can be used. Also, cell type can influence or dictate
the type of media used.
[0087] The Collagenase II and DNase cocktail of the invention can
optionally contain compounds to reduce the likelihood of microbial
contamination. For instance, the composition can contain one or
more antibiotics, including, but not limited to, gentamicin,
streptomycin, kanamycin and penicillin. The composition can also
contain one or more fungicides, including, but not limited to,
nystatin and amphotericin B.
[0088] The tumor explants treated with a Collagenase II and DNase
cocktail can be incubated under conditions appropriate for the cell
type. For instance, the effect of treatment may be enhanced by
incubating the tissue explant for about 3 minutes, about 5 minutes,
about 10 minutes, about 15 minutes, about 20 minutes, about 25
minutes, about 30 minutes, about 35 minutes, about 40 minutes,
about 45 minutes, or about an hour or more with the Collagenase II
and DNase cocktail. Incubation coupled with gentle agitation may
further increase the release of cells from the treated tissue
explants. Cells can be mechanically agitated by gently shaking
cells on a shaker during incubation.
[0089] After the treatment of tumor explants with Collagenase II
and DNase, explants should preferably be washed one or more times
to remove the enzymes. Cells can be washed by methods known in the
art such as by adding cell culture media to explants, centrifuging
explants, and removing the resulting supernatant. It may be
necessary to wash cells two or three times to remove the
Collagenase II and DNase.
[0090] The use of both of the above procedures 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.
[0091] A. Primary Culture
[0092] Once a primary culture is established from a patient's
malignancy, the primary culture can be maintained without any
treatments beside normal feedings, as indicative of the growth of
the malignancy absent treatment with a therapeutic regimen.
Subcultures of the primary culture are prepared so that the cells
of the primary culture are not affected by any subsequent testing
or treatments. Although the primary culture is preferably left
untreated, either the primary culture or a subculture thereof can
be propagated as a reference culture. The reference culture is a
culture which is treated with an agent or agents reflective of a
patient's actual treatment regimen. For instance, if a patient is
treated with a pharmaceutical agent, the reference culture is
treated with the same agent in the same concentration. The
reference culture can be monitored genotypically or phenotypically,
using molecular predictors of response or markers, 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, and without limitation, thrombolytic or anti-thrombogenic
treatments, can be applied to the reference culture to reflect a
patient's treatment.
[0093] Subcultures of either the primary 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.
[0094] B. Tissue Explants
[0095] The explant is removed prior to the emergence from the
explant of a substantial number of non-target cells, resulting in a
monolayer of cells that is enriched for a tumor cell population of
interest. For example, it has been discovered that cells emerge as
a monolayer from a culture tumor tissue explant in an orderly
fashion, the tumor cells emerging first, followed by stromal cell
populations. If the tumor cell explant remains in culture, the
stromal cells have been found to dominate the tumor cells in
culture. This creates a culture that is enriched from non-target
stromal cells and that is not reflective of the in vivo cell
population. Thus, in a tumor cell culture, the explant is removed
from the growth medium prior to the emergence of a substantial
number of stromal cells from the explant. The time at which an
explant is removed from its culture medium depends upon the type of
cell being cultured, the rate of emergence of various cell types
and the desired purity of the resulting cell culture monolayer.
This can be determined empirically for a given cell type. In the
case of tumor cells, the multicellular tissue explant is preferably
removed when the cell culture monolayer is at about 10 to about 50
percent (or more) confluent. In one aspect of the method, the
multicellular tissue explant is removed at about 15 to about 25
percent confluency. In another aspect of the method, the explant is
removed at about 20 percent confluency. Percent confluency is the
estimate of the area occupied by the cells divided by the total
area in an observed field.
[0096] One method of minimizing phenotypic and/or genotypic drift
in cultures is to limit the passaging of cells and testing the
cells at the earliest moment of reaching clinical number.
Occasionally, explants are "replanted" in another culture as a
rescue technique if the first culture was not successful.
[0097] C. Methods of Determining Cell Viability
[0098] Enhanced growth of actual tumor cells is only one aspect of
the present methods. A growth rate monitoring system may also be
used to oversee growth of the monolayer once formed. Once a primary
culture and its, derived secondary monolayer tissue culture have
been initiated, the growth of the cells is monitored to ascertain
the time to initiate the chemotherapy assays and to determine the
growth rate of the cultured cells.
[0099] 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. 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, although such methods are optionally included in the
practice of the methods included in the invention disclosed
herein.
[0100] Protocols for monolayer growth rate generally use a
phase-contrast inverted microscope to examine culture flasks
incubated in a humidified 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 (monitoring cell viability) flasks, an average cell count for
the total patient sample should be calculated.
[0101] 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. See, for
example, U.S. Pat. Nos. 5,728,541; 6,887,680 and 6,416,967 and U.S.
patent application Ser. Nos. 09/040,161; 09/039,957; 09/095,993;
09/691,492 and 60/616,851.
[0102] D. Segregated Sites
[0103] The performance of the chemosensitivity assays 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.
[0104] The first step in preparing the microtiter plates is
preparing and monitoring the monolayer as described above. The
following protocol is exemplary [all protocols herein are
exemplary] and variations are apparent to one skilled in the art.
Other methods employing microtiter plates and plating cells are
publicly available, well known to those of skill in the art and are
intended to be used as an option in the practice of, or in
conjunction with, the assays, methods, tools and systems disclosed
herein.
[0105] 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 30 .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 30 .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.
[0106] After the desired concentration of cells in medium has been
determined, the resulting cell solution is placed in a channel of a
deep well plate. An automated liquid handling system delivers the
appropriate amount of cell solution to each well of a 384 well
microtiter plate. A plurality of plates may be prepared from a
single cell suspension as needed.
[0107] After the microtiter plates have been prepared, exposure of
the cells therein to one or more pharmaceutical agents is conducted
according to the following exemplary protocol. During this portion
of the assay, the appropriate amount of specific pharmaceutical
agent or agents is transferred into the microtiter plates prepared
using an automated liquid handling device.
[0108] A general protocol, which may be adapted, follows. Each
microtiter plate is 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 being tested are administered into
progressively higher numbered rows in the plate. The plates are
then incubated in a humidified incubator at 37.degree. C. under 5%
CO.sub.2. After a predefined exposure time, the plates are fixed
and stained for evaluation.
[0109] E. Types of Wells or Culture Plates Used
[0110] Standard tissue culture plates can be utilized for the assay
comprising 384 equivalent wells. Each well is capable of holding
approximately 120 .mu.l of solution. As can be appreciated by a
skilled artisan, various sizes of tissue culture plates can be
used. For instance, wells may be reduced in size to hold only 80
.mu.l. In one embodiment, the plates are made of molded plastic.
Glass bottom plates of standard coverslip thickness may be used. In
such a case, the glass bottom plates may be pretreated with a thin
layer of extracellular matrix material such as collagen, vitrogen,
fibronectin or the like.
[0111] IV. Treatment Protocols
[0112] For each drug tested as a single agent, an initial 10 dose
range of concentrations to be used in the assay is determined (see
below). Patient-derived tumor cells are treated with the indicated
drug(s) at their indicated dosages for a period of about 25 to 200
hours. In one embodiment, the treatment period is 72 hours.
However, the agent tested can dictate a shorter or longer treatment
period. For instance, biological agents may require longer
treatment periods than traditional pharmaceutical agents.
[0113] Beginning with Dose 10 (the highest dose tested), serial
dilutions of the same magnitude are repeated to create Doses 9
through 1. Dilutions are prepared in the medium type or balanced
solution that is appropriate for the tumor type and drug being
tested. The initial dosages may be adjusted so that 0% cell kill is
evident at Doses 1-2, and maximal cell kill is evident at Doses
9-10. Dosages are preferably validated on at least 15
patient-derived tumor cell cultures of the appropriate tumor
type(s) for each drug.
[0114] Following the establishment of 25 to 200 hour dosing levels
for several of the drugs tested as single agents, a new method of
dealing with combination treatments was developed. The 25 to 200
hour combination drug dosing developed is consistent and flexible
for numerous combinations (including 2, 3 or 4 drug combinations).
Essentially the highest doses of each drug in the combination are
mixed resulting in the same concentration of each drug when tested
as a single agent and a serial dilution series is created to give
10 dose levels for the drugs.
[0115] A combination of immunotherapy and/or radiation treatment
and/or chemotherapy is also useful for treatment of tumors that are
resistant to one or more chemotherapeutic agents. Chemotherapy
alone has limitations in that the cancer cells often become
resistant to a broad spectrum of structurally unrelated
chemotherapeutic agents. Such resistance, termed "multidrug
resistance" (MDR), is not an uncommon problem in the treatment of
patients with cancer and while significant efforts have been made
to understand the mechanisms responsible for MDR, that
understanding has not fulfilled the expectations for eradicating
chemoresistant cancer cells.
[0116] Immunotherapy, alone or in combination with radiotherapy,
has also been investigated as a method for inhibiting or
eradicating cancer cells. Such methods are useful for treating
patients whose tumors are chemoresistant to one or more
chemotherapeutic agents. Immunotherapy, alone or in combination
with radiotherapy, and in conjunction with the assays, methods and
tools described herein are suitable for use with patients whose
tumors are chemoresistant.
[0117] V. Preparation and Determination of Dose Levels
[0118] The ordinarily skilled artisan may select an appropriate
amount of each individual pharmaceutical agent in the combination
for use in the aforementioned assays or similar assays. Changes in
chemotherapeutic drug metabolism, drug transport, cell apoptosis,
cell proliferation, DNA repair or other biological activity
(including gene expression) are used to determine whether the
selected amounts are "effective amounts" for the particular
combination of agents/compounds.
