Stromal cells support the survival of human primary chronic lymphocytic leukemia (CLL) cells through Lyn-driven extracellular vesicles

Abstract

IntroductionIn chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support.MethodsWe compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells.ResultsLyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context.ConclusionOur data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix.

Analysis of Primary Chronic Lymphocytic Leukemia Cells’ Signaling Pathways

“…In our previous work on CLL, we focused our attention on modulators of lymphocyte differentiation such as members of the Ikaros family of zinc finger proteins [ 18 ]. These transcription factors modify the expression of genes by binding and remodeling chromatin, thereby regulating nuclear activity and controlling the lineage specification of B cells [ 31 ].…”

“…Despite this feature, CLL cells die rapidly when cultured in vitro alone without stromal cells. Co-cultures of leukemic cells with supporting stroma in vitro emphasize the importance of cellular interactions and microenvironmental signals for the proliferation and resistance of neoplastic cells to cell death [ 17 , 18 ].…”

B cell receptor signaling and associated pathways in the pathogenesis of chronic lymphocytic leukemia

“…Although LYN is well-known to balance BCR signaling, LYN activation seems to be dispensable in the development of CLL, since B cell specific gain- and loss-of-function mutations of LYN showed no significant changes in CLL progression in Eµ-TCL1 mice ( 61 , 62 ). However, several studies suggest an emerging new role of LYN as a crucial regulator within the CLL tumor microenvironment supporting leukemic cell growth and CLL progression ( 61 , 63 , 64 ). For example, LYN-deficiency in macrophages reduces their capability to support CLL cell survival ( 61 ).…”

Extracellular Vesicles in Chronic Lymphocytic Leukemia: Tumor Microenvironment Messengers as a Basis for New Targeted Therapies?

“…Hence, CLL EVs activate stromal cells and cause them to differentiate into CAFs. The EVs secreted by these activated cells then provide the CLL cells with survival signals, this vesiculation being dependent on LYN kinase [ 68 ]. MSC-derived EVs protect CLL cells from spontaneous apoptosis by inducing not only the expression of anti-apoptotic proteins but also cell migration, drug resistance, and BCR signaling activation [ 54 ].…”