chemosensitivity vol 1

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M E T H O D S I N M O L E C U L A R M E D I C I N ETM

Chemosensitivity

Edited by

Rosalyn D. Blumenthal

Volume 1

In Vitro Assays

Chemosensitivity

Edited by


Volume 1

In Vitro Assays


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Chemosensitivity


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M E T H O D S I N M O L E C U L A R M E D I C I N E

John M. Walker, SERIESEDITOR

118. Antifungal Agents:Methods and Protocols,edited byErika J. Ernst and P. David Rogers,2005

117. Fibrosis Research:Methods and Protocols,edited byJohn Varga, David A. Brenner,and Sem H. Phan, 2005

116. Inteferon Methods and Protocols, edited byDaniel J. J. Carr, 2005

115. Lymphoma:Methods and Protocols, edited byTimothy Illidge and Peter W. M. Johnson,2005

114. Microarrays in Clinical Diagnostics, edited byThomas Joos and Paolo Fortina, 2005

113. Multiple Myeloma:Methods andProtocols,edited byRoss D. Brown and P. Joy Ho, 2005

112. Molecular Cardiology:Methods andProtocols,edited byZhongjie Sun, 2005

111. Chemosensitivity: Volume 2, In Vivo Mod-els, Imaging, and Molecular Regulators,editedbyRosalyn D. Blumethal, 2005

110. Chemosensitivity: Volume 1, In Vitro Assays,edited byRosalyn D. Blumethal, 2005

109. Adoptive Immunotherapy:Methods andProtocols,edited byBurkhard Ludewig and

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108. Hypertension:Methods and Protocols,edited byJrme P. Fennell and Andrew

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107. Human Cell Culture Protocols, SecondEdition,edited byJoanna Picot, 2005

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96. Hepatitis B and D Protocols: Volume 2,Immunology, Model Systems, and ClinicalStudies,edited byRobert K. Hamatake and

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Chemosensitivity

Volume 1

In Vitro Assays

Edited by

Rosalyn D. BlumenthalGarden State Cancer Center, Belleville, NJ

M E T H O D S I N M O L E C U L A R M E D I C I N E


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Cover illustrations: Foreground illustration: Figure 3, from Chapter 10, Chemosensitivity Testing UsingMicroplateAdenosine TriphosphateBased Luminescence Measurements, by Christian M. Kurbacher andIan A. Cree. Background illustration: Figure 4, from Chapter 22 (Volume 2), Assessing Growth andResponse to Therapy in Murine Tumor Models, by C. P. Reynolds et al.

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E-ISBN 1-59259-869-2Library of Congress Cataloging in Publication DataChemosensitivity / edited by Rosalyn D. Blumenthal.

v. ; cm. (Methods in molecular medicine ; 110-111)Includes bibliographical references and index.Contents: v. 1. In vitro assays v. 2 In vivo models, imaging, andmolecular regulators.ISBN 1-58829-345-9 (hardcover : alk. paper)1. CancerChemotherapyLaboratory manuals. 2. AntineoplasticagentsEffectivenessLaboratory manuals. 3. Cancer cellsLaboratorymanuals. 4. Cancer--Molecular aspectsLaboratory manuals.

[DNLM: 1. Antineoplastic Agentspharmacology. 2. Drug Screening Assays,Antitumormethods. 3. Drug Resistance, Neoplasm. 4. Models, Animal. 5.Neoplasmsdrug therapy. QV 269 C5177 2005] I. Blumenthal, Rosalyn D. II.Series.

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v

Preface

Chemotherapy is used to treat many types of cancer. A large number of drug

classes are in use, including the vinca alkaloids, taxanes, antibiotics, anthracy-

clines, DNA alkylators, other DNA damaging agents, hormones, and interfer-

ons. More potent analogs of existing drugs and novel agents directed at new

targets are continuously being developed. Over the last few years, agents that

affect COX-2, PPAR, and various signal transduction pathways have received

much attention. To identify which agents are effective for which types of tumors,

it is important to develop accurate in vitro and preclinical in vivo screening sys-

tems that can identify the cytotoxic and/or cytostatic potential of an agent on

established tumor cell lines or cells isolated from individual fresh cancer biopsy

specimens removed from cancer patients. Chemosensitivity testing allows the

selection of drugs that appear sensitive in the laboratory, thus offering patients a

better chance of response.

One of the main problems associated with chemotherapy has been that

patient tumors with the same histology do not necessarily respond identically

to the same agent or dose schedule of multiple agents. Identifying the presence

of resistance mechanisms and other determinants for drug sensitivity in orderto classify tumors into response categories has been an ongoing research

effort. Advances in our understanding of the genetic and protein fingerprints of

primary tumors and their metastases has opened a door to the possibility of

customizing therapy to individuals. There is accumulating evidence suggest-

ing that laboratory screening of samples from a patients tumor may help select

the appropriate treatment(s) to administer, thereby avoiding ineffective drugs,

and sparing patients the side effects normally associated with these agents.

The aim of these two volumes on Chemosensitivity of theMethods in Molecu-lar Medicineseries, is to comprehensively present protocols that can be used to

(a) assess chemosensitivity in vitro and in vivo, and (b) assess parameters that

modulate chemosensitivity in individual tumors. Volume I presents an overview

in Chapter 1 and then covers In Vitro Measures of Chemosensitivity, includes

clonogenic, colorimetric, fluorometric, and histochemical approaches. Volume

II, Part I,Measurements of DNA Damage, Cell Death, and Regulators of Cyto-

toxicity, includes methods to detect chromosome loss and breakage, changes in

cell cycle, expression of members of the bcl-2 family of proteins, expression of

caspases and PARP cleavage, metabolic factors influencing sensitivity, measure-

ments of drug retention, expression of drug resistance proteins, and measure-

ments of ceramide and sphingolipids associated with drug sensitivity. Volume


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vi Preface

II, Part II, Genomics, Proteomics, and Chemosensitivity, addresses DNA

microarrays for gene profiling, genetic manipulation to identify genes regu-

lating chemosensitivity, proteomics using 2D-PAGE and mass spectrometry,and bioinformatics approaches. The last part, In Vivo Animal Modeling of

Chemosensitivity,covers protocols to establish clinically meaningful metastatic

and orthotropic models of solid and liquid tumors, statistical approaches to ana-

lyze preclinical data, and animal imaging approaches that can be used to assess

chemosensitivity such as GFP-tagged genes, SPECT using 99mTc-annexin, PET

imaging with 18FDG, and magnetic resonance imaging.

Each chapter is written by someone experienced with the methodology and

contains a detailed introductory section with references of how the technique

has been used in the past, a list of materials and equipment needed to perform

the assay, and a step-by-step set of instructions for each method. At the end of

each chapter a Notes section is included with useful information, helpful

hints, and problems and pitfalls to be aware of, in order to make the assay run

smoothly and allow for easy interpretation of data.



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Contents

Preface ..............................................................................................................v

Contributors ..................................................................................................... ix

Contents of Volume 2 ......................................................................................xi

PARTI. OVERVIEW

1 An Overview of Chemosensitivity Testing

Rosalyn D. Blumenthal ......................................................................... 3

PARTII. INVITROMEASURESOFCHEMOSENSITIVITY

2 Clonogenic Cell Survival Assay

Anupama Munshi, Marvette Hobbs, and Raymond E. Meyn.............. 21

3 High-Sensitivity Cytotoxicity Assays for Nonadherent Cells

M. Jules Mattes ................................................................................... 29

4 Sulforhodamine B Assay and Chemosensitivity

Wieland Voight ................................................................................... 39

5 Use of the Differential Staining Cytotoxicity

Assay to Predict ChemosensitivityGertjan J. L. Kaspers ........................................................................... 49

6 Collagen Gel Droplet Culture Methodto Examine In Vitro Chemosensitivity

Hisayuki Kobayashi ............................................................................. 59

7 The MTT Assay to Evaluate Chemosensitivity

Jack D. Burton .................................................................................... 69

8 Histoculture Drug Response Assay to Monitor Chemoresponse

Shinji Ohie, Yasuhiro Udagawa, Daisuke Aoki,and Shiro Nozawa .......................................................................... 79

9 In Vitro Testing of Chemosensitivity in Physiological Hypoxia

Rita Grigoryan, Nino Keshelava, Clarke Anderson,and C. Patrick Reynolds.................................................................. 87

10 Chemosensitivity Testing Using Microplate AdenosineTriphosphateBased Luminescence Measurements

Christian M. Kurbacher and Ian A. Cree .......................................... 101

11 High-Throughput Technology:Green Fluorescent Protein to Monitor Cell Death

Marylne Fortin, Ann-Muriel Steff, and Patrice Hugo ..................... 121

vii


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viii Contents

12 DIMSCAN: A Microcomputer Fluorescence-Based CytotoxicityAssay for Preclinical Testing of Combination Chemotherapy

Nino Keshelava, Tom

s

Frgala, Jir

Krejsa, Ondrej Kalous,and C. Patrick Reynolds ................................................................ 139

