Engineered and Encapsulated Stem Cells for Resected Brain Tumors

总结 胶质母细胞瘤（GBM）是成人中最常见的原发性脑肿瘤，预后极差。给出了 肿瘤切除术在GBM治疗临床护理中发挥的中心作用，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 基于免疫的治疗GBM。我们最近开发了同基因原位小鼠GBM模型， 肿瘤切除术，并显示肿瘤减积导致髓源性抑制因子的实质性减少， 细胞（MDSC）和同时募集CD 4/CD 8 T细胞进入切除腔。在本提案中，我们 将首先从遗传上不同的目前可用的小鼠GBM系产生GBM切除模型， 分析肿瘤减积前后浸润到肿瘤微环境中的免疫细胞的概况。而 切除原发性肿瘤已显示出临床益处，全身递送或直接注射治疗性肿瘤的药物组合物， 在肿瘤切除腔中的药物提供了有限的额外益处。在我们以前的研究中，我们有 广泛证明了局部递送的受体靶向的工程化成体干细胞具有治疗性 干细胞的合成细胞外基质（sECM）包封是必要的，以防止它们的快速生长。 小鼠GBM肿瘤切除腔中移植后的“洗出”。在我们最近发表的研究中，我们 已经表明sECM包裹的间充质干细胞（MSC）介导双功能的局部递送， 免疫调节和细胞毒性蛋白干扰素（IFN）β增强术后CD 8 T细胞选择性浸润 细胞并直接诱导肿瘤细胞的细胞周期停滞。然而，已知IFN γ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IFN γ的免疫调节功能。 基于最近的研究结果：阻断IFNβ治疗诱导的PD-L1可根除已建立的肿瘤; 肿瘤抑制因子，磷酸酶和张力蛋白同源物（PTEN）的损失促进免疫抵抗;和我们令人兴奋的 关于MSC-IFNβ介导的PD-L1体内上调和ScFv-PDL 1局部递送的初步数据，我们将 创建表达ScFv-PDL 1和IFNβ的双峰MSC，并在同基因PTEN野生型（wt.）和 切除的突变GBM模型。为了简化临床翻译并确保我们方法的安全性，我们将 最终工程化临床级人MSC以共表达ScFv-PDL 1/IFN β和HSV-胸苷激酶（TK） 并在人源化NSG小鼠中从患者来源的GBM系产生的GBM肿瘤中测试我们的方法。的 将遗传工程成像标记物标记物掺入MSC和GBM将使我们能够跟踪 MSC在体内的命运和功效，从而微调所提出的方法。本提案的总体目标是 因此，免疫概况遗传上不同的GBM切除模型，并评估基于治疗的基本原理， 免疫调节剂的功效。一旦证实，我们将启动一项临床研究，在大脑 在肿瘤手术中，主要肿瘤块将被移除，并且包封的双峰MSC将被引入， 增强肿瘤细胞根除。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.