Control of Glioma Cell Invasion By Immunotherapy

描述（申请人提供）：目前，还没有已知的治疗方法可以靶向侵袭性胶质瘤细胞。为了在分子水平上研究入侵胶质瘤细胞的生物学特性，我们利用绿色荧光蛋白（GFP）标记的小鼠GL261胶质瘤细胞建立了体内动物模型。这使我们能够使用GFP免疫组织化学技术识别侵入主要肿瘤肿块附近大脑的胶质瘤细胞。微阵列基因技术突出了侵袭性和非侵袭性胶质瘤细胞之间基因表达谱的差异，确定了MHC I类分子作为新型免疫治疗方法的潜在靶点。GL261胶质瘤细胞系由于其MHC分子的低表达而具有较差的免疫原性。然而，我们已经证明，低剂量全脑放疗（WBRT）在体内可上调入侵GL261胶质瘤细胞的MHC表达。在目前的应用中，我们将继续验证这样一个假设，即低剂量的WBRT对GL261颅内肿瘤会上调MHC表达，为t细胞介导的针对入侵细胞的抗肿瘤免疫反应提供靶标，并提高免疫治疗的疗效。在Aim 1中，我们将分别确定上调侵袭胶质瘤细胞MHC表达的最佳剂量和时间过程，以及提高长期存活率的最佳疫苗接种次数（1a）；确定WBRT与疫苗联合引起的宿主免疫应答(V)。仅4gy的WBRT计划可诱导与肿瘤部位TILs流入相关的炎症反应。在治疗后1周和2周，通过FACS分析和冷冻切片的免疫组织化学来表征大脑的TILs。(1 b)。在Aim 2中，我们将在体外和体内确定GL261肿瘤细胞中MHC过表达对其生长、侵袭和免疫原性的影响。GL261肿瘤细胞将被改造成稳定表达一种可诱导构建体（Tet-On），该构建体编码小鼠CIITA cDNA，这是一种调节MHC复合物表达的基因。在Aim 3中，我们将确定是否需要上调体内入侵GL261肿瘤细胞的MHC表达，以提高WBRT后接种疫苗的治疗效果。利用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局部照射相结合的基本原理。

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.