13 The ChemoFxAssay: An Ex Vivo Cell Culture Assayfor Predicting Anticancer Drug Responses

Robert L. Ochs, Dennis Burholt, and Paul Kornblith ....................... 155

14 Evaluating Response to Antineoplastic DrugCombinations in Tissue Culture Models

C. Patrick Reynolds and Barry J. Maurer .......................................... 173

15 Image Analysis Using the Fluochromasia Assayto Quantify Tumor Drug Sensitivity

John F. Gibbs, Youcef M. Rustum, and Harry K. Slocum ................. 185

16 Immunohistochemical Detection of Ornithine Decarboxylaseas a Measure of Chemosensitivity

Uriel Bachrach .................................................................................. 197

17 Immunohistochemistry of p53, Bcl-2 and Ki-67as Predictors of Chemosensitivity

Mitsuyoshi Itaya, Jiro Yoshimoto, Kuniaki Kojima,

and Seiji Kawasaki ........................................................................ 213

Index ............................................................................................................ 229


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ix

Contributors

CLARKE ANDERSONDivision Hematology-Oncology, Keck School of Medicine,

University of Southern California, Los Angeles, CA, USA

DAISUKEAOKIDepartment of Obstetrics-Gynecology, Keio University

School of Medicine, Keio, Japan

URIELBACHRACHDepartment of Molecular Biology, Hebrew University

Hadassah Medical School, Jerusalem, Israel

ROSALYND. BLUMENTHAL Garden State Cancer Center, Belleville, NJ, USA

DENNISBURHOLT Precision Therapeutics, Pittsburgh, PA, USA

JACKD. BURTON Garden State Cancer Center, Belleville, NJ, USA

IANA. CREEDepartment of Histopathology, Queen Alexandria Hospital,

Portsmouth, UK

MARYLENEFORTIN Topigen Pharmaceuticals, Montreal, Quebec, Canada

TOMS FRGALA USC-CHLA Institute for Pediatric Clinical Research,

University of Southern California and Childrens Hospital Los Angeles,

Los Angeles, CA, USA

JOHNF. GIBBSDepartment of Surgical Oncology, Roswell Park Cancer

Institute, Buffalo, NY, USA

RITAGRIGORYANDevelopmental Therapeutics Section, Childrens Hospital

of Los Angeles, Los Angeles, CA, USA

MARVETTEHOBBSDepartment of Experimental Radiology, University of

Texas M.D. Anderson Cancer Center, Houston, TX, USA

PATRICE HUGO Caprion Pharmaceuticals Inc., Saint Laurent, QC, Canada

MITSUYOSHIITAYADepartment of Hepato-Biliary-Pancreatic Surgery,

Juntendo University, Tokyo, Japan

ONDREJ KALOUS USC-CHLA Institute for Pediatric Clinical Research,University of Southern California and Childrens Hospital Los Angeles,


GERTJANJ. L. KASPERSDepartment of Pediatric Hematology Oncology, VU

University Medical Center, Amsterdam, Netherlands

SEIJIKAWASAKIDepartment of Hepato-Biliary-Pancreatic Surgery,


NINOKESHELAVA USC-CHLA Institute for Pediatric Clinical Research,

University of Southern California and Childrens Hospital Los Angeles,Los Angeles, CA, USA

HISAYUKIKOBAYASHI Biochemical Laboratory, Nitta Gelatin Inc.,

Futamata, Yao-City, Japan


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KUNIAKIKOJIMADepartment of Breast and Endocrine Surgery, Juntendo

University, Tokyo, Japan

PAULKORNBLITHPrecision Therapeutics, Pittsburgh, PA, USA

JIR KREJSA USC-CHLA Institute for Pediatric Clinical Research, University of

Southern California and Childrens Hospital Los Angeles, Los Angeles, CA,

USA

CHRISTIANM. KURBACHERDepartment of Gynecology & Obstetrics, University

of Cologne, Cologne, Germany

M. JULESMATTES Center for Molecular Medicine and Immunology,

Belleville, NJ, USA

BARRYJ. MAURER USC-CHLA Institute for Pediatric Clinical Research,



RAYMONDE. MEYNDepartment of Experimental Radiology, University of


ANUPAMAMUNSHIDepartment of Experimental Radiology, University of


SHIRONOZAWADepartment of Obstetrics-Gynecology, Keio University

School of Medicine, Keio, Japan

ROBERTL. OCHS Precision Therapeutics, Pittsburgh, PA, USA

SHINJIOHIE Cancer Research Laboratory, Hanno Research Center, Taiho

Pharmaceutical Co., Saitama, Japan

C. PATRICKREYNOLDS USC-CHLA Institute for Pediatric Clinical Research,



YOUCEFM. RUSTUMDepartment of Experimental Therapeutics, Roswell

Park Cancer Institute, Buffalo, NY, USA

HARRYK. SLOCUMDepartment of Experimental Therapeutics, RoswellPark Cancer Institute, Buffalo, NY, USA

ANN-MURIELSTEFF World Anti-Doping Agency, Montreal, Quebec, Canada

YASUHIROUDAGAWADepartment of Obstetrics-Gynecology, Fujita Health

University School of Medicine, Japan

WIELANDVOIGHT Klinik fr Innere Medizin IVMartin Luther Universitt

Halle, Halle/Saale, Germany

JIROYOSHIMOTODepartment of Hepato-Biliary-Pancreatic Surgery,


x Contributors


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xi

Contents of Volume 2

Preface ..............................................................................................................v

Color Plate .......................................................................................................xi

Contributors .................................................................................................. xiii

Contents of Volume 1 ...................................................................................xvii

PARTI. MEASUREMENTSOFDNA DAMAGE, CELLDEATH,ANDREGULATORSOFCYTOTOXICITY

1 In Vitro Micronucleus Technique to Predict ChemosensitivityMichael Fenech ..................................................................................... 3

2 Cell Cycle and Drug Sensitivity

Aslamuzzaman Kazi and Q. Ping Dou ................................................ 33

3 TUNEL Assay as a Measure of Chemotherapy-Induced Apoptosis

Robert Wieder..................................................................................... 43

4 Apoptosis Assessment by the DNA Diffusion Assay

Narendra P. Singh ............................................................................... 55

5 PARP Cleavage and Caspase Activity to Assess ChemosensitivityAlok C. Bharti, Yasunari Takada, and Bharat B. Aggarwal ................ 69

6 Diphenylamine Assay of DNA Fragmentationfor Chemosensitivity Testing

Cicek Gercel-Taylor ............................................................................ 79

7 Immunodetecting Members of the Bcl-2 Family of Proteins

Richard B. Lock and Kathleen M. Murphy.......................................... 83

8 Correlation of Telomerase Activity

and Telomere Length to ChemosensitivityYasuhiko Kiyozuka .............................................................................. 97

9 Application of Silicon Sensor Technologies to Tumor Tissue In Vitro:Detection of Metabolic Correlates of Chemosensitivity

Pedro Mestres-Ventura, Andrea Morguet, Anette Schofer,Michael Laue, and Werner Schmidt ............................................. 109

10 Overview of Tumor Cell Chemoresistance Mechanisms

Laura Gatti and Franco Zunino ........................................................ 127

11 Flow Cytometric Monitoring of Fluorescent DrugRetention and Efflux

Awtar Krishan and Ronald M. Hamelik ............................................ 149


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12 Flow Cytometric Measurement of Functionaland Phenotypic P-Glycoprotein

Monica Pallis and Emma Das-Gupta ................................................ 16713 Measurement of Ceramide and Sphingolipid Metabolism in Tumors:Potential Modulation of Chemosensitivity

David E. Modrak ............................................................................... 183

PARTII. GENOMICS, PROTEOMICS, ANDCHEMOSENSITIVITY14 Gene Expression Profiling to Characterize Anticancer

Drug Sensitivity

James K. Breaux and Gerrit Los ........................................................ 197

15 Identifying Genes Related to ChemosensitivityUsing Support Vector Machine

Lei Bao .............................................................................................. 233

16 Genetic Manipulation of Yeast to Identify GenesInvolved in Regulation of Chemosensitivity

Giovanni L. Beretta and Paola Perego .............................................. 241

17 Real-Time RT-PCR (Taqman) of Tumor mRNAto Predict Sensitivity of Specimens to 5-Fluorouracil

Tetsuro Kubota .................................................................................. 25718 Use of Proteomics to Study Chemosensitivity

Julia Poland, Silke Wandschneider, Andrea Urbani,Sergio Bernardini, Giorgio Federici, and Pranav Sinha ............... 267

PARTIII. INVIVOANIMALMODELINGOFCHEMOSENSITIVITY19 Clinically Relevant Metastatic Breast Cancer

Models to Study Chemosensitivity

Lee Su Kim and Janet E. Price ........................................................... 285

20 Orthotopic Metastatic (MetaMouse) Modelsfor Discovery and Development of Novel Chemotherapy

Robert M. Hoffman ........................................................................... 297

21 Preclinical Testing of Antileukemic DrugsUsing an In Vivo Model of Systemic Disease

Richard B. Lock, Natalia L. Liem, and Rachael A. Papa ................... 323

22 Assessing Growth and Responseto Therapy in Murine Tumor Models

C. Patrick Reynolds, Bee-Chun Sun, Yves A. DeClerck,and Rex A. Moats .......................................................................... 335

xii Contents of Vol. 2


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23 Evaluation of Chemosensitivity of Micrometastatseswith Green Fluorescent ProteinGene-Tagged Tumor Models in Mice

