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