[0119] The regimen of administration also can affect what
constitutes an effective amount. Further, several divided dosages,
as well as staggered dosages, can be administered daily
sequentially to the microtiter plates, or the dose can be
proportionally increased or decreased as indicated by the
exigencies of the therapeutic situation. See, for example, U.S.
Pat. No. 6,599,912. In another embodiment, a first agent and a
second agent are administered to the cells at the same time or in
overlapping time periods; the first agent and the second agent are
administered at different times; the first agent is administered
first and the second agent is administered subsequently; the second
agent is administered first and the first agent is administered
subsequently.
[0120] Dosages of drugs are initially determined based on
concentration of drug determined to be present in the extracellular
fluid surrounding a tumor in vivo (information is extracted from
the literature) and/or the range of concentrations of the drug
reported to elicit an anticancer effect in similar in vitro models.
Once use of one or more agents is indicated, the initial dosages
are determined and a series of dilutions is prepared from each
agent such that the range of the dilutions covers the range of
initially determined doses and also includes dose levels resulting
in 0% and up to and including maximal cell killing. Thus, the upper
and lower levels of each agent are determined. This procedure is
used on chemoresistant cells as well as cells untested for
chemotherapeutic agent sensitivity.
[0121] In one embodiment of the methods and assays disclosed
herein, an agent is used at a dose level where the lowest dose had
a minimal effect on cell viability and the highest dose had a
moderate to strong effect on cell viability. In another embodiment,
the methods further include repeated dosages of the same, or a
different agent. In therapeutic applications, the dosages of the
agents used in the methods herein vary depending on the agent and
clinical condition of the recipient patient, and the experience and
judgment of the clinician or practitioner administering the
therapy, among other factors affecting the selected dosage.
Generally, the dose should be sufficient to result in slowing, and
preferably regressing, the growth of the tumor cells and also
preferably causing complete regression of the cancer in vitro or in
vivo. In one embodiment, an effective amount of a pharmaceutical
agent is that amount which provides an objectively identifiable
slowing, or death or regression of the tumor cells in vitro. See,
for example, U.S. Pat. No. 6,875,745.
[0122] In one embodiment of the assay methods included in the
invention disclosed herein, the cells to be assayed are grown on
microliter 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.
[0123] VI. Phenotypic and Genotypic Drift
[0124] It has been recognized that when patient cells are removed
from their in situ locations in tissues and cultured in vitro, the
cells are subject to phenotypic and genotypic drift, i.e., they
begin to lose some of the morphological features (and components)
of some characteristic of their tissue or organ of origin,
sometimes as a result of changes in expression of a gene, or
expression of a mutated gene. This instability is the result of
culture condition variations, selective overgrowth of some cells in
the population, and genetic variation. As it is important to
standardize the culture so that the cell population remains as
stable as possible over time, explants and seed stocks of the cell
culture are often preserved. Cell preservation minimizes the
genetic and phenotypic drift in cultures, serves to avoid
senescence, guards against contamination and provides a stock
culture, should the "working" culture become contaminated, change,
or otherwise unusable. See, for example, U.S. Pat. No. 5,587,297
and U.S. Pat. No. 6,528,309.
[0125] In one embodiment of the invention, the adherent cells are
analyzed prior to fixation and staining. Such analysis may include
but is not limited to treating the remaining adherent cells with
additional drugs to determine response to a second regiment of
chemotherapeutic agents. Such analysis may include but is not
limited to analysis of different vital stains to measure cell
viability, membrane integrity, cell signaling pathways, apoptosis,
multi-drug resistance (MDR) ability, etcetera. Such analysis may
include but is not limited to genotypic analysis for gene
expression or genome mutations, phenotype analysis, such as
expression of surface proteins, cell viability, immunohistochemical
analysis and pathological analysis. Subsequent to analysis of
adherent cells as mentioned above, the cells are fixed and stained
for counting/analysis as described in Version 2 assay method.
[0126] A. Phenotypic Changes
[0127] Changes in phenotype are monitored by a variety of ways,
using techniques and methods publicly available and well known to
those of skill in the art. In one aspect included in the invention,
a phenotypic assay is employed to assess response to culture
conditions and may also be employed to assess sensitivity and/or
resistance to chemotherapeutic agents and as an indicator of the
occurrence of phenotypic drift. The phenotypic assay is performed
in vitro using the cultured cells. The phenotype assay also allows
for identification and separation of tumor cells from other cells
found in a tissue sample, as well as direct measurement and
monitoring of tumor cells in response to chemotherapeutic and/or
radiation treatment and/or immunotherapy treatment. Direct
measurements and monitoring of live tumor cells are performed using
known methods in the art including, for example, the measuring of
doubling rate, proliferative assays, monitoring of cytostasis, cell
death, cell adhesion, gene expression, cell motility, cell
invasiveness and others. Direct measurements also include known
assays, such as those directed to measurement and monitoring of
apoptosis, senescence and necrosis. Phenotypic assays may also
measure changes in a molecular predictor of response.
[0128] Once a primary culture and its derived secondary monolayer
tissue culture have been initiated, the growth of the cells is
monitored to oversee growth of the monolayer and ascertain the time
to initiate the phenotypic assay. Prior to the phenotypic assay,
monitoring of the growth of cells may be conducted by visual
monitoring of the flasks on a periodic basis, without killing or
staining the cells and without removing any cells from the culture
flask. Data from periodic counting or measuring is then used to
determine growth rates or cell motility, respectively, using
methods known to those of skill in the art.
[0129] One embodiment of the present methods contemplates a
phenotypic assay that assesses whether chemotherapeutic agents
affect cell growth. Monolayer growth rate is monitored using, for
example, a phase-contrast inverted microscope. Following initial
culturing of the multicellular tissue explant, the tissue explant
is removed from the growth medium at a predetermined time. In one
embodiment of the invention, the predetermined time is 1 to 40 days
after disaggregation, i.e., agitation, of the tumor specimen. In
another embodiment, the predetermined time is 6 to 30 days or 6 to
28 days after disaggregation of the tumor specimen. In another
embodiment, the predetermined time is 7 to 21 or 7 to 15 days after
disaggregation of the tumor specimen. In yet another embodiment,
the predetermined time is 7 to 10 days after disaggregation of the
tumor specimen.
[0130] The predetermined time for removal of the tissue explant
from growth medium can also be calculated from the time of initial
culturing of the tissue explant, i.e., preparation of cell culture
monolayer. In one embodiment of the invention, the predetermined
time is 1 to 40 days after initial culturing of the tumor explant.
In another embodiment, the predetermined time is 6 to 30 days or 6
to 28 days after initial culturing of the tumor explant. In another
embodiment, the predetermined time is 7 to 21 or 7 to 15 days after
initial culturing of the tumor explant. In yet another embodiment,
the predetermined time is 7 to 10 days after initial culturing of
the tumor explant.
[0131] In one embodiment of the methods disclosed herein, the
explant is removed from the growth medium prior to the emergence of
a substantial number of stromal cells from the explant.
Alternatively, the explant may be removed according to the percent
confluence of the cell culture. In another embodiment of the
methods disclosed herein, the explant is removed at about 10 to
about 50 percent confluence. In yet a different embodiment, the
explant is removed at about 15 to about 25 percent confluence. In
another embodiment, the explant is removed at about 20 percent
confluence. By removing the explant in any of the above manners, a
cell culture monolayer predominantly composed of tumor cells is
produced.
[0132] In another embodiment, a phenotypic assay assesses whether
chemotherapeutic agents affect cell motility. Methods for measuring
cell motility are known by persons skilled in the art and any
method for assessing cell motility is optionally used in
conjunction with the assays and methods disclosed herein.
Generally, these methods monitor and record the changes in cell
position over time. Examples of such methods include, but are not
limited to video microscopy, optical motility scanning (for
example, see U.S. Pat. No. 6,238,874) and impedance assays. In one
embodiment, cell motility assays are carried out using monolayer
cultures of tumor cells as described herein.
[0133] An important aspect included in the present invention is to
provide a system for screening specific tissue samples from
individual patients for expressed or non-expressed cellular
markers, such as receptors, secreted factors, or antigens,
including tumor antigens, characteristic of the tissue sample. For
instance, a tumor 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. 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.
[0134] 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, and at least one
molecular predictor of response as described herein. By screening
tumor samples in this manner, for production of such factors,
markers or antigens, the cultured cells may be further identified
and monitored, aiding the physician in treatment strategies and as
a prognosis indicator. Furthermore, by combining the use of the
culture technique with assaying for markers, factors or antigens as
described herein, a treatment strategy for a cancer or disease
state may be optimized and treatment progression may be monitored.
High throughput phenotypic analysis of cells can be accomplished
through the use of automated processes available commercially.
[0135] B. Genotypic Changes
[0136] Genomics is defined as the study of genes, their composition
and their expression patterns. As such, genomic analysis
encompasses genetic variability such as DNA mutations or single
nucleotide polymorphisms (SNPs), as well as alterations in gene
expression at the RNA level. More specifically, pharmacogenomics
encompasses the effect of genetic variability on drug response.
Although a relatively new practice, pharmacogenomics can be applied
to patients with a variety of different disease types, such as
hypertension, asthma and cancer. In the case of cancer
pharmacogenomics, there are a variety of molecular factors which
may influence chemoresponse, including, for example, pathways
involved in drug metabolism, uptake, efflux, activation and
detoxification, as well as target gene expression and DNA repair
mechanisms. Thus, multiple genes play a role in response to
chemotherapy, and genetic variations within drug pathway genes are
often associated with clinical resistance to the drug's effects.
