Immunoglobulin Free Light Chains and GAGs Mediate Multiple Myeloma Extracellular Vesicles Uptake and Secondary NfκB Nuclear Translocation

Abstract

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. Monoclonal plasma cells often secrete high amounts of immunoglobulin free light chains (FLCs) that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles (EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS) serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs with anti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished after anti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure. The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular re-distribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches.

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

“…Moreover, heparin has been shown to directly interact with extracellular vesicles and heparin inhibits vesicle binding to target cells (11,14). In another study, exogenous heparin or treatment of cells with heparan sulfate degrading enzymes reduced exosome-target cell interaction by ϳ50% (12).…”

“…Although the functional effects of exosomes rely on their interaction with, and subsequent delivery of their cargo to target cells, the mechanisms mediating exosome-cell interactions remain unclear. Studies have shown that heparin can block interactions between extracellular vesicles and cells and that heparan sulfate proteoglycans on glioblastoma cells facilitates extracellular vesicle uptake (11)(12)(13)(14). This uptake was blocked by the addition of exogenous heparin (a highly sulfated glycosaminoglycan mimetic of heparan sulfate), which can compete with cell surface heparan sulfate to bind exosomes.…”

Interaction of Extracellular Vesicles with Si Surface Studied by Nanomechanical Microcantilever Sensors

“…Ten mL of serum free RPMI 1640, supplemented with 1% penicillin/streptomycin and 1% glutamine, was added to each plate. A serial centrifugation protocol previously described [21] was used to isolate EVs produced by 2 × 10 7 cells from serum free medium, after 24 h incubation. The quantity of isolated EVs obtained by serum-free culture medium is greatly increased while retaining EV biophysical and size properties [22].…”

Extracellular vesicle cross-talk in the bone marrow microenvironment: implications in multiple myeloma

“…through the measurement of their acetylcholinesterase activity. The highest EV amount was found in MM patients in contrast to healthy donors and patients with monoclonal gammopathy of undetermined significance (MGUS) [112]. Moreover, exosome secretion from MM cells was shown to be dramatically increased by heparanase whose expression is up-regulated in aggressive cancer cells including MM cells, implying that heparanase may be one of the factors causing elevated secretion of exosomes [113].…”

“…Umezu et al [88] found that MM cells under hypoxic conditions secreted more exosomes with increased levels of miR-135b which induced increased expression of HIF-1α in endothelial cells, leading to enhanced angiogenesis. Moreover, serum EVs obtained from MM patients clearly promoted the proliferation of human vascular endothelial cells as compared to those from healthy donors or MGUS patients [112]. A very recent paper has suggested that fibronectin on the surface of MM cell-derived exosomes binds to heparin sulfate on endothelial cells thereby mediating MM exosome-BM cell interactions [131].…”