Radiation Induced Changes in Tumor Derived Exosomes Promotes Metastasis

Abstract

Radiation is an extremely effective therapy in cancer treatment. Unfortunately, relapses and metastasis are common impediments to its efficiency. Several studies have demonstrated that radiation might support migration and invasion of tumor cells by altering the microenvironment. Our objective in this study was to link radiation induced exosome released from the tumor cells and their effect on the metastatic potential of recipient irradiated or control MC38 colon tumor cells. In addition, we investigated the known metastasis promoting miRNA load of these exosomes. MC38 colon tumor cells syngeneic to C57BL/6 mouse was grown in flank and then exposed to irradiation. Irradiated tumor was then removed and primary cell cultures were prepared. Irradiated tumor cells were cultured for 48 h post radiation and then exosomes were isolated. These exosomes were then injected in C57BL/6 mice 3 times per week for 3 weeks followed by tail vein injection of non-irradiated/irradiated MC38 cells. The mouse was killed on day 28 for analysis of metastatic lesion in lung. Total RNA was extracted from the exosomes and miRNA expression was analyzed by qPCR. The lungs from the mice were scored for metastatic lesions and we found that the group that received the exosomes from the irradiated tumor cells had significantly higher number of metastatic nodules in the lung as compared to the group that received exosomes from non-irradiated tumor cells. This was true for both sets of mice receiving non- irradiated (p<1.64E05) or irradiated MC38 cells (p<9.66E-06) tail vein injection. A comprehensive list of miRNA shown to have an effect on the metastatic or oncogenic ability of cancers was made using literature search and their expression was quantified using qPCR. We found upregulation of miR-92a, miR-200c and miR-29a, all of which have been shown to promote progression and metastasis in colorectal cancers. In addition, the exosomes from irradiated tumor had significantly higher amount of protein relative to the exosomes from the non-irradiated tumor. Taken together these results prove that exosomes released from irradiated tumor cells can alter the microenvironment such that they promote tumor cell seeding and growth in distant organs Our data provides evidence that exosomes from irradiated tumor cells enhance the capacity of tail vein injected MC38 colon tumor cells to induce metastasis lesions in mice lung. Further analysis of these exosomes confirmed that they carried increased copies of oncogenic miRNA often correlated with poor prognosis in cancer patients and higher protein load than non-irradiated samples. It lays important groundwork for future studies aimed to abate the side effects of radiation therapy and characterization of earlier prognostic markers for chance of metastasis in patients after undergoing radiation therapy.