These genetic alterations are often detected as SNPs and/or altered
gene expression.
[0137] In the clinical setting, the goal of cancer pharmacogenomics
is to develop a "predictor" of an individual patient's response to
a given chemotherapeutic agent. The predictor is most commonly in
the form of a subset (generally, less than 60) of SNPs and/or tumor
expression of genes which is correlated with patient response to
chemotherapy. From a bioinformatics perspective, the potential
components of a predictor can be obtained in a variety of ways.
[0138] First, a "discovery" approach takes into consideration as
many SNPs and/or genes in the human genome as possible. With the
advent of the Human Genome Project, the discovery approach has
become possible because of the availability of prefabricated
microarrays which comprise over 30,000 human genes; additionally,
"SNP chips" are also available.
[0139] Alternatively, a "candidate" gene approach may be taken. In
this instance, specific genes are chosen based on
biologically-based findings in the literature and/or scientific
intuition; and the expression of these candidate genes can then be
analyzed using more quantitative methods, such as RT-PCR. However,
any method known in the art to quantitatively or qualitatively
analyze gene expression is optionally used in the practice of the
methods included in the invention.
[0140] Lastly, a "hybrid" of the previous two approaches may be
utilized in which gene classes (e.g., cell cycle genes, apoptosis
genes, cell proliferation genes, drug transport genes and drug
metabolism genes) may be used as a starting point to identify genes
with which to begin. Microarrays or RT-PCR may be used to determine
the genes which are most predictive of patient response.
[0141] A genotype assay is performed to determine whether cells
from a patient comprise a genetic characteristic associated with
resistance to the chemotherapeutic agents. Genotype assays reveal
latent resistance to chemotherapeutic agents not observed by
phenotypic assays. Genotypic assays may measure characteristics,
such as, for example, drug metabolism, drug transport, cell
proliferation, apoptosis, DNA repair, toxic effects, cell
invasiveness and extracellular matrix formation.
[0142] In addition, the assays often involve the evaluation of
nucleic acids in tumor cells and/or the presence of polymorphisms
(SNPs). The methods for evaluating the nucleic acids of tumor cells
are many, and include without limitation, hybridization studies
(e.g., Southern and northern blots), nucleic acid sequencing,
fingerprinting (e.g., the analysis of restriction fragment length
polymorphisms) and PCR-based protocols, which may be quantitative
and/or qualitative in nature. These protocols may be performed in a
traditional manner, i.e., by running the results on a gel, or by
newer methods known in the art. Newer methods may involve
miniaturization and multiplication of the traditional protocols and
include nucleic acid micro arrays and a variety of applications
that may be performed in connection with such micro arrays, for
instance, DNA:DNA or DNA:RNA hybridizations and competitive
hybridizations. Arrays permit the rapid analysis of a nucleic acid
sample for the presence of or absence of hundreds to many tens of
thousands independent nucleic acids. These independent nucleic
acids may be from genes of known function or from nucleic acids of
unknown function, i.e., expressed sequence tags (EST).
[0143] The nucleic acid can be any nucleic acid that is present in
the proliferating cells, i.e., RNA or DNA. The analyzing step
typically involves use of one or more analytical methods known to
have the capacity to characterize the nucleic acids, including,
without limitation quantitative methods that identify the amount of
specific RNAs in a cell as well as qualitative methods that
determines the presence of or absence of specific genetic markers,
such as DNA or RNA sequence insertions, deletions or
substitutions.
[0144] The data generated by the analytical process of the present
invention (hereinafter, the "genetic data") can be gathered to form
a record that is part of a data set in a data structure. Data
indicating the phenotype of the tumor cells also may be included.
This phenotypic data includes, without limitation, histochemical,
immunohistochemical, biochemical and growth characteristics of the
cells and/or tumor, including the production of secreted compounds,
whether or not the tumor cells were cultured according to the
methods of the present invention. Non-genetic analysis, i.e.,
phenotypic analysis, is also of value in the diagnosis of tumor
diseases. The resultant non-genetic data can be combined with the
above described genetic data to provide a complete and accurate
profile of the tumor cells of the tissue sample.
[0145] Once a primary culture is established from a patient's
abnormal proliferating cells, the primary culture can be maintained
without any treatments beside normal feedings and passage
techniques, as indicative of the growth of the cells absent
treatment with a therapeutic regimen. Subcultures of the primary
culture are prepared so that the cells of the primary culture are
not affected by any subsequent testing or treatments. Although the
primary culture is preferably left untreated, either the primary
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
and/or phenotypically to reflect actual progress of the tumor or
disease or condition 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 and to indicate a possible
interaction of other drugs with the chemotherapeutic agent(s).
Subcultures of either the primary culture or the reference culture
can be used for further analysis, such as the genotypic analysis or
phenotypic analysis techniques of the present invention.
Preferably, since the reference culture is indicative of the
current state in a patient of a tumor or disease, subcultures of
the reference culture are analyzed.
[0146] Molecular predictors of response may be monitored by
genotypic assays, phenotypic assays or both using patient tumor
cells obtained or cultured by one or more of the methods disclosed
herein at any stage of the culture process. The cells to be
monitored include untested tumor cells, known chemoresistant tumor
cells and known chemosensitive tumor cells.
[0147] 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. Tumor cells prepared according
to the culture methods of-the present invention are then
genetically analyzed for markers specific to the tumor or disease
state of the cells. The cells that are analyzed typically are from
subcultures of the primary or reference cultures. In this process,
nucleic acid is isolated from the cells and is analyzed to identify
markers that are characteristic of abnormal proliferating cells.
The isolated nucleic acid is DNA or RNA. The nucleic acid,
preferably, is analyzed in a microarray for expression of one or
more genes.
[0148] Preferably, the microarray contains nucleic acids that are
characteristic of known proliferative or tumor disease states, as
well as nucleic acids, that are not correlated with known
proliferative or tumor disease states, so that previously unknown
relationships between gene expression and a cancer, proliferative
disease or condition may be identified. Methods for isolation and
analysis of the nucleic acids of the cells are varied and typically
differ from laboratory to laboratory. Further, certain analytical
methods may require that the nucleic acid is prepared in a specific
manner. Nucleic acid purification methods may be found in anyone of
a number of molecular biology laboratory texts. Purification
products or systems also are commercially available.
[0149] The presence of known proliferation markers, such as the
expression of one or more genes, may be determined by
methodologies, without limitation, by northern blotting or
quantitative polymerase chain reaction (PCR) methods, i.e.,
RT-PCR.
[0150] Microarrays of either known DNAs or unknown DNAs, i.e.,
partially identified or unidentified expressed sequence tags
(ESTs), are now commercially available from a large number of
commercial sources. Custom microarrays may be prepared in the
laboratory with commercially available robotic devices or can be
purchased from one or more commercial sources for microarrays. DNA
microarrays can include hundreds to many thousands of unique DNA
samples covalently bound to a glass slide or other substrate in a
very small area. By hybridizing labeled cDNA or other labeled
nucleic acid or ligand that can be hybridized specifically to the
covalently-bound nucleic acid to the array, the altered expression
of one of more genes may be identified.
[0151] It is possible to label cDNA from two cell types, i.e.,
normal and tumor cells, and hybridize equivalent amounts of both
probe populations to a single microarray to identify differences in
RNA expression for both normal and tumor cells. Tools for
automating preparation and analysis of micro array assays, such as
micro array scanners and readers, are available commercially. The
automation of the microarray analytical process is desirable and,
for all practical purposes necessary, due to the huge number and
small size of discrete sites on the micro array that must be
analyzed.
[0152] DNA microarrays are possibly the more powerful tools to
utilize in combination with the cell culturing method of the
present invention due to the increased sensitivity of mRNA
quantification protocols when a substantially pure population of
tumor cells is used. For their ease of use and their ability to
generate large amounts of data, microarrays are preferred, when
practicable. However, certain other or additional qualitative
assays may be preferred in order to identify certain markers.
[0153] The presence of, or absence of, specific RNA or DNA species
also may be identified by PCR procedures. Known genetic
polymorphisms (SNPs), translocations, or insertions (e.g.,
retroviral insertions or the insertion of mobile elements, such as
transposons) often can be identified by conducting PCR reactions
with DNA isolated from cells cultured by the methods of the present
invention.
[0154] Where the sequence anomalies are located in exons, the
genetic polymorphisms may be identified by conducting a PCR
reaction using a cDNA template. Aberrant splicing of RNA precursors
also may be identified by conducting a PCR reaction using a cDNA
template. An expressed translocated sequence may be identified in a
microarray assay. Small or single nucleotide substitutions may be
identified by the direct sequencing of a given gene by the use of
gene-specific oligonucleotides as sequencing primers. Single
nucleotide mutations also may be identified through the use of
allelic discrimination molecular beacon probes. See, for example,
those described in Tyagi, S. and Kramer, F. R. (1996) Nature
Biotech. 14:303-308 and in Tyagi, S. et al., (1998) Nature Biotech.
16:49-53. Mass spectrometry (MALDI-TOF) may also be used to
identify mutations.
[0155] While the above-mentioned assays are useful in the analysis
of nucleic acids derived from cells produced by the culture methods
included in the invention, numerous additional methods are known in
the general fields of molecular biology and molecular diagnostics
that may be used in place of the above-referenced methods.
[0156] Any or all of the steps of the unified assays and culturing
techniques included in the present invention may be automated. 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 programmed to reduce the
data to the indices described above, or to any other relevant form,
i.e., graphical or figurative representations of the data.
[0157] The methods of the invention further include the step of
characterizing the tumor cells by analyzing the genetic and/or
phenotypic data in connection with a set of corresponding clinical
data for statistically significant commonalities and/or trends to
generate one or more profiles which link one or more proliferative
cell disease states with phenotypic and/or genotypic
characterizations, diagnoses and/or prognoses. These data and/or
profiles may be encoded in a computer storage medium and stored in
a data base. The contents of these databases include, but are not
limited to, observed in vitro phenotypes (disease factors) and
genotypes (host factors). A method for diagnosing proliferative
diseases is also provided that compares either 1) the genetic and
corresponding clinical data and/or 2) the profiles generated
therefrom, to data generated in connection with a new tissue
sample. By applying analytical techniques to the stored phenotypic
and genotypic information, predictions of chemotherapeutic efficacy
can be made. A computer system containing the data and/or profiles
is also provided that, optionally, allows dissemination and/or
analysis of the data over a computer network.
[0158] VII. Method of Generating a Dose Response Curve
[0159] Cells harvested from patient tumor explants can be seeded
into a black-walled, clear bottom 384 microplate at a concentration
of about 4,000 to 12,000 cells/ml and 30-50 .mu.l of cell
suspension per well. In one embodiment, explants are seeded at a
concentration of about 8,000 cells/ml and 40 .mu.l of cell
suspension per well. Cells can be seeded according to the number of
drug treatments requested by the oncologist with three replicates
per dose of each drug. To prevent evaporation of medium, the
outermost ring of wells on the microplate can be filled with
buffer, for instance, about 80 .mu.l of Hank's Balanced Salt
Solution (HBSS). Cells can then be allowed to attach to the bottom
of the microplate during a 24 hour incubation period. After the
initial incubation, doses of each drug, or combination of drugs,
can be added to the patient plate with about 30 to 50 .mu.l of a
dose per well and one control well treated with growth medium per
10 doses.
[0160] In one embodiment, immediately prior to drug application,
images of the cells are taken with an automated cell imaging system
using visible light. The microtiter plate is placed on the
automated cell imaging system. Each well of a microtiter plate is
scanned to capture images. Alternatively, only previously selected
wells are imaged. Images are analyzed to determine the number of
cells in each well. Then each well is treated with the appropriate
amount of drug, and the cells are incubated with drug for a set
about of time. In one embodiment, the cells are incubated with drug
for about 25-200 hours. At about a specified time after plating,
for instance, at about 96 hours after plating, the cells are again
imaged with visible, fluorescent and UV light using the automated
cell imaging system.
[0161] Cellular imaging after 96 hours can be accomplished by
visible light or through fluorescent light utilizing the
appropriate cellular fluorescent dyes. Such fluorescent dyes may
label the nucleus, the cell membrane, organelles, or constituents
of the cytoplasm. Alternatively, the fluorescent dye may require
activation in metabolically active cells, in which case only living
cells would fluoresce. The wavelengths of fluorescent light range
from 250 nm to 800 nm depending on the characteristics of the
fluorescent dye. Using an automated imaging system to capture
images enables the unique identification of each cell or cell
confluency based on visible imaging or fluorescent light in each
well.
[0162] The cells can then be fixed in the plate using ethanol or
other standard fixative used in the art and stained with a nuclear
stain, such as DAPI. The automated cell imaging system can then be
used to take fluorescent images of the patient cells so that cell
nuclei can be counted. The data generated from the visible and
fluorescent images can then be used to generate dose response
curves for each drug the patient cells were treated with.
[0163] In one embodiment of the method, prior to fixation of the
cells, the cells are treated with two fluorescent dyes which are
able to distinguish living from dead cells. The integrity of the
stains survives the fixation process. After fixation, the cells are
stained with a nuclear stain, such as DAPI. The automated imaging
system can then capture images with three different wavelengths of
light. Subsequent analysis of the images can enable the
determination for each cell of a live or dead status. The data
generated from the visible and fluorescent images can then be used
to generate dose response curves for each drug the patient cells
were treated with.
[0164] VIII. Methods of Cell Fixing and Staining
[0165] Fixing and staining may be conducted according to a number
of suitable procedures; the following is representative. Other
methods of fixing and staining cells are publicly available, well
known to those of skill in the art and are intended to be used as
an option in the practice of the methods disclosed herein.
[0166] For example, when fluorescent light is used to quantitate
cells (e.g., to determine viability or confluence), a stain such as
calcein AM or a cytotracker dye can be used. Fluorescent images can
be taken of the cells at predetermined intervals or at any time
throughout the course of the experiment to track the cell counts
(for example, viability due to the effects of the one or more
agents on confluence). The dyes chosen for this procedure should
not affect either cell growth characteristics or drug efficacy
characteristics.
[0167] In one embodiment of the invention, after removal of the
plates from the incubator box, culture medium/drug is removed by an
automated liquid handler. Continuing to use the automated liquid
handler, the plates are rinsed with about 40-60 .mu.l of HBSS, and
about 40-80 .mu.l of ethanol is added to each well of the plate for
at least 10 minutes. Ethanol is removed, and staining is
accomplished with approximately about 50-70 .mu.l of a DAPI
solution per well for at least 20 minutes. The automated liquid
handler removes the DAPI solution from each well, followed by the
addition of about 50-70 .mu.l of water. The plates are now prepared
to be scanned.
[0168] Alternatively, this procedure may be performed manually, in
the absence of an automated liquid handler. In that instance,
plates are removed from the incubator and culture medium/drug is
removed from each well with a manually operated pipette. 60-80
.mu.l of ethanol is added to each well for at least 10 minutes.
Ethanol is removed by plate inversion and vigorous shaking. 60-80
.mu.l of DAPI solution is added to each well for at least 20
minutes, followed by removal via plate inversion and vigorous
shaking. After about 60-80 .mu.l of water is added to each well,
the plates can be scanned.
[0169] 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.
Other techniques for counting cells are publicly available, well
known to those of skill in the art, and intended to be used as an
option in the practice of the methods disclosed herein.
[0170] The techniques disclosed herein for fixing and counting
cells is intended as exemplary; other methods are known in the art
and intended to be used as an option in the practice of the methods
disclosed herein. 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
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 or other
non-epithelial cell markers as a stain to confirm the
non-epithelial nature of the cells.
[0171] The importance of having a stain or stain cocktail (for
example, a cocktail comprised of at least one antibody), as well as
an overall protocol, for identifying epithelial cells in explants
or 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, can be confirmed with independent proof
before the cells are 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.
[0172] In general, 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. The methods disclosed herein improve the
cytotoxicity assay by adding the epithelial staining protocol with
any known epithelial stain and a 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.
[0173] 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.
[0174] IX. Targeting Agents
[0175] Binding of the targeting agent to the epithelial marker is
revealed with associated staining procedures and reactions which
give a visual indication that the marker binding took place.
Various techniques already available to reveal whether marker
binding took place. 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.
[0176] There is an advantage in using one or more binding reagents
together. The combination of two general binding reagents
(containing a total of three monoclonal antibodies) for
cytokeratin, for example, admixed with a general binding reagent
for EMA glycoprotein, for example, is advantageous. 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.
[0177] Although the binding reagents and other reagents identified
in the Examples are the preferred reagents for use in the practice
of the methods disclosed herein, 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. 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. Vimentin can be considered a binding reagent
which is specific to non-epithelial cells of mesenchymal
origin.
[0178] In a further aspect of the present methods, immunological
markers may be monitored in applications requiring up- or
down-regulation of such markers, such as, for example, Major
Histocompatibility Complex (MHC) molecules. This aspect can be
especially useful in monitoring phenotypic or genotypic drift.
[0179] X. Pharmaceutical Agents
[0180] In conventional therapy, residual tumor cells are left
undamaged due to chemoresistance or due to the fact that these
cells are located in hypoxic areas poorly vascularized and not
accessible to conventional treatments. The genetic instability and
heterogeneity of tumors allows them to adapt and to develop
resistance to therapies. The beneficial effects of chemotherapy can
be compromised by cellular mechanisms that allow tumor tissue to
evade the toxicity of drugs. In some cases, pleiotropic resistance
to a variety of unrelated drugs has been observed, and this
phenomenon has been called multidrug resistance. To combat
multidrug resistance and to increase efficacy of treatment,
therapies comprised of one or more agents (combination therapies)
have been developed.
[0181] A combination therapy includes one or more of the following
chemotherapeutic agents: anthracyclins, daunorubicin, adriamycin,
taxoid derivatives, vinca alcaloids, vincristine, carmustine,
cisplatin, fluorouracils, cytostatic compounds such as polyamine
inhibitors, topoisomerase inhibitors, tamoxifene, prodasone, or
sandostatine, or compounds inducing apoptosis such as sodium
butyrate or mitomycin C, protease inhibitors or foscarnet. An agent
in the combination therapy may also be an antimicrotubule agent, a
topoisomerase I inhibitor, a topoisomerase II inhibitor, an
antimetabolite, a mitotic inhibitor, an alkylating agent, an
intercalating agent, an agent capable of interfering with a signal
transduction pathway, a selective estrogen receptor modulator, an
aromatase inhibitor, an agent that promotes apoptosis and/or
necrosis, an interferon, an interleukin, a tumor necrosis factor,
and radiation. In one embodiment of the methods disclosed herein,
the agent is one or more of paclitaxel, interferon alpha,
gemcitabine, fludarabine, carboplatin, cisplatin, doxorubicin,
epirubicin, 5-fluorouracil, leucovorin, UFT, tamoxifen, goserelin,
ketoconazole, leuprolide (Lupron) or flutamide. In one embodiment,
an agent is vinblastine, vincristine, vindesine, vinorelbine,
docetaxel (e.g., Taxotere), camptothecin, topotecan, irinotecan
hydrochloride (e.g., Camptosar), etoposide, mitoxantrone,
daunorubicin, idarubicin, teniposide, amsacrine, merbarone,
piroxantrone hydrochloride, methotrexate, 6-mercaptopurine,
6-thioguanine, fludarabine phosphate, cytarabine (Ara-C),
trimetrexate, acivicin, alanosine, pyrazofurin,
N-Phosphoracetyl-L-Asparate, Phosphoracetyl-L-Asparate (PALA),
pentostatin, N-Phosphoracetyl-L-Asparate, pentostatin,
5-azacitidine, 5-azacitidine, 5-Aza-5-Aza-2'-deoxycytidine,
adenosine arabinoside (Ara-A), cladribine, ftorafur, UFT
(combination of uracil and ftorafur), 5-fluoro-2'-deoxyuridine,
5-fluorouridine, 5'-deoxy-5-fluorouridine, hydroxyurea,
dihydrolenchlorambucil, tiazofurin, oxaliplatin, mitomycin C,
melphalan, thiotepa, busulfan, chlorambucil, plicamycin,
dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen
mustard, uracil mustard, pipobroman, 4-ipomeanol, dihydrolenperone,
spiromustine, geldenamycin, cytochalasins, depsipeptide, tamoxifen,
4'-cyano-3-(4-(e.g., Zoladex) and
4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-3-methyl-3'-(trifluorometh-
yl)propionanilide, pemetrexed and radiation. In one embodiment, an
agent is the biologically active metabolite of any of the above
listed agents.
[0182] Biological response modifiers may also be used. Such agents
include for example, anti-Her2/neu antibodies (e.g., Herceptin),
anti-EGFR antibodies (e.g., Erbitux), other growth factor receptor
antibodies (e.g., Avastin), small molecule inhibitors (e.g.,
Tarceva, Iressa), anti-CD20 (e.g., Rituxan), interferon alpha,
interferon beta, interferon gamma, interleukin 2, interleukin 4,
interleukin 12 and tumor necrosis factors.
[0183] The agents described here may be used in the cell culture
methods singly or in a cocktail containing two or more agents or
one of the agents with other therapeutic agents, including but not
limited to, immunosuppressive agents, potentiators and side-effect
relieving agents.
[0184] The therapeutic agents may be compositions also including,
depending on the formulation desired, pharmaceutically-acceptable,
nontoxic carriers or diluents. Many pharmaceutically acceptable
carriers are known in the art (See, for example, Remington's
Pharmaceutical Sciences) and are optionally used in the practice of
any of the methods or assays of the invention. The diluent is
selected so as not to affect the biological activity of the
combination. Examples of such diluents are distilled water,
physiological saline, Ringer's solution, dextrose solution, and
Hank's balanced salt solution. In addition, the pharmaceutical
composition or formulation may also include other carriers,
adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers
and the like. Effective amounts of such diluent or carrier will be
those amounts which are effective to obtain a pharmaceutically
acceptable formulation in terms of solubility of components, or
biological activity, or desired chemoresponse.
[0185] XI. Apoptosis Assay
[0186] It is now well documented that the induction of apoptosis in
tumor cells is a key mechanism for most anti-tumor therapies,
including chemotherapy, radiation, immunotherapy and cytokines.
More recently, studies have applied measurement of the apoptotic
response to the determination of chemo-sensitivity. These studies
indicate that drug-induced apoptosis but not antiproliferative
measurement, can predict tumor response to chemotherapeutic drugs.
Furthermore, the in vitro response of tumor cells exposed to
physiological doses of chemotherapeutic agents can be tested for
sensitivity or resistance by employing markers of apoptosis which
correlate with tumor cell death. Methods of inducing, measuring and
monitoring apoptosis are known in the art (See, for example,
International Appl. No. PCT/US04/039650) and are optionally used in
conjunction with the assays, methods, tool and systems included in
the invention disclosed herein.
[0187] XII. Choice of Agent(s)
[0188] In one aspect included in the invention, a course of
chemotherapy is selected based on results obtained from the
chemosensitivity, phenotypic, genotypic and/or apoptosis assays.
The present invention includes the assessment of the likelihood of
whether chemotherapeutic agents will be effective in treating a
malignancy in a patient. Assessment of results from phenotypic
assays optionally in combination with genotypic assays optionally
in combination with apoptosis assays, as well as assessment of at
least one molecular predictor of response, operates to minimize the
risk of administering to a patient a chemotherapeutic agent or
combinations of chemotherapeutic agents to which the tumor is
resistant. In one aspect of the invention, chemotherapeutic agents
or combinations of chemotherapeutic agents are selected for
treatment where an effect on cellular phenotype is observed and the
genotypic characteristics associated with resistance are not
observed. In another aspect of the invention, chemotherapeutic
agents or combinations of chemotherapeutic agents are selected for
treatment where an effect on cellular phenotype is not observed and
the genotypic characteristics associated with resistance are
observed. In a different aspect of the invention, chemotherapeutic
agents or combinations of chemotherapeutic agents are selected for
treatment where an effect on both cellular phenotype and cellular
genotype is observed or is not observed.
[0189] XIII. Molecular Predictors of Response
[0190] As used herein, a molecular predictor of response is the
expression of, or expression product of, one or more genes in one
or more biochemical pathway. Nearly 60 genes have been identified
whose expression and/or related SNPs are believed to play a role in
response to chemotherapy. This candidate gene list includes genes
involved in chemotherapeutic drug metabolism (for example, YP3A4,
CYP3A5, CYP2D6, CYP2C8 and CYP2C9), drug transport (for example,
ABCB1, ABCC2 and ABCG2), cell apoptosis (for example, BCL2, BAD,
BAX and BAKI), cell proliferation (for example, EGRI, CYR61,
p21/WAF and TP53), and DNA repair (for example, RCC1, ERCC2, MLH1
and MSH2). Molecular predictors used are selected from the group
consisting of: ABCB1; ABCC1; ABCC2; ABCG2; ABL1; ACLY; ADH1A;
ADPRT; ADSS; AKAP2; AKT1; AKT2; ALDH1A1; ALDH4; ANK3; ANXA8; AP2B1;
APAF-1; APH-1A; API5; APOE; ATF5; ATP7B; B4-2; BAD; BAG1; BAK1;
BARX2; BAX; BBC3; BCL2; BCL2L1; BCL2L2; BNIP3; BRCA1; BRCA2; BRF2;
BTF3; BUB1; BUB3; C8orf2; CASP2; CBR1; CCNL2; CCNB1; CCNE2; CD44;
CD68; CDA; CDC45L; CDK9; CEACAM6; CEGP1; CENPA; CES1; CFFM4; CFLAR;
COL1A1; COL4A2; COX17; CPR2; CREM; CSNK2B; CTSL2; CUL1; CYP1B1;
CYP2A6; CYP2B6; CYP2C8; CYP2C9; CYP2C19; CYP2D6; CYP3A4; CYP3A5;
CYR61; DC13; DCK; DCTD; DD96; DDB1; DIA4; DLC1; DNAJD1; DPYD; DPYS;
ECGF1; ECT2; EFEMP1; EGR1; EMP-1; EPB42; EPRS; ER; ERBB2; ERCC1;
ERCC2; ERCC4; ERG; ESM1; EXT1; FAAH; FCGRT; FDXR; FGF18; FGFR2;
FLJ10948; FLJ11190; FLJ11196; FLJ13855; FLJ14299; FLJ20323;
FLJ20585; FLNA; FLT1; FN 1; GADD34; GADD153; GBX2; GJB1; GNAZ;
GMPS; GRB7; GSR; GSTM1; GSTM3; GSTP1; GTF2H3; HBOA; HCFC1; HEC;
HER2; HLA-C; HMG1; HN1; HSPC134; IGFBP5; IL4R; ISGF3G; ITGA5; Ki67;
KIAA0175; KIAA0281; KIAA0303; KIAA1041; KIAA1067; KIAA1442; KIP2;
KIT; KLK4; KNTC2; KPNA2; KRT13; L2DTL; LAMB1; LCHN; LDHA; LOC51061;
LOX; MAD2L1; MAP2K4; MAP4; MAPT; MCM2; MCM6; MGMT; MGST1; MLH1;
MMP9; MMP11; MP1; MPO; MSH2; MSN; MUC1; MYBL2; MYC; NDP; NFAT5;
NFATC3; NFKB1; NME1; NME2; NMT1; NMU; NPM 1; NR1I2; ORC6L; ORM1/2;
OXCT; p21/WAF; PAPPA; PB1; PCDHB2; PCSK7; PECI; PGK1; PGR; PK428;
PLD3; POLA2; POLB; POLE; POLH; POR; PP591; PPP2RIA; PRC1; PRKDC;
PRPSAP1; PSME 1; PTK2; PTPRC; RAB6B; RAB11FIP1; RALGDS; RFC4; RNF2;
RPL27; RRM1; RRM2; RTKN; SCARA3; SCUBE2; SEC61A1; SERF1A; SIAH2;
SLC2A3; SLC7A10; SLC28A1; SLC28A2; SLC29A1; SLC29A2; SLC35B1; SM20;
SOD1; SPARC; STK15; STOML1; SURF4; SURVIVIN; TBPL1; TCEB3; TDP1;
TFRC; TGFB3; TIMP1; TIMP3; TLOC1; TNC; TNF; TNFSF6; TOP1; TOP2A;
TP53; TRAG3; TUBB/TUBA2; TWIST; TXN; TYMS; UBE2M; UBCH10; UBPH;
UCH37; UMP-CMPK; UMPS; UP; UPB1; USP22; WISP1; XIAP; XIST; XPA; XPB
and XRCC1. The GenBank Accession number for each gene or gene
fragment is provided in Table 1. All the accession numbers are
incorporated herein by reference.
