The present study sought to dissect the effects of combined blockage of VEGF and PDGF signaling on tumor blood vessels in two mouse tumor models, RIP-Tag2 tumors and LLC tumors. We found that combination of Ad-VEGFR1 and Ad-PDGFRβ strongly reduced tumor size, blood vessels and pericytes in both tumor models. In RIP-Tag2 tumors, single Ad-VEGFR1 or Ad-PDGFRβ treatment caused a lower reduction in tumor area, blood vessels and pericytes compared to the combination treatment. In contrast, in LLC tumors blocking VEGF or PDGF signaling alone reduced tumor size, pericytes and endothelial cells density to the same extent than the combination treatment. Similar results were observed by inhibition of VEGF and PDGF signaling using the tyrosine kinase inhibitors AG-013736 and Imatinib.
Pericytes in tumors are loosely associated with endothelial cells and have cytoplasmic processes that extend away from the vessel wall (12
). Pericytes express different markers in different organs and tumors (12
). The lack of a single unique marker for all pericytes presents a challenge for their identification. The absence of a marker could reflect absence of expression by pericytes or absence of pericytes. In the present study, to assess the presence of pericytes we used four markers: α-SMA, desmin, NG2 and PDGFR-β (23
). Pericytes in RIP-Tag2 tumors and LLC tumors expressed all four markers, with minor differences in cellular localization due to the association of α-SMA and desmin with the cytoskeleton and NG2 and PDGFR-β with the plasma membrane. By looking at four markers we found that Ad-VEGFR1 as well as AG-013736 did not affect the overall number of pericytes in RIP-Tag2 tumors but did reduce the amount of α-SMA and desmin immunoreactivity. Because many blood vessels regressed in these tumors after VEGF signaling inhibition, many pericytes were left without endothelial cells. The reduction in the two-cytoskeletal markers may be a consequence of the reorganization of the pericyte cytoskeleton, which could occur after loss of contact with endothelial cells. Treatment with Ad-PDGFRβ led to pericyte loss in both RIP-Tag2 tumors and in LLC tumors, but the reduction was much greater in LLC tumors. In RIP-Tag2 tumors the combined adenoviral vectors had a complex effect on pericytes, involving loss of about half of the pericyte population and change in phenotype of the remaining pericytes, as reflected by decreased expression of α-SMA. LLC tumors differed in this regard from RIP-Tag2 tumors. In LLC tumors, the combined viral vectors reduced by 75% pericyte density detected by all four markers, a reduction comparable to the one obtained by treatment with Ad-VEGFR1 or Ad-PDGFRβ alone.
After combination of Ad-PDGFRβ and Ad-VEGFR1 almost all the surviving blood vessels were associated with pericytes. It has been reported that in RIP-Tag2 tumors after blocking PDGFR and VEGFR using the tyrosine kinase inhibitor SU5416 and Imatinib or SU5416 and SU6668 the remaining blood vessels were covered by few pericytes (24
). These differences could be attributed to the following reasons: the different specificities of the inhibitors used, the duration of the treatment and the stage of the tumors analyzed.
The difference of responsiveness between RIP-Tag2 tumors and LLC tumors could be attributed to the level and/or localization of VEGF and PDGF expression. Indeed RIP-Tag2 tumors had significantly higher VEGF and PDGF transcript levels and immunoreactivity compared to LLC tumors. By analyzing the distribution of VEGF within tumors, we found that both tumor cells and pericytes produced VEGF in RIP-Tag2 tumors. Those results are in line with previous studies showing that VEGF is highly expressed in the tumor cells of tumorigenic islets as well as in the normal islets in RIP-Tag2 mice (45
). Our data suggest that in RIP-Tag2 tumors the cellular sources targeted by the Ad-VEGFR1 treatment are mainly the tumor cells and that are responsible for the reduction of the endothelial cells. This hypothesis is confirmed by the fact that the elimination of pericytes by Ad-PDGFRβ did not lead to reduction of tumor blood vessels.
In contrast, in LLC tumor VEGF immunoreactivity was located in cells close to blood vessels and most of these cells were immunoreactive for pericyte markers. VEