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SUMMARYRecent data suggests that colon tumors contain a subpopulation of therapy resistant quiescent cancer stem cells (qCSCs) that are the source of relapse following treatment. Here, using colon cancer patient-derived organoids (PDOs) and xenograft (PDX) models, we identify a rare population of long-term label-retaining (PKH26Positive) qCSCs that can re-enter the cell cycle to generate new tumors. RNA-sequencing analyses demonstrated that these cells are enriched for stem cell associated gene sets such as Wnt and hedgehog signaling, epithelial-to-mesenchymal transition (EMT), embryonic development, tissue development and p53 pathway but have downregulated expression of genes associated with cell cycle, transcription, biosynthesis and metabolism. Furthermore, qCSCs are enriched for p53 interacting negative regulators of cell cycle, includingAKAP12, CD82, CDKN1A, FHL2, GPX3, KIAA0247, LCN2, TFF2, UNC5BandZMAT3, that we show are indicators of poor prognosis and may be targeted for qCSC abolition. Interestingly, CD82, KIAA0247 and UNC5B proteins localize to the cell surface and may therefore be potential markers for the prospective isolation of qCSCs. These data support the temporal inhibition of p53 signaling for the elimination of qCSCs and prevention of relapse in colorectal cancer.

Human cell line models have been widely used for testing of novel anticancer compounds and for predicting clinical response to monotherapies and combinatorial therapies. For many years, standard monolayer culture conditions were used as gold standard, only surpassed by in vivo testing of mouse models. Recently, the incorporation of three-dimensional culture has been shown to further improve predictive compound testing. In view of the renewed interest in anti-RAS cancer therapy, we provide a protocol for establishing colorectal cancer organoids which are characterized by a high prevalence of KRAS mutations.

Summary Recent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer., Graphical Abstract, Highlights • Colon tumors contain therapy-resistant quiescent cancer stem cells (qCSCs) • qCSC gene expression mirrors that of quiescent stem cells of the regenerating gut • qCSCs are enriched for p53 signaling genes • qCSC elimination may be achieved by inhibiting downstream targets of p53 signaling, Cancer stem cells ; Quiescence; Organoids; Colon cancer; p53 signaling; Transcriptomics

Summary: Colon cancer is a heterogeneous tumor driven by a subpopulation of cancer stem cells (CSCs). To study CSCs in colon cancer, we used limiting dilution spheroid and serial xenotransplantation assays to functionally define the frequency of CSCs in a panel of patient-derived cancer organoids. These studies demonstrated cancer organoids to be enriched for CSCs, which varied in frequency between tumors. Whole-transcriptome analysis identified WNT and Hedgehog signaling components to be enhanced in CSC-enriched tumors and in aldehyde dehydrogenase (ALDH)-positive CSCs. Canonical GLI-dependent Hedgehog signaling is a negative regulator of WNT signaling in normal intestine and intestinal tumors. Here, we show that Hedgehog signaling in colon CSCs is autocrine SHH-dependent, non-canonical PTCH1 dependent, and GLI independent. In addition, using small-molecule inhibitors and RNAi against SHH-palmitoylating Hedgehog acyltransferase (HHAT), we demonstrate that non-canonical Hedgehog signaling is a positive regulator of WNT signaling and required for colon CSC survival. : Colon cancer is a heterogeneous tumor driven by a subpopulation(s) of therapy-resistant cancer stem cells (CSCs). Regan et al. use 3D culture models to demonstrate that CSC survival is regulated by non-canonical, SHH-dependent, PTCH1-dependent Hedgehog signaling, which acts as a positive regulator of WNT signaling to block CSC differentiation. Keywords: WNT pathway, non-canonical Hedgehog signaling, cancer stem cell, colon cancer, cancer organoid, PTCH1, HHAT, SHH

Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients.

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.

