Abstract 3875: Functional and molecular characterization of colon cancer stem cells in tumor heterogeneity and disease relapse using a 3D-model of patient-derived tumors

Abstract

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