BMP4 Engineered Mesenchymal Stem Cell Therapy for Glioblastoma

 描述(申请人提供)：胶质母细胞瘤(GBM)是成人最常见的原发脑肿瘤，占所有原发脑肿瘤的20%。尽管目前最好的治疗方法包括手术和化疗，但GBM的中位存活率仅为14.6个月。这种发病率和死亡率的一个重要原因是基底膜侵犯正常脑实质的能力，使局部治疗无效。为了使治疗有效，需要针对这些入侵细胞。一种有希望的方法涉及使用间充质干细胞(MSCs)，这是我们和其他人发现的，可以优先迁移到癌细胞。此外，骨髓间充质干细胞可以合成和释放抗肿瘤蛋白，如骨形态发生蛋白4(BMP4)，它已被发现影响脑肿瘤起始细胞(BTICs)。MSCs可从骨髓(BM-MSC)和脂肪组织(AMSC)获得。BM-MSCs的应用受到了限制，因为这些细胞很难获得，体外增殖能力有限，而且随着供者年龄的增加，效果会下降。因此，资产证券化可能是一个更好的选择。在这项资助中，我们建议使用一种新的人MSCs来源，来自我们患者的脂肪组织，并对这些细胞进行基因修饰，以分泌BMP4用于治疗GBM。与BM-MSCs相比，人AMSCs(HAMSCs)提供了一种治疗上可比的细胞来源，更容易获得，并具有更好的体外扩张性。我们的总体假设是，表达BMP4的病毒修饰的hAMSCs联合辅助放射治疗构成了治疗颅内GBM的有效方法。为了实现这些目标，我们将追求以下特定目标：(目标1)确定病毒修饰的分泌BMP4的原代hAMSCs在体外的肿瘤趋向性、内皮黏附、血脑屏障穿越能力和抗胶质瘤反应-我们已经用商业的hAMSCs证明了这一点，我们建议现在用新鲜提取的脂肪组织(F.A.T.)来做这项工作；(目的2)在活体小鼠模型中确定病毒修饰的分泌BMP4的hAMSCs联合靶向放射治疗对人GBM的安全性和有效性。我们的团队和合作者已经开发出了在体外和体内使用的技术，并进一步确定了这些技术的特征。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。活体研究将采用哺乳动物异种移植模型，植入人类BTIC来源的GBM，这是最好的概括人类GBM。此外，我们将使用小动物辐射研究平台(SARRP)，这是我们的团队和合作者开发和使用的一种新型设备，它可以向荷瘤小鼠提供靶向放射治疗，类似于人类的共聚焦射线治疗。SARRP能够以0.2毫米的精度聚焦辐射束，在老鼠的规模上为人类重现放射治疗。除了我们对商业hAMSCs的实验外，我们还将在手术中从人类患者那里获得原代hAMSCs，并测试它们的抗肿瘤效果，以最大限度地提高本研究的临床可译性。这项研究的结果将证明hAMSCs是否可以提供一种安全有效的治疗方法，不仅适用于GBM患者，而且适用于多种类型的原发和转移性脑癌。这项研究的结果可能会导致临床试验，为治疗布林癌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.