Genetic heterogeneity between and within tumours is a major factor determining cancer progression and therapy response. Here we examined DNA sequence and DNA copy-number heterogeneity in colorectal cancer (CRC) by targeted high-depth sequencing of 100 most frequently altered genes. In 97 samples, with primary tumours and matched metastases from 27 patients, we observe inter-tumour concordance for coding mutations; in contrast, gene copy numbers are highly discordant between primary tumours and metastases as validated by fluorescent in situ hybridization. To further investigate intra-tumour heterogeneity, we dissected a single tumour into 68 spatially defined samples and sequenced them separately. We identify evenly distributed coding mutations in APC and TP53 in all tumour areas, yet highly variable gene copy numbers in numerous genes. 3D morpho-molecular reconstruction reveals two clusters with divergent copy number aberrations along the proximal–distal axis indicating that DNA copy number variations are a major source of tumour heterogeneity in CRC., The contribution of intra-tumour heterogeneity is increasingly associated with resistance to therapy. Here, the authors use genomic analyses to study heterogeneity in colorectal cancer and perform in-depth reconstruction of heterogeneity in one sample.

For decades of molecular cancer research, immortal cancer cell lines have served as an easily accessible source for basic cancer biology research and preclinical testing of anticancer drugs. However, numerous studies have suggested that these cell lines poorly recapitulate the diversity, heterogeneity, and resulting drug resistance or relapse in patients. The derivation and (short-term) culture of primary cells from solid tumors have thus gained significant importance in personalized cancer therapy. This chapter focuses on our current understanding and the pros and cons of different preclinical and prospective clinical models of three-dimensional primary tumor cultures. We will discuss cell culture approaches, such as biomimetic scaffolds and growth factor supplemented, chemically defined media for various forms of solid tumors. Complex culture models of primary tumor cells could finally provide a key missing link between compound screening and clinical trials and ultimately will help redefining therapeutic intervention with high translational relevance at the level of the individual patient.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab., The heterogeneity of colorectal cancer has important clinical and therapeutic implications. Here the authors analysed the responses of a large biobank of organoids and xenografts derived from colorectal patients to a panel of clinically relevant therapeutic agents to identify genes signatures associated with drug response.

Analysis of the signaling response of colon cancer cells to systematic perturbations reveals an EGF receptor-mediated cross-talk between the MAPK and AKT pathways. Accordingly, the predicted combinatorial treatment is shown to inhibit tumor growth in vivo., A modular response analysis model trained on perturbation data in a panel of colon cancer cells revealed a negative feedback from MAPK signaling to the EGF receptor, which leads to AKT cross-activation after MEK inhibition. The model predicted that successful inhibition of growth-factor signaling in colon cancer requires combined inhibition of MEK and EGF receptor to prevent AKT activation and tumor cell survival, which was confirmed by growth assays. A xenograft tumor model of KRAS-mutant colon cancer showed that EGFR receptor inhibition alone has no effect, but in combination with a MEK inhibitor successfully reduces tumor growth., The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model-based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR-dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF- as well as KRAS-mutated tumor cells, which we confirmed using a xenograft model.

During recent years, circumstantial evidence has suggested that tumor development and cancer heterogeneity are triggered by so-called cancer stem cells (CSCs). These cells comprise a distinct, yet rare population found in both hematological and solid tumors. CSCs initiate tumor formation when ectopically transplanted into immune-deficient mice and share properties of tissue stem cells and partially reprogrammed induced pluripotent stem cells (iPSCs). CSCs are capable of self-renewal and differentiation into every cell type of the primary tumor they reside in. CSCs are responsible for tumor recurrence given that they often develop resistance mechanisms against chemotherapeutic drugs, e.g., an increased expression of ABC transporters or increased DNA-repair activity. Understanding the mechanisms that maintain CSCs in their niche and the development of strategies for specific targeting are essential to improve cancer therapy.

