Tumour microenvironment and radiation response in sarcomas originating from tumourigenic human mesenchymal stem cells.

BACKGROUND: Resistance to radiation therapy remains a serious impediment to cancer therapy. We previously reported heterogeneity for clonogenic survival when testing in vitro radiation resistance among single-cell derived clones from a human mesenchymal cancer stem cell model (hMSC). Here we aimed to determine whether this heterogeneity persisted in tumours established from these clones, and whether the response to radiation treatment was principally governed by cell-intrinsic qualities or by factors pertaining to the tumour microenvironment, such as the degree of hypoxia and vascularization. METHODS: Immune-deficient female mice were implanted on the backs with cells from one of the clones. The subsequent tumours were subjected to either radiation treatment or had the tumour microenvironment assayed, when they reached 400 mm³. Radiation was given as a single fraction of 0-15 Gy and the degree of tumour control and time to three times the treatment volume were noted. Tumours used for the microenvironmental assay had intratumoral hypoxia measured by the Eppendorf oxygen electrode and pimonidazole staining, and the extent of vascularization determined by a microvasculature density assay using endothelial-specific staining. RESULTS: All microenvironmental assays indicated a similar degree of hypoxia and vascularization for the selected clones. Nonetheless, the tumours responded differently to radiation treatment since the BB3 clone displayed tumour control at 5, 10 and 15 Gy, whereas tumour control was not seen below 15 Gy with the CE8 clone. CONCLUSION: For tumours that displayed similar degrees of oxygenation and vascularization, the clone-specific in vitro radiation resistance could predict the in vivo response to radiation treatment. These results favor the hypothesis that intrinsic genetic factors can govern radiation resistance in this cancer stem cell model.

Tumourigenicity and radiation resistance of mesenchymal stem cells.

BACKGROUND: Cancer stem cells are believed to be more radiation resistant than differentiated tumour cells of the same origin. It is not known, however, whether normal nontransformed adult stem cells share the same radioresistance as their cancerous counterpart. MATERIAL AND METHODS: Nontumourigenic (TERT4) and tumourigenic (TRET20) cell lines, from an immortalised mesenchymal stem cell line, were grown in culture prior to irradiation and gene expression analysis. Radiation resistance was measured using a clonogenic assay. Differences in gene expression between the two cell lines, both under nontreated and irradiated conditions, were assessed with microarrays (Affymetrix Human Exon 1.0 ST array). The cellular functions affected by the altered gene expressions were assessed through gene pathway mapping (Ingenuity Pathway Analysis). RESULTS: Based on the clonogenic assay the nontumourigenic cell line was found to be more sensitive to radiation than the tumourigenic cell line. Using the exon chips, 297 genes were found altered between untreated samples of the cell lines whereas only 16 genes responded to radiation treatment. Among the genes with altered expression between the untreated samples were PLAU, PLAUR, TIMP3, MMP1 and LOX. The pathway analysis based on the alteration between the untreated samples indicated cancer and connective tissue disorders. CONCLUSION: This study has shown possible common genetic events linking tumourigenicity and radiation response. The PLAU and PLAUR genes are involved in apoptosis evasion while the genes TIMP3, MMP1 and LOX are involved in regulation of the surrounding matrix. The first group may contribute to the difference in radiation resistance observed and the latter could be a major contributor to the tumourigenic capabilities by degrading the intercellular matrix. These results also indicate that cancer stem cells are more radiation resistant than stem cells of the same origin.

Cancer stem cell overexpression of nicotinamide N-methyltransferase enhances cellular radiation resistance.

BACKGROUND: Cancer stem cells are thought to be a radioresistant population and may be the seeds for recurrence after radiotherapy. Using tumorigenic clones of retroviral immortalized human mesenchymal stem cell with small differences in their phenotype, we investigated possible genetic expression that could explain cancer stem cell radiation resistance. METHODS: Tumorigenic mesenchymal cancer stem cell clones BB3 and CE8 were irradiated at varying doses and assayed for clonogenic surviving fraction. Altered gene expression before and after 2 Gy was assessed by Affymetric exon chip analysis and further validated with q-RT-PCR using TaqMan probes. RESULTS: The CE8 clone was more radiation resistant than the BB3 clone. From a pool of 15 validated genes with altered expression in the CE8 clone, we found the enzyme nicotinamide N-methyltransferase (NNMT) more than 5-fold upregulated. In-depth pathway analysis found the genes involved in cancer, proliferation, DNA repair and cell death. CONCLUSIONS: The higher radiation resistance in clone CE8 is likely due to NNMT overexpression. The higher levels of NNMT could affect the cellular damage resistance through depletion of the accessible amounts of nicotinamide, which is a known inhibitor of cellular DNA repair mechanisms.