Reciprocal Interaction between Megakaryocytes and Myeloma Cells Results in Increased Survival and Proliferation of Myeloma Cells.
Authors: Yaccoby Shmuel , Saha Rinku , Kozlowska Ewa , Ge Yun , Ling Wen , Cottler-Fox Michele , Barlogie Bart , Tricot Guido 
Published: April 2020

Abstract
Megakaryocytes are often expanded in bone marrow (BM) areas infiltrated with myeloma (MM) cells while thrombocytopenia is uncommon in untreated MM patients. The aim of this study was to investigate the reciprocal interaction between primary megakaryocytes and MM cells. Cultures of megakaryocytes and their precursors were prepared by incubating mobilized peripheral blood in IMDM media supplemented with BSA, thrombopoeitin (TPO), IL-6 and IL-3. Following removal of all non-adherent cells, the remaining adherent megakaryocytes (&gt;90%) highly expressed c-MPL, CD41a and factor VIII, and had various degree of ploidy. We initially demonstrated that purified MM cell-conditioned media from 7 patients increased migration of megakaryocyte precursors across a 8 μM pore size membrane 3.6±0.9 fold (p=0.001), an effect that was inhibited by anti-CXCR4 neutralizing antibody (p&lt;0.003). To study whether TPO is produced in myelomatous BM, mesenchymal stem cells (MSCs) were cultured on the backside of 1 μM pore-size transwell inserts’ membranes while MM cells were incubated in the upper chamber of the inserts. Histologic examination demonstrated that the MSC’s cytoplasmic villi pass through the membrane pores, allowing contact with tumor cells. Using qRT-PCR, we demonstrated upregulation of TPO expression by 5±3 and 3±1 folds in co-cultured MSCs and MM cells, respectively. Co-culture of megakaryocyte precursors with CD138-selected MM cells stimulated their differentiation into mature megakaryocytes as determined by flow cytometry DNA content. This suggests that megakaryopoiesis and platelet maintenance are locally and systemically regulated in MM. To study the effect of megakaryocytes on tumor cells, purified MM cells from 14 patients were co-cultured with megakaryocytes in media containing FBS and TPO at an estimated ratio of 1000:1 for 5–7 days. Myeloma cells did not firmly adhere to megakaryocytes and were easily removed by gentle pipetting. Survival and proliferation of MM cells were determined by trypan blue exclusion, annexin V/PI flow cytometry, MTT assay and BrdU labeling index. MM cells in co-cultures with megakaryocytes had more viable cells (225,000±22,000 vs. 482,000±46,000, p&lt;0.0001), higher MTT OD reading (0.4±0.1 vs. 1.1±0.2, p&lt;0.03), fewer annexin V positive cells (79%±6 vs. 33%±5, p&lt;0.0008) and higher labeling index (2.1±0.5 vs. 6.7±1.3, p&lt;0.01) compared to MM cells cultured alone. Culture of MM cells in transwell inserts and megakaryocytes on the bottom of wells (non-contact conditions), partially supported survival and proliferation of MM cells, indicating that both soluble factors and physical contact are involved in MM-megakaryocyte interactions. Co-culture of MM cells from 5 patients with either megakaryocytes or osteoclasts resulted in similar stimulatory effects on MM cells. Since bortezomib is increasingly used for MM treatment and known to induce thrombocytopenia in patients with MM we evaluated its effect on isolated megakaryocytes using MTT assay. Bortezomib at concentrations of 0.2–1 nM effectively inhibited growth of megakaryocytes and MM cell lines, CAG and ARP-1, but not MSCs, suggesting an additional mechanism for its anti-MM response. We conclude that megakaryocytes are an important microenvironmental component directly supporting MM growth. Therapeutic interventions that interfere with MM-megakaryocyte interaction may help control MM.