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models of cancer

Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents
Sreenath V. Sharma, Daniel A. Haber and Jeff Settleman

Abstract | Efforts to discover new cancer drugs and predict their clinical activity are limited by the fact that laboratory models to test drug efficacy do not faithfully recapitulate this complex disease. One important model system for evaluating candidate anticancer agents is human tumour-derived cell lines. Although cultured cancer cells can exhibit distinct properties compared with their naturally growing counterparts, recent technologies that facilitate the parallel analysis of large panels of such lines, together with genomic technologies that define their genetic constitution, have revitalized efforts to use cancer cell lines to assess the clinical utility of new investigational cancer drugs and to discover predictive biomarkers.
The discovery and development of effective cancer medicines has historically been hampered by the lack of reliably predictive preclinical models to assess the therapeutic efficacy of candidate agents. Such models have largely consisted of human tumour-derived cell lines propagated either in culture or as xenografts in mice, and more recently of genetically engineered mouse models of human tumorigenesis. Although the physiological relevance of each of these models as well as their usefulness for assessing drug efficacy remain controversial, and each approach is associated with important caveats, most investigators agree that these remain our best, and possibly only tools for the identification and characterization of medicinal agents that can potentially produce clinical benefit in cancer patients. Tumour-derived cell lines have been used for many years as drug discovery tools (Timeline). However, it is only recently that investigators have begun to appreciate the enormous degree of genomic heterogeneity across the human cancer patient population, and therefore across tumour-derived cell lines, and the crucial role that this diversity has in the variable clinical response to treatment. This realization has reinvigorated efforts to exploit these lines for the distinct purpose of capturing genotype–response relationships. The rapidly expanding use of cancer cell lines to predict the clinical efficacy of new agents is already affecting the course of drug development and is now becoming an important tool for the biotechnology and pharmaceutical industries, in which efforts that are focused on molecularly targeted cancer therapies are accelerating. In the following sections, we review the application of cancer cell lines to the discovery and evaluation of potential new anticancer agents. We also describe the relatively recent use of large cell line panels to capture the genomic diversity of human cancer, with the goal of identifying biomarkers that might allow the stratification of patients for appropriate drug treatment. We also compare such approaches to the various other preclinical model systems that are currently being explored to evaluate cancer drug efficacy.

Center for Molecular Therapeutics, Massachusetts General Hospital Cancer Center and Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA. Correspondence to J. S. e‑mail: settleman@helix.mgh. harvard.edu doi:10.1038/nrc2820 Published online 19 march 2010

Historical perspective The establishment of the National Cancer Institute 60 (NCI60) platform (BOX 1; Timeline) , the first highthroughput cancer cell line screening programme of its kind, was a scientific tour de force that required the development of many new technologies, such as the development of assays for measuring cytotoxicity, introduction of miniaturization in the form of microplates, automation of liquid handling and high volume data analysis on an unprecedented scale (reviewed in Ref. 1). The technologies developed as a direct result of this programme remain the cornerstones of many cancer drug-screening programmes today and will probably remain so for the foreseeable future. However, as described below, the limitations imposed by the use of only 60 cell lines have become increasingly apparent in recent years. The decision to use 60 cell lines has to be considered from the perspective that in the period from 1984 to 2005, during which the development, implementation and use of the NC160 programme was in effect,
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Given the high rates of clinical responses that were observed (25–70%).0% for gemcitabine and a placebo)16 (reviewed in Ref. remains a limitation of these systems.6% (compared with 8. lapatinib (Tykerb. pfizer)28. Similar response rates of ~10% were observed with an alternative eGFrtargeting antibody.nature. which elicit responses in a larger proportion of treated patients. • Although throughput. respond to a therapeutic monoclonal antibody directed against the receptor (cetuximab (erbitux. which introduced the concept of high-throughput cell-based profiling. melanocyte-specific genes in melanomas). limited clinical response to TKIs targeting other tumour-associated kinase pathways is a recurrent theme in numerous cancer settings (TABle 2) as exemplified by trastuzumab (Herceptin. clinical activity is often limited to small subsets of treated patients. most of which showed some activity in a considerable proportion of treated cancer patients2–10 (TABle 1). It has been proposed that the clinical response to targeted TKIs potentially reflects a state of oncogene addiction that arises at a low frequency in specific disease settings36. in metastatic colorectal cancers21. In these oncogene-addicted cancers. The experience with gefitinib and erlotinib has taught us that. highlighting the enormous genomic heterogeneity in human cancer and its role in the response to therapy. 20). in which acute inactivation of an oncoprotein is associated with differential attenuation rates of pro-survival and pro-apoptotic signals emanating from the oncoprotein. Lineage addiction The strict requirement for certain lineage-specific genes in tumorigenesis (for example.25. but also to the discovery of several agents that have subsequently been found to demonstrate therapeutic efficacy. • Consequently. has resulted in a new generation of anticancer agents with fewer side effects and impressive clinical activity. only around 9% of patients with chemotherapy-refractory colorectal cancer and 13% of patients with head and neck cancer. although receptor tyrosine kinases (rTKs) such as eGFr are important targets for therapeutic intervention. Bucking this trend. This is particularly well exemplified in the case of tyrosine kinase inhibitors (TKIs) that target epidermal growth factor receptor (eGFr) (TABle 2). Alternative hypotheses to explain the specific death of oncogeneaddicted cancer cells after acute inactivation of the driving oncogene include synthetic lethality40. resulting in the death of the cancer cell38.27. Beyond eGFr. gefitinib (Iressa.REVIEWS at a glance • Human tumour-derived cell lines