Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and <em>Ex vivo</em> Fibroblasts

Abstract

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions. Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC. Video LinkThe video component of this article can be found at

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“…The remodeling process includes the degradation of preexisting ECM molecules by proteolytic enzymes and the neosynthesis of ECM secreted by tumor cells and/or stromal cells [29][30][31]. As previous studies have suggested that FN could be produced by various types of cells including tumor cells [32], we identified a positive association between stromal FN expression and the levels of FN expressed in GBC epithelial cells.…”

A novel ex vivo Huntington’s disease model for studying GABAergic neurons and cell grafts by laser microdissection

“…Also, in our protocol organotypic slices were incubated in ethanol and it has been reported that ethanol conserves tissue morphology and RNA integrity [ 66 ]. Other protocols of LMD on organotypic slices imply performing a supplemental cut of the organotypic slices with a cryostat to obtain 10–20μm thick slices [ 67 , 68 ], while in this study mRNA expression was analysed directly after the LMD of 300–400μm thick tissue. Thus, this protocol allows having easily reproducible results.…”