Abstract 1649: Deadly fuel: Fibroblasts mediate cancer cell death through tunneling nanotubes in response to ionizing radiation

Abstract

Abstract Despite surgery or chemo radiotherapy the five-year survival rate of esophageal carcinoma (eSCC) patients remains around 15% indicating the need for new therapy regimes. Recently the role of the tumor microenvironment has been recognized as an important determinant of radiation responses. Here we investigate the influence of stromal fibroblasts (hF) on radiation induced tumor cell death. To evaluate functional consequences of ionizing radiation hF and eSCC cells were either cultured separately and irradiated (2Gy) or co-cultured after irradiation to investigate radiation dependent tumor-stroma interactions. Subsequently cells were monitored by time-lapse microscopy and cell death was assessed visually as well as by PARP cleavage, live cell staining with Hoechst33342/propidium iodide and quantification of sub-G1 cell cycle. The rate of cell death was barely altered in monocultures. Of note significantly increased cell death of eSCC was observed in co-culture with irradiated hF compared to co-cultures with mock-irradiated fibroblasts (16.10±4.25 fold of control). Microscopic evaluation of co-cultures revealed that hF and cancer cells are connected via tunnelling nanotubes (TNT). Furthermore, TNT formation was found to be dependent on the extracellular matrix component hyaluronan. eSCC cell death was prevented by pharmacological inhibition of TNT formation by cytochalasin B as well as inhibition of hyaluronan by 4-MU. As TNTs are known to mediate intercellular protein or mitochondrial transfer we investigated which stromal proteins are shuttled into eSCC cells by trans-SILAC experiments. For this purpose the proteome of fibroblasts was labeled with heavy amino acids. After co-culturing hF and eSCC were separated by FACS-sorting and labeled stromal proteins were identified in the cancer cells by mass spectrometry. Four consistently shuttled proteins were identified in the cancer cells, which were exclusively transferred after irradiation of both cell lines. Knock-down experiments of these proteins in hF were performed and SHANK1, which is not expressed in eSCC cells, was found to be responsible for the hF induced cell death of eSCC cells in response to radiation. In conclusion, by usage of a novel trans-SILAC approach, we provide evidence that in response to ionizing radiation stromal hF transfer SHANK1 to eSCC cells, thereby inducing cellular death. SHANK1 might therefore serve as potential new pharmacological target to sensitize cancer cells to radiation therapy. Citation Format: Katharina Röck, Alexandra Schütze, Janna Morawitz, Verena Jendrossek, Klaus Pantel, Jens W. Fischer. Deadly fuel: Fibroblasts mediate cancer cell death through tunneling nanotubes in response to ionizing radiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1649.