Hayao Nakanishi, Seiji Ito, Yoshinari Mochizuki,and Masae Tatematsu ................................................................... 351

24 99mTc-Annexin A5 Uptake and Imaging to Monitor Chemosensitivity

Tarik Z. Belhocine and Francis G. Blankenberg ............................... 363

25 Magnetic Resonance Imagingof Tumor Response to Chemotherapy

Richard Mazurchuk and Joseph A. Spernyak ................................... 381

26 Metabolic Monitoring of Chemosensitivity with 18FDG PET

Guy Jerusalem and Tarik Z. Belhocine ............................................. 417

Index ............................................................................................................ 441

Contents of Vol. 2 xiii


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I

OVERVIEW


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3

1

An Overview of Chemosensitivity Testing


SummaryThis overview chapter presents the importance of chemosensitivity testing for screening new

therapeutic agents, identifying patterns of chemosensitivity for different types of tumors, estab-

lishing patterns of cross-resistance and sensitivity in treatment naive and relapsing tumors; iden-

tifying genomic and proteomic profiles associated with sensitivity; correlating in vitro response,

preclinical in vivo effect, and clinical outcome associated with a particular therapeutic agent,

and tailoring chemotherapy regimens to individual patients. Various assays are available to

achieve these end points, including several in vitro clonogenic and proliferation assays, cell

metabolic activity assays, molecular assays to monitor expression of markers for responsiveness,

development of drug resistance and induction of apoptosis, in vivo tumor growth and survival

assays in metastatic and orthotopic models, and in vivo imaging assays. The advantages and dis-

advantages of the specific assays are discussed. A summary of research areas related to chemo-

sensitivity testing is also included.

Key Words

Dose-response curve; IC50 values; imaging; metabolic assays; molecular markers; proliferation

assays.

1. Introduction

Chemosensitivity testing is an ex vivo means of determining the cytotoxicand/or cytostatic, or apoptosis-inducing effect of anticancer drugs. The empha-

sis on screening new agents derived from synthetic compound archives, and

from pure natural products and their extracts, for antitumor activity necessi-

tates in vitro evaluation in cell culture and then in vivo in appropriate tumor-

bearing animal models. If the agent appears effective in this system, then the

drug may be further evaluated in clinical trials. This paradigm seeks to identify

the single best treatment to administer to the average patient with a given form

of cancer through the use of prospective, randomized trials.

From: Methods in Molecular Medicine, vol. 110: Chemosensitivity: Vol. 1: In Vitro AssaysEdited by: R. D. Blumenthal Humana Press Inc., Totowa, NJ


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In addition to the standard, DNA-damaging and metabolic inhibiting agents

in use, many new target classes now exist, such as kinase and nonkinase

enzymes, transcriptional regulators, growth factor receptors, chemokines, angio-

genic regulators, and proteinprotein interactions. Identifying which types of

tumors (e.g., breast, lung, pancreas) and which subtypes (e.g., based on mor-

phology, differentiation status, growth rate, drug resistance status, p53 expres-

sion) within each tumor category will respond to each new agent is essential.

Experience has shown that individual patients with a similar tumor histology do

not necessarily respond identically to a given agent or set of agents. Defining

the best treatment options, combinations, and schedules using standard agents

alone or combined with new agents for an individual patient is an area of active

investigation that requires both in vitro and in vivo testing. Chemosensitivityassays can potentially facilitate the individualization of patient treatment plans.

Many retrospective and prospective studies for leukemia and for solid tumors

have shown that drug resistance/sensitivity can be determined accurately by in

vitro drug-response assays. As a consequence, knowing the drug sensitivity of

a given tumor for a particular agent can significantly impact decision making

and treatment planning. By identifying inactive drugs, patients can be spared

standard chemotherapy regimens and their associated toxicities. In cases in

which the patients tumor is unresponsive to chemotherapy, the oncologist maybe able to offer alternative or experimental treatments much sooner, when they

may have a better chance of succeeding. Cell culture drug resistance testing

refers to testing the resistance of a patients own cancer cells in the laboratory

to drugs that may be used to treat the patients cancer (1). Table 1 summarizes

the potential value of predictive chemosensitivity assays.

When addressing the usefulness of an assay method, several questions must

be addressed: (1) Are the drug sensitivity patterns observed in vitro with tumors

of a particular histological type similar to those observed clinically for the sametumor type? (2) Can chemotherapy, which is selected on the basis of in vitro

studies, improve patient survival? Efforts have been made to correlate in vitro

drug sensitivity at initial diagnosis with 4-yr survival results, or in vitro resis-

tance and early relapse. If this can be achieved, it may allow one to select more

intensive regimens. (3) Do the results obtained in vitro predict those obtained

in vivo? Since the process of tumor resection, transport, and processing for

culture (mechanical or enzymatic disaggregation) introduces tissue stress and

damage, which can perturb cell function and potentiate drug sensitivity, obtain-

ing good correlations can be problematic. Another important consideration isthat tumor cell growth rate in vitro is likely to be much faster than in vivo.

Therefore, the in vitro assay might indicate chemosensitivity in a situation

of rapid cell division, which may be a false positive result, when attempting

to translate results in vivo. Test systems using single-cell suspensions may

4 Blumenthal


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overestimate sensitivity. Finally, the ability to shrink tumors in vivo with a given

treatment does not necessarily translate into a significant survival benefit.

Prediction of chemosensitivity in the clinic is particularly challenging

because drug responses reflect not only properties intrinsic to the target cell, but

also host metabolic properties. Pharmacokinetic and pharmacodynamic vari-

ables that affect drug action in vivo are not considered by in vitro assays.

Because each patient has a unique pharmacogenetic makeup, leading to sig-nificant interpatient variations in drug half-life, volume of distribution, types of

metabolites formed, and routes of elimination, correlating in vitro and in vivo

results is often not a straightforward process. Furthermore, because some ther-

apeutic agents (e.g., cyclophosphamide or CPT-11) are prodrugs that require

metabolic activation, the in vitro modeling of in vivo tumor cell drug exposure

becomes even more complex. However, by using in vitro models to address

questions of chemosensitivity, one limits the study to cell-intrinsic properties

found in cultured cells, which simplifies the system and focuses the initialinvestigation on tumor cell responsiveness.

Many different methods are available for assessing chemosensitivity (24).

In general, all assays generate dose-response curves where the dose of the drug

is related to the percentage effect, such as tumor cell kill (Fig. 1). Determining

the molar concentration that results in a 50% reduction in cell survival (IC50)

can be used to compare the efficacy of different drugs in one tumor cell system

or the same drug in different cell systems.

The most common in vitro assays can be divided into one of three cate-

gories: (1) clonogenic/proliferation assays, (2) assessment of cell metabolicactivity, and (3) measurement of cell membrane integrity. There has been much

debate as to the characteristics of the best chemosensitivity assay. For exam-

ple, should the assay measure colony formation or tumor cell proliferation?

Should the assay be short term (hours to days) vs long term (days to weeks)?

Chemosensitivity Testing 5

Table 1

Summary of Potential Value of Chemosensitivity Assays

Conducting an initial screening of new therapeutic agentsTailoring chemotherapy regimens to the individual patient: determining tumors that

are likely to respond to a particular agent and eliminating ineffective drugs

Identifying patterns of chemosensitivity for different types and subtypes of tumors

Establishing patterns of cross-resistance and sensitivity in treatment-naive and in

relapsing patients

Identifying genomic and proteomic profiles associated with sensitivity

Correlating in vitro, preclinical in vivo, and clinical response to a therapeutic agent


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Should the assay measure cytostatic or cytotoxic end points? Should the assay

use a cell suspension or tumor microorgans? Should metabolic or morpholog-

ical end points be used? Various assays measure different end points of cellu-

lar damage. Morphological appearance is generally considered too insensitive.

Measurements of biochemical parameters or reproductive capacity are likelyto be more reliable. Short-term assays may suggest that an agent is cytotoxic,

but cells may recover. Thus, early indicators of drug-induced cell damage may

provide misleading results. For an assay to be useful, the results must correlate

with clinical response and survival; the end point of the assay must detect

effects on cancer cells exclusive of other cellular elements such as fibroblasts,

mesothelial cells, and endothelial cells; the turnaround time must meet clinical

requirements; the test information must be easily interpreted and applied; and

the test must be cost-effective.

2. Clonogenic and Proliferation Assays

The clonogenic method such as the human tumor colony-forming assay (5)

is analogous to antibiotic sensitivity testing in bacteria. Single untreated or

treated tumor cells are grown in Petri dishes in a soft agar system and colonies

are counted after about 2 to 3 wk. Automated image analyzers now make this a

much faster procedure. The use of agar allows most tumor cells to grow but

prevents fibroblast proliferation. A reduction in colony number in treated groups

reflects cytotoxicity of the agent toward the tumor cells. This is the gold stan-dard to which all other predictive assays have been compared for positive pre-

dictive reliability (predicting patient response). Controlling the number of

colonies per plate and including only colonies with at least 4050 cells is

6 Blumenthal

Fig. 1. Theoretical dose response curve.