TABLE-US-00001 TABLE 1 Genes and GenBank Accession Numbers Gene
Accession No. ABCB1 NM_000927 ABCC1 NM_004996, NM_019862,
NM_019898, NM_019899, NM_019900, NM_019901, NM_019902 ABCC2
NM_000392 ABCG2 NM_004827 ABL1 NM_005157, NM_007313 ACLY NM_001096,
NM_198830 ADH1A NM_000667 ADPRT NM_001618 ADSS NM_001126 AKAP2
NM_001004065 AKT1 NM_001014431, NM_001014432, NM_005163 AKT2
NM_001626 ALDH1A1 NM_000689 ALDH4 NM_003748, NM_170726 ANK3
NM_001149, NM_020987 ANXA8 NM_001630 AP2B1 NM_001030006, NM_001282
APAF-1 NM_001160, NM_013229, NM_181861, NM_181868, NM_181869 APH-1A
NM_016022 API5 NM_006595 APOE NM_000041 ATF5 NM_012068 ATP7B
NM_000053, NM_001005918 B4-2 NM_006813 BAD NM_004322, NM_032989
BAG1 NM_004323 BAK1 NM_001188 BARX2 NM_003658 BAX NM_004324 BBC3
NM_014417 BCL2 NM_000633, NM_000657 BCL2L1 NM_001191, NM_138578
BCL2L2 NM_004050 BNIP3 NM_004052 BRCA1 NM_007294, NM_007295,
NM_007296, NM_007297, NM_007298, NM_007299, NM_007300, NM_007301,
NM_007302, NM_007303, NM_007304, NM_007305, NM_007306 BRCA2
NM_007296 BRF2 NM_018310 BTF3 NM_001207 BUB1 NM_004336 BUB3
NM_001007793, NM_004725 C8orf2 NM_001003790, NM_001003791,
NM_007175 CASP2 NM_001224, NM_032982, NM_032983 CBR1 NM_001757
CCNL2 NM_030937 CCNB1 NM_031966 CCNE2 NM_004702, NM_057735,
NM_057749 CD44 NM_000610, NM_001001389, NM_001001390, NM_001001391,
NM_001001392 CD68 NM_001251 CDA NM_001785 CDC45L NM_003504 CDK9
NM_001261 CEACAM6 NM_002483 CEGP1 NM_020974 CENPA NM_001809 CES1
NM_001025194, NM_001025195, NM_001266 CFFM4 NM_021201, NM_206938,
NM_206939, NM_206940 CFLAR NM_003879 COL1A1 NM_000088 COL4A2
NM_001846 COX17 NM_005694 CPR2 NM_004749, NM_030900, NM_199122 CREM
NM_001881, NM_181571, NM_182717, NM_182718, NM_182719, NM_182720,
NM_182721, NM_182722, NM_182723, NM_182724, NM_182725, NM_182769,
NM_182770, NM_182771, NM_182772, NM_182850, NM_182853, NM_183011,
NM_183012, NM_183013, NM_183060 CSNK2B NM_001333 CTSL2 NM_001333
CUL1 NM_003592 CYP1B1 NM_000104 CYP2A6 NM_000762 CYP2B6 NM_000767
CYP2C8 NM_000770, NM_030878 CYP2C9 NM_000771 CYP2C19 NM_000769
CYP2D6 NM_000106, NM_001025161 CYP3A4 NM_017460 CYP3A5 NM_000777
CYR61 NM_001554 DC13 NM_020188 DCK NM_000788 DCTD NM_001012732,
NM_001921 DD96 NM_005764 DDB1 NM_001923 DIA4 NM_000903,
NM_001025433, NM_001025434 DLC1 NM_006094, NM_024767 DNAJD1
NM_013238 DPYD NM_000110 DPYS NM_001385 ECGF1 NM_001953 ECT2
NM_018098 EFEMP1 NM_004105, NM_018894 EGR1 NM_001964 EMP-1
NM_001423 EPB42 NM_000119 EPRS NM_004446 ER NM_000125 ERBB2
NM_001005862, NM_004448 ERCC1 NM_001983, NM_202001 ERCC2 NM_000400
ERCC4 NM_005236 ERG NM_004449, NM_182918 ESM1 NM_007036 EXT1
NM_000127 FAAH NM_001441 FCGRT NM_004107 FDXR NM_004110, NM_024417
FGF18 NM_003862, NM_033649 FGFR2 NM_000141, NM_022969, NM_022970,
NM_022971, NM_022972, NM_022973, NM_022974, NM_022975, NM_022976,
NM_023028, NM_023029, NM_023030, NM_023031 FLJ10948 NM_018281
FLJ11190 NM_018354 FLJ11196 NM_018357, NM_197958 FLJ13855 NM_023079
FLJ14299 NM_025069 FLJ20323 NM_019005 FLJ20585 XM_371575, XP_371575
FLNA NM_001456 FLT1 NM_002019 FN 1 NM_002026, NM_054034, NM_212474,
NM_212475, NM_212476, NM_212478, NM_212482 GADD34 NM_014330 GADD153
NM_004083 GBX2 NM_001485 GJB1 NM_000166 GNAZ NM_002073 GMPS
NM_003875 GRB7 NM_001030002, NM_005310 GSR NM_000637 GSTM1
NM_000561 GSTM3 NM_000849 GSTP1 NM_000852 GTF2H3 NM_001516 HBOA
NM_007067 HCFC1 NM_005334 HEC NM_006101 HER2 NM_001005862,
NM_004448 HLA-C NM_002117 HMG1 NM_002128 HN1 NM_001002032,
NM_001002033, NM_016185 HSPC134 NM_014169 IGFBP5 NM_000599 IL4R
NM_000418, NM_001008699 ISGF3G NM_006084 ITGA5 NM_002205 Ki67
NM_002417 KIAA0175 NM_014791 KIAA0281 NM_014800, NM_130442 KIAA0303
XM_291141 XP_291141 KIAA1041 NM_014947 KIAA1067 NM_001013839,
NM_015219 KIAA1442 XM_044921 XP_044921 KIP2 NM_000076 KIT NM_000222
KLK4 NM_004917 KNTC2 NM_006101 KPNA2 NM_002266 KRT13 NM_002274,
NM_153490 L2DTL NM_016448 LAMB1 NM_002291 LCHN AB032973, AF116707,
AF136629, BC012493 LDHA NM_005566 LOC51061 NM_015914 LOX NM_002317
MAD2LI NM_002358 MAP2K4 NM_003010 MAP4 NM_002375, NM_030884,
NM_030885 MAPT NM_005910, NM_016834, NM_016835, NM_016841 MCM2
NM_004526 MCM6 NM_005915 MGMT NM_002412 MGST1 NM_020300, NM_145764,
NM_145791, NM_145792 MLH1 NM_000249 MMP9 NM_004994 MMP11 NM_005940
MP1 NM_021970 MPO NM_000250 MSH2 NM_000251 MSN NM_002444 MUC1
NM_001018016, NM_001018017, NM_001018021, NM_002456 MYBL2 NM_002466
MYC NM_002467 NDP NM_000266 NFAT5 NM_006599, NM_138713, NM_138714,
NM_173214, NM_173215 NFATC3 NM_004555, NM_173163, NM_173164,
NM_173165 NFKB1 NM_003998 NME1 NM_000269, NM_198175 NME2
NM_001018136, NM_001018137, NM_001018138, NM_001018139, NM_002512
NMT1 NM_021079 NMU NM_006681 NPM 1 NM_002520, NM_199185 NR1I2
NM_003889, NM_022002, NM_033013 ORC6L NM_014321 ORM1/2 NM_000607,
NM_000608 OXCT NM_000436 p21/WAF NM_000389, NM_078467 PAPPA
NM_002581 PB1 NM_018165, NM_018313, NM_181041, NM_181042 PCDHB2
NM_018936 PCSK7 NM_004716 PECI NM_006117, NM_206836 PGK1 NM_000291
PGR NM_000926 PK428 NM_003607 PLD3 NM_001031696, NM_012268 POLA2
NM_002689 POLB NM_002690 POLE NM_006231 POLH NM_006502 POR
NM_000941 PP591 NM_025207, NM_201398 PPP2R1A NM_014225 PRC1
NM_003981, NM_199413, NM_199414 PRKDC NM_006904 PRPSAP1 NM_002766
PSME 1 NM_006263, NM_176783 PTK2 NM_005607, NM_153831 PTPRC
NM_002838, NM_080921, NM_080922, NM_080923 RAB6B NM_016577
RAB11FIP1 NM_001002233, NM_001002814, NM_025151 RALGDS NM_006266
RFC4 NM_002916, NM_181573 RNF2 NM_007212
RPL27 NM_000988 RRM1 NM_001033 RRM2 NM_001034 RTKN NM_001015055,
NM_001015056, NM_033046 SCARA3 NM_016240, NM_182826 SCUBE2
NM_020974 SEC61A1 NM_013336 SERF1A NM_021967 SIAH2 NM_005067 SLC2A3
NM_006931 SLC7A10 NM_019849 SLC28A1 NM_004213, NM_201651 SLC28A2
NM_004212 SLC29A1 NM_004955 SLC29A2 NM_001532 SLC35B1 NM_005827
SM20 NM_022051 SOD1 NM_000454 SPARC NM_003118 STK15 NM_003600,
NM_198433, NM_198434, NM_198435, NM_198436, NM_198437 STOML1
NM_004809 SURF4 NM_033161 SURVIVIN NM_001012270, NM_001012271,
NM_001168 TBPL1 NM_004865 TCEB3 NM_003198 TDP1 NM_001008744,
NM_018319 TFRC NM_003234 TGFB3 NM_003239 TIMP1 NM_003254 TIMP3
NM_000362 TLOC1 NM_003262 TNC NM_002160 TNF NM_000594 TNFSF6
NM_000639 TOP1 NM_003286 TOP2A NM_001067 TP53 NM_000546 TRAG3
NM_004909 TUBB/TUBA2 NM_178014, NM_006001, NM_079836 TWIST
NM_000474 TXN NM_003329 TYMS NM_001071 UBE2M NM_003969 UBCH10
NM_007019, NM_181799, NM_181800, NM_181801, NM_181802, NM_181803
UBPH NM_019116 UCH37 NM_015984 UMP-CMPK NM_016308 UMPS NM_000373 UP
NM_003364, NM_181597 UPB1 NM_016327 USP22 XM_042698 XP_042698 WISP1
NM_003882, NM_080838 XIAP NM_001167 XIST NR_001564 XPA NM_000380
XPB NM_000122 XRCC1 NM_006297
[0191] Analysis of the expression of one or more of the molecular
predictors of response includes analysis of at least one gene in at
least one pathway whose expression is activated to a higher or
lower level in a patient suffering from a cancer relative to the
expression in a normal or control subject. A differentially
expressed gene may be activated or inhibited at the nucleic acid
level or protein level, or may be subject to alternative splicing
to result in a different polypeptide product. Such differences may
be evidence by a change in RNA levels, surface expression;
secretion or other cellular polypeptide expression patterns.