Precision medicine approaches seeking to predict individual susceptibility to single or combinatorial drugs for advanced and/or metastasized solid cancers heavily depend on biological model systems that maintain key morphological, phenotypic and genomic features of the original tumor. Here, we present a case study of a female patient with metastasized colorectal cancer. Using tissue from five distinct areas of the primary tumor and its metastasis of the synchronously removed primary tumor and liver metastasis, we generated cell cultures. To address cell morphology and phenotypic features we performed immunohistochemistry (IHC) and immunofluorescence imaging showing that these cell cultures express colon-specific marker proteins/combinations CDX2 and CK20+/CK7-. Ultra-deep panel sequencing of original tumor tissue samples revealed damaging mutations in KRAS (G12D), PIK3CA (H1047R) and TP53 (C242F). Two cell cultures showed an additional SNP in the tumor suppressor SMAD4 (R361H), which was not detected in the original tissue, thus hinting at the existence of a small sub-population of cancer cells with a divergent mutation pattern. In parallel we established patient-derived xenografts (PDX) models from each region and investigated the response to targeted therapeutics, aiming at key molecules of the MAPK, PI3K/AKT and mTOR pathways in these genetically heterogeneous populations. drug response in single and mixed cell populations to address differential growth characteristics and response to mono- and combinatorial drug treatment. To distinguish the cells-of-origin and to calculate distributions/contribution of each population to the in vivo tumor growth in this model, sub-populations were color-coded using lentiviral constructs, expressing either a green or red tag. FACS analyses after treatment were performed and ratios of SMAD4mut(green) vs. SMAD4wt(red) were determined for each PDX tumor. These analyses, together with confirmatory IHC staining for the respective tag, showed significant effects on both tumor growth and population distributions in a treatment-dependent manner, advocating for systematic evaluation of combinatorial treatment in patients. Citation Format: Dirk Schumacher, Karsten Boehnke, Marlen Keil, Alessandra Silvestri, Maxine Silvestrov, Jens Hoffman, Iduna Fichtner, Reinhold Schaefer, Christian RA Regenbrecht. Reconstructing intratumor heterogeneity: lessons from therapeutic intervention in patient-specific models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2412.

Recent data support a hierarchical model of colon cancer in which tumor growth is driven by a subpopulation of cancer stem cells (CSCs) that may also be the source of relapse following treatment. Elucidation of the cellular heterogeneity within a tumor would therefore facilitate better characterization of patient subtypes and lead to more personalized and effective treatments. Here, as part of the OncoTrack* consortium, we report the use of Matrigel-based 3D in vitro models of patient-derived colon cancer for the characterization of molecular pathways important in the regulation of CSC self-renewal and survival. Cellular subpopulations were isolated from patient-derived 3D-culture models based on expression of the CSC marker aldehyde dehydrogenase (ALDH) and then functionally tested for tumor initiation and self-renewal capacity by limiting dilution serial xenotransplantation. These studies demonstrate CSCs to be ALDH+. ALDH+ and ALDH- cells from 3D-culture and xenograft models were then subjected to RNA sequencing for whole transcriptome analysis. These analyses demonstrated ALDH+ CSCs cells to be enriched for stem cell associated genes (ALDH1A1, LGR5, BMI1, CD44, CD166), developmental processes (embryonic, tissue development, EMT) and signaling pathways (Wnt signaling). In particular, the Hedgehog signaling pathway was found to be enriched in both 3D in vitro and xenograft models. The role of Hedgehog signaling in colon cancer remains controversial. It has been reported as a negative regulator of Wnt signaling and to be inactive in colon cancer cells lines. Here, using small molecule inhibitors and lentivirus mediated gene knockdown, we report on the role of Hedgehog signaling in the regulation of colon CSC self-renewal and identify non-canonical Hedgehog signaling as a positive regulator of Wnt signaling. *The research leading to these results has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n°115234. Citation Format: Joseph L. Regan, Stephanie Staudte, Dirk Schumacher, Ulrich Keilholz, Johannes Haybaeck, Hans Lehrach, Marie-Laure Yaspo, David Henderson, Andreas Steffen, Joern Toedling, Ralf Lesche, Reinhold Schaefer, Christian R. Regenbrecht, Dominik Mumberg, Martin Lange. The role of Hedgehog signaling in the regulation of human colon cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1714.