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important for obtaining accurate information from this assay. However, the

method is complicated by the ability to obtain a single cell tumor suspension,

adequate plating efficiency, proper growth in agar, and sufficient cell numbers

to test multiple concentrations of drug. Variations on this assay include micro-

clonogenic assays on tumor cells grown in a 96-well plate or in suspension

(6). IC50 values are determined based on dose-response curves that fit the data

to a linear quadratic equation.

Several other in vitro short-term growth inhibition assays are in use, which

are based on survival of tumor cell populations that have been in contact with

a chemotherapeutic agent. In these assays, which are experimentally simpler

than clonogenic assays, growth inhibition might not reflect true cell kill and can

result in higher false positive results. However, a measure of chemosensitivitycan be obtained even when plating efficiencies are low. One such assay is the

differential staining cytotoxicity assay (DiSC; [7]), which consists of incubat-

ing dissociated cells from biopsy specimens in the presence or absence of a

drug for 46 d, and using a dye such as fastgreen, which permeates only

through dead cells. The ratio of dead cells to total cells is a measure of cell kill.

In general, there has been good qualitative agreement between the DiSC assay

and the clonogenic assay. Duration of the assay is relatively short, and the assay

can be used on the majority of tumor specimens but is labor intensive and sub-ject to individual interpretation. Another method, the Kern assay (8), relies on

uptake of radiolabeled precursor such as 3H-thymidine into the DNA of prolif-

erating cells and is an example of similar assays that measure drug-induced

inhibition of radioactive precursor incorporation into cellular macromole-

cules (DNA, RNA, or protein) of single-cell suspensions or tumor slices. The

assumption is that thymidine incorporation into DNA reflects cell division.

There is a difference in view as to whether the assay is considered a reliable

means of quantifying drug sensitivity. Some believe that it is not an accuratemeasure in biopsies because of contaminating nontumor cells and, in general,

may be problematic because the effect on thymidine incorporation is not the

same as measuring cell kill or growth inhibition (2). Others have reported sim-

ilar results with the Kern assay to that obtained with clonogenic assays and

significant correlations between clinical responses and depression of DNA syn-

thesis in vitro (9). In general, this assay excels at negative predictive reliability

(predicting drug resistance). A third approach based on proliferation is the col-

lagen gel droplet drug sensitivity test, which is a quick and simple colorimet-

ric quantitative approach using neutral red staining within collagen gel dropsthat are imaged on a videomicroscope (10). It affords the advantage of being

able to eliminate the influence of fibroblasts in a mixed tumor/stroma popula-

tion derived from a biopsy specimen.



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3. Cell Metabolic Activity Assays

Assays that use various surrogate markers of cell number and viability have

been used to measure cell survival in response to a therapeutic. These methodsinclude bioluminescence of adenosine triphosphate (ATP) (ATP-based tumor

chemosensitivity assay [ATP-TCA]) (11) using the firefly luciferin-luciferase

reagent; fluorometric microculture cytotoxicity assay (FMCA), which measures

fluorescence generated from cellular hydrolysis of nonfluorescent fluorescein

diacetate to fluorescein by viable cells in microtiter plates (12); coloration with

sulforhodamine B (SRB), a general aminoxanthene dye for proteins, which

binds electrostatically to basic amino acids; and coloration with reduction of a

yellow tetrazolium dye such as MTT or MTS (13), substrates for mitochon-

drial dehydrogenases, resulting in a blue-purple formazan product that reflects

metabolic activity of living cells.

The ATP-TCA is extremely sensitive because ATP levels are linear with the

number of viable cells and correlate with cytotoxicity to a number of antineo-

plastic agents in vitro. Assay results have also been used to predict clinical

responses (14). This assay is done in a 96-well microplate and can use cell lines,

or specimens from surgical needle biopsies, pleural or ascites fluid, and requires

only 10,00020,000 cells/well (15). The FMCA is also a useful assay in that it

is simple, is rapid, and seems to report clinically relevant cytotoxic drug sensi-tivity data. An alternative high-throughput fluorescence assay using enhanced

green fluorescent protein (GFP) is available that can be analyzed by flow cytom-

etry or by fluorescence microplate reader (16). It is a sensitive and rapid method

to detect drug-induced cell death that provides results comparable to those

obtained by other traditional apoptosis assays such as annexin-V binding, pro-

pidium iodide incorporation, or reactive oxygen species production.

The SRB assay provides a sensitive index of cellular protein content that is

linear over a cell density range of two orders of magnitude, has a stable endpoint that does not require immediate range, and compares favorably with the

MTT assay. However, the MTT assay is able to discriminate live cells from

cellular debris, which the SRB assay cannot do. The drawbacks of assays such

as the MTT are the sensitivity to the pH and glucose content of the media;

chemical interferences with dye reduction; the quality of reagents used to sol-

ubilize the formazan crystals; the need to read the assay immediately before the

color fades; and the limited dynamic range, with a sensitivity of only 1-log cell

kill (17). In addition, such assays do not differentiate between cytostatic and

cytotoxic effects. In spite of these limitations, the MTT assay has been effec-

tive at predicting the sensitivity of patients who have attained remission and of

patient resistance (18). Both SRB and MTT assays provide rapid results and

hold more promise in the screening and evaluation of potential new agents in

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established tumor cell lines than in evaluating chemosensitivity of primary

tumor specimens.

Some researchers believe that results derived from single-cell preparations

are not as accurate as those obtained from tumor fragments in which the three-

dimensional structure of the tumor tissue is maintained (19). Two methods to

assess chemosensitivity using tumor fragments in place of single cells have

been utilized. The first is a variation on the MTT assay, called the histoculture

drug response assay (20). The second approach, the fluorescent cytoprint assay

(FCA), can be directly used on biopsy tissue without cell dissociation (21). In

this assay, the tumor fragments are maintained in a collagen matrix during drug

exposure. They are then treated with a fluorescent dye, which is visualized and

photographed as cytoprints before and after drug treatment. This method hasbeen shown to have a very high positive and negative predictive accuracy.

Another marketed in vitro assay that has been reported to have a high pre-

dictive value is the extreme drug resistance assay (22). Tumor cells are exposed

to suprapharmacological drug concentrations and either precursor incorporation

or colony formation is measured. A drug resistance profile (extreme, intermedi-

ate, or low) can be determined based on statistical comparison to a historical

database of tumor specimens tested against the same panel of chemotherapeutic

agents (23).An alternative approach to measuring cell proliferation or metabolic viabil-

ity is to measure drug-induced DNA damage at the chromosome level using

morphological criteria. The cytokinesis block micronucleus assay can reliably

measure chromosome loss, chromosome breakage, chromosome rearrangement

(nucleoplasmic bridges), cell division inhibition, necrosis, and apoptosis (24).

All the previously summarized in vitro assays, and which are detailed in the

chapters that follow, must ultimately provide a drug sensitivity index to put the

results into the context of other similar assay results, or calculate a probability

of response. Table 2 provides a summary of correlations of in vitro results with

patient response (25). The most sensitive assays are the DiSc and the FCA,

and the most specific are the clonogenic, MTT, and ATP assays. The ATP assay

also gives the highest positive prediction, and the clonogenic assay, thymidine

incorporation assay, DiSc and FCA are all highly accurate.

4. In Vivo Assays

In vitro assays in general are limited in that they do not take into account insuf-

ficient drug absorption, inadequate drug distribution to the tumor owing to poor

vascularization, pharmacological barriers, the need for drug activation, detoxifi-

cation of drug by general metabolic pathways, differences in tumor growth rate in

vitro and in vivo, or the problem of tumor heterogeneity. Therefore, assessing



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chemosensitivity in preclinical tumor-bearing animal models is essential. Pri-mary tumor models that have been utilized include human tumors grown sub-

cutaneously in athymic nude mice, severe combined immunodeficient mice, or

triple-deficient mice (bg/nu/xid mutations; [26]). Examples of metastasis

models of human tumors include those grown orthotopically (27) or introduced

systemically via iv, ip, intracardiac, intrasplenic, or intrahepatic injection or via

inoculation of a cell suspension or tumor fragment into the mammary pad (28)

or subrenal capsule (29). For primary growth, tumor size is followed by caliper

measurement and tumor growth curves are constructed. Various formulas tomeasure tumor volume, area, and diameter have been used (30). Absolute tumor

size, change in tumor size, percentage of growth inhibition, and area under the

tumor growth curve have been reported. Using appropriate statistical methods

to analyze tumor growth experiments that have sufficient power and low type

I error rates is essential when performing in vivo chemosensitivity experiments

(31). For metastasis models, median animal survival time, number and/or size

of metastases after a fixed time, and weight of an organ containing metastases

have been used as measures of response to a therapeutic agent. In addition todirect measurements of tumor growth, imaging approaches have been devel-

oped to assess tumor chemosensitivity (32). These tools include the use of18FDG positron emission tomography (33), 99mTc-annexin-V scintigraphy (34),

magnetic resonance imaging (35) and GFP (36).

