Control of Glioma Cell Invasion By Immunotherapy

描述（由申请人提供）：目前，没有已知的可以靶向侵入的神经胶质瘤肿瘤细胞的疗法。为了在分子水平上研究侵袭性胶质瘤细胞的生物学特性，我们利用绿色荧光蛋白（GFP）标记的鼠胶质瘤细胞GL 261建立了体内动物模型。这使我们能够使用GFP免疫组织化学鉴定侵入脑内邻近主要肿瘤块的胶质瘤细胞。微阵列基因技术突出了入侵和非入侵的人类胶质瘤细胞之间的基因表达谱的差异，确定MHC I类分子作为新的免疫方法的潜在目标。GL 261胶质瘤细胞系由于其低表达MHC分子而免疫原性差。然而，我们已经表明，低剂量全脑放射治疗（WBRT）的一个既定的GL 261肿瘤上调MHC的表达入侵GL 261胶质瘤细胞在体内。在本申请中，我们将继续测试对GL 261颅内肿瘤的低剂量WBRT将上调MHC表达的假设，为针对入侵细胞的T细胞介导的抗肿瘤免疫应答提供靶标，并提高免疫疗法的功效。在目的1中，我们将分别确定上调入侵胶质瘤细胞上的MHC表达的最佳剂量和时程以及增加长期存活的最佳疫苗接种次数（1a）;以及确定由WBRT和疫苗接种的组合引起的宿主免疫应答（V）。单独4戈伊的WBRT方案诱导与肿瘤部位的TIL流入相关的炎症反应。在处理后1周和2周，通过FACS分析和冷冻切片的免疫组织化学表征脑的TIL。（1b）。目的二：研究MHC过表达对GL 261肿瘤细胞生长、侵袭和免疫原性的影响。GL 261肿瘤细胞将被工程化以稳定表达编码鼠CIITA cDNA的诱导型构建体（Tet-On），所述鼠CIITA cDNA是调节MHC复合物表达的基因。在目标3中，我们将确定是否需要在体内上调侵袭性GL 261肿瘤细胞上的MHC表达来改善WBRT后疫苗接种的治疗效果。将使用siRNA技术抑制MHC表达。越来越多的人认识到，低剂量照射可以使肿瘤更容易被患者的免疫系统识别，这构成了我们将癌症疫苗与脑肿瘤的局部照射相结合的理论基础。

DESCRIPTION (provided by applicant): Presently, there is no known therapy that can target invading glioma tumor cells. In order to study the biological properties of invading glioma cells at the molecular level, we have developed an in vivo animal model using green fluorescent protein (GFP)-tagged murine GL261 glioma cells. This allows us to identify invading glioma cells into the brain adjacent to the main tumor mass using GFP immunohistochemistry. Microarray gene technology high-lighted differences in gene expression profiles between invading and non-invading human glioma cells identifying MHC class I molecules as a potential target for novel immunotherapeutic approaches. The GL261 glioma cell line is poorly immunogenic due to its low expression of MHC molecules. However, we have shown that low dose whole brain radiation therapy (WBRT) of an established GL261 tumor up-regulates MHC expression on invading GL261 glioma cells in vivo. In the present application we will continue to test the hypothesis that low dose WBRT to the GL261 intracranial tumor will up-regulate MHC expression providing a target(s) for a T-cell mediated antitumor immune response directed towards the invading cells and improve the efficacy of immunotherapy. In Aim 1 we will determine the optimal dose and time course to up-regulate MHC expression on invading glioma cells and optimal number of vaccinations to increase long-term survivals, respectively (1a); and to determine host's immune response elicited by the combination of WBRT and vaccination (V). The WBRT schedule of 4 Gy alone induces an inflammatory response associated with an influx of TILs at the tumor site. Brains will be characterized for TILs by FACS analysis and immunohistochemistry of frozen section at 1 week and 2 weeks following treatments. (1b). In Aim 2, we will determine the effects of MHC overexpression in GL261 tumor cells on their growth, invasion and immunogenicity in vitro and in vivo. GL261 tumor cells will be engineered to stably express an inducible construct (Tet-On) encoding the murine CIITA cDNA, a gene that regulates expression of the MHC complex. In Aim 3, we will determine whether up-regulation of MHC expression on invading GL261 tumor cells in vivo is required for the improved therapeutic effect of vaccination following WBRT. MHC expression will be inhibited using siRNA technology. The growing awareness that low dose irradiation can make tumors more amenable to recognition by the patients' immune system forms the basis of our rationale combining cancer vaccines with local irradiation of the brain tumor.