Cell separation and gene expression analysis in a tumor-stroma interaction model

english

2004

volume 23, issue 3

Coculture   Fibroblasts    Tumor Cells, Cultured   Neoplasm Invasiveness   Gene Expression    Immunohistochemistry    Microscopy, Confocal 

Društvo za stereologijo in kvantitativno analizo slike
(Obvezni izvod spletne publikacije)

A novel technique for co-culturing and separating fibroblasts and carcinoma cells in a 2-D model of tumor stroma interaction is presented. The methodology is based on cell co-cultivation on an 1.35 microm 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 wasamplificable 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.

16.09.2009