have historically had a very important role in the discovery and development of new cancer therapeutics. Amgen). This might also be true for BRAF kinase mutations. it would be reasonable to assume that the 6–9 human tumour-derived cell lines representative of each cancer that comprise the NCI60 panel would be adequate to capture such frequencies of drug response. response to the clinically approved eGFr TKIs. Genentech/ oSI pharmaceuticals). but feature prominently in melanomas (70%)35. Oncogenic shock A mechanism to explain oncogene addiction. and www. Non-oncogene addiction Cancer cells might harbour potential therapeutic targets that do not correspond to oncogenes but constitute proteins to which the cancer cell is similarly addicted. • Subsequently developed genomic analysis technologies have provided an opportunity to link variable treatment responses to specific underlying genotypes. a transient imbalance in pro-survival and pro-apoptotic signals acutely following kinase inhibition. lilly)) for the treatment of chemotherapy-naive locally advanced and metastatic pancreatic cancers. rendering the composition of the NCI60 panel somewhat inadequate for the task of capturing such low frequency responses. sorafenib (Nexavar. Genentech)22. Bristol–myers Squibb) and nilotinib (Tasigna. erlotinib. All rights reserved . which occur in approximately 7% of human cancers. are TKIs such as imatinib (Gleevec. the mainstay of cancer treatment largely consisted of nonspecific cytotoxic agents. which seems to underlie much of the variable response to TKI therapy. as with the traditional cytotoxic chemotherapy agents. together with additional technological developments involving various three-dimensional culture systems and more sophisticated xenograft models. was crucial not only to the development of technologies that are still being used in various high-throughput discovery platforms.39. Be that as it may. their use in the clinical setting has revealed that.19 (reviewed in Ref. it is fair to say that the NCI60 programme was key to establishing cell line-based screening platform technologies as a foundation for anticancer drug discovery and development efforts. Thus. cell line panels for cancer drug profiling Rationale. Synthetic lethality Two genes are synthetic lethal if mutation of either one of the two genes is compatible with viability but mutation of both genes results in cell lethality. Genentech). However. Similarly. with the recent advent of so-called ‘rationally targeted’ anticancer therapeutics. such that apoptotic signals become predominant and kill the cancer cell. much larger panels of cancer cell lines are beginning to be exploited for the purpose of identifying genomic determinants of drug sensitivity. which has also been uS Food and Drug Administration (FDA)-approved (in combination with gemcitabine (Gemzar. Bayer HealthCare/onyx pharmaceuticals)26. Oncogene addiction The hypothesis that tumours arising as a result of a particular oncogenic lesion remain dependent on the continued expression of that oncogene. GlaxoSmithKline)24. primarily in chronic myeloid leukaemia (Cml). sunitinib (Sutent. lineage addiction41 and even non-oncogene addiction42. • This development.29 and bevacizumab (Avastin. the acute inhibition of the oncoprotein by the targeted inhibitor may induce oncogenic shock. AstraZeneca) and erlotinib (Tarceva. panitumumab (vectibix. Genomic heterogeneity and cancer therapeutics The advent of rationally targeted therapeutics. there seems to be little doubt that tumour-derived cell lines will continue to have a vital role in the preclinical assessment of new candidate anticancer agents. apparently reflecting the larger proportion of patients who have genomic lesions affecting the drug target in Cml30–34. Novartis). Bristol–myers Squibb/merck/ImClone Systems Incorporated))18. • The National Cancer Institute 60 (NCI60) platform. their inhibition may be clinically efficacious in a small subset of patients only. 17). particularly those directed against oncogenic kinases. and several studies have validated the usefulness of this approach to reveal clinically informative biomarkers. has recently reinvigorated the application of cancer cell lines to the analysis of drug efficacy in cancer. which express eGFr. the clinical response to treatment with these agents varies substantially between patients — even among those with histologically indistinguishable disease. it is anticipated that BrAF inhibitors will provide clinical benefit in this particular setting. the generally observed paucity of clinical response to targeted therapeutics underscores the genomic heterogeneity that is inherent in cancer and strongly argues for greater representation of cell lines derived from various cancers to adequately capture this genetic diversity.com/reviews/cancer 242 | AprIl 2010 | volume 10 © 2010 Macmillan Publishers Limited. However. and revealing the need to reconsider the scale of cell line-based studies to assess the activity of candidate anticancer agents.23. shows a minimal increase in objective response rate of 8. dasatinib (Sprycel. is restricted to around 10% of patients with non-small-cell lung cancer (NSClC)11–15.37. as well as the physiological relevance of some of these approaches. Such anachronistic criticism notwithstanding. Novartis).

Given the current cell-based screening strategies (for example.200 cell lines. platelet-derived growth factor receptor (pDGFr). In most cases. rare cell lines derived from other tumour volume 10 | AprIl 2010 | 243 © 2010 Macmillan Publishers Limited. as an empirical determination of their properties suggests that only ~80% of such lines are amenable to profiling for drug sensitivity.44). Therefore.000–6. one of the early surprises that emerged from this large data set was the remarkable degree to which human tumour-derived cell lines recapitulate clinical findings.46 further bolster the validity of this approach in uncovering clinically meaningful correlations between tumour cell genetics and drug sensitivity. A similar case could easily be made for all tumour types. this profiling platform involves a 72-hour assay of cells plated on plastic under standard culture conditions to detect changes in cell number as a consequence of treatment with various experimental agents. Sensitivity profiling with this platform commenced in may 2006 and. 127 candidate and established anticancer agents have been interrogated for cytostatic and/or cytotoxic activity against approximately 700 human tumourderived adherent cell lines. cumulatively corresponding to around 70. Briefly.000 cell lines (assuming conservatively that there are 20 different tissue origins with equal representation).000 drug–cell line pairings. JFCR. Developmental Therapeutics Program. and cell lines with exquisite sensitivity to eGFr. Center for Molecular Therapeutics (CMT)-related events are indicated with red boxes. Although in vitro analysis of cultured cell lines is certainly associated with caveats related to effects potentially attributed to a non-physiological