10 Blumenthal

Table 2

Correlation of In Vitro Results with Patient Responsea

Type of Total Positive Accuracy Sensitivity Specificityassay (N) TP TN FP FN prediction (%) (%) (%) (%)

Clonogenic 2300 512 1427 226 135 69 91 79 86

Thymidine 494 123 232 119 20 51 92 86 66

DiSC 510 247 175 72 16 77 92 94 71

MTT 326 187 74 37 28 83 73 86 86

ATP 129 74 37 6 12 93 76 86 86

FCA 333 154 116 52 11 75 91 93 69

Total 4092 1297 2061 512 222 75 86 87 74

aSummary of clinical correlations from Table 7 in ref. 25. TP = true positivepatients who are

sensitive in vitro and respond to therapy; TN = true negativepatients who are resistant in vitro and

do not respond to therapy; FP = false positivepatients who are sensitive in vitro but resistant clin-

ically; FN = false negativepatients who are resistant in vitro but respond clinically; positive pre-

diction = TP/(TP + FP); accuracy = TN/(TN + FN); sensitivity = tests ability to detect clinically

responsive patients = TP/(TP + FN); specificity = tests ability to detect clinically unresponsive

patients = TN/(TN + FP).


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5. Molecular Assays of Chemosensitivity

The range of response to a particular cytotoxic agent can be quite substantial,

and studies to understand the molecular parameters at the gene and at the pro-tein level that regulate chemosensitivity or resistance are an active area of inves-

tigation (37). The molecular basis of sensitivity to chemotherapy is a complex

product of cellular and tissue factors (3841). These include expression of the

P-glycoprotein family of membrane transporters (e.g., MDR1, MRP, LRP), which

decrease the intracellular accumulation of drug; changes in cellular proteins

involved in detoxification (e.g., glutathione S-transferase , metallothioneins,

NADP cytochrome P-450 reductase); changes in expression of molecules

involved in DNA repair (e.g., O6-methylguanine DNA methyltransferase, DNA

topoisomerase II, hMLH1, p21WAF1/CIP1); and activation oncogenes such as

Her-2/neu, bcl-2, bcl-XL, c-myc, ras, c-jun, c-fos, MDM2, p210, BCR-abl, or

mutant p53. In addition, expression of growth factors and receptors, proliferation

markers (42), telomerase (43), enzymes that regulate intracellular ceramide

levels (44), positive and negative regulators of apoptosis, and cell-cycle check-

point controls are all important (45,46). For example, the form of p53 expression

(null, wild type, or mutant and the location of the mutation) is known to affect

chemosensitivity (47). Drug resistance can occur at the onset of the disease or

can be acquired after previous chemotherapy. Methods that identify and detectexpression of drug resistance genes (48) or proteins (49) by flow cytometry

(50), Western blotting, or immunohistochemistry (IHC) (51); or by induction of

apoptosis by the DNA diffusion assay (52), diphenylamine assay to evaluate

DNA fragmentation (53), TUNEL assay (54), flow cytometry with fluorescein

isothiocyanate-annexin-V (34), PARP assay (55), or expression of BCl-2 family

proteins including Bcl-2, Bcl-X(L), Bax, Bad, and Bak (56); or by changes in

cell cycle (57) can be used as indirect measures of chemosensitivity.

6. Genomic and Proteomic Approachesto Understand Chemosensitivity

Previous efforts to use genetic information to predict drug sensitivity primar-

ily have focused on individual genes that have broad effects, such as the mul-

tidrug resistance genes mdr1 and mrp1 (58). There has been an effort to develop

a genomics- and proteomics-based approach for the prediction of drug response

(5961). An example of the clinical application of single-gene analysis as a pre-

dictor of chemosensitivity is the evaluation of thymidylate synthase mRNAexpression from tumor core needle biopsies using real-time polymerase chain

reaction analysis. Those patients with a clinical response to 5-fluorouracil (5-FU)

therapy had a significantly lower level of expression of this gene. Expanding

beyond single-gene effects on treatment response, the recent development of



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DNA microarrays permits large-scale screening of genes and the simultaneous

measurement of the expression levels of thousands of genes and raises the pos-

sibility of an unbiased, genomewide approach to understand the genetic basis

of drug response (62). Algorithms have been developed for the classification

of cell line chemosensitivity based on gene expression profiles. Using oligonu-

cleotide microarrays, the expression levels of genes can be measured in a panel

of cancer cell lines with known chemosensitivity profiles for various chemical

compounds. Genes associated with repair processes, cell-cycle checkpoints,

apoptosis, signal transduction, and metabolism can all be studied. Microarray

analysis of baseline gene expression (63,64) and drug-induced changes in gene

expression (65) have been successfully applied to predicting chemotherapy

response. Further development of this technology will enable those responsiblefor treatment planning not only to predict chemosensitivity prior to therapy, but

to answer whether the classifiers are dependent on tumor type or tumor class.

Toward that goal, chemosensitivity prediction studies are being extended beyond

cell line models to include the analysis of primary patient material.

The addition of proteomic studies to genomic studies will further facilitate

the ability to identify a priori sensitive and resistant tumors (66). In the past,

protein analysis of formalin-fixed paraffin-embedded tumor tissue or Western

blotting was used to assess prediction of therapeutic response. Techniques suchas IHC provide semiquantitative information on the level of expression of key

proteins. Similar to mRNA results, thymidylate synthase expression at the pro-

tein level has been a consistent predictor of response to 5-FU-based chemother-

apy of metastatic colorectal cancer (67). Other protein markers that have been

useful for predicting chemosensitivity include ornithine decarboxylase (68),

HER-2/neu (69), the p27 cell-cycle regulatory protein (70), and Bcl-2 (71).

Other proteins that have been studied but have not been predictive include p53

and cerbB-2(72)

. The use of tissue arrays allows IHC to be performed on0.6-mm core tissue sections from a large panel of tumor samples of varying his-

totypes (73). The field is now expanding beyond detection and quantification of

single proteins, to include evaluation of a sizable panel of proteins by combin-

ing the tools of laser capture detection of tumor cells with silver stained two-

dimensional gel electrophoresis and generate a tumor phenotype (74). Much

of this effort has focused on the mass spectral identification of the thousands of

proteins that populate complex biosystems. Protein patterns can be analyzed

in hundreds of clinical samples per day utilizing this technology.

7. Future Directions

The availability of predictive in vitro and in vivo assays provides a mean of

addressing many important issues in tumor biology. Several areas of investiga-

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tion related to chemosensitivity testing are worthy of consideration. First, work

is required to establish gene and protein profiles associated with drug sensitiv-

ity (59). Studies are needed to address whether baseline gene/protein expression

or drug-induced changes in expression are most predictive of response to treat-

ment. Second, developing a better understanding of how to use drug combina-

tions and schedules is essential. In vitro (75) and in vivo (76) models have

been developed to assess interactions between multiple therapeutic agents.

Toward that end, studies to determine how the use of one agent influences the

biology of the surviving tumor cells and environmental milieu, and thus affects

sensitivity to the second agent, are needed. Third, how chemosensitivity can

be modulated, such as by using compounds that affect DNA repair (77); phar-

macological agents (e.g., small molecule [78]); or genetic methods such as anti-sense, ribozyme, or RNA interference technology to overcome drug resistance

(79) or antiapoptotic states must be determined. Fourth, the role that circadian

rhythms play in chronotherapy (80) is just beginning to receive attention. In

vivo designs to address how light/dark cycles impact the optimal time of day to

administer a particular drug to achieve maximal efficacy are under investiga-

tion. Fifth, tumor heterogeneity within a patient is important. Clinically, this is

seen when a tumor mass in one area responds to chemotherapy, while a sepa-

rate site may remain stable or progress. Intrapatient response variations havebeen evaluated by comparing the in vitro drug response of primary vs metasta-

tic sites from the same patient. Thus, in vitro test results for solid tumors from

one site may not be representative for other sites within the same patient. Addi-

tional research to understand the molecular differences in chemosensitivity

between primary and secondary sites is needed. Finally, developing approaches

to individualize drug treatment as a function of tumor cell sensitivity (as has

been done in infectious disease) requires further attention.

References

1. Kaspers, G., Zwaan, C., Pieters, R., and Veerman, A. (1999) Cellular drug resis-

tance in childhood acute myeloid leukemia: a mini-review with emphasis on cell

culture assays.Adv. Exp. Med. Biol. 457, 415421.

2. Robert, J. (1999) Chemosensitivity testing: prediction of response to anticancer

drugs using in vitro assays.Electronic J. Oncol. 2, 198210.

3. Bellamy, W. (1992) Prediction of response to drug therapy of cancer: a review of

in vitro assays.Drugs 44, 690708.

4. Gercel-Taylor, C., Ackermann, M., and Taylor, D. (2001) Evaluation of cell pro-liferation and cell death based assays in chemosensitivity testing. Anticancer Res.

21, 27612768.

5. Von Hoff, D., Clark, G., Stogdill, B., et al. (1983) Prospective clinical trial of a

human tumor cloning system. Cancer Res. 43, 19261936.



29/247

6. Engblom, P., Rantanen, V., Kulmala, J., and Grenman, S. (1996) Paclitaxel and

cisplatin sensitivity of ovarian carcinoma cell lines tested with a 96-well plate

clonogenic assay.Anticancer Res. 16, 17431747.

