BMP4 Engineered Mesenchymal Stem Cell Therapy for Glioblastoma

 描述（由申请人提供）：胶质母细胞瘤（GBM）是成人中最常见的原发性脑肿瘤，占所有原发性脑肿瘤的20%。GBM的中位生存率仅为14.6个月，尽管目前最好的治疗方法包括手术和放化疗。这种发病率和死亡率的一个重要原因是GBM侵入正常脑实质的能力，使得局部治疗无效。为了使治疗有效，需要靶向这些入侵细胞。一种有希望的方法涉及使用间充质干细胞（MSC），我们的小组和其他人发现它优先迁移到癌细胞。此外，MSC可以被工程化以合成和释放抗肿瘤蛋白，例如骨形态发生蛋白4（BMP 4），其已被发现影响脑肿瘤起始细胞（BTIC）。MSC可以从骨髓（BM-MSC）和脂肪组织（AMSC）获得。BM-MSC的使用受到限制，因为这些细胞难以获得，具有有限的离体增殖能力，并且随着供体年龄的增加有效性降低。因此，AMSC可能是更好的选择。在这项授权中，我们建议使用一种新的人类MSC来源，即来自我们患者的脂肪组织，并对这些细胞进行遗传修饰以分泌BMP 4用于治疗GBM。与BM-MSC相反，人AMSC（hAMSC）提供了治疗上相当的细胞来源，其更容易获得并且具有更好的离体扩增性。我们的总体假设是，表达BMP 4的病毒修饰的hAMSC与辅助放疗组合构成了针对颅内GBM的有效治疗。（目的1）确定病毒修饰的分泌BMP 4的原代hAMSC在体外的肿瘤向性、内皮粘附、血脑屏障穿越能力和抗胶质瘤反应-我们已经用商业化的hAMSC证明了这一点，我们现在建议用新鲜提取的脂肪组织（F.A.T.）进行; (Aim 2）在体内鼠模型中确定病毒修饰的分泌BMP 4的hAMSC与靶向放射疗法组合对人GBM的安全性和功效。我们的团队和我们的合作者已经开发了本提案中体外和体内使用的技术，并对其进行了进一步表征。将利用微流体和纳米生物技术领域的新进展进行体外研究。体内研究将采用移植人BTIC衍生的GBM的哺乳动物异种移植物模型，其最好地概括了人GBM。此外，我们将使用小动物放射研究平台（SARRP），这是一种由我们的团队和合作者开发和使用的新型设备，它允许向荷瘤小鼠提供靶向放射治疗光束，类似于人类的共聚焦光束治疗。SARRP能够以0.2 mm的精度聚焦辐射束，在小鼠的规模上为人类重现放射治疗。除了我们对商业hAMSC的实验外，我们还将在手术中从人类患者中获得原代hAMSC，并测试其抗肿瘤功效，以最大限度地提高本研究的临床可转化性。这项研究的结果将证明hAMSC是否可以提供一种不仅对GBM患者安全有效的治疗方法，而且对许多类型的原发性和转移性脑癌也是如此。这项研究的结果可能会导致临床试验，为治疗癌症患者提供一种革命性的新方法。

 DESCRIPTION (provided by applicant): Glioblastoma (GBM) is the most common primary brain tumor in adults, and accounts for 20% of all primary brain tumors. GBM has a median survival rate of only 14.6 months despite current best treatment practices which include surgery and chemoradiation. A significant reason for this morbidity and mortality is the ability of GBM to invade normal brain parenchyma, making localized treatment ineffective. In order for treatment to be effective, these invading cells need to be targeted. One promising approach involves the use of mesenchymal stem cells (MSCs), which have been found by our group and by others to migrate preferentially to cancer cells. Moreover, MSCs can be engineered to synthesize and release anti-tumor proteins, such as bone morphogenic protein 4 (BMP4), which has been found to affect brain tumor initiating cells (BTICs). MSCs can be obtained from bone marrow (BM-MSC) and adipose tissue (AMSC). The use of BM-MSCs has been limited because these cells are difficult to obtain, have limited ex vivo proliferation capacity, and decrease in effectiveness with increasing donor age. AMSCs may therefore be a better option. In this grant, we propose to use a novel source for human MSCs, adipose tissue from our patients, and genetically modify these cells to secrete BMP4 for the treatment of GBM. In contrast to BM-MSCs, human AMSCs (hAMSCs) provide a therapeutically comparable source of cells which are more readily accessible and have better ex vivo expansibility. Our overall hypothesis is that virally-modified hAMSCs expressing BMP4 in combination with adjuvant radiotherapy constitute an effective treatment against intracranial GBM. To achieve these goals, we will pursue the following specific aims: (Aim 1) To determine the tumor tropism, endothelial adherence, blood brain barrier crossing capability, and anti-glioma response of virally-modified BMP4-secreting primary hAMSCs in vitro-we have shown this with commercial hAMSCs and we propose to do it now with Freshly extracted Adipose Tissue (F.A.T.); (Aim 2) To determine the safety and efficacy of virally-modified BMP4-secreting hAMSCs in combination with targeted radiation therapy on human GBM in an in vivo murine model. The techniques to be used in vitro and in vivo in this proposal have been developed and further characterized by our team and by our collaborators. In vitro studies will be conducted using new advancements in the fields of microfluidics and nanobiotechnology. In vivo studies will employ a mammalian xenograft model that engrafts human BTIC- derived GBM, which bests recapitulates human GBM. Additionally, we will use the small animal radiation research platform (SARRP), a novel device developed and used by our team and collaborators, which allows the delivery of targeted beams of radiation therapy to tumor-bearing mice analogous to confocal beam therapy in humans. The SARRP is capable of focusing a beam of radiation with an accuracy of 0.2 mm, recreating radiotherapy for humans on the scale of a mouse. In addition to our experiments on commercial hAMSCs, we will obtain primary hAMSCs intraoperatively from human patients and test their anti-tumor efficacy to maximize the clinical translatability of this study. The results of this stuy will demonstrate whether hAMSCs can provide a treatment that is safe and effective for not only patients with GBM, but many types of primary and metastatic brain cancers. The results of this study may likely lead to clinical trials, with a revolutionary new way of treating patients with brin cancer.