We have discovered a population of blood-vessel-associated PDGFRβ+ cells in pancreatic Rip1Tag2 tumours that elicit pericyte maturation and vascular stabilization and survival. Interestingly, only 15–20% express the mature pericyte markers desmin, NG2 or αSMA, whereas mature pericytes in these tumours are devoid of PDGFRβ. Our in vitro and in vivo evidence supports the conclusion that perivascular PDGFRβ+ cells are progenitors (PDGFRβ+ PPCs) that have the capacity to differentiate into desmin+, NG2+ and αSMA+ pericytes/vSMCs. Interestingly, we were able to dissect distinct differentiation pathways for pericytes. Whereas PDGFRβ+ PPCs induced NG2 and αSMA when cultured alone, co-culture of PDGFRβ+ PPCs with endothelial cells was crucial for the induction of desmin. We therefore propose that paracrine signalling circuits between endothelial cells and PDGFRβ+ PPCs drive induction to desmin+ pericytes in a cell-contact-dependent manner.
The proposition of distinct key factors triggering pathways that give rise to various pericyte subtypes raises the question of the identity of such factors. TGFβ seems to be required for differentiation into αSMA+
cells, because inhibiting TGFβ blocked differentiation to αSMA-positive pericytes, but not to desmin- and NG2-positive pericytes. Indeed, TGFβ appears to be involved in smooth muscle differentiation, since it induces αSMA in the immortalized neural crest stem cell line Monc-1 (ref. 32
) and αSMA and SM22α, another contractile smooth muscle protein, in the mesenchymal cell line 10T1/2 (refs 31, 34
). Another crucial factor is PDGF-B. When we abrogated the PDGFRβ-signalling pathway by treating tumour-bearing mice with a neutralizing PDGFRβ antibody, we depleted the tumours not only of PDGFRβ+
PPCs but also of mature pericytes.
Our data demonstrate that tumours share striking similarities with embryos. Similar to pancreatic islet tumours, PDGF-B is expressed by sprouting capillary endothelial cells during developmental angiogenesis, whereas its receptor PDGFRβ is localized on pericytes indicative of a paracrine signalling circuit. As with anti-PDGFRβ-treated tumours, PDGF-B- or PDGFRβ-deficient mice exhibit a marked reduction in the number of pericytes/vSMCs10,11,15,16
, leading to hyperdilated blood vessels. In addition, fibrosarcomas transplanted into mice that cannot retain PDGF-B on the cell surface or in the extracellular matrix show fewer pericytes attached to the tumour vessels35
, confirming the critical role of PDGFRβ signalling in pericyte activation and/or recruitment. In summary, these results support the hypothesis that pericytes in tumours, similar to those in the embryo, are formed de novo
by maturation of undifferentiated perivascular cell progenitors recruited to the newly formed vasculature from bone marrow or from a pre-existing pool of pericytes. Abrogation of PDGFRβ signalling reduced the number of activated pericytes in tumours, whereas quiescent pericytes in normal tissues were not affected. A second implication from these data is that activated PDGFRβ+
PPCs are inhibited from multiplying and differentiating into mature pericytes. This hypothesis also reflects the situation in development where disruption of PDGF-B/PDGFRβ signalling hinders pericytes to expand and spread along the newly formed vessels due to their reduced proliferative and migratory capability2
Where do PDGFRβ+
PPCs in the tumours come from? A small pool of these cells may exist in the tissue that then becomes activated when blood vessels need to be formed. Alternatively or additionally, a subset of PDGFRβ+
PPCs could be recruited from a different organ. Our novel finding that PDGFRβ+
PPCs are also bone-marrow-derived parallels the concept that tumours may recruit endothelial cell precursors from bone marrow36,37
PPCs express haematopoietic stem cell markers including Sca1, CD11b and c-Kit. Bone marrow transplant experiments and cultures of bone-marrow- or tumour-derived Sca1+
cells with endothelial cells revealed that it is the Sca1+
cell population from the bone marrow that is recruited to angiogenic sites of the tumour and then matures into pericytes. Bone-marrow-derived cells that cover blood vessels and are either positive for NG2, CD11b or CD45 were detected in a subcutaneous B16-melanoma xenograft tumour model33
suggesting that the tumour type or location dictates which type of bone-marrow-derived cells are recruited to angiogenic sites. Taken together, these studies demonstrate that tumours are not only able to recruit endothelial precursor cells, but also pericyte precursor cells from the bone marrow during vascular remodelling.
Our results from anti-PDGFRβ-treated tumours also underline the functional importance of tumour pericytes. Pericyte-deprived tumour vessels were enlarged and hyperdilated, reminiscent of the phenotype in PDGF-B- or PDGFRβ-deficient embryos. These results imply that tumour pericytes, albeit less abundant or more loosely attached, still regulate vessel integrity, maintenance and function. The high increase in endothelial cell apoptosis, when pericytes are depleted from blood vessels, further strengthens the function of tumour pericytes in protecting endothelial cells, probably by expressing potent endothelial survival factors. One such factor, VEGF, which we found to be highly expressed in PDGFRβ+
PPCs, harks back to the observation that in vitro
pericyte-associated endothelial tubes are more stable and viable than endothelial cords without them. In agreement with this finding, immature blood vessels without pericytes are more sensitive to anti-VEGF therapy38
. Moreover, our group and others demonstrated that receptor tyrosine kinase inhibitors that target both pericytes and endothelial cells by combinatorial inhibition of PDGFR and VEGFR signalling, are very efficacious even in late-stage disease, disrupting the established tumour vasculature and effecting tumour regression20,22,39