CELL SEPARATION AND GENE EXPRESSION ANALYSIS IN A TUMOR-STROMA INTERACTION MODEL

Abstract

A novel technique for co-culturing and separating fibroblasts and carcinoma cells in a 2-D model of tumorstroma interaction is presented. The methodology is based on cell co-cultivation on an 1.35 μm thin membrane followed by rapid immunostaining and microdissection of the different cell compartments using a laser microdissection system (P.A.L.M. Microlaser Technologies AG, Germany). For identifying the tumor cell compartment, immunolabeling for a marker that is expressed only in epithelial tumor cells is performed. The RNA quality from the microdissected co-cultured cells was successfully proved by RT-PCR for a housekeeping gene transcript and for the laminin gamma 2 chain gene transcript used before in the tumor cell immunostaining. Laminin cDNA was amplificable only in tumor cells and not in the co-cultivated fibroblasts indicating no cell-cross-contamination during microdissection. Microdissected tumor and stroma cells from the presented membrane based co-culture model can be used for gene expression profiling and DNA based analysis in the investigation of tumor-stroma interactions.