7. Weisenthal, L., Marsden, J., Dill, P., and Macaluso, C. (1983) A novel dye exclu-sion method for testing in vitro chemosensitivity of human tumors. Cancer Res.43,

749757.

8. Kern, D., Drozemuller, C., Kennedy, M., et al. (1985) Development of a minia-

turized improved nucleic acid precursor incorporation assay for chemosensitivity

testing of human solid tumors. Cancer Res. 45, 54365441.

9. Sondak, V., Bertelson, C., Tanigawa, N., et al. (1984) Clinical correlations with

chemosensitivities measured in a rapid thymidine incorporation assay. Cancer Res.

46, 17251728.

10. Kobayashi, H., Higashiyami, M., Minamigawa, K., et al. (2001) Examination of invitro chemosensitivity test using collagen gel droplet culture method with colori-

metric endpoint quantitation.Jpn. J. Cancer Res. 92, 203210.

11. Kangas, L., Gronroos, M., and Nieminen, A. (1984) Bioluminesence of cellular

ATP: a new method for evaluating cytotoxic agents in vitro. Med. Biol. 62,

338343.

12. Csoka, K., Larsson, R., Tholander, B., Gerdin, E., de la Torre, M., and Nygren, P.

(1994) Cytotoxic drug sensitivity testing of tumor cells from patients with ovarian

carcinoma using the fluorometric microculture cytotoxicity assay (FMCA).

Gynecol. Oncol. 54, 163170.13. Rubinstein, L., Shoemaker, R., Pacell, K., Simon, R., and Tosini, S. (1990) Com-

parison of in vitro anticancer-drug-screening data generated with a tetrazolium

assay versus a protein assay against a diverse panel of human tumor cells lines.

J. Natl. Cancer Inst. 82, 11131118.

14. Sevin, B., Peng, Z., Perras, J., Panalver, G., and Averette, H. Application of an

ATP bioluminescence assay in human tumor chemosensitivity testing. Gynecol.

Oncol. 31, 191204.

15. Andreotti, P., Cree, I., Kurbacher, C., et al. (1995) Chemosensitivity testing of

human tumors using a microplate adenosine triphosphate luminescence assay: clin-ical correlation for cisplatin resistance of ovarian carcinoma. Cancer Res., 55,

52765282.

16. Steff, A., Fortin, M., Arguin, C., and Hugo, P. (2001) Detection of a decrease in

green fluorescent protein fluorescence for the monitoring of cell death: an assay

amenable to high-throughput screening technologies. Cytometry 45, 237243.

17. Vistica, D., Skehan, P., Scudiero, D., et al. (1991) Tetrazolium-based assays for

cellular viability: a critical examination of selected parameters affecting formazan

production. Cancer Res. 51, 25152520.

18. Sargent, J. and Taylor, C. Appraisal of the MTT assay as a rapid test of chemosen-sitivity in acute myeloid leukemia.Br. J. Cancer60, 206210.

19. Hoffman, R. (1991) Three-dimensional histoculture: origins and applications in

cancer research. Cancer Cells 3, 8692.

14 Blumenthal


30/247

20. Singh, B., Li, R., Xyu, L., et al. (2002) Prediction of survival in patients with head

and neck cancer using the histoculture drug response assay.Head Neck24, 437442.

21. Meitner, P. (1991) The fluorescence cytoprint assay: a new approach to in vitro

chemosensitivity testing. Oncology 5, 7581.22. Kern, D. and Weisenthal, L. (1990) Highly specific prediction of antineoplastic

drug resistance with an in vitro assay using supra-pharmacologic drug exposures.

J. Natl. Cancer Inst. 82, 582588.

23. Haroun, R., Clatterbuck, R., Gibbons, M., et al. (2002) Extreme drug resistance in

primary brain tumors: in vitro analysis of 64 resection specimens.J. Neurooncol.

58, 115123.

24. Fenech, M. (2000) The in vitro micronucleus technique.Mutation Res.455, 8195.

25. Fruhauf, J. and Bosanquet, A. (1993) In vitro determination of drug response: a

discussion of clinical applications. PPO Updates 7, 121.26. Clarke, R. (1996) Human breast cancer cell line xenografts as models of breast

cancer: the immunobiologies of recipient mice and the characteristics of several

tumorigenic lines.Breast Cancer Res. Treat. 39, 6986.

27. Hoffman, R. (1999) Orthotopic metastatic mouse models for anticancer drug dis-

covery and evaluation: a bridge to the clinic.Invest. New Drugs 17, 343359.

28. Price, J. (1996) Metastasis from human breast cancer cell lines. Breast Cancer

Res. Treat. 39, 93102.

29. Konovalova, N., Diatchkovskaya, R., Ganieva, L., Volkova, L., Lapshin, I.,

Rudakov, B., Shaposhnikov, Y., and Shapiro, A. (1991) Subrenal capsule assay ofhuman tumor chemosensitivity.Neoplasma 38, 275284.

30. Tomayko, M. and Reynolds, C. (1989) Determination of subcutaneous tumor size

in athymic (nude) mice. Cancer Chemother. Pharmacol. 24, 148154.

31. Heitjan, D., Manni, A., and Santen, R. (1993) Statistical analysis of in vivo tumor

growth experiments. Cancer Res. 53, 60426050.

32. Gibbs, J., Slocum, H., Cao, S., and Rustum, Y. (1999) Image analysis for quanti-

tation of solid tumor drug sensitivity. Int. J. Surg. Invest.1, 133138.

33. Kubota, K. (2001) From tumor biology to clinical PET: a review of positron

emission tomography (PET) in oncology. Ann. Nucl. Med. 15, 471486.34. Belhocine, T., Steinmetz, N., Hustinx, R., et al. (2002) Increased uptake of the

apoptosis imaging agent (99m)Tc recombinant human Annexin V in human tumors

after one course of chemotherapy as a predictor of tumor response and patient

prognosis. Clin. Cancer Res. 8, 27662774.

35. Mazurchuk, R., Glaves, D., and Raghavan, D. (1997) Magnetic resonance imaging

of response to chemotherapy in orthotopic xenografts of human bladder cancer.

Clin. Cancer Res. 3, 16351641.

36. Nakanishi, H., Mochizuki, Y., Kodera, Y., et al. (2003) Chemosensitivity of peri-

toneal micrometastases as evaluated using green fluorescence protein(GFP)tagged human gastric cancer cell line. Cancer Sci. 94, 112118.

37. Tewari, K. and Manetta, A. (1999) In vitro chemosensitivity testing and mecha-

nisms of drug resistance. Curr. Oncol. Rep. 1, 7784.



31/247

38. el-Deiry, W. S. (1997) Role of oncogenes in resistance and killing by cancer ther-

apeutic agents. Curr. Opin. Oncol. 9, 7987.

39. Fan, S., el-Deiry, W., Bae, I., et al. (1994) p53 gene mutations are associated with

decreased sensitivity of human lymphoma cells to DNA damaging agents. CancerRes. 54, 58245830.

40. Bosken, C., Wei, Q., Amos, C., and Spitz, M. (2002) An analysis of DNA repair as

a determinant of survival in patients with non-small-cell lung cancer. J. Natl.

Cancer Inst. 94, 10911099.

41. Zunino, F., Perego, P., Pilotti, S., and Pratesi, G. (1997) Role of apoptotic response

in cellular resistance to cytotoxic agents. Pharmacol. Ther. 76, 177185.

42. Wang, Y., Ashkenazi, Y., and Bachrach, U. In vitro chemosensitivity of hemato-

logical cancers: immunohistochemical detection of ornithine decarboxylase.Anti-

cancer Drugs 10, 797805.43. Park, K., Rha, S., Kim, C., et al. (1998) Telomerase activity and telomere lengths

in various cell lines: changes of telomerase activity can be another method for

chemosensitivity evaluation.Int. J. Oncol. 13, 489495.

44. Modrak, D., Rodriguez, M., Goldenberg, D., Lew, W., and Blumenthal, R. (2002)

Sphingomyelin enhances chemotherapy efficacy and increases apoptosis in human

colonic tumor xenografts.Int. J. Oncol. 20, 379384.

45. Epstein, R. (1990) Drug-induced DNA damage and tumor chemosensitivity.

J. Clin. Oncol. 8, 20622084.

46. Xia, F. and Powell, S. (2002) The molecular basis of radiosensitivity andchemosensitivity in the treatment of breast cancer. Semin. Radiat. Oncol. 12,

296304.

47. Manahan, K., Taylor, D., and Gercel-Taylor, C. (2001) Clonal heterogeneity of

p53 mutations in ovarian cancer. Int. J. Oncol. 19, 387394.

48. Granjean, F., Bremaud, L., Verdier, M., Robert, J., and Ratinaud, M. (2001)

Sequential gene expression of P-glycoprotein (P-gp), multidrug resistance associ-

ated protein (MRP) and lung resistance protein: functional activity of P-gp and

MRP present in the doxorubicin-resistant human K562 cell lines.Anticancer Drugs

12, 247258.49. Michieli, M., Damiani, D., Ermacora, A., et al. (2000) P-glycoprotein (PGP), lung

resistancerelated protein (LRP) and multidrug resistanceassociated protein

(MRP) expression in acute promyelocytic leukemia. Br. J. Haematol. 108,

703709.