environment and long-term passage in culture. the small proportion of treated patients who benefit from TKIs points to the genetic heterogeneity that is a characteristic of cancer and highlights some deficiencies in the currently used cell-based drug screening platforms as tools for discovering biomarkers that predict clinical response. both qualitatively and quantitatively. anaplastic lymphoma kinase (AlK) or BrAF kinase inhibitors were typically marked by activating mutations or amplification of the gene encoding the drug target. although. suggesting that an idealized human tumour cell line profiling panel should consist of 2. with regard to their response to targeted inhibitors. DTP. This panel of cell lines is referred to as the Center for molecular Therapeutics 1000 (CmT1000). we suggest that cell line-based drug screening platforms should ideally be designed with the capacity to detect responses in the 1–10% range in a particular cancer type and should adequately represent the genetic heterogeneity of cancer. as discussed above. EGFR mutations in around 10% of NSClC). a cellbased screen would require a minimum of 10 and ideally >100 different NSClC-derived cell lines. as of may 2009. recent findings demonstrating that tumour-derived cell lines largely retain the genomic features of the primary tumour 45. that activating mutations in the genes encoding these rTKs constitute one of the useful predictors of clinical response to TKIs (reviewed in Refs 43. largely reflecting technical limitations such as insufficient doubling times or atypical culture requirements. polyvinylidene fluoride. meT.158 Culturing human tumour cells in PVDF hollow fibres JFCR39 launched Human tumour cell lines retain genomic features of the primary tumour from which they were derived National Cancer Institute (NCI)-related events are indicted with blue boxes. NATure revIeWS | CanCer The cmT1000 platform With this goal in mind. However. PVDF. Japanese Foundation for Cancer Research. a simple calculation reveals that to recapitulate the 10% response rate to eGFr TKIs such as gefitinib or erlotinib.000. multicellular tumour spheroids. All rights reserved . Methodology and early findings. it is unlikely that sensitivity to drugs such as gefitinib or erlotinib (which yield responses in ~10% of NSClCs) would ever have been linked to a clinically relevant drug-sensitizing genotype. This platform is now being used to probe the genetic basis for sensitivity to approved and investigational anticancer agents. Details of the methodologies used in the CmT1000 cancer cell line-based screening platform have previously been reported47. NCI60) that typically involve a small number of cell lines (less than 10) representative of each tumour type.500 to 2. a human tumour cell line platform with as broad a representation as possible was recently established. Her2. MCTS.REVIEWS Timeline | History of the development of cell-line platforms for evaluating anticancer agents Animal cell culture becomes routine DTP established156 Cell-free cell culture media developed Culturing human tumour cells as MCTS COMPARE algorithm reported Clustered heat maps reported DTP external review (2000–present) NCI60 operates as service screen for drug profiling CMT1000 launched 1950 1951 1955 1963 1977 1986 1989 1990 1992 1995 1997 1998 2000 2006 HeLa: first human cell line developed from a cancer patient Methods for cryopreservation of mammalian cells developed (1986–1990) NCI60 model development (1990–2000) NCI60 as a drug discovery screen157. our own search for human tumour-derived cell lines in all publicly available repositories suggests that the total number of relevant cell lines is around 1. For example. This is beyond the capacity of currently available cell-based screening platforms. such mutations were correlated with particular tumour types (for example. early studies revealed that the responses of cell lines to agents targeting protein kinases were highly restricted. which currently includes 1.

cancers of the lung (9 non-small-cell lung cancer lines). In addition to the genomic heterogeneity present across tumours from different patients. pDGFrA is activated in approximately 13% of NSClCs53. the National Cancer Institute 60 (NCI60) platform consists of 60 human tumour cell lines. to a test compound with the sensitivity profile of all drugs previously tested against the nCi60 panel. The observation that specific genotypes are tightly associated with drug sensitivity was further explored in the context of NSClC. is the Cancer Cell line Project. as is currently the standard practice in medical oncology. One of the first revelations to emerge from the NCI60 screening programme was that drugs with similar profiles of cell line sensitivity tend to function through a common mechanism. Such an analysis can reveal unanticipated similarities between compounds with putatively distinct targets48. can have distinct drug sensitivity (reviewed in Refs 54. which compares the pattern of drug sensitivity. Given this high throughput. brain (6 lines). researchers at the Cancer Chemotherapy Center of the Japanese Foundation for Cancer Research (JFCR) established the JFCR-39 in 1999. Although typical short-term treatment assays are likely to yield readouts that are not affected by the potential presence of a small subpopulation of cells with distinct drug sensitivity. Such studies revealed that the broad chemotherapeutic resistance of several cell lines in the NCI60 panel was strongly correlated with the expression of multidrug resistance 1 (MDR1.000 pure compounds and 34. A similar approach could be broadly applicable in a wide variety of human cancers. gastric cancers) were also found to harbour the same genetic lesion and exhibit sensitivity to that particular inhibitor 48. Therefore. a major re-sequencing effort that is focused on the most common cancer-associated genes in human tumour-derived cell lines. additional applications of cancer cell line panels Tissue-specific cell line panels. it is now possible to begin to integrate mutational information that is associated with these cell lines with drug sensitivity data in an effort to better understand the genomic determinants of clinical response to various cancer therapeutics. JFCr‑39 Building on the NCI60 experience. this is analogous to the COmPARe algorithm and this approach has been used to compare 14 different kinase inhibitors on the basis of their activity against 500 human tumour-derived cell lines. Hierarchical clustering analysis of sensitivity profiles that emerge from the CmT1000 cell line screen can also reveal unanticipated relationships between distinct inhibitors. it is possible to subdivide NSClCs into genetically discrete subsets on the basis of the activating mutations that they harbour. this ongoing effort has led to the re-sequencing of 51 ‘cancer genes’ in 785 human tumour-derived cell lines. An in-depth review of the NCI60 programme has previously been published1. Clustered heat map A data matrix display in which the values of a variable in a two-dimensional map are represented as colours. such heterogeneity could certainly contribute to the nature of clinical treatment responses in patients.55). colon (7 lines). ovary (7 lines). 