Microvesicle Dynamics in B-Cell Chronic Lymphocytic Leukemia Tumor Microenvironme

描述(由申请人提供)：肿瘤细胞影响其邻近的间质，为生存和临床进程的进展创造有利的生态位。到目前为止，恶性细胞如何改变周围组织中其他类型细胞的功能在很大程度上是未知的。新的证据表明，由恶性细胞产生的微泡(MV)具有生物学活性，因为它们能够对宿主基质细胞产生亲密和远程影响。最近，我们发现来自B细胞慢性淋巴细胞白血病血浆的循环MV是CLL微环境中独特的通讯媒介。我们检测到：(A)大多数CLL血浆中含有升高的MV；以及(B)CLL早期以“血小板来源”(PD)MV(CD61+)为主的表型转变为晚期以“白血病B细胞来源”(LD)MV(CD19+)为主。最近，我们检测到CLL B细胞可以自发地或在刺激下在体外产生LD MV，而不受疾病阶段(RAI阶段)的影响。此外，我们发现CLL MV和CLL基质细胞的相互作用导致CLL-骨髓基质细胞(BMSC)AKT信号通路的强健激活，导致促进白血病B细胞生存的血管内皮生长因子(VEGF)的产生，以及Cyclin D1和c-myc的持续增加，至少部分是通过传递Axl受体酪氨酸激酶(RTK)。相比之下，增加了 血管内皮生长因子在MV暴露时也会发生在正常的骨髓间充质干细胞中，但发生在非常微小或降低的水平。有趣的是，我们检测到了正常和CLL BMSC之间的内在功能差异，这些差异包括：(I)CLL BMSC对MV介导的细胞内信号调节高度反应；(Ii)表达异常激活的信号成分，例如Ax1及其下游的c-Src，参与细胞的增殖和生存。此外，CLL BMSC表达更高水平的Src同源磷酸酶-2(SHP-2)，据报道，SHP-2对Src信号通路具有正向调节作用。根据我们的发现，这一建议的中心假设是CLL中MV的产生是一个动态过程，循环MV在CLL发病中起着关键作用，这是由于MVS通过促进CLL的进展而重新编程BMSC功能的独特能力的结果。因此，中和循环中的MV或抑制白血病B细胞产生MV和/或阻止MV与CLL间质的相互作用可能会阻止疾病的进展，最终可能适用于其他人类恶性肿瘤。 我们的假设将通过三个特定的目标来验证：(1)研究CLL B细胞在体外产生MV的动力学；(2)研究体内MV产生的动力学，并建立CLL MV参数与CLL进展和治疗结果的关系；(3)探讨CLL BMSC功能异常的机制。我们将研究纯化的CLL B细胞体外产生MV的动力学，以及CLL预后参数或其他体外刺激是否对LD-MV的产生至关重要。我们将对CLL血浆中MV产生的动力学及其与预后和治疗时间的关系进行连续的研究。我们还将研究在两个化学免疫治疗(CIT)临床试验中，血浆MV水平和表型参数是否可以预测CLL患者的治疗结果。最后，利用生化和遗传学的方法，我们将剖析CLL BMSC中异常信号的机制以及Axl RTK在BMSC功能调节中的作用。使用这些方法，我们将能够通过更明确地了解CLL在体外和体内产生MV的动力学、循环血浆MV预测CLL患者治疗结果的能力、CLL BMSC中异常信号的性质以及MV参数如何与疾病进展相关联来解决我们的中心假设。

DESCRIPTION (provided by applicant): Tumor cells influence their neighboring stroma to create a favorable niche for survival and advancement of the clinical course. To date, how the malignant cells alter the function of other cell types in surrounding tissues is largely unknown. Emerging evidence suggests that microvesicles (MV) produced by malignant cells are biologically active as a result of their ability to have both intimate and remote influences on hos stromal cells. Recently, we discovered circulating MV from B-Cell Chronic Lymphocytic Leukemia plasma are unique mediators of communication within the CLL microenvironment. We detected: (a) majority of CLL plasma contained elevated levels of MV; and (b) a phenotypic shift from predominantly "platelet-derived" (PD) MV (CD61+) in early stages of CLL toward a more "leukemic B-cell derived" (LD) MV (CD19+) during advanced stages. Most recently, we have detected that CLL B-cells can spontaneously or with stimulation produce LD MV in vitro irrespective of disease stages (Rai stage). In addition, we have shown that CLL MV and CLL stromal cell interaction results in robust activation of the AKT signaling pathway in CLL-bone marrow stromal cells (BMSC), leading to enhanced production of vascular endothelial growth factor (VEGF), a facilitator of leukemic B-cell survival, and sustained increases in cyclin D1 and c-myc, at least in part, by delivering Axl receptor tyrosine kinase (RTK). In contrast, increase in VEGF upon MV exposure also occurs with normal BMSC but at very subtle or diminished levels. Interestingly, we have detected intrinsic functional differences between normal and CLL BMSC that include: (i) CLL BMSC are hyper responsive to MV-mediated modulation of intracellular signaling; and (ii) express aberrantly activated signaling components, e.g., Axl and its downstream c-Src, involved in cell proliferation and survival. In addition, CLL BMSC express higher levels of the Src homology phosphatase-2 (SHP-2), reported to positively regulate the Src-signaling pathway. Based on our findings, the central hypothesis of this proposal is that MV generation in CLL is a dynamic process and that the circulating MV play a critical role in CLL pathogenesis as a result of MVs' unique ability to reprogram BMSC function with facilitation of CLL progression. Thus, neutralization of MV from circulation or inhibition of MV generation by the leukemic B-cells and/or prevention of MV interaction with the CLL stroma may halt disease progression and ultimately may apply to other human malignancies. Our hypothesis will be tested by addressing three specific aims: (1) Study the dynamics of MV generation in vitro by CLL B-cells; (2) Study the in vivo dynamics of MV generation and establish the relationship of CLL MV parameters to CLL progression and therapeutic outcome; (3) Interrogate the mechanism of aberrant function in CLL BMSC. We will study dynamics of in vitro generation of MV by purified CLL B-cells and whether CLL prognostic parameters or other in vitro stimulations are critical for the generation of LD-MV. We will study sequentially the dynamics of MV generation in CLL plasma and their relationship with prognostics and time to therapy. We will also study whether plasma MV levels and phenotype parameters predict therapeutic outcome in CLL patients being treated on two chemoimmunotherapy (CIT) clinical trials. Finally, using biochemical and genetic approach we will dissect mechanism of aberrant signaling in CLL BMSC and role of the Axl RTK in modulation of BMSC functions. Using these approaches we will be able to address our central hypothesis by acquiring more definitive knowledge of the dynamics of MV generation in CLL both in vitro and in vivo, ability of the circulating plasma MV to predict therapeutic outcome in CLL patients, nature of aberrant signaling in CLL BMSC and how the MV parameters associate with disease progression.