Interactions Between Megakaryocytes and Tumour Cells at the Bone Marrow Vascular Stem Cell Niche Promote Tumour Growth and Metastasis.

Specialized microenvironments, or niches, of the bone marrow (BM) maintain and regulate physiological haematopoietic stem/progenitor cells and have been implicated as areas of preferential engraftment and ‘sanctuary sites' for leukaemic cells and metastasizing cells of solid tumours. The cellular and molecular factors that comprise the BM vascular niche have not been well described. Megakaryocytes (MKs) reside in close association with the BM sinusoidal endothelium. The aim of this study was to investigate whether MKs play a role in the homing and engraftment of malignant cells in the BM.

C57Bl/6 wild type and thrombopoietin (TPO)^-/- mice (which have <10% of the normal number of MKs and platelets) received flank or tail vein injections of the syngeneic B16-F10 melanoma or EL4 lymphoma cell lines fluorescently labelled with mCherry or GFP. The MK-vascular niche was examined using citrulline, MECA32, and TSP1 immunostaining. MKs were cultured from the Lin^-Sca1⁺cKit⁺-enriched fraction of BM cells from flushed femurs and tibias, using 50ng/ml TPO + 20ng/ml SCF. Costar transwell co-culture plates and Neuroprobe chemotaxis chambers were used; cellular proliferation was assessed using the CellTitre MTS assay. RNA was extracted from flushed BM cells and in vitro cell cultures using Qiagen RNeasy columns/Trizol and gene expression was analyzed by RT-qPCR.

In wild type mice, the majority of BM sinusoids were surrounded by one or more large MKs forming the MK-vascular niche, with MKs tightly abutting the vascular endothelium. In TPO^-/- mice, MKs were largely absent from the BM, blood vessels appeared more tortuous and the mean vessel diameter in the BM was significantly larger than in wild type mice (P<0.01). Expression of the angiogenic regulatory proteins platelet factor 4 (PF4) and thrombospondin 1 (TSP1) was markedly lower in BM from TPO^-/- mice than wild type; expression of VEGF and TGFb was also reduced. Together these findings suggest that MKs support the integrity of the vascular niche and that homeostasis of the niche may be disrupted in the absence of MKs. Wild type mice injected with either B16-F10 melanoma or EL4 lymphoma had increased numbers of MKs and a larger mean vessel diameter. Although there was no increase in platelet count, the mean platelet volume was significantly increased by day 18 (p=0.002), suggesting increased thrombopoiesis. Furthermore, there was a linear decrease in PF4 in response to tumour, reaching a 3-fold reduction by late-stage tumour growth. This finding was consistent with the increase in MK-vascular niches as PF4 normally acts as an autocrine inhibitor of megakaryopoiesis and inhibits endothelial cell proliferation and migration. Consistent with a modulatory effect of MKs and/or the MK-vascular niche on tumour phenotype, tumour growth in TPO^-/- mice was markedly retarded and there was reduced metastasis to the BM and lung. To investigate the mechanism of these effects, MK-conditioned medium (MCM) was added to in vitro cultures of B16 cells. MCM significantly enhanced the proliferation rate of B16 melanoma cells (P<0.001). Further, MCM was highly chemotactic for B16 cells (P<0.001). This effect was found to be mediated by pertussis toxin-sensitive Gi-protein receptors and reduced but not entirely abrogated in the absence of TSP1 (using MCM generated from TSP1^-/- mice). To investigate the interactions between tumour cells and MKs, MKs were cocultured with B16 cells. Coculture increased MK expression of proangiogenic factors VEGF and TGFb while cocultured B16 cells displayed increased expression of alpha integrins, a4, a5 and a6. Moreover, coculturing B16 cells with MKs prior to tail vein injection enhanced tumour cell engraftment in the lung. A pilot study of BM trephine biopsies from 8 patients with carcinoma (breast, lung, prostate, bladder and kidney) supported these preclinical findings. MKs in 3/3 patients with BM metastasis and 3/5 patients without BM metastasis showed a variable excess of MKs, some in loose clusters, with abnormal morphology and localization.

These findings suggest that MKs contribute to the integrity and functionality of the BM vascular niche in homeostasis and in malignancy, and that cellular/molecular cross-talk between MKs and tumour cells at the vascular niche may promote metastasis. Targeting these interactions may be a useful as adjunctive therapy to prevent dissemination of cancer to the BM.

No relevant conflicts of interest to declare.