101 melanoma-derived cell lines59 and 84 NSClC cell lines46 reached the seminal conclusion that the genetic landscape of tumour-derived cell lines is remarkably www. thereby representing a paradigm shift from the current approach to evaluating and treating cancer patients. Since 1997. Therefore. most notably bortezomib. recent advanced genetic techniques have revised the number of unique cell lines in the NCI60 panel to 57. see Further information). an NSClC cell line with sensitivity to a pDGFr kinase inhibitor was found to harbour co-amplification of genes encoding the receptor (pDGFrA) and one of its ligands (pDGFC)52. other studies have used smaller cancer cell line panels consisting either of short-term tumour-derived cultures or established cell lines to recapitulate the genomic diversity of cancer. As of 2005. as revealed by the nCi60 analysis. resulting in the development of clustered heat maps132–135. can exhibit such heterogeneity. it is becoming increasingly evident that heterogeneity of cell types in tumour cell populations is also likely to have an important role in drug sensitivity. and each of these subsets may correspond to patient cohorts that are likely to benefit from treatment with specific inhibitors that target the products of these specific genetic lesions (BOX 2). melanoma (8 lines). It is a panel of 39 human tumour-derived cell lines that included a subset of the NCI60 cell lines as well as additional cell lines derived from gastric cancer (owing to its prevalence in the Japanese population)141. During the 1990s several new anticancer agents were identified by the NCI60 screening programme137. the NCI60 programme has been a compound evaluation resource for the research community. This algorithm enabled the rapid comparison of newly screened compounds with previously screened compounds to determine whether the compound exhibited a new or previously described mode of action. tumour cell subsets exhibiting distinct states of differentiation or ‘stem cell’ properties. For example. To date. A large proportion of human tumour cell lines in the CmT1000 are represented in the Cancer Cell line project and efforts are underway to include all of the cell lines in the CmT1000 in the Sanger Institute’s Cancer Cell line project. prostate (2 lines) and kidney (8 lines)130. genetically defined cancer subsets. the NCI60 represents a true drug discovery platform (as opposed to a drug development tool). Details regarding the NCI60 screen can be found at the Developmental Therapeutic Program website (see Further information). rather than on tissue of origin. sensitivity to an AlK inhibitor was well correlated with AlK-activating chromosomal translocations that arise at low frequency (3–7%) in these tumours49–51. leukaemia (represented by 6 cell lines). which was approved by the US Food and Drug Administration for the treatment of multiple myeloma138. Drugs with similar cell line sensitivity profiles tend to have a similar mode of action. COMPARE algorithm A computer program. one of the components of the Cancer Genome project at the Wellcome Trust’s Sanger Institute.REVIEWS Box 1 | The ncI60 and Jfcr-39 screening platforms nCI60 Launched in 1990. approximately 88. seem to be associated with response to specific kinase inhibitors. Similarly. irrespective of the tissue of origin. breast (6 lines). Conceptually. and using the CmT1000 platform. which encodes a P-glycoprotein136. Using the COMPARE algorithm and advanced data mining techniques this platform has been successful in identifying several new anticancer agents142–146 as well as tumour biomarkers147. However. Therefore. and in vitro studies have revealed that such cell heterogeneity can affect the response to treatment with anticancer agents (reviewed in Refs 56–58).com/reviews/cancer 244 | AprIl 2010 | volume 10 © 2010 Macmillan Publishers Limited. moreover. using the CmT1000 platform. gene expression information was integrated with the screening data. representing 9 cancer types. as well as tumour cells from patients. also known as ABCB1).139. All rights reserved . Such studies using 51 breast cancer cell lines45. highlighting the potential benefit from the stratification of patients based on genotype. yielding a rich database of mutations in common oncogenes in human cancer cell lines (details of the genes and the cell lines that are part of this database can be found at the Cancer Cell line project website. In the early 1990s. types (for example.000 crude extracts have been screened against the NCI60 panel140. namely. a finding that led to the development of the COMPARE algorithm131. cultured cancer cell lines.nature. uK.

Although this study demonstrates the feasibility of this approach using one human tumourderived cell line. Although the early clinical development of combination treatment strategies was painstakingly slow.000 combinations) for growth inhibitory activity against one human lung tumour-derived cell line. All rights reserved . Tumour cell killing by inhibition of meK in breast cancer cells was antagonized by concomitant activation of the pI3K pathway. further supporting the use of cancer cell lines to uncover genotypes that potentially predict drug vulnerabilities60.10 2. Lilly) Fluouracil Doxorubicin Etoposide Topotecan (Hycamtin. Similarly.9 8. recent innovations have enabled the use of cell line-based screening platforms to explore combinatorial space in a high-throughput fashion.6 8. tumour-derived cell lines have also been useful models in the refinement of other potentially important therapeutic targets. Systems approaches.7 6. A549 (Ref. as revealed through mechanistic studies of phenomena such as non-oncogene addiction and lineage addiction41. Such efforts have recently yielded several new candidate therapeutic targets that remain to be validated in patients64–69. monotherapy with anticancer agents is not curative and most currently successful anticancer therapeutic strategies involve a combination of drugs. Food and Drug Administration. involving trial-and-error in patients. and possibly to gain insights into the mechanism of action of less well-characterized agents. NATure revIeWS | CanCer © 2010 Macmillan Publishers Limited. fibroblast growth factor receptor (FGFR)-activating mutations were associated with sensitivity to meK inhibitors in melanomas59. suggesting that in this setting the dual inhibition of meK and pI3K might be more efficacious than inhibition with single agents62. Tumour-derived cell lines have also begun to be used to identify genes that are in a synthetic lethal relationship with activated oncogenes (for example.42. mutated KRAS) or disrupted tumour suppressors (for example. The analysis includes loss of heterozygosity and copy number analysis. these studies revealed several drug-sensitizing genotypes. In one study. Group Alkylating agent Alkylating agent Alkylating agent Mitotic