Proper patient-tailoring strategy and the validation of novel therapeutic targets remain enormous challenges during drug discovery processes. Patient-derived three-dimensional organoid cell culture models possess great potential to associate compound sensitivity and disease complexity in order to provide a key missing link between compound screening and clinical trials. Abemaciclib is a reversible, ATP competitive, selective inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing advanced clinical testing. In this study, we established and characterized three-dimensional organoid cultures from primary colorectal cancer patients and validated their use as drug sensitivity models. We aimed to explore the antitumor activity of abemaciclib in colon cancer organoid cultures by assessing markers for cell viability, proliferation, cell cycle, senescence and apoptosis. Single cell suspension of patient-derived samples were precultured for four days to allow for complete morphogenesis of three-dimensional organoid structures. Subsequently, the cultures were treated for at least two population doubling times and analyzed by luminescent cell viability, immunohistochemistry and flow cytometry assays. Our data suggest that abemaciclib treatment decreased the cell viability of patient-derived colorectal cancer organoid cultures characterized by G1 cell cycle arrest and reduced Ki-67-positive cells. Furthermore, treated cultures showed elevated levels of reactive oxygen species and increased markers for early and late apoptosis. In summary, complex organoid models have the potential to further evaluate the antitumor activity of abemaciclib in various tumor types by enabling mechanistic studies in a patient-specific preclinical setting. Citation Format: Karsten Boehnke, Bruna Calsina, Joaquín Amat, Ana Hermoso, Raquel Torres, Christoph Reinhard, Juan A. Velasco, Philip W. Iversen, Alfonso De Dios, Sean Buchanan, Richard P. Beckmann, Dirk Schumacher, Christian RA Regenbrecht, Marie-Laure Yaspo, Hans Lehrach, María José Lallena. Preclinical analysis and characterization of abemaciclib using three-dimensional patient-derived colorectal cancer organoid cultures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2829.

Colon cancer is a heterogeneous tumour entity. Growing evidence supports a subpopulation of cancer stem cells (CSCs) as both the drivers of tumour growth and the source of relapse following treatment. Elucidation of the cellular heterogeneity within a tumour would therefore facilitate better characterization of patient subtypes and lead to more personalized and effective treatments. Here, as part of the OncoTrack* consortium, we report the use of a Matrigel-based 3D in vitro model for the identification of genetic factors important in the regulation of CSCs. The identity and frequency of CSCs within each patient-derived culture model was determined by separating cells using fluorescence assisted cell sorting (FACS) based on expression of previously defined markers of colon CSCs (ALDH+ CD44+ CD166+). Isolated subpopulations of cells are functionally tested for CSC properties in vitro (limiting dilution serial passage and population dynamics analysis) and in vivo (limiting dilution serial xenotransplantation). These studies reveal ALDH+ colon cancer cells to be highly enriched for CSCs and demonstrate large levels of variation in CSC frequency between patients. To identify genes and pathways that could be used to specifically target CSC function, ALDH+ tumour cell subpopulations were subjected to genome-wide analyses using next-generation sequencing for detailed characterization at the level of the transcriptome and methylome. To date, genes important in the regulation of normal stem cells and CSCs, such as BMI1, EPHB2 and the WNT signalling pathway, were found to be highly expressed in CSCs. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) mediated gene technology was used to knock out genes of interest and their role in regulating CSC function was confirmed using established functional assays. These studies provide insight into the relationship between variable tumour composition and the variable response of tumours to treatment. Ultimately, these studies will facilitate the more accurate classification and personalized treatment of colon tumours. *The research leading to these results has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n°115234. Citation Format: Joseph L. Regan, Dirk Schumacher, Stephanie Staudte, Karsten Boehnke, Ulrich Keilholz, Johannes Haybaeck, Hans Lehrach, David Henderson, Reinhold Schaefer, Christian R. Regenbrecht, Dominik Mumberg, Martin Lange. 3D-models of patient-derived colon tumors for the identification of genetic factors important in the regulation of cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 977. doi:10.1158/1538-7445.AM2015-977

