Microenvironmental alterations by radiation in murine hepatocarcinoma

Abstract

Background: Radiotherapy (RTx) technology for hepatocellular carcinoma (HCC) has rapidly developed and enables more successful treatment. However, RTx has been challenging due to the potential for intrahepatic and extrahepatic metastasis.These occurrences suggest that the tumor microenvironment (TME) and surrounding adjacent normal tissues might be important modulators of RTx. For murine hepatocarcinoma, heterotopic tumor implantation into the thigh or dorsal skin has become a standard method for establishingmurine hepatocarcinomamodels. Although these types of models help in understanding the nature of cancers and their therapeutic approaches, they are not sufficient for investigating the interactions of tumor cells with the surrounding microenvironment of adjacent normal tissues, including immunological responses. For this reason, we established a syngenic murine hepatocarcinoma model via orthotopic implantation. The goal of this study was to evaluate the biological features of orthotopic murine hepatocarcinoma and to determine the response of TME molecules to radiation.Methods: Murine hepatocarcinoma (HCa-I) models were established in the liver (orthotopic) and thigh muscle (heterotopic) of male C3H/HeJ mice. In these models, tumor growth and lung metastasis were assessed. To evaluate the tumor microenvironmental alterations by different TME and radiation, the tumor models were irradiated with 10 Gy. Immunohistochemical and Western blot analysis were then performed for vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), transforming growth factor beta1 (TGF-β1) and clusters of differentiation 31 (CD31). Serum sampling was conducted for evaluation of VEGF, TGF-β1 and interleukin-6 (IL-6) level. The presence of immune cells was evaluated inperitumor liver tissuesand in tumors in the orthotopic TME after irradiation.Results: The number of lung metastasis was higher in the orthotopic tumor model than in the heterotopic tumor model. VEGF, CD31, COX-2, and TGF-β1 expression increased in the orthotopic tumor model. This expression was prominent at the peripheral tumor margin. The expression of angiogenic factor (VEGF) and key regulatory molecules (TGF-β1 and COX-2)in the tumor tissue decreased following radiation. However, the expression in peritumoral normal liver also increased after radiation. Levels of secreted factors (VEGF and TGF-β1) decreased after radiation, but IL-6 levels increased. The immune cell response of CD8 cells and programmed death 1 (PD-1)in CD8 T-cellswere increased after radiation. However, numbers of regulatory T cellsdecreased after radiation.Conclusion:The tumor microenvironment in murine hepatocarcinoma was more aggressive for orthotopic than for heterotopic tumors in terms of lung metastasis. Radiation enhanced the expression of VEGF, COX-2, and TGF-β1 at peritumoral normal tissue in the orthotopic tumor model. This model is useful for investigating tumor microenvironments and radiation effects.