Myeloma Derived Extracellular Vesicles Containing Endoplasmic Reticulum Remodel the Bone Marrow Microenvironment Towards a Pro-Tumoral Senescent Phenotype

Multiple myeloma (MM) is a tumor characterised by the generation of large quantities of immunoglobulin which undergoes protein folding and secretion through the endoplasmic reticulum (ER). Many studies have shown that primary MM cells have high ER stress (Lee et al, 2003 PNAS, Obeng et al 2006 Blood and Mimura et al, 2012 Blood,). Moreover, it has also been shown that serum from MM contains significantly more extracellular vesicles (EVs) than serum from normal patients (Caivano et al, 2015 Tumour Biol). Here we explore if these two observations are connected and determine whether primary MM cells export ER inside EVs and the impact this has on the tumor microenvironment. Primary MM and primary MM bone marrow stromal cells (BMSC) were isolated from the bone marrow of patients. To determine if MM secrete ER inside EV, patient derived MM and MM cell lines were transduced with rLV.EF.mCherry-ER lentivirus which fluorescently tags the ER. High-resolution imaging combined with image cytometry shows that CD38+ vesicles containing ER are formed by budding from the MM cell surface. Analysis of MM derived EVs using a combination of proteomics, confocal microscopy, image cytometry and dynamic light scattering shows that MM actively export ER in the large EVs, approximately 0.6micron-1.5micron in diameter. To track the recipient cell for large EV packaged ER in vivo, U266 MM cell line (transduced with rLV.EF.mCherry-ER9 lentivirus) was injected into the tail of NSG mice. BM was extracted from engrafted mice and various cell populations were analysed for increases in mCherry fluorescence, as evidence of MM derived ER uptake. Murine CD45-/CD31-Ter119-/CD105+/CD140a+ BMSC had increased mCherry fluorescence but not F4/80+/GR1-/CD115int BM macrophages or CD45-/Ter119-/CD31+ endothelial cells or CD45+ leukocytes. Two proteins detected at high levels in the proteomics analysis of large EV were endoplasmic reticulum oxidoreductin 1 (ERO1) and protein disulfide-isomerase (P4B4) which functionally catalyze the formation, breakage and rearrangement of disulfide bonds resulting in the production of superoxide. Next, we analysed large EVs containing ER for superoxide. MM derived ER+ and ER- large EVs were isolated by sorting for mCherry fluorescence (MM transduced with rLV.EF.mCherry-ER9 lentivirus) and Amplex Red assay confirmed that ER+ large EVs had increased levels of H2O2. In vivo analysis of the BMSC from MM engrafted NSG mice confirmed high oxidative stress as measured by increased H2DCFDA fluorescence. To determine the impact of MM derived ER containing EVs on the function of the BMSC isolated ER containing EVs were incubated with BMSC repeatedly for up to 7 days and senescent markers were assessed. Beta-galactosidase staining, p16ink4a gene expression and a senescence associated secretary phenotype (SASP) were all upregulated in BMSC cultured with MM derived ER+ large EV and not ER- large EV. To determine if MM induced BMSC senescence in vivo we injected U266 and primary MM into NSG mice, humanised NSG mice were used as a control. Post MM engraftment, animals were sacrificed and BMSC were isolated by cell sorting for CD45-/CD31-Ter119-/CD105+/CD140a+ cells and senescent markers were analysed by real-time PCR. p16ink4a and p21 were both upregulated in BMSC from U266 and primary MM engrafted NSG mice and not from humanised NSG mice. Knockdown of p16 in BMSC prevents ER+ large EV from inducing a SASP and conditioned media had no effect on MM proliferation compared to conditioned media from ER+ large EV treated BMSC. Finally, we used an NSG mouse model whereby we transplanted p16ink4a KD BMSC or control KD BMSC with MM cells subcutaneously into the flank. MM combined with p16ink4a KD BMSC has reduced tumor volume compared with animals with control KD BMSC. Data indicates that MM secrete ER in large EV and that MM derived EVs containing ER function as a signal which then changes the physiology of BMSC, towards a senescent phenotype which in turn promotes malignant plasma cell survival and proliferation.