50. Pallis, M., Turzanski, J., Langabeer, S., and Russell, N. (1999) Reproducible flow

cytometric methodology for measuring multidrug resistance in leukaemic blasts.

Adv. Exp. Med. Biol. 457, 7788.

51. Pall, G., Spitaler, M., Hofmann, J., Thaler, J., and Ludescher, C. (1997) Multidrug

resistance in acute leukemia: a comparison of different diagnostic methods.Leukemia 11, 10671072.

52. Singh, N. (2000) A simple method for accurate estimation of apoptotic cells.Exp.

Cell. Res. 256, 328337.

16 Blumenthal


32/247

53. Munshi, A., McDonnell, T., and Meyn, R. (2002) Chemotherapeutic agents

enhance TRAIL-induced apoptosis in prostate cancer cells. Cancer Chemother.

Pharmacol. 50, 4652.

54. Maciorowski, Z., Klijanienko, J., Padoy, E., et al. (2001) Comparative image andflow cytometric TUNEL analysis of fine needle samples of breast carcinoma.

Cytometry 46, 150156.

55. Ogata, S., Okumura, K., and Taguchi, H. (2000) A simple and rapid method for the

detection of poly(ADP-ribose) by flow cytometry. Biosci. Biotechnol. Biochem.

64, 510515.

56. Nita, M., Nagawa, H., Tominago, O., et al. (1998) 5-Fluorouracil induces apopto-

sis in human colon cancer cell lines with modulation of Bcl-2 family proteins.

Br. J. Cancer78, 986992.

57. Smith, D., Gao, G., Zhang, X., Wang, G., and Dou, Q. (2000) Regulation of tumorcell apoptotic sensitivity during the cell cycle [review].Int. J. Mol. Med.6, 503507.

58. Sonneveld, P. (2000) Multidrug resistance in hematological malignancies.J. Intern.

Med. 247, 521534.

59. Staunton, J., Slonim, D., Coller, H., et al. (2001) Chemosensitivity prediction by

transcriptional profiling. Proc. Natl. Acad. Sci. USA 98, 10,78710,792.

60. Bao, L. and Sun, Z. (2002) Identifying genes related to drug anticancer mecha-

nisms using support vector machine. FEBS Lett. 521, 109114.

61. McLeod, H. Individualized cancer therapy: molecular approaches to the predic-

tion of tumor response.Expert Rev. Anticancer Ther. 2, 113119.62. Amundson, S., Myers, T., Scudiero, D., Kitada, S., Reed, J., and Fornace, A.

(2000) An informatics approach identifying markers of chemosensitivity in human

cancer cell lines. Cancer Res. 60, 61016110.

63. Alizadeh, A., Eisen, M., and Dasvis, R. (2000) Distinct types of diffuse large

B-cell lympoma identified by gene expression profiling.Nature403, 503511.

64. Kihara, C., Tsunoda, T., Tanaka, T., et al. (2001) Prediction of sensitivity of

esophageal tumors to adjuvant chemotherapy by cDNA microarray analysis of

gene-expression profiles. Cancer Res. 61, 64746479.

65. Sotirious, C., Powles, T., Dowsett, M., et al. (2002) Gene expression profilesderived from fine needle aspiration correlate with response to systemic chemother-

apy in breast cancer.Breast Cancer Res. 4, R3.

66. Poland, J., Schadendorf, D., Lage, H., Schnolzer, M., Celis, J., and Siha, P. (2002)

Study of therapy resistance in cancer cells with functional proteome analysis. Clin.

Chem. Lab. Med. 40, 221234.

67. Aschele, C., Debernardis, D., Casazza, S., et al. (1999) Immunohistochemical

quantitation of thymidylate synthase expression in colorectal cancer metastases

predicts for clinical outcome to fluorouracil-based chemotherapy. J. Clin. Oncol.

17, 17601770.68. Bachrach, U. and Wang, Y. (2003) In vitro chemosensitivity testing of hematolog-

ical cancer patients: detection of ornithine decarboxylase.Rec. Results Cancer Res.

161, 6270.



33/247

69. Konecny, G., Fritz, M., Untch, M., et al. (2001) Her-2/neu overexpression and in

vitro chemosensitivity to CMF and FEC in primary breast cancer. Breast Cancer

Res. Treat. 69, 5363.

70. Yang, Q., Sakurai, T., Yoshimura, G., et al. (2000) Overexpression of p27 proteinin human breast cancer correlates with in vitro resistance to doxorubicin and mit-

omycin C.Anticancer Res. 20, 43194322.

71. Yang, Q., Sakurai, T., Yoshimura, G., et al. (2000) Expression of Bcl-2 but not

Bax or p53 correlates with in vitro resistance to a series of anticancer drugs in

breast carcinoma.Breast Cancer Res. Treat. 61, 211216.

72. Rozan, S., Vincent-Salomon, A., Zafrani, B., et al. (1998) No significant predictive

value of c-erbB-2 or p53 expression regarding sensitivity to primary chemotherapy

or radiotherapy in breast cancer. Int. J. Cancer79, 2733.

73. Ginestier, C., Charafe-Jauffret, E., Bertucci, F., et al. (2002) Distinct and comple-mentary information provided by use of tissue and DNA microarrays in the study

of breast tumor markers. Am. J. Pathol. 161, 12231233.

74. Jones, M., Krutzsch, H., Shu, H., et al. (2002) Proteomic analysis and identifica-

tion of new biomarkers and therapeutic targets for invasive ovarian cancer. Pro-

teomics 2, 7684.

75. Konecny, G., Untch, M., Slamon, D., et al. (2001) Drug interactions and cytotoxic

effects of paclitaxel in combination with carboplatin, epirubicin, gemcitabine, or

vinorelbine in breast cancer cell lines and tumor samples. Breast Cancer Res.

Treat. 67, 223233.76. Lopez, A., Pegram, M., Slamon, D., and Landaw, E. (1999) A model-based

approach for assessing in vivo combination therapy interactions. Proc. Natl. Acad.

Sci. USA 96, 13,02313,028.

77. Heim, M., Eberhardt, W., Seeber, S., and Muller, M. (2000) Differential modula-

tion of chemosensitivity to alkylating agents and platinum compounds by DNA

repair modulators in human lung cancer cell lines. J. Cancer Res. Clin. Oncol.

126, 198204.

78. Kondratov, R., Komarov, P., Becker, Y., Ewenson, A., and Gudkov, A. (2001)

Small molecules that dramatically alter multidrug resistance phenotype by modu-lating the substrate specificity of P-glycoprotein. Proc. Natl. Acad. Sci. USA 98,

14,07814,083.

79. Henewisch-Becker, S. (1996) MDR1 reversal: criteria for clinical trials designed to

overcome the multidrug resistance phenotype. Leukemia 10, S32S38.

80. Levi, F., Giacchetti, S., Zidani, R., et al. (2001) Chronotherapy of colorectal cancer

metastases.Hepatogastroenterology 48, 320322.

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II

IN VITRO MEASURES OF CHEMOSENSITIVITY


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Clonogenic Cell Survival Assay

Anupama Munshi, Marvette Hobbs, and Raymond E. Meyn

SummaryThe clonogenic cell survival assay determines the ability of a cell to proliferate indefinitely,

thereby retaining its reproductive ability to form a large colony or a clone. This cell is then said

to be clonogenic. A cell survival curve is therefore defined as a relationship between the dose

of the agent used to produce an insult and the fraction of cells retaining their ability to reproduce.

Although clonogenic cell survival assays were initially described for studying the effects of radi-

ation on cells and have played an essential role in radiobiology, they are now widely used to

examine the effects of agents with potential applications in the clinic. These include, in addition

to ionizing radiation, chemotherapy agents such as etoposide and cisplatin, antiangiogenic agents

such as endostatin and angiostatin, and cytokines and their receptors, either alone or in combi-

nation therapy. Survival curves have been generated for many established cell lines growing in

culture. One can use cell lines from various origins including humans and rodents; these cells can

be neoplastic or normal. Because survival curves have wide application in evaluating the repro-

ductive integrity of different cells, we provide here the steps involved in setting up a typical

experiment using an established cell line in culture.

Key Words

Survival curve; cell survival; plating efficiency; radiation.

1. IntroductionClonogenic cell survival is a basic tool that was described in the 1950s for

the study of radiation effects. Much of the information that has been generated

on the effect of radiation on mammalian cells has been obtained from clono-

genic cell survival assays.

Various mechanisms have been described for cell death; however, loss

of reproductive integrity and the inability to proliferate indefinitely are the

most common features. Therefore, a cell that retains its ability to synthesize pro-

teins and DNA and go through one or two mitoses, but is unable to divide and

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produce a large number of progeny is considered dead. This is very commonly

referred to as loss of reproductive integrity or reproductive death and is the

end point measured with cells in culture. On the other hand, a cell that is not

reproductively dead and has retained the capacity to divide and proliferate

indefinitely can produce a large clone or a large colony of cells and is then

referred to as clonogenic. A cell survival curve describes a relationship

between the insult-producing agent and the proportion of cells that survive.