Differential gene expression may include a comparison of the ratios
of the expression between two or more genes or their gene products
or a comparison of two differently processed products of the same
gene which differ between normal subjects and subjects suffering
from a cancer. Differential expression includes quantitative and
qualitative differences in the temporal or cellular expression
patterns in a gene or its expression product among normal and tumor
cells.
[0192] Any one or more of the methods, assays, tools, systems or
any subpart of any method, assay, tool or system, disclosed herein
may be used alone, combined with, or used optionally with, any
other method, assay, tool or system, or subpart of any method,
assay, tool or system, disclosed herein. As a non-limiting example,
the cell culture protocol of Sections II and III, or any subsection
thereof (for example, III(a)-III(e)), may optionally be used
together with any one or more of Sections IV (Combination
Treatment), Section V (Preparation and Determination of Dose
Levels), Section VI (Determination of Phenotypic and Genotypic
Drift), Section VII (Methods of Generating Dose Response Curves),
Section VIII (Methods of Cell Fixing and Staining), Section IX
(Targeting Agents), Section X (Pharmaceutical Agents), Section XI
(Apoptosis Assay), Section XII (Choice of Agents) and Section XIII
(Molecular Predictors of Response). As another non-limiting
example, the cells obtained from the cell culture protocol of
Sections II and III, or any subsection thereof, may optionally be
used in the methods of one or more of Sections IV (Combination
Treatment), Section V (Preparation and Determination of Dose
Levels), Section VI (Determination of Phenotypic and Genotypic
Drift), Section VII (Methods of Generating Dose Response Curves),
Section VIII (Methods of Cell Fixing and Staining), Section IX
(Targeting Agents), Section X (Pharmaceutical Agents) and Section
XI (Apoptosis Assay). In another non-limiting example, the cells
obtained from the culture protocols of Sections II and III may be
treated as in Section IV (Combination Treatment) or the cells may
be examined as in Section VI for determination of phenotypic and/or
genotypic drift and optionally used as in Section XI, the apoptosis
assay. As stated above, cells from any of the foregoing methods may
be assayed with respect to changes in one or more molecular
predictors of response.
[0193] In addition, any one or more of the assays, methods, tools
or systems disclosed herein may optionally be substituted by one or
more assays, methods, tools or systems known in the art and
publicly available. As a non-limiting example, the cells generated
from the protocol of Sections II and/or III may be contacted by any
stain or molecule known in the art and visualized, and/or imaged
and/or counted using any means for visualization and/or imaging
and/or counting of cells known in the art.
[0194] The foregoing examples and limitations related therewith are
intended to be illustrative and not exclusive. The practice of the
methods included in the invention disclosed herein use, unless
otherwise indicated, conventional techniques in molecular biology
(including recombinant techniques), microbiology, cell biology, and
biochemistry which are within the skill in the art. Such techniques
are explained fully in the literature, such as "Molecular Cloning:
A Laboratory Manual," 2nd edition (Sambrook et al., 1989);
"Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell
Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology"
(Academic Press, Inc.); "Handbook of Experimental Immunology" 4th
edition (D. M. Weir & C. C. Blackwell, eds., Blackwell Science,
Inc., 1987); "Gene Transfer Vectors for Mammalian Cells" (J. M.
Miller & M. P. Calos, eds., 1987); "Current Protocols in
Molecular Biology" (F. M. Ausubel et al., eds., 1987); and, "PCR:
The Polymerase Chain Reaction," (Mullis et al., eds., 1994). See,
for example, WO 2004/065583.
EXAMPLES
Example 1
Initiation of a Primary Culture
[0195] A tumor biopsy of approximately 100 mg of non-necrotic,
non-contaminated tissue was harvested from a 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 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, either agitated or non-agitated, were plated
in culture flasks using sterile pasteur pipettes (approximately 9
explants per T-25 or approximately 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 humidified 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 humidified 37.degree. C., 5%
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 appropriate volume of growth medium, the
explants grew out into a monolayer.
Example 2
Establishment of Concentrations for Each Dose
[0196] A multiple step drug dilution procedure that included 384
well microtiter plates and four ATCC cell lines to establish proper
dose ranges to obtain cell killing from 0% up to and including
maximal cell kill was used. Drugs were diluted in medium specific
for each cell line and serial dilutions were made to provide ten
drug concentrations. Drug dosages from step 1 were then validated
on at least twenty patient-derived cell lines isolated from each of
four major tumor types: ovarian, breast, lung and colon. Dosages
were adjusted both on the low and the high end to get 0% up to and
including maximal cell killing. Finally the newly determined
dosages were further validated on patient cells using 384 well
microplates and were further adjusted to get the whole spectrum of
possible responses, from 0% up to and including maximal cell
killing. Following the establishment of 72 hour dosing levels for
several of the drugs that we test as single agents, a new method of
dealing with combination treatments was developed.
Example 3
Combination Treatment
[0197] 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 minced samples were placed into at least one,
possible multiple, tissue culture flasks with complete medium, and
visual confirmation was made that the particulates were evenly
distributed along the bottom of each flask and the flasks were
placed in a 37.degree. C., 5% 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 growth medium, the
particulates grew into monolayers.
[0198] Enough cells were then removed from the monolayers grown in
the flasks for centrifugation into standard size cell pellets for
each flask. 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 30 .mu.l 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 placed on each side of a hemocytometer for
examination using a standard light microscope. Cells were counted
in five out of nine hemocytometer quadrants on each side, under
10.times. magnification--only those cells which had not taken up
the trypan blue dye were counted. 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.
[0199] Accommodating the above calculations, additional cell
aliquots from the other culture flasks were separately suspended in
growth medium via vortex and rocking and then were loaded into
separate channels of an 8-channel deep well plate. Aliquots of the
prepared cell suspension were delivered into the 384 well
microtiter plates using an automated liquid handler with techniques
known in the art. Cells were plated into each well of the
microtiter plates at a concentration of 320 cells per well.
[0200] Approximately twenty-four (24) hours post-plating, the
chemotherapeutic agents paclitaxel and carboplatin were applied to
the wells in the microtiter plates in increasing dosages. The first
columns of the plate served as control wells with no treatment. The
tumor cells in the wells were then incubated with the
chemotherapeutic drugs for another 72 hours.
[0201] Fraction surviving treatment calculated as the cell number
relative to control. For the cells from the tumor specimens a dose
response relationship was observed for paclitaxel/carboplatin
treatment schema.
Example 4
Digestion of Ovarian Tumor Specimen and Preparation of a Cell
Culture Monolayer
[0202] Ovarian tumor tissue was received and minced into pieces
approximately 5 mm.sup.3. Each cut specimen was placed in a 15 ml
conical tube containing 10 ml of 0.25% Collagenase II and 0.001%
DNase I in Hank's Balanced Salt Solution (HBSS) with Ca.sup.2+ and
Mg.sup.2+. Specimens were then incubated for about 15 to 30 minutes
in a 37.degree. C. incubator on a rocking platform.
[0203] After the thirty minute incubation, specimens were
centrifuged for 3 minutes at 2200 RPM. The sample media (HBSS with
Ca.sup.2+ and Mg.sup.2+) was poured off of the specimens, and
specimens were rinsed with 10 ml of 10% McCoy's media. Samples were
centrifuged again for 3 minutes at 2200 RPM, followed by removal of
sample media. After pouring off the sample media, samples were
centrifuged again to remove media and residual Collagenase II and
DNase from the cells.
[0204] Each sample was then placed in a non-vitrogen coated flask
in 10% McCoy's media and placed in a 37.degree. C. incubator. The
media was changed as necessary (twice a week or more) depending on
the growth of cells. Once the cells began to grow, the media
changes involved a rinse step to remove residual Collagenase II and
DNase I.
