Nanoparticle modified Human Fat Derived Mesenchymal Stem Cells for Brain Cancer

 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。越来越多的证据表明，一小部分细胞，即脑肿瘤起始细胞（BTIC），负责疾病的治疗抗性。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），已发现这些细胞优先迁移并定位在癌细胞上。此外，MSC可以被工程化以合成和释放抗肿瘤蛋白，如影响BTIC的骨形态发生蛋白4（BMP 4）。MSC可以从骨髓（BM-MSC）和脂肪组织（AMSC）获得。BM-MSC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长而有效性降低。与BM-MSC不同，AMSC的供应更丰富，更容易从脂肪组织获得，表达更高水平的与细胞迁移有关的表面标志物，并且已显示出抵抗致癌转化。因此，AMSC可能是更好的选择。病毒基因递送方法虽然通常用于修饰AMSC，但与插入诱变和免疫原性相关，因此，用于人类患者的翻译能力可能有限。可生物降解的聚合物纳米颗粒能够有效地将非病毒基因递送到多种细胞类型，包括人类AMSC（hAMSC），同时避免了病毒的典型问题。在这项资助中，我们提出了一种新的技术，将联合收割机新鲜提取的脂肪组织（F.A.T.）和纳米颗粒，以非病毒工程化包含在F.A. T中的原代hAMSC，而无需预先培养以分泌抗癌蛋白，同时保持细胞向肿瘤细胞迁移的能力。我们的总体假设是，从F.A.T.在临床相关的体内人GBM模型中保持其肿瘤抑制特性。为了验证这一假设，我们将追求以下具体目标：（1）将感兴趣的外源基因有效地递送到新鲜提取的脂肪组织（F.A.T.）通过冻干的可生物降解的纳米颗粒从患者体内回收。(2)确定纳米颗粒修饰的分泌BMP 4的hAMSC是否保留体外抗胶质瘤作用。(3)在体内鼠模型中，确定纳米颗粒修饰的BMP 4分泌型hAMSC治疗与靶向放射疗法组合对人GBM的安全性和有效性。目的1涉及研究和优化将纳米颗粒与F.A.T.结合的独特技术。从我们的病人。对于目标2和3，使用已经在商业hAMSC中测试的纳米颗粒，我们现在将研究在添加纳米颗粒之前已经分离和培养的原代hAMSC的修饰。我们的团队已经开发出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GSC衍生的GBM的哺乳动物异种移植模型，其最好地概括了人GBM。此外，在体内研究中，动物受试者将使用小动物放射研究平台（SARRP）进行放射治疗，从而在小鼠规模上为人类重现传统的适形射束放射治疗。这项研究的结果将确定纳米颗粒修饰的hAMSC是否可以提供一种不仅对GBM患者，而且对许多类型的原发性和转移性脑癌安全有效的治疗。对于未来的临床应用，纳米颗粒可以在培养几天后给予从患者脂肪中获得的hAMSC，或者然后IV给予作为治疗，或者给予F.A.T.并在手术过程中重新给予所得到的工程化hAMSC。这可能会导致临床试验，从而为治疗脑癌患者提供革命性的新方法，并促进个性化医疗。

 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 including 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. There is increasing evidence of a small subset of cells, brain tumor initiating cells (BTICs) that are responsible for the disease's treatment resistance. 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 to migrate preferentially to and home in on cancer cells. Moreover, MSCs can be engineered to synthesize and release anti-tumor proteins, like bone morphogenic protein 4 (BMP4), which affects BTICs. MSCs can be obtained from bone marrow (BM- MSC) and adipose tissue (AMSCs). BM-MSCs are difficult to obtain, have limited ex vivo proliferation capacity, and decrease in effectiveness with donor age. Unlike BM-MSCs, AMSCs are more abundant in supply, easier to obtain from fat tissue, express higher levels of surface markers implicated in cell migration, and have been shown to resist oncogenic transformation. AMSCs may therefore be a better option. The viral gene delivery method, though commonly used to modify AMSCs, is associated with insertional mutagenesis and immunogenicity, and, therefore, has potentially limited translational ability for use in human patients. Biodegradable, polymeric nanoparticles enable effective non-viral gene delivery to multiple cell types, including human AMSCs (hAMSCs), while avoiding the problems typical of viruses. In this grant, we propose a novel technology to combine Freshly-extracted Adipose Tissue (F.A.T.) and nanoparticles to non-virally engineer the primary hAMSCs contained within F.A.T without prior culture to secrete anti-cancer proteins while maintaining the cells' ability to migrate toward tumo cells. Our overall hypothesis is that nanoparticle-modified hAMSCs obtained from F.A.T. retain their tumor suppressive characteristics in a clinically relevant in vivo human GBM model. To test this hypothesis, we will pursue the following specific aims: (1) To effectively deliver exogenous genes of interest to Freshly-extracted Adipose Tissue (F.A.T.) from patients via lyophilized biodegradable nanoparticles. (2) To determine if nanoparticle-modified BMP4-secreting hAMSCs retain an anti-glioma effect in vitro. (3) To determine the safety and efficacy of nanoparticle-modified BMP4-secreting hAMSC treatment in combination with targeted radiation therapy on human GBM in an in vivo murine model. Aim 1 involves investigation and optimization of a unique technology of combining nanoparticles with F.A.T. from our patients. For aims 2 and 3, using nanoparticles already tested in commercial hAMSCs, we will now investigate the modification of primary hAMSCs that have been isolated and cultured prior to adding the nanoparticles. The techniques to be used in vitro and in vivo in this proposal have been developed and further characterized by our teams. 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 GSC-derived GBM, which bests recapitulates human GBM. Further, in the in vivo studies, animal subjects will be treated with radiation using Small Animal Radiation Research Platform (SARRP), thus recreating traditional conformal beam radiotherapy for humans on the scale of a mouse. The results of this study will determine whether nanoparticle-modified 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. For future clinical application, the nanoparticles could be administered either to hAMSCs obtained from patient fat after culturing for a few days or then given IV as a treatment or to F.A.T. with the resulting engineered hAMSCs re- administered during surgery. This may lead to clinical trials, with a revolutionary new way of treating patients with brain cancer and facilitating personalized medicine.