Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133 + CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133 + CSCs and CD133 -bulk, which can be cultivated and functionally analyzed thereafter.
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 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
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