Investigate the role of extracellular vesicles in promoting the progression of pulmonary lymphangioleiomyomatosis

项目摘要。肺淋巴管肌瘤病(LAM)是一种转移性、未被充分研究的肉瘤。 主要影响女性，表现为肿瘤内平滑肌样细胞的增殖 肺部。LAM是由导致mTOR过度激活的TSC1/2功能突变丢失引起的，因此， MTOR抑制剂雷帕霉素被批准用于LAM。然而，尽管雷帕霉素可以稳定肺功能， 治疗停止后，肺功能恢复进行性下降，导致呼吸功能不全。 也没有其他药物被批准用于LAM。因此，LAM新的治疗靶点的发现是 这是急需的。 该项目的目标是：(1)确定细胞外小泡(EV)在LAM转移中的作用；(2) 为LAM提供针对电动汽车的概念验证。 电动汽车在LAM中的作用尚未被探索，但我们已发表的和初步的研究表明， (A)EV货物通过增强肿瘤干细胞(CSC)的特性和存活率促进LAM转移 循环LAM细胞和肺重塑以及(B)雷帕霉素影响EV，可能导致增强 LAM细胞在肺中的种植。 这些数据导致了我们的中心假设TSC1/2-零细胞衍生的EV货物，含有SOX-10， 整合素和c-Src通过增加CSC的可塑性和循环LAM的存活率增加LAM的转移 细胞和通过驻留肺成纤维细胞介导的肺细胞外基质(ECM)重塑，从而增强 LAM细胞肺种植。雷帕霉素通过TSC1/2缺失细胞提高EV的产量和质量 也许可以解释接受雷帕霉素类似物治疗的患者循环中LAM细胞清除更快的原因 同时，对LAM肺转移有不利的促进作用。这一假设的一个关键翻译推论是 LAM对抑制EV途径和靶向EV可能会改善预后 雷帕霉素治疗的患者。 为了验证这一假设，我们将确定：目的1.EV-Cargo对CSC可塑性和LAM存活率的影响 目的2.EV-Cargo对LAM细胞肺重塑和肺种植的影响；以及目的3.对EV-Cargo的影响 EV摄取或EV生物生成的抑制剂单独或与雷帕霉素联合应用对肺重塑的影响 和LAM体内转移。 我们的方法包括：测试EV-Cargo对循环LAM细胞影响的培养系统，新型LAM 结合纵向无创活体成像的小鼠模型，并对患者样本进行研究 证实体外和小鼠的数据，并建立EV货物作为LAM进展的生物标志物。 该项目将通过建立EV是LAM的新治疗靶点来影响该领域并提供 为LAM改变已批准的EV靶向药物的用途的基金会。

Project Summary. Pulmonary lymphangioleiomyomatosis (LAM) is metastatic, understudied sarcoma, which predominantly affects women, and manifests as proliferation of tumor smooth muscle-like cells within the lungs. LAM is caused by TSC1/2 loss of function mutations that lead to mTOR hyperactivation, therefore, mTOR inhibitor rapamycin is approved for LAM. However, although rapamycin stabilizes the lung function, after therapy cessation the progressive decline in the lung function resumes leading to respiratory insufficiency and there are no other drugs approved for LAM. Thus, the discovery of new therapeutic targets for LAM is critically needed. The objectives of this project are: (1) determine a role of extracellular vesicles (EV) in LAM metastasis and (2) provide the proof-of-concept for targeting EV for LAM. A role of EV in LAM has not yet been explored, however, our published and preliminary studies indicate that (a) EV cargo contribute to LAM metastasis via enhancing cancer stem-cell (CSC) characteristics and survival of circulating LAM cells and lung remodeling and (b) rapamycin affects EV, possibly, leading to enhanced seeding of the lungs by LAM cells. These data lead to our central hypothesis that TSC1/2-null-cell-derived EV cargo, containing SOX-10, integrins, and c-Src, increases LAM metastasis via increasing CSC plasticity and survival of circulating LAM cells and via lung extracellular matrix (ECM) remodeling mediated by resident lung fibroblasts, which enhances lung seeding by LAM cells. Rapamycin enhances EV production by TSC1/2-null cells and their quality, which may explain faster clearance of LAM cells from the circulation of patients treated with rapamycin analogs but simultaneously and adversely enhances LAM lung metastasis. A key translational corollary of this hypothesis is that LAM will be sensitive to inhibition of the EV pathway and targeting EV may improve outcomes in rapamycin treated patients. To test this hypothesis we will determine: Aim 1. The effect of EV-cargo on CSC plasticity and survival of LAM cells; Aim 2. Impact of EV-cargo on lung remodeling and lung seeding by LAM cells; and Aim 3. The effect of inhibitors of EV uptake or EV biogenesis, individually or in combination with rapamycin, on lung remodeling and LAM metastasis in vivo. Our approaches include: the culture systems to test impact of EV-cargo on circulating LAM cells, novel LAM mouse models in conjunction with longitudinal non-invasive live imaging, and studies with patient samples to corroborate in vitro and mouse data and establish EV cargo as biomarker of LAM progression. This project will impact the filed by establishing EV is a new therapeutic target for LAM and providing foundation for repurposing approved EV-targeting drugs for LAM.