Interactions between megakaryocytes and tumour cells in the bone marrow vascular stem cell niche promote tumour growth and metastasis

Abstract

Specialized bone marrow microenvironments (vascular and osteoblastic 'niches') regulate normal haematopoietic stem/progenitor cells. Recently, the vascular niche has also been implicated as an area for preferential engraftment of malignant cells. The cellular and molecular factors that regulate the vascular niche and, in particular, the role of megakaryocytes are poorly understood. The aim of my work was to investigate the role of megakaryocytes in homing and engraftment of malignant cells to the bone marrow vascular niche using mouse models. C57Bl/6 wild-type and megakaryocyte-deficient, thrombopoietin (TPO)-/- mice were injected with B16 melanoma or EL4 lymphoma cell lines and the megakaryocyte-vascular niche investigated by immunohistochemistry, confocal microscopy, in vitro culture, co-cultures and gene expression by RT-PCR. In wild-type mice injected with B16 melanoma, platelet size and megakaryocyte numbers significantly increased (P<0.02). B16 tumour cells were found to produce the thrombopoietic factors VEGF, SCF and IL11. Bone marrow sinusoids were almost universally surrounded by one of more megakaryocytes tightly abutting the vascular endothelium, forming the megakaryocyte-vascular niche. Metastatic B16 cells were observed in close association with megakaryocytes in the vascular niche, consistent with this being a port of entry to the bone marrow. In TPO-/- mice, tumour growth and metastasis was markedly retarded and no tumour cells were seen in the bone marrow, suggesting that megakaryocytes play a functional role in metastasis. In TPO-/- bone marrow, vessels were more tortuous and larger in diameter (P=0.01); and expression of PF4, TSP1, VEGF and TGFβ was 70%- 90% lower, suggesting that a major proportion of angiogenic regulatory factors is producted by megakaryocytes in the bone marrow in wild-type mice. Furthermore, in wild-type mice, expression of VEGF and TGFβ significantly increased during tumour growth and metastasis while PF4 expression decreased (P<0.05). Megakaryocyte-conditioned medium (MCM) enhanced the proliferation rate of B16 cells (P<0.001) and also was highly chemotactic for B16 cells (P<0.001), an effect mediated by pertussis toxin-sensitive Gi-protein receptors and reduced in the absence of TSP1. Co-culture with B16 cells increased megakaryocyte expression of VEGF, TGFβ and TSP1 and decreased PF4, consistent with the in vivo observations, while cocultured B16 cells displayed increased expression of VEGF and TGFβ and adhesion integrins. Moreover, pretreating B16 cells with MCM prior to tail vein injection enhanced metastatic engraftment. To investigate the role of megakaryocytes in human malignancy, trephine bone marrow biopsies from patients with metastatic carcinoma were examined. Increased megakaryocyte numbers and abnormal megakaryocyte clustering were observed in the majority of patients, suggesting that megakaryocyte-tumour interactions may also occur in the setting of human metastatic disease. In conclusion, my findings suggest that megakaryocytes contribute to the integrity and function of the bone marrow vascular niche and that cellular/molecular cross talk between megakaryocytes and tumour cells may promote metastasis. Targeting these interactions may be useful as adjunctive therapy in metastatic disease.