Engineered and Encapsulated Stem Cells for Resected Brain Tumors

摘要 胶质母细胞瘤(GBM)是成人最常见的原发脑肿瘤，预后极差。在给定情况下， 肿瘤切除在GBM治疗临床护理中的核心作用，了解肿瘤的具体影响 切除肿瘤微环境中的免疫反应为开发有效的治疗提供了新的平台 基于免疫的基底膜治疗。我们最近建立了同基因原位小鼠肾小球基底膜移植模型。 肿瘤切除后发现，肿瘤切除会导致髓系抑制因子的显著减少。 细胞(MDSCs)和CD4/CD8T细胞同时募集到切除腔内。在这项提案中，我们 将首先从遗传上截然不同的当前可用的小鼠GBM系和 分析肿瘤消融前后肿瘤微环境中免疫细胞的分布情况。而当 原发肿瘤切除已显示出临床效益，全身注射或直接注射治疗 肿瘤切除腔内的药物提供的额外好处有限。在我们之前的研究中，我们有 广泛证明局部递送受体靶向的工程化成人干细胞具有治疗作用 干细胞的优点和合成细胞外基质(SECM)的包裹是必要的，以防止其迅速 小鼠肾小球基底膜肿瘤切除腔内“洗出”。在我们最近发表的研究中，我们 研究表明，SECM包裹的间充质干细胞(MSC)介导的双功能、 免疫调节和细胞毒蛋白干扰素β促进术后CD8T的选择性渗透 并直接诱导肿瘤细胞的细胞周期停滞。然而，已知干扰素可上调程序 细胞死亡配体1(PD-L1)在肿瘤细胞上的表达，从而阻碍干扰素的免疫调节功能。 基于最近的发现：阻断干扰素β治疗诱导的PD-L1可根除已建立的肿瘤； 肿瘤抑制因子、磷酸酶和紧张素同源物(PTEN)的缺失可增强免疫抵抗力；我们的兴奋 关于：间充质干细胞-干扰素β在体内介导PD-L1的上调；以及单链抗体-PDL1的局部传递的初步数据，我们将 建立表达ScFv-PDL1和干扰素β的双峰间充质干细胞，并在同基因PTEN野生型(wt.)和 突变型基底膜切除模型的建立。为了简化临床翻译并确保我们方法的安全性，我们将 最终使临床级人骨髓间充质干细胞共表达单链抗体-PDL1/干扰素β和单纯疱疹病毒胸苷激酶(TK) 并在人源化NSG小鼠的患者来源的GBM系产生的GBM肿瘤中测试我们的方法。这个 将基因工程成像标记整合到MSC和GBM中将使我们能够跟随 MSC在体内的命运和疗效，从而微调拟议的方法。这项提案的总体目标是 因此，免疫图谱基因不同的基底膜切除模型，并评估基于治疗的理论基础 免疫调节剂的疗效。一旦被证实，我们将启动一项临床研究，在大脑时间 肿瘤手术后，主要肿瘤肿块将被切除并被包裹的双峰MSC将被引入 加强对肿瘤细胞的清除。这将对挽救脑癌患者的生命产生重大影响。

SUMMARY Glioblastoma (GBM) is the most common primary brain tumor in adults with a very poor prognosis. Given, the central role tumor resection plays in GBM therapy clinical care, understanding the specific influence of tumor resection on immune response in the tumor microenvironment offers a new platform for developing effective immune based therapies for GBM. We have recently developed syngeneic orthotopic mouse GBM-model of tumor resection and shown that tumor debulking results in substantial reduction of myeloid-derived suppressor cells (MDSCs) and simultaneous recruitment of CD4/CD8 T cells into the resection cavity. In this proposal, we will first generate GBM resection models from genetically distinct currently available mouse GBM lines and analyze profiles of immune cells infiltrated into tumor microenvironment pre- and post-tumor debulking. While resection of primary tumor has shown clinical benefit, systemically delivered or direct injection of therapeutic agents in tumor resection cavities has provided limited additional benefit. In our previous studies, we have extensively demonstrated that locally delivered receptor targeted engineered adult stem cells have therapeutic benefits and synthetic extracellular matrix (sECM) encapsulation of stem cells is necessary to prevent their rapid “wash- out” post-transplantation in mouse GBM tumor resection cavity. In our recently published studies, we have shown that sECM encapsulated mesenchymal stem cell (MSC) mediated local delivery of bifunctional, immunomodulatory and cytotoxic protein, interferon (IFN) β enhances selective post-surgical infiltration of CD8 T cells and directly induces cell-cycle arrest in tumor cells. However, IFN has been known to upregulate program cell death ligand 1 (PD-L1) expression on tumor cells, thus hindering the immunomodulatory function of IFN. Based on the recent findings that: blocking PD-L1 induced by IFNβ treatment eradicates established tumors; tumor suppressor, phosphatase and tensin homolog (PTEN) loss promotes immune resistance; and our exciting preliminary data on: MSC-IFNβ mediated upregulation of PD-L1 in vivo; and local delivery of ScFv-PDL1, we will create bimodal MSC expressing ScFv-PDL1 and IFNβ and test them in syngeneic PTEN wild type (wt.) and mutant GBM models of resection. To ease clinical translation and ensure safety of our approach, we will ultimately engineer clinical grade human MSC to co-express ScFv-PDL1/IFN β and HSV-thymidine kinase (TK) and test our approach in GBM tumors generated from patient derived GBM lines in humanized NSG mice. The incorporation of genetically engineered imaging markers markers into MSC and GBMs will allow us to follow MSC fate and efficacy in vivo and thus to fine tune the proposed approaches. The overall goal of this proposal is thus to immune profile genetically distinct GBM resection models and to assess rationale based therapeutic efficacy of immunomodulatory agents. Once validated, we will initiate a clinical study in which at the time of brain tumor surgery, the main tumor mass will be removed and encapsulated bimodal MSC will be introduced to enhance tumor cell eradication. This will have a major impact in saving the lives of brain cancer patients.