Colon cancer is a heterogeneous disease. Growing evidence supports a subpopulation of cancer stem cells (CSCs) as both the drivers of tumour growth and the source of relapse following treatment. Elucidation of the cellular heterogeneity within a tumour would therefore facilitate better characterization of patient subtypes and lead to more personalized and effective treatments. Here we report the use of a Matrigel-based 3D in vitro model for the study of tumour heterogeneity and therapeutic resistance in patient derived colon tumours. To date, this system has enabled the establishment and long term culture of more than 53 patient-specific primary and metastasis derived colon tumours. Tumour heterogeneity and the prospective isolation of CSCs within each patient-derived culture model is determined by separating cells using fluorescence assisted cell sorting (FACS) based on expression of previously defined markers of colon CSCs (e.g. ALDH+, CD133+, CD44+, CD166+, EpCAMHigh). To date, these analyses have been performed on ten of the patient derived tumour models and demonstrate both inter- and intra- tumour heterogeneity as each tumour contains varying subpopulations of cells either positive or negative for expression of CSC markers. In addition, immunofluorescence staining and 3D confocal analysis of genes important in the regulation of both normal and CSCs (e.g. Notch, Wnt and Hedgehog signalling genes) has also revealed heterogeneity of expression. Functional characterization using in vitro and in vivo assays to determine the tumorigenic and differentiation capacity of the separated subpopulations are currently underway. Validated CSC subpopulations will be subjected to genome-wide analyses using next-generation sequencing for detailed characterization at the level of the transcriptome and methylome. To study the effect of drug treatment on CSCs we have determined the IC50 values for standard of care drugs (e.g. 5-fluorouracil, oxaliplatin, irinotecan, receptor tyrosine kinase inhibitors) for each patient derived colon tumour. Tumour cells were treated for 72 - 96 hrs after which time treatment was terminated and the cells were allowed to recover. Surviving cells were then analyzed by FACS to determine the effect of treatment on the frequency of CSCs. Molecular characterization of the therapy resistant CSCs was then used to identify possible biomarkers and targets for more effective treatments. These studies will provide insight into the relationship between variable tumour composition and the variable response of tumours to treatment. Ultimately, these studies will facilitate the more accurate classification and personalized treatment of colon tumours. OncoTrack is a project funded by the Innovative Medicines Initiative Joint Undertaking (IMI JU). Citation Format: Joseph L. Regan, Dirk Schumacher, Karsten Boehnke, Cathrin Davies, Ulrich Keilholz, Johannes Haybaeck, Christoph Reinhard, Hans Lehrach, David Henderson, Reinhold Schaefer, Christian Regenbrecht, Martin Lange. Functional and molecular characterization of colon cancer stem cells in tumor heterogeneity and disease relapse using a 3D-model of patient-derived tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3875. doi:10.1158/1538-7445.AM2014-3875

*OncoTrack is a project funded by the Innovative Medicines Initiative Joint Undertaking (IMI JU). New therapies for colon cancer barely improved cure rates over the last decade and typically only a subset of patients benefits from them. The reasons for these disappointing outcomes are likely related to the inherent genetic and cellular heterogeneity and plasticity of tumors. Therefore only comprehensive approaches that correlate data from systematic molecular tumor tissue characterization with preclinical and clinical responses could improve our understanding of this disease and help to design diagnostic procedures allowing optimal therapy for individual patients. OncoTrack is an international consortium funded by the Innovative Medicines Initiative (www.OncoTrack.eu) that has launched one of Europe's largest collaborative public-private research projects to implement novel approaches of systems biology into colon cancer therapy. Tumor tissue and circulating tumor cells from a cohort of more than 120 patients with primary or metastatic colon cancer were subjected to high-throughput protein, epigenome, and gene expression analysis as well as next generation sequencing. Three- dimensional (3D) in vitro cultures and in vivo patient-derived xenografts (PDX) in immunodeficient mice were generated from each tissue sample and used to determine response to more than 14 approved drugs, investigational drug candidates, and tool compounds. These wet-lab data, combined with clinical data were used for in silico modeling to identify new predictors for tailored therapies. Here we are presenting data from the subproject tumor models and therapy response. Tumor tissues from 118 colon cancer patients (87 primary tumors and 31 metastases) were transplanted into mice. To date, 44 permanent new PDX models have been generated from the primary tumors and 13 from the metastases; (establishment of 25 additional models is ongoing). These models are being used to determine sensitivity of the tumors to a broad range of drugs, targeting signaling pathways frequently activated in cancer. In parallel, in vitro cultures using a Matrigel-based, serum-free 3D system were established and adapted for high-throughput 384-well assay format for testing against a similar drug panel. Drug response data generated from more than 30 patient specimens using both the specific 3D in vitro assays and the in vivo PDX experiments are available and will be shown and compared. These experimental results about drug sensitivity together with the molecular signatures of the tumor tissues provide an exceptionally broad basis to analyze the relationship between tumor biology and treatment response. Such approaches may hold promise in the design of rational therapy for colon cancer in the future. Citation Format: Maria Rivera, Marlen Keil, Karsten Boehnke, Martin Lange, Dirk Schumacher, Reinhold Schäfer, Christian RA Regenbrecht, David Henderson, Ulrich Keilholz, Alexander Kuehn, Amin El-Heliebi, Tabea Hohensee, Johannes Haybäck, Christoph Reinhard, Juan A. Velasco, Hans Lehrach, Pilar Garin-Chesa, Garry Beran, Jens Hoffmann. Generation of drug response data from 57 new patient-derived colon cancer xenografts and 3D cell cultures for systematic correlation with tumor biology within the OncoTrack* project. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2978. doi:10.1158/1538-7445.AM2014-2978