Example 5
Assays of the Chemoresistant Cell Population
[0205] In one embodiment of the methods disclosed herein, tumor
cells determined to be chemoresistant by the methods disclosed
herein may be cultured according to ChemoFx.RTM. Assay V1 or V2
protocols. The chemoresistant cells obtained by the methods
disclosed herein may be used in, or with, any of the other methods,
assays, tools or systems disclosed herein, or with any methods,
assays, tools or systems known in the art. Tumor cells determined
to be chemoresistant by other methods known in the art may also be
cultured according to ChemoFx.RTM. Assay V1 or V2 protocols
disclosed herein. In either instance, additional chemosensitivity
testing and/or genotypic and/or phenotypic and/or apoptotic assays
and/or evaluation of one or more molecular predictors of response
will be subsequently performed on the cultured chemoresistant tumor
cells.
[0206] For example, and not by way of limitation, according to
Version 1 of the ChemoFx.RTM. Assay, chemoresistant cells can be
seeded into 60 well microtiter plates at a density of about 100-150
cells per well and allowed to attach and grow for about 24 hours.
After about 24 hours in culture the cells can be exposed for about
2 hours to a battery of chemotherapeutic agents. At the end of the
incubation with the chemotherapeutic agents, the plates will be
washed to remove non-adherent cells. The remaining cells can be
fixed with 95% ethanol and stained with the DNA intercalating blue
fluorescent dye, DAPI, or 6-diamidino 2-phylindole dihydrochloride
(Molecular Probes, Eugene, Oreg., USA). The surviving cells are
then counted using an operator-controlled, computer-assisted image
analysis system (Zeiss Axiovision, Thornwood, N.Y., USA). A
cytotoxic index can be calculated using methods known in the art.
The data can be presented graphically as the Cytotoxicity Index
(CI). A dose-response curve can be generated for each drug
evaluated.
[0207] In another nonlimiting example, according to Version 2 of
the ChemoFx.RTM. Assay, a cell suspension of primary tumor cells
can be prepared at a concentration of about 8,000 cells/ml and
delivered in a large basin to the stage of a liquid handling
machine. The machine then seeds about 320 cells in 40 .mu.l of
medium into the wells of a 384 well microplate in replicates of 4,
after which the cells are allowed to adhere to the plate and grow
for about 24 hours at 37.degree. C. Following the 24 hour
incubation, the liquid handling machine prepares ten doses of each
drug, in the appropriate growth medium, via serial dilutions in a
96 well deep-well microplate. When the drugs are ready, the liquid
handling machine dispenses 40 .mu.l of 2.times. drug into the
appropriate wells of the deep well plate. Proprietary software,
named Resource Allocator, ensures that the cells are treated with
the correct drugs and dosages. Resource Allocator determines the
settings and layout for the liquid handler based on the number of
patient specimens that are ready for processing (achieved required
confluence in the culture flasks) and the number of drugs required
for each patient specimen. After processing the information,
Resource Allocator provides a script to the operator indicating
where each plate, basin, and deepwell plate must be put.
Subsequently to configuring the liquid handler, the operator
initiates the Resource Allocator software to plate cells or dilute
drug or treat specimens. After treatment, the drugs are left on the
cells for about a 72 hour incubation, thus necessitating their
preparation in growth medium. During this period, cell viability
can be maintained in a standard tissue culture incubator, and
visible, UV or fluorescent light images are taken at predetermined
intervals using proprietary software.
[0208] At the end of the 72 hour incubation period, the liquid
handling machine is used to remove the media and any non-adherent
cells. Then, the remaining cells will be fixed 5 minutes in 95%
ethanol containing the DNA intercalating blue fluorescent dye,
DAPI. Following fixation and staining, the automated microscope can
be used to take UV images of the stained cells in every well.
Afterwards, the number of cells per well in both visible and UV
light can be quantified using proprietary software named Cell
Counter. Cell Counter identifies cells from the background of
images mathematically manipulating the images to increase contrast.
Subsequent processing uses the threshold based on the pixel
histogram of the image to determine the number of cells within the
image.
[0209] A complete dose response curve can be generated for each
drug evaluated by comparing cells remaining at each dose to the
untreated control wells. An image analysis system is used in
analysis of the cells. Here, chemoresistant cells grown in plates
are imaged on a Nikon TE300 Eclipse inverted microscope equipped
with a motorized stage and a Photometrics Cool Snap FX CCD
camera.
[0210] In one embodiment of this invention, the non-adherent cells
are collected from the microtiter plate for subsequent analysis.
Such analysis could include genotypic or phenotypic measurements,
such as cell viability, genetic stability analysis, ability to form
secondary cultures either in a ChemoFx assay Version 1 or version
2, or other analysis of someone skilled in the art.
[0211] In one embodiment of the invention, the adherent,
chemoresistant cells are analyzed prior to fixation and staining.
Such analysis may include but is not limited to treating the
remaining adherent cells with additional drugs to determine
response to a second regiment of chemotherapeutic agents. Such
analysis may include but is not limited to analysis of different
vital stains to measure cell viability, membrane integrity, cell
signaling pathways, apoptosis, multi-drug resistance (MDR) ability,
etcetera. Such analysis may include but is not limited to genotypic
analysis for gene expression or genome mutations, phenotypic
analysis, such as expression of surface proteins, cell viability,
immunohistochemical analysis and pathological analysis. Subsequence
to analysis of adherent cells as mentioned above, the cells are
fixed and stained for counting/analysis as described in Version 2
assay methodology.
[0212] Modification of ChemoFx.RTM. assays Versions 1 and Versions
2 disclosed herein are within the ordinary skill in the art.
Inclusion of other assays, methods, procedures, tools, materials,
drugs, systems, compounds and equipment known in the art is
intended to be an option in the practice of the assays, methods,
tools and systems included in the invention disclosed herein.
[0213] Chemoresistant cells may be cultured and subcultured
repeatedly using one or more methods of the invention in order to
determine an effective amount of an agent or combination of agents
to provide a desired chemoresponse.
Example 6
Digestion of Colon Tumor Specimen and Preparation of a Cell Culture
Monolayer
[0214] Colon tumor tissue (in shipping medium) was received and
gently shaken three to four times. Shipping medium was poured off
of the tumor and transferred to a 50 ml conical tube which was
centrifuged for 3 minutes at 800.times.g. After centrifugation, the
supernatant was poured off the resulting pellet. The tube
containing the pellet were set aside for later use.
[0215] The solid tumor was transferred to an open, sterile Petri
dish using sterile forceps as needed. Disposable sterile scalpels
were used to mince the tumor into smaller explants to a size
equivalent to one capable of being sucked up by a 10 ml pipette.
Using 5 ml of antibiotic wash, small explants and floating cells
were aspirated with a pipette and transferred to the tube
containing the pellet.
[0216] The remaining larger explants were minced further to the
size equivalent to one capable of being sucked up by a 5 ml
pipette. Depending on the size of the colon tumor explant, 5 or 10
ml of antibiotic wash containing 1 or 2 ml of cocktail of 0.025%
Collagenase II and 0.001% DNase was added to each sample. The
antibiotic wash is Hanks solution containing penicillin,
streptomycin, gentamicin, nystatin and ciprofloxacin. Explants were
aspirated and transferred to a 15 ml conical tube. Once in the 15
ml tube, the explants were pipetted in and out to disaggregate the
big explants. The tube was capped and shaken 2-3 times and then
incubated for 15 minutes on a rocker in a 37.degree. C. and 5%
CO.sub.2 incubator.
[0217] Both the conical tube containing the small explants and the
tube which had contained the larger explants were centrifuged_for
three minutes at 800.times.g. After centrifugation, supernatants
were removed from the resulting pellets. The pellet resulting from
the smaller explants was resuspended in 3 ml of RPMI-1640 cell
culture medium containing 2% FBS and antibiotics and transferred to
a labeled T-25 flask. The pellet resulting from the larger explants
(the explants treated with Collagenase II and DNase) were treated
with 3 ml or 6 ml of RPMI-1640 containing 2% FBS (depending on the
size of the pellet) and transferred to a labeled T-25 or T-75
flask.
[0218] Both flasks were swirled evenly to distribute the explants.
Flasks were propped on an angle in the hood for ten minutes to
allow as much media but as few explants as possible drain to the
bottom edge of the flask.
[0219] The explants were transferred to new flasks such that 30-50%
of the bottom of the flask(s) was covered with explants. The flasks
containing the explants were incubated at 37.degree. C. and 5%
CO.sub.2 to allow for cell growth. Cell media was changed as
needed.
Example 7
Determination of Normalized Cytotoxicity Index
[0220] Cytotoxicity Index scores were normalized to account for
variations in the starting number of cells assayed. Twenty-four
hours after cells were placed in wells, i.e., segregated sites,
plates were removed from the incubator and placed on an imaging
system. Each well of the plate was imaged by the imaging system to
capture visible and fluorescent images. Each image was analyzed and
the number of cells in each well was determined.
[0221] Cells were then treated with an agent. Untreated cells were
used as a control. At the completion of the assay, cells were
counted again. The Cytotoxicity Index (CI) was calculated using
cell counts pre-treatment and post-treatment (test and control
groups) as follows:
C I = T end treated T end untreated .times. T 24 untreated T 24
treated ##EQU00001##
[0222] wherein T.sub.end is the post-treatment cell count and
T.sub.24 is the pre-treatment cell count.
[0223] All cited patent, patent applications, publications and
documents mentioned in the above specification are herein
incorporated by reference in their entirety. Various modifications
and variations of the described method and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in the art of cell biology,
and/or related fields are intended to be within the scope of the
following claims.
* * * * *