Platelets/Megakaryocytes Are Critical Regulators of Tumor Progression in Multiple Myeloma
Authors: Takagi Satoshi , Tsukamoto Shokichi , Kawano Yawara , Moschetta Michele , Mishima Yuji , Kokubun Katsutoshi , Manier Salomon , Salem Karma , Huynh Daisy , Sacco Antonio , Roccaro Aldo M , Johnson Kelly Elizabeth , Battinelli Elisabeth M. , Ghobrial Irene M. 
Published: October 2019

Abstract
Platelets play important roles in both physiological and pathological conditions. For instance, platelets may facilitate cancer metastasis by protecting circulating tumor cells (CTCs) from shear stress and immunological assault during their intravascular phase and by supporting CTCs extravasation. Moreover, soluble factors released from activated platelets enhance proliferation and migration of endothelial cells, thereby promoting tumor angiogenesis. Correlations between increased platelet counts and shorter survival time have been described for many solid tumors. However, the role of platelets/megakaryocytes (MKs) in regulating tumor progression and dissemination in Multiple myeloma (MM) has not been previously examined.
We measured the platelet aggregation-inducing abilities of MM cell lines and found MM cell lines (MM.1S, OPM-2, KMS-11, U266, and H929) induced platelet aggregation. This was not observed using leukemia cells (K562) and primary healthy donor-derived peripheral blood mononuclear cells. We next investigated whether platelets interact with MM cells within the bone marrow (BM) niche, in vivo. In order to identify platelets/MKs, femurs were harvested from MM.1S GFP+-harboring mice and stained with a DyLight649-conjugated anti-GPIb-beta antibody. The femurs were rendered transparent by the CUBIC method as previously described (Cell 159, 911-24 (2014)) and examined using confocal microscopy; and found that platelets/MKs co-localized within MM.1S GFP+-infiltrated BM niches. Further confirmation of the co-localization of MM cells and MKs was performed using immunohistochemistry (CD138+ and GPIb-alpha+). We next investigated the effect of platelets on MM cell proliferation: MM cells, where co-cultured with platelets and found that platelets enhanced the proliferation rate of MM cells in a platelet number-dependent manner, as shown by using BrdU (p<0.001). The observed platelet-dependent induction of MM cells proliferation was also demonstrated by using platelet aggregation releasate.
We next performed RNA sequencing studies using MM cells and platelet-interacting MM cells, and demonstrated that platelet-interacting MM cells presented with enrichment for signaling pathways such as TGF-beta as compared to MM cell control. We performed cytokine array analysis and confirmed that several cytokines, including TGF-beta, ENA-78, SDF-1, and TARC, were released from platelets activated by co-culture with MM cells.
We next investigated the effect of platelets on MM tumor growth in vivo, by using bioluminescence imaging (BLI). MM cells were pre-cultured with platelets for 2 days, washed and then intravenously injected into SCID/Beige mice (n=6). We found a significantly higher MM tumor burden in mice injected with MM cell pre-cultured with platelets, as compared to control mice where buffer treated MM cells were used (p<0.01), together with reduced survival (p=0.0011). These findings indicate the ability of platelets to enhance MM cell growth in vivo. To further confirm the role of platelets in mediating MM tumor growth, we performed platelet depletion by treating mice with an anti-platelet antibody (R300, 4 ug/g IP injection), and subsequently injected MM cells intravenously. We found that platelet-depleted mice presented with decreased MM tumor growth (p<0.05) and improved survival rate compared to control mice treated with IgG isotype control (p=0.0044).
These findings indicate that MM cells induce platelet aggregation and interact with platelets/MKs within the BM. Importantly, MM cell-platelet interaction is responsible for the activation of pro-survival signaling pathways in MM cells at transcriptome level, leading to enhanced MM cell proliferation, as shown both in vitro and in vivo.

Disclosures
No relevant conflicts of interest to declare.