inhibitor Mitotic inhibitor Mitotic inhibitor Antimetabolite Antimetabolite Anthracycline Topoisomerae II inhibitor Topoisomerase I inhibitor Cancer and response rates 67% for ovarian cancer and 69% for breast cancer 67% for ovarian cancer and 47% for breast cancer 24% for ovarian cancer and 53% for breast cancer 31% for ovarian cancer and 39% for breast cancer 30% for ovarian cancer and 41–60% for breast cancer 42% for ovarian cancer and 27% for breast cancer 49% for ovarian cancer and 36% for breast cancer 36% for breast cancer 61% for ovarian cancer and 45% for breast cancer 48% for ovarian cancer and 26% for breast cancer 14% for ovarian cancer refs 9.9 9. GlaxoSmithKline) FDA.10 4 9. for example.REVIEWS Table 1 | response rates of breast and ovarian cancers to different Us fda-approved cancer chemotherapeutic agents US FDa‑approved chemotherapeutic Cyclophosphamide Cisplatin Carboplatin Docetaxel (Taxotere. and one of those combinations was tested and shown to be active in vivo using A549 cells grown as xenografts in nude mice70. except in rare cases. 70). Drug combinations. Synthetic lethality.10 10 similar to that of the primary tumours from which they originated. transcriptomic. implying that cultured tumour-derived cell lines are valid genetic surrogates of tumours in vivo. Such an integrated analysis of 30 breast cancer cell lines showed that overexpression of pAK1 confers hypersensitivity to meK inhibition61. A similar systems analysis approach using 48 breast cancer cell lines yielded an expression signature of 13 genes that was associated with the sensitivity of these cells to the polyamine analogue pG-11047. detection of microsatellite instability and deep resequencing of all known cancer genes. the project involves 784 cell lines and 51 of the most common cancer genes. but has now been extended to the treatment of solid tumours. 600 FDA-approved drugs were combinatorially analysed (yielding approximately 100. such an undertaking on a larger panel of human tumour-derived cell lines would be volume 10 | AprIl 2010 | 245 Cancer Cell Line Project A project within the Cancer Genome Project. one of the most important challenges in deriving biomarkers that are associated with drug sensitivity is the integration of data from the various ‘systems’ platforms that yield genomic. Such studies highlight the potential value of systems approaches to integrate complex molecular information that describes cellular states that are associated with drug sensitivity or resistance to identify predictive biomarkers of clinical activity. Such signatures might be useful for both predicting drug sensitivity in treated patients and elucidating mechanisms of drug action. an investigational clinical compound63. it is focused on genetic characterization of all human cancer cell lines currently used in laboratories.10 3. To this end. Such an unbiased in vitro analysis revealed unanticipated synergistic interaction between drugs that are used in the treatment of unrelated diseases. Combination chemotherapy was first used in the treatment of leukaemias and lymphomas.9 5. p53) in cancer cells. ERBB2 gene amplification was linked to sensitivity to trastuzumab in breast cancer cells45 and KRAS mutations were found to associate with sensitivity to HSp90 inhibitors in NSClC46. Sanofi Aventis) Vinorelbine Paclitaxel (Taxol. Currently.71. systems biology approaches have been applied to panels of cell lines derived from a particular tumour type to discover meaningful molecular correlates of drug sensitivity. proteomic and epigenomic information to establish molecular ‘signatures’ that may be clinically useful. Bristol–Myers Squibb) Gemcitabine (Gemzar. In addition.

CML. Philadelphia chromosome positive.REVIEWS Table 2 | objective response rates in patients to different Us fda-approved kinase inhibitors US FDa‑approved kinase inhibitor Gefitinib (Iressa. Therefore. Therefore. Ph+.23 25 26 27 28 32 Trastuzumab ERBB2 (Herceptin. Food and Drug Administration. Genentech) Lapatinib (Tykerb.nature. EGFR. GlaxoSmithKline) Sorafinib (Nexavar. Given the current state of the technology this is still only a hope but it is likely that technical advances in the future could enable such an ambitious endeavour. FDA. Amgen) EGFR 19 EGFR EGFR 9% 10% 15–26% 39% 10% 2% 31% 7% 18 21 22. Genentech/OSI Pharmaceuticals) Target EGFR EGFR EGFR Cancer Non-small-cell lung cancer Non-small-cell lung cancer Pancreatic cancer (in combination with gemcitabine (Gemzar. PDGFRb and RET VEGFA Bevacizumab (Avastin. Novartis) BCR–ABL. epidermal growth factor receptor. All rights reserved . The ability to explore combinatorial space with large 246 | AprIl 2010 | volume 10 numbers of human tumour cell lines would be one of the greatest challenges for the future. KIT and PDGFR BCR–ABL Ph+ CML and Ph+ AML Metastatic or unresectable KIT+ gastrointestinal stromal tumour Imatinib-intolerant or Imatinib-resistant Ph+ CML Imatinib-intolerant or imatinib-resistant Ph+ CML 20–26% 76% 38–54% 52% (major cytogenetic response) 47% (haematological response) 33 34 AML.com/reviews/cancer © 2010 Macmillan Publishers Limited. VEGFA.32 Glioblastoma Imatinib (Gleevec.6% (compared with 8% with gemcitabine and placebo) 13% refs 11. VEGFR3. carrying out such an analysis on limited sets of combinations for which there is a scientific rationale is not only feasible but would also be invaluable in the development of more effective therapeutic strategies for cancer treatment. Lilly)) Advanced squamous cell carcinoma of the head and neck Metastatic colorectal cancer Metastatic colorectal cancer ERBB2-overexpressing metastatic breast cancer Trastuzumab-refractory breast cancer Advanced renal cell carcinoma Unresectable hepatocellular carcinoma Advanced renal cell carcinoma Imatinib-intolerant gastrointestinal stromal tumour Metastatic colorectal cancer Objective response rates 9–19% 10% 8.12 15 16 Cetuximab (Erbitux. Bristol–Myers Squibb) Nilotinib (Tasigna. vascular endothelial growth factor A. Bristol–Myers Squibb/ Merck/ImClone Systems Incorporated ) Panitumumab (Vectibix. VEGFR3 and PDGFRb VEGFR1. PDGFR. PDGFR and KIT BCR–ABL. monumental. Novartis) ABL. most cancer patients who demonstrate a response to drug therapy. It is clear that exploring all possible drug combinations is virtually impossible. chronic myeloid leukaemia. but for now. VEGF receptor. VEGFR1. Pfizer) ERBB2 RAF1. modelling drug resistance in cancer cell lines one of the major limitations to the clinical benefit derived from cancer drug therapies is the problem of rapidly acquired drug resistance. VEGFR. AstraZeneca) Erlotinib (Tarceva. will eventually — and often rapidly — relapse with drug-resistant disease. VEGFR 2. SRC. platelet-derived growth factor receptor. but one which promises to yield rich rewards. acute myeloid leukaemia. it is important www. Bayer HealthCare/ Onyx Pharmaceuticals) Sunitinib (Sutent. albeit theoretically possible. VEGFR2. PDGFR and KIT Dasatinib (Sprycel. Genentech) 3% ECOG E3200 study (bevacizumab monotherapy group) NCI 06-C-0064E and AVF3708g studies 30 31.