Complex sets of driver mutations critically control clinical course and treatment response in colorectal cancer (CRC). It is expected that multiple mutations in different signaling pathways interact to establish an individual cancer cell phenotype. Furthermore, mutational patterns can evolve under selective pressure, for instance during tumor metastasis or targeted therapy. Yet only few mutations, such as these in KRAS, NRAS and BRAF, are routinely assessed in the clinic today. With the introduction of next generation sequencing (NGS) many more genes can be simultaneously sequenced, allowing detailed analyses of mutational patterns. We have assembled the CRC5.2 NGS panel for application with Ion Torrent PGM covering 100 frequently mutated genes with 784 amplicons (covering 21,000 COSMIC mutations), going well beyond the scope of commercially available cancer panels. In particular, exon coverage was optimized to embrace all genomic information related to drug sensitivity, using several drug and signaling network databases. The German Consortium for Translational Cancer Research (DKTK) comprises the top cancer centers in Germany. As a part of this group, we employ our custom CRC5.2 panel for the analysis of mutational patterns in CRC. We address several key questions relevant for current diagnostics and treatment in colorectal cancer. In retrospective and prospective clinical studies, we assess mutational heterogeneity in primary tumors, synchronous and metachronous metastases of individual patients. In-depth NGS mutational analyses, transcriptome analysis and methylation studies will identify (epi-)genetic alterations occurring as a consequence of selective pressure during tumor evolution e.g. during metastasis, chemotherapy or targeted tumor therapy. Thus, we will uncover new links between cancer-specific mutations, clinical course and treatment response as well as novel mechanisms of secondary resistance. In addition we aim to identify novel predictive and prognostic mutational markers for the benefit of future CRC patients Citation Format: Soulafa Mamlouk, Liam Childs, Thorben Redmer, Pawel Durek, Dirk Schumacher, Hendrik Bläker, Daniela Aust, Moritz von Winterfeld, Volker Heinemann, Dominik Modest, Kerstin Möhr, Markus Morkel, Reinhold Schäfer, Christine Sers. Linking tumor evolution and therapy response using diagnostic targeted next generation sequencing in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2014-5170

