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.

Public Health Relevance

This grant will develop new and comprehensive approaches to further dissect the mechanism of how cancer cells influence the microenvironment so as to favor the disease progression in B-cell Chronic Lymphocytic Leukemia (CLL). This leukemia is the most common leukemia in North America and is thus an important health issue for this country. We were the first to identify a novel component in CLL, namely the circulating small, extracellular vesicles termed microvesicles in CLL plasma. In this proposal we hypothesize that leukemic B-cells from high risk CLL patients spontaneously generate higher level of microvesicles in contrast to low risk CLL. However, other known prognostic parameters and/or other stimuli may also induce CLL B-cells to generate microvesicles. Moreover, interaction of the leukemic B-cells with other cell types in bone marrow stromal microenvironment including megakaryocytes may increase MV levels in CLL circulation. We will continue to study the total plasma levels and phenotype populations of the microvesicles in sequential, follow up (at every 6 month interval) plasma samples of CLL patients and their potential for use as predictors of disease progression and time to therapy. In addition, we will also assess whether plasma levels and subtypes of MV in CLL patients entering onto chemoimmunotherapy clinical trials can predict therapeutic response and duration of therapy by conducting sequential studies. Finally, we will address why CLL BMSC are hyper reactive to microvesicle-mediated modulation and express aberrantly activated signaling components which we believe is related the generation of a more favorable environment for disease progression. Overall, this study will generate knowledge that could help direct and/or modify our approaches for understanding and preventing disease progression and to better predict clinical outcomes for our CLL patients. Because plasma MV likely exist in many other tumors, we are also hopeful that these studies will ultimately impact on our understanding of the functional consequences of increased microvesicle generation in the host microenvironment in human malignancies, strategies for interrupting the influence of microvesicles on disease progression and thus improve the outcome for many other types of cancer patients.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Tumor Microenvironment Study Section (TME)
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Howcroft, Thomas K
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Mayo Clinic, Rochester
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