The ability of a single cell to grow into a large colony that can be visualized

with the naked eye is proof that it has retained its capacity to reproduce. The

loss of this ability as a function of dose of radiation or chemotherapy agent is

described by the dose-survival curve. Most laboratories now extensively use

established cell lines for studying the effects of various agents either alone orin combination. Therefore, the aim of this chapter is to go through the steps

involved in setting up a typical clonogenic cell survival experiment using estab-

lished cells lines growing as monolayer cultures.

In brief, cells from an actively growing stock culture in monolayer are pre-

pared in a suspension by the use of trypsin, which causes the cells to detach

from the substratum. The number of cells per milliliter in this suspension is

then counted using a hemocytometer or a Coulter counter. From this stock cul-

ture, if 50 cells are seeded into a dish, e.g., and the dish is incubated for approx2 wk, each single cell divides many times and forms a colony that is easily

visible with the naked eye, especially if it is fixed and stained (the steps

involved in this process are briefly outlined in Fig. 1). All the cells that make

up the colony are the progeny of a single cell. For the 50 cells seeded into

the dish, the number of colonies counted may be anywhere from 0 to 50. One

would ideally expect the number to be 50, but that is rarely the case for several

possible reasons, including suboptimal growth medium, errors in counting the

number of cells initially plated, and the loss of cells by trypsinization and gen-eral handling. The term plating efficiency (PE) indicates the percentage of cells

seeded into a dish that finally grow to form a colony. Therefore, in the previ-

ous example, if there are 25 colonies in the dish, then the PE becomes 50%. If

a parallel dish is seeded with cells exposed to a dose of 6 Gy of gamma rays

and incubated for approx 2 wk before being fixed and stained, then the fol-

lowing may be observed:

1. Some cells may remain single, not divide, and, in some cases, may show evi-

dence of nuclear deterioration as they die by apoptosis. These cells would bescored as dead.

2. Some cells may go through one or two divisions and form small colonies of

just a few cells. These cells would be scored as dead.

3. Some cells may form large colonies, indicating that the cells have survived the

treatment and have retained the ability to reproduce indefinitely.

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2. Materials

2.1. Preparation of Cell Lines Prior to Setting Up Clonogenic Assays

1. Cell lines that need to be tested for their ability to form colonies.

2. Complete growth medium as recommended by the manufacturer typically con-

taining 10% fetal bovine serum plus antibiotics (penicillinstreptomycin) and glu-

tamine. For every 500 mL of medium, add 55 mL of serum, 5 mL of 200 mM

L-glutamine, and 5 mL of 10,000 U/mL penicillinstreptomycin solution. Medium

should be stored at 4C but warmed to 37C prior to use.

3. Trypsin-EDTA, to make single-cell suspensions from monolayer cultures. Store

at 4C.

4. Plasticware, for carrying out tissue culture including flasks (T-25 and T-75);

100-mm dishes; and 5-, 10-, and 25-mL pipets.

5. Micropipets and corresponding tips.

6. 70% ethanol, for wiping the surface of the hood as well as the surface of all

medium bottles prior to bringing them into the hood.7. Cidecon (detergent disinfectant with bactericidal and virucidal properties), to wipe

the surface of the shelf on which the dishes will be incubated.

8. Phosphate-buffered saline (PBS) (calcium magnesium free). Store at 4C.

9. Isoton II (diluent for counting cells using a Coulter counter). Store at room

temperature.

Clonogenic Cell Survival Assay 23

Fig. 1. Schematic representation of steps involved in setting up a clonogenic cell sur-

vival assay.


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2.2 Staining of Plates

1. 0.5% Gentian Violet (made up in methanol). Store at room temperature in a dark

bottle. Do not pour it down the sink.

3. Methods

3.1. Cells Growing in Monolayers or Attached Cells

The procedure that we outline in this chapter is a basic protocol for setting

up radiation clonogenic assays. However, this protocol can be modified to test

the effect of different agents on the cell line of interest, either alone or in com-

bination. These could include gene therapy vectors, chemotherapy agents, tyro-

sine kinase inhibitors, antiangiogenic agentsbasically any agent that has to betested for its ability to affect reproductive cell death.

3.2. Preparation of Cell Lines Prior to Setting UpClonogenic Cell Survival

1. Label six T-25 flasks in preparation for setting each flask with a known number

of cells as 0, 2, 4, and 6 Gy (depending on the experiment) for the various doses

of radiation to be given. Add 5 mL of growth medium to the flasks and keep them

aside in a hood.

2. Trypsinize the stock flask of cells containing the cells that have to be tested fortheir radiosensitivity. Make sure that the cells are in single-cell suspension and

obtain an accurate cell count. We use a Coulter counter to obtain a cell count. If

a Coulter counter is not available, cells can be counted using a hemocytometer.

Using a Pipettman, add 250,000 cells (the cell number can vary depending on the

cell type) to the 5 mL of medium in each T-25 flask. Shake gently to distribute the

cells evenly.

3. Place the flasks in a 37C incubator set at 5% CO2 and be sure to leave the cap

one thread loose so as to allow CO2 exchange (see Note 1).

4. Allow the cells to settle and attach as a monolayer.

3.3. Irradiation of Flasks and Performance of Plating Experimentfor Clonogenic Assay

1. Prepare the hood and clean an incubator shelf. Because these cells are going to be

left untouched in an incubator for up to 2 wk, and the possibility of contamination

is high, clean the shelf thoroughly with Cidecon and 70% ethanol. Keep the

cleaned shelf in the hood. Make sure that more than one bottle of complete

medium is available; this experiment may require >500 mL of medium.

2. Prepare the 100-mm dishes and 15-mL tubes in advance. One will need 100-mm

dishes in triplicate, and two cell numbers will be plated for each dose of radiation.

Because cells will be exposed to six doses of radiation, 36 dishes will be needed.

Label the bottom of each dish as the lid, for the dishes will be loose during stain-

ing, and the bottom is where the colonies will form, which is what will actually

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be counted. Label the first set of triplicate dishes as 0 Gy A and 0 Gy B (A and

B for the two cell numbers to be used). Repeat this for all dose levels: 2, 4, 6, 8,

and 10 Gy. Place 10 mL of complete medium in each dish. Place all dishes,

stacked in threes, on the incubator shelf and put back in the incubator until readyfor plating.

3. Place 18, 15-mL tubes on a clean rack three deep. Label the first tube 0Gy, 11;

the next one 0 Gy 110; and the next 0 Gy 1100. Repeat this for all dose levels.

Place 4.5 mL of complete medium in the back two tubes but not in the 11 dilu-

tion tubes.

4. Put the flasks on ice. Clean a rectangular tub and fill it halfway with ice. Remove

the flasks from the incubator and close the caps tightly. Place the flasks on ice,

and insert half depth into the ice. Tilt the flasks to the bottom so that the medium

does not rest against the cap. Start a timer for 20 min.5. While waiting, prepare the Coulter counting vials. Label each as 0, 2, 4, and 6 Gy.

Place 9.9 mL of Isoton (to be used for counting cells if using a Coulter counter)

into each vial. Place Isoton in a control vial and run through the counter to get a

background measurement; repeat until a satisfactory low background is obtained.

Make sure that the Coulter is set to the appropriate size parameter for the cell line.

6. When the 20-min time is up, irradiate the flasks using an appropriate irradiator

according to the desired dose.

7. Return to the hood, keeping the flasks on ice outside the hood. Begin the

trypsinization procedure for each flask. Start with the 0-Gy flask. Aspirate themedium, rinse the cells gently with PBS, and then trypsinize. Place the harvested

cells in the 15-mL tube labeled 0 Gy, 11. Make sure that you have a good single-

cell suspension. From this cell suspension, take 100 L and place in the appro-

priate counting vial for 0 Gy. Now trypsinize the next flask. While the flask is on

the warming tray, count the previous counting vial and record the counts on a

dilution sheet (see Fig. 2 for a setup of a dilution sheet that we commonly use in

the laboratory). This expedites the experiments and lessens the chance of cell divi-

sions taking place unequally during the time of trypsinization and counting.

8. Continue this procedure until all the flasks have been trypsinized and counted.There should now be cells in each of the 11 dilution tubes, with a known number

of cells/milliliter, all documented on the dilution sheet.

9. Perform serial dilutions for each radiation dose so that the desired number of cells

will be obtained by adding between 100 and 1000 L of volume to the dishes. If

the number of cells needed requires a volume exceeding 1000 L, use a more

concentrated dilution. Plate a number of cells consistent with obtaining a colony

count of 50100. This may require only 100 cells for the control plate whereas at

6 Gy this may require 4000 cells or more. Remember that the larger the insult to

the flask (i.e., increasing radiation dose or increasing drug concentration if usingchemotherapy agents), the lower the plating efficiency and the more cells are

needed to obtain the desired colony count (see Note 2).

10. It is important to resuspend the cell pellet, which has probably settled to the bottom

of the tube by the time all the flasks have been counted. Place 0.5 mL of the 11

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dilution into the 110 dilution tube (contai

chemosensitivity vol 1

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