48 ALK MET Oncogene Effective inhibitor KRAS EGFR ALK MET PDGFR ROS ERBB2 BRAF PIK3CA MEK1 None Gefitinib or erlotinib TAE684 PF-2341066 Sunitinib CEL-1869 Lapatinib (GW572016) PLX4032 GDC-0941 AZD6244 or PD-325901 2% 2% 1% 2% 1% 6% 10% 1% 49. In some cases. Limitations and perspective. for example.151 152. yielding growth inhibition and/or apoptosis of cancer cell lines with the corresponding mutated oncogenes. NSCLC (139 lines) Gene Oncogenic activation Frequency Patients Cell lines 5% refs EGFR Deletion 10–40% (DE746-A750). The logistical challenges associated with the culture and sensitivity profiling of large cancer cell line panels considerably limits the throughput of the platform with respect to the number of compounds that can be realistically tested in a given time period. By continuously exposing such drug-sensitive cells to treatment in vitro over a period of time. such mechanisms of acquired drug resistance might also underlie some cases of de novo drug resistance. Consequently. This is exemplified by p53 mutations. it is possible to identify specific molecular mechanisms of drug resistance. platelet-derived growth factor. Furthermore. most cell-based screens to detect cytostatic or cytotoxic activities are geared to rapidly dividing tumour cell lines (those that have a doubling time of less than the duration of the assay) and therefore some slow-growing tumour cell lines will not be amenable to screening on this platform.REVIEWS Box 2 | stratification of non-small-cell lung cancers on the basis of activating mutations The pie chart (see the figure) shows the distribution of various reported activating oncogenic mutations in a survey of 139 non-small-cell lung cancer (NSCLC)-derived cell lines. This approach has been successfully used. which are more frequent in cell lines compared with primary haematopoietic tumour cells 74. All rights reserved . one of the experimental strategies that has been sucNature Reviews the | Cancer cessfully used to address this issue involves use of drug-sensitive cancer-derived cell lines as a model for establishing mechanisms of acquired drug resistance. This potentially reflects the fact that the tumours that carry p53 mutations may be much more suitable for in vitro establishment as volume 10 | AprIl 2010 | 247 © 2010 Macmillan Publishers Limited. and so agents that potentially function to affect the interaction of tumour cells with their environment (for example. epidermal growth factor receptor. to develop a better understanding of the molecular mechanisms underlying acquired drug resistance.53 53 150. raising the possibility that analogous efforts to model acquired drug resistance in larger panels of cell lines with established drug sensitivity could also be useful for identifying additional mechanisms of de novo drug resistance. By comparing various properties of the parental drug-sensitive cells and the selected drugresistant cells. PDGFR.148 48. The composition of culture media and the presence of fetal bovine serum in standard culture almost certainly fail to precisely recapitulate the features of tumour cell growth in vivo. Similarly. There are currently no inhibitors that target oncogenic KRAS. it is often possible to eliminate the majority of cells while selecting for the expansion of relatively rare drugresistant clones. and CrAF overexpression as a potential mechanism of acquired resistance to BrAF inhibitor therapy in melanomas73. Also shown for all the activating mutations (except KRAS) are inhibitors that selectively target the activated oncoproteins. angiogenesis inhibitors or inhibitors of tumour–stromal interaction) cannot be assessed with these platforms. another limitation of such analysis is that only cell autonomous sensitivities to tested compounds can be scored. As with all cell line-based screens involving monocultures.50% 43. the range of mutational changes in tumours may be somewhat distinct from that of the cell lines established from these tumours. which has provided a system for a relatively NATure revIeWS | CanCer high-throughput analysis of a large number of candidate agents. point mutation (L858R) and amplification Translocation (EML4–ALK) Amplification Amplification Translocation (CD74–ROS) Insertion Point mutation (exon 11) Point mutation Point mutation 3–7% 11% 13% 1% 2–4% 3% 2% 0. unlike the NCI60 platform. the comparatively low throughput of the much larger cell line platforms is more suited to function as a drug development tool. to establish MET amplification as a mechanism of acquired resistance to eGFr kinase inhibitor therapy in non-small-cell lung cancer 72. the non-physiological oxygen levels typically used in cell culture studies may have an important impact on response to agents that target DNA damage pathways or hypoxia-dependent pathways.149 52.153 154 155 PDGFR ROS ERBB2 BRAF PIK3CA MEK1 EGFR. largely to provide information about the activity of a much smaller number of wellcharacterized agents that have already demonstrated promise as candidate anticancer agents. In both of these cases.

