Cellular and molecular phenotypes of proliferating stromal cells from human carcinomas

Abstract

BACKGROUND: Stromal cells are a functionally important component of human carcinomas. The aim of this study was to obtain and characterise primary cultures of stromal cells from human carcinomas and the corresponding surrounding normal tissue. METHODS: Primary stromal cell cultures from tumours of lung, oesophagus and pancreas were obtained using a mild tissue dissociation method and a medium for culturing mesenchymal cells. Immunofluorescence staining and western blotting were used to analyse the expression of differentiation markers and selected known oncoproteins in the cell cultures obtained. RESULTS: A panel of stromal primary cultures was prepared from different human tumours and from matched normal cancer-free tissues. The in vitro proliferative potential of tumour-associated fibroblasts was shown to be higher than that of matched normal stromal cells. A mutational analysis of the TP53 and KRAS2 genes in a number of stromal cultures did not reveal known mutations in most cells of the cultures studied. Western blot analysis showed that stromal cells of lung tumours were characterised by a statistically significantly lower expression level of the p16 protein as compared with that in normal lung stromal cells. An important finding of our study was that, according to immunofluorescence assay, a fraction of fibroblast-like vimentin-positive cells in some tumour and normal stromal cell cultures expressed an epithelial marker -cytokeratins. CONCLUSION: Proliferating stromal cells from the carcinomas studied proved to be genetically normal cells with altered expression profiles of some genes involved in carcinogenesis, as compared with normal stromal cells. Epithelial-mesenchymal transition may lead to the emergence of transdifferentiated fibroblast-like cells in tumour stroma and in the tumour-surrounding tissue.

Stromal LRP1 in Lung Adenocarcinoma Predicts Clinical Outcome

“…First, therapies activating LRP1 in the stroma may produce a durable effect on tumor behavior, since the tumor stroma could be more genetically stable (46) and less likely to evolve drug resistance during therapy (47); analysis of the LRP1 locus in the validation set of tumors failed to demonstrate a high rate of genetic amplification or deletion (Supplementary Fig. 6A).…”

Construction of a Combinatorial Library of Chimeric Tumor-Specific Promoters

“…A431 cells (epidermoid carcinoma of skin), HT1080 cells (fibrosarcoma), and HEK 293T cells (human embryonic kidney cells stably expressing the SV40 large T antigen) were purchased from ATCC (Manassas, VA). ICL-7NS fibroblasts were prepared from normal lung tissue adjacent to a tumor according to the standard protocol (23,24). …”

Cancer Specificity of Promoters of the Genes Involved in Cell Proliferation Control

“…We used the plasmid clones containing the cloned promoters to transfect the following cell lines: A375 (malignant melanoma, ATCC ), A431 (epidermoid carcinoma of the skin, ATCC ), A549 (lung carcinoma, ATCC ), Calu1 (lung epidermoid carcinoma, EC ACC ), HepG2 (hepatocellular carcinoma, ATCC ), HT1080 (fibrosarcoma, ATCC ), Panc-1 (epithelioid pancreatic carcinoma, ATCC ), and normal fibroblasts IVL-7C. Fibroblasts IVL-7C were obtained from the morphologically normal tissue of the lung of a patient who had undergone surgical resection of his lung cancer at the Blokhin Cancer Center, Russian Academy of Medical Sciences, using the previously described procedure [11]. Co-transfection with the plasmid pRL-TK (Promega, WI, USA) expressing the Rluc gene was used as an internal control of the transfection.…”