OncoTrack is an international consortium funded by the Innovative Medicines Initiative (www.OncoTrack.eu) that has launched one of Europe's largest collaborative public-private research projects to implement novel approaches of systems biology in colon cancer therapy. Within OncoTrack, tumor tissue and circulating tumor cells from a cohort of more than 120 patients with primary or metastatic colon cancer are subjected to whole genome-, exome-, epigenome- and transcriptome sequencing, as well as high-throughput protein analysis. Alongside the systematic analysis of colon cancer tissues, in vivo xenografts in immunodeficient mice (patient-derived xenografts, PDX) and in vitro 3D cell cultures are generated from each tissue sample and used to determine response to more than 14 approved drugs, investigational drug candidates and tool compounds. These wet-lab data, combined with clinical data, will serve as a basis for in silico modeling to identify new predictors for tailored therapies. Although early diagnosis and molecular characterization of colon cancer has improved significantly, rapid and cost-effective means to address molecular genotyping and therapeutic options on the level of the individual patient are still in high demand. Here, we present an experimental pipeline within the OncoTrack project starting from Matrigel-based Patient-tumor-derived 3D cell cultures (PT3DC). To date, we were able to establish 50 long-term 3D cell culture strains originating from 35 primary tumors, 9 metastases and 6 PDX-derived tissues as suitable models for basic and translational research. On the basis of immunohistochemistry, we show that our in vitro cultures preserve an in vivo like architecture, preventing tumor cells from differentiating and allowing the investigation of intra-tumor heterogeneity and cancer stem cell like sub-populations. To interrogate the mutation status of selected clinically relevant oncogenes and tumor suppressors in PT3DC cultures, we applied cost-efficient benchtop sequencing and show the preservation of putative driver mutations found in the original tumor. IC50 data generated by automated 384-well based dose-response experiments of approved drugs are then used to link individual genotypes with drug sensitivity phenotypes and serve as a source of comparison and complementation to drug response data of the corresponding in vivo PDX models, where applicable. Citation Format: Dirk Schumacher, Karsten Boehnke, Martin Lange, Yvonne Welte, Cathrin Davies, Maria Rivera, Marlen Keil, Ulrich Keilholz, Johannes Haybaeck, Juan Angel Velasco, Marie-Laure Yaspo, Hans Lehrach, David Henderson, Christoph Reinhard, Jens Hoffmann, Reinhold Schaefer, Christian Regenbrecht. A pipeline within the OncoTrack project for generating Patient-tumor-derived 3D cell cultures (PT3DC) and their application for individualized, targeted drug sensitivity assays. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2018. doi:10.1158/1538-7445.AM2014-2018

Colon cancer is the 3rd most common type of cancer and the 4th leading cause of cancer-related deaths worldwide. OncoTrack is an international consortium that has launched one of Europe's largest collaborative academic-industry research projects to develop and assess novel approaches for identification of new markers for colon cancer (1). To this end, colorectal tumors and metastases, as well as isolated circulating tumor cells and cancer stem cells are subjected to high-throughput sequencing of DNA and RNA. The laboratory data are combined with clinical data and used for in silico modeling of the patients' tumors and prediction of biomarkers as well as a tailored therapy. These predictions are validated using patient-specific in vitro (primary cells) and in vivo (xenografts) models. Recent data show that a small subpopulation of cells within different tumors, including colon cancer, is responsible for tumor growth and maintenance. Due to their functional similarities to adult tissue stem cells and their likely origin, these cells have been termed cancer stem cells. We aim at characterizing patient-derived primary cell cultures and cancer stem cells isolated from these. Primary tumors and metastases from colon cancer patients are cultivated in vitro using a matrigel-based, serum-free culture system. This system allows for long-term expansion of primary colon cancer cells in vitro (2). Using this methodology, we were able to establish over 35 novel, patient-specific colon cancer cell lines, both from primary tumors and metastases. Using previously established markers of colon cancer stem cells (CD44+, CD133+, CD166+, EpCAM-high, Wnt-high), cells are sorted by flow cytometry and subsequently subjected to genome-wide analyses using next-generation sequencing. These experiments allow for a detailed characterization of colon cancer stem cells at the level of transcriptome and methylome. It has been shown that tumors are heterogeneous with regard to gene mutations and expression of cancer stem cell markers (3). Using molecular tools such as padlock probes (4), we are analyzing the distribution of gene mutations identified by next-generation sequencing within primary cells as well as primary tumors. Moreover, we are addressing the expression pattern of cancer stem cell markers in these cell types. These data will help in a better understanding of individual tumors and assist in design of a personalized therapy. OncoTrack is a project funded by the Innovative Medicines Initiative Joint Undertaking (IMI JU). 1 Elsner M, Nature Biotechnology 29, 378 (2011); www.oncotrack.eu 2 Sato T el al., Gastroenterology. 2011 Nov;141(5):1762-72. 3 Gerlinger M et al., N Engl J Med. 2012 Mar 8;366(10):883-92. 4 Larsson C, et al., Nat Methods. 2010 May;7(5):395-7. Citation Format: Martin Lange, Dirk Schumacher, Thomas Hauling, Christian Regenbrecht, Oliver Politz, Mats Nilsson, Jens Hoffmann, Reinhold Schaefer, David Henderson. Molecular characterization of cancer stem cells isolated from primary colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2013-252