greater awareness of the importance of the tumour microenvironment and the three-dimensional (3D) aspects of solid tumours in the tumorigenic process and the response to therapy has prompted efforts to model these features of tumour cell growth in vitro more precisely. has been considered but has not yet been implemented83. the hollow fibre assay. hollow fibre-based approaches. and are therefore likely to be under-represented. 83 ). and sensitivity to drugs and radiation81. There is also a growing awareness among cancer researchers of cell line identity issues that can complicate the interpretation of findings. changes in polarity. reviewed in Ref. Typically. fewer than 100 human tumour cell lines have been shown to have the capacity to grow in spheroid cultures94. and the hollow fibre assay as it is currently implemented uses only 12 human cancer cell lines89.REVIEWS permanent cell lines. it facilitates the in vivo analysis of drug effects on human tumour cell lines that do not form tumours in animals. mice are treated with the candidate anticancer agent for 4 days. Tumour cells growing in 3D cultures are generally believed to more closely mimic their counterparts in vivo 75–80. testing with a small number of cell lines limits the potential to capture the genomic heterogeneity of cancer. overall. This shortterm assay greatly reduces the time and amount of drug required for standard in vivo efficacy testing.nature. the protocol involves short-term in vitro culture (24–48 hours) of a panel of 12 human tumour cell lines in biocompatible hollow fibres. Another area that requires further investigation is the comparison between drug responses in two-dimensional (2D) and 3D cell culture systems to determine how they www. 3D cultures have begun to uncover aspects of tumour biology and tumour metastases (for example. drugs that show efficacy in hollow fibre assays generally show good anti-tumour activity in human xenografts88–90. invasive potential. Therefore. regardless of the basis for such differences. as it is currently used at the NCI. and so this system is generally used as a pre-screen before more expensive and time-consuming human xenograft testing is undertaken (TABle 3). Despite these limitations. However. and is believed to accurately simulate in vivo growth of tumour cells both in terms of their pathophysiology and response to therapy 91–94. and despite the many advantages that they potentially offer. the various 3D cell culture systems are irrelevant for cancers of the blood that comprise approximately one-third of all human cancers. one of the technological innovations emerging from the NCI60 programme was the development of the hollow fibre assay 84 that was based on previous techniques for microencapsulation and the cultivation of mammalian cells in hollow fibres85–87. As alluded to earlier. Around half the cell lines in the NCI60 panel do not form mTCS83. moreover. gene expression information from microarrays and gene copy number from single nucleotide polymorphism (SNp) arrays) constitutes a powerful and feasible approach to facilitate the identification of signatures or biomarkers of drug sensitivity that can potentially be used to identify patients who are likely to derive benefit from a particular targeted therapeutic. good correlation was observed between the sensitivity of drugs in the NCI60 and hollow fibre assays88. The mTCS system (fiG. intraperitoneally in nude mice. To date. matrixindependent survival. followed by the implantation of these structures subcutaneously or 248 | AprIl 2010 | volume 10 . and chemoresistance and radioresistance95–100.101. Therefore. 1a).82). In the following sections. multilayer cell systems. These emerging technologies are currently hampered by substantial technical challenges that are associated with the generation. moreover. matrix-embedded 3D cultures. prostate) are difficult to propagate in vitro. over the past few years. 1b) is among the most well characterized 3D culture systems. these genotype distinctions potentially constitute an important basis for drug sensitivity differences between primary tumour cells and established cancer cell lines. Additional caveats associated with the use of cell line panels as models of the human disease include the fact that some tumour types (for example. precluding its use as a high-throughput platform. after which the fibres are removed and tumour cell viability is analysed by standard cell viability assays (fiG. use of the mTCS system to evaluate drug efficacy. is not a stand-alone technology for drug discovery. we describe various 3D platforms and their application in studies aimed at elucidating the basis for cancer drug sensitivity and resistance in vivo. MTCS. there is likely to be some bias in cell line representation resulting from the possibility that tumour cells from various disease stages and cancer subtypes may not adapt equivalently in culture. The mTCS system has also been used to examine various aspects of cancer therapeutics such as metabolic and chemical gradients. propagation and testing of these organotypic cultures that severely limits their use. 3D culture techniques are still in their infancy. and the requirement for implantation in nude mice with the hollow fibre assay system greatly increases the cost and time of the assay. Instead. the ability to correlate drug sensitivity data from large numbers of human cancer cell lines with various forms of genomic information (mutation data from re-sequencing studies. Furthermore. ex vivo tumour cultures and multicellular tumour spheroids (mTCS. this system examines the ability of the administered drug to access two pharmacological compartments and can be used to assess tumour responses to drug treatment in these two compartments. tumour hypoxia. Limitations and perspective. In addition. examples of 3D culture systems include.com/reviews/cancer © 2010 Macmillan Publishers Limited. All rights reserved 3d cell culture systems and drug development Rationale. cell–cell and cell–matrix interactions. Hollow fibre assays. it will be some time before these methodologies enter the mainstream of largescale drug discovery and development programmes. as a supplement to monolayer-based assays and before whole-animal studies.

it is possible to generate homotypic or heterotypic spheroids. These hollow fibres with growing cells are then implanted subcutaneously and intraperitoneally into mice. All rights reserved . the predictability of discovering clinically useful agents is likely to improve in the future. After 3–4 days of drug treatment. Numerous studies have documented differences in cancer drug sensitivity between cells cultured in monolayers and those grown in 3D cultures102–106. mouse xenografts remain a volume 10 | AprIl 2010 | 249 NATure revIeWS | CanCer © 2010 Macmillan Publishers Limited. Thus.112. With improved animal models for human tumours such as orthotopic models . it is worth noting that it remains to be definitively demonstrated that drug sensitivity data derived from 3D culture models captures clinically relevant responses more faithfully than standard 2D culture. tumours growing as xenografts in mice responded similarly to the original tumours in humans 124–126. it is possible to implant several different human tumour cell lines simultaneously into a single mouse. As the cells are isolated in the hollow fibres. Autochthonous model An endogenous or in situ tumour that evolves from normal cells in an animal (for example. Mice are then allowed to recover for 3–4 days and subsequently treated with either the drug or vehicle. 123) (TABle 3). and animal models will continue to have an important role in anticancer drug development. respectively. poly HEMA and poly lysine Hanging drop method • as for embryoid bodies Count cells in hollow fibres in drug-treated compared with vehicle-treated mice b Enzymatic or mechanical dissociation Homotypic spheroids Tumour cells Heterotypic spheroids Tumour cells or homotypic spheroids and endothelial cells or tumour-derived stromal cells Endothelial cells Single-cell suspension Smooth muscle cells or osteoblasts Figure 1 | Three‑dimensional cell culture assays. but others have questioned this88. relate to drug responses in vivo. roller flasks and spinner flasks Mechanical methods to promote aggregation • centrifugal compression into a cell pellet followed by gently raising the pellet into culture medium Coating tissue culture surface with non-adhesive surfaces • for example.113. a | In hollow fibre assays human tumour cells are flushed into Nature Reviews | Cancer semi-permeable polyvinylidene fluoride (PVDF) fibres with an internal diameter of 1 mm and a 500 kDa molecular weight exclusion. as well as genetically engineered cancer models (reviewed in Refs 127. This is in contrast to tumour models in which exogenous tumour cells are implanted into an animal (xenografts). one of the potential issues associated with xenografts as models of drug efficacy relates to inappropriate dosing. chemically induced tumours). BD Matrigel. gyratory shakers. Orthotopic model Tumours generated by the introduction of human tumour fragments or cells into the same anatomical sites in an animal as those from which the tumour arose in humans. overall. The fibres are heat sealed at 2 cm intervals and these tubular bags of cells are grown under standard tissue culture conditions for 24–48 hours.121.128). previous studies have shown that some drugs are more effective in 3D cell culture systems (compared with 2D systems)107–111 although other drugs show greater activity in the 2D cell culture systems100. moreover. injected intraperitoneally. potentially reflecting differential gene expression between the two states114–116. recent studies demonstrated that at clinically relevant doses. However. although xenografts of human cancer cell lines have been useful for establishing the pharmacological properties of new agents. Several studies have documented the overall usefulness of these systems in predicting clinical activity 117–119. metastatic models and autochthonous models.REVIEWS a Human tumour cells Heat-seal the ends 24–48 h 3–4 d Drug or vehicle (intraperitoneal injection) 3–4 d PVDF fibre Cells growing in hollow fibres • Subcutaneous implants • Intraperitoneal implants Seeding conditions Mechanical methods that prevent attachment • for example.120. animal models to evaluate drug efficacy The question of whether or not human tumour xenografts are good surrogates for the tumours from which they originate remains highly controversial. b | In multicellular tumour spheroids (MCTS) single cell suspensions of human tumour cells are seeded under conditions that prevent attachment to plastic but promote attachment to each other (see the seeding conditions box in the figure). It is likely that future technological advances will overcome many of these obstacles and that 3D cell cultures will gradually be integrated into high-throughput drug discovery and development platforms (TABle 3). they have been less reliable as a readout for drug efficacy. the hollow fibres are surgically removed and the surviving cells in the hollow fibres are quantified in drug-treated mice compared with vehicle-treated mice to determine drug efficacy. This is highlighted by the disappointing clinical performance of many candidate anticancer agents that showed great promise in xenograft models122 (reviewed in Ref. agarose. By seeding tumour cells singly or by mixing them with other cells.

Gloucester Pharmaceuticals) • Using the COMPARE algorithm and advanced data mining techniques. recent studies have highlighted important differences in drug efficacy in mice with transplanted tumours compared with genetically engineered mouse models of the same tumour type129 (TABle 3). time and cost (TABle 3). NCI.com/reviews/cancer © 2010 Macmillan Publishers Limited. obvious limitations of all animal models that preclude their use in large-scale drug discovery programmes include their unsuitability for high-throughput drug screening owing to considerations of space. Developmental Therapeutics Program. large-scale cancer cell line-based drug sensitivity screens are emerging as an important component of drug discovery and development www. 59–63 Hollow fibre assays Low throughput for both drugs and cell lines No Yes 84–88 Xenografts Low throughput for both drugs and cell lines Low throughput for both drugs and cell lines No Yes 156 Genetically engineered mouse models No Yes 126–129 CMT. good correlation was observed when comparing the sensitivity of drugs in the NCI60 screen and hollow fibre assays • Originally used by the NCI DTP for anticancer drug screening. vital tool — particularly in the biotechnology and pharmaceutical industries — for assessing the ability of various investigational agents to effectively penetrate tumour tissue in vivo and to suppress the function of their target. each of these models is associated with important caveats. but abandoned for lack of predictability of drug sensitivity in solid tumours • Discovery of paclitaxel (Taxol. flavopiridol (Alvocidib.46. copy number. the tumours in these mice. Therefore. National Cancer Institute. fail to adequately capture the additional genomic heterogeneity that seems to be a hallmark of human cancer. 250 | AprIl 2010 | volume 10 Although the studies mentioned above argue in favour of genetically engineered mouse models. MET.nature. breast cancer. moreover. Millenium Pharmaceuticals). Limitations and perspective. platelet-derived growth factor receptor. Sanofi Aventis) and romidepsin (Istodax. which by definition are largely genetically homogenous. proteomic and pathway activation) to derive signatures predictive of drug sensitivity • Demonstrates that cultured tumour-derived cell lines have genetic alterations that reflect primary tumours • Used in conjunction with nude mice. In summary. across the various animal models. Center for Molecular Therapeutics. mutations. epidermal growth factor receptor. ALK and BRAF kinase inhibitors have activating mutations in these genes • Captures the genetic heterogeneity of a specific tumour type by enlarging the number of cancer cell lines of that particular tumour type (for example. melanoma and non-small-cell lung cancer) • Integrates data from multiple platforms (including. UCN-01. DTP. All rights reserved . JFCR. this platform has been successful in identifying several new anticancer agents as well as biomarkers • Captures the genetic heterogeneity of cancer by enlarging the number of cancer cell lines in the panel • Discovery that cell lines with sensitivity to EGFR. EGFR. and it remains unclear which of them will ultimately prove to be the most useful for predicting the clinical activity of new investigational agents in the treatment of human cancer. Japanese Foundation for Cancer Research. PDGFR. Bristol–Myers Squibb) • Uncovered differences in drug efficacy in mice with transplanted tumours compared with genetically engineered mouse models of the same tumour type • Failure to adequately capture the genetic heterogeneity that is a hallmark of human cancer and an important determinant of variable response to treatment refs 131–139 JFCR36 High throughput for drugs but low throughput for cell lines High throughput for cell lines but low throughput for drugs Yes Yes 141–147 CMT1000 No Yes 47–53 Tissue-specific Low throughput for cancer cell both drugs and cell line panels lines No Yes 45.REVIEWS Table 3 | cell line platforms for assessing anticancer therapeutics Platform NCI60 Throughput High throughput for drugs but low throughput for cell lines Drug discovery Yes Drug Hallmarks development Yes • Discovery that drugs with similar profiles have a similar mode of action led to the development of the COMPARE algorithm • Development of clustered heat maps to show inter-relatedness of large molecular biological data sets • Discovery that broad chemotherapeutic resistance correlates with expression of MDR1 • Discovery of bortezomib (Velcade. PDGFR. ERBB2. this assay facilitates the in vivo analysis of drug effects on human tumour cell lines that do not form tumours in animals • Overall. and which represents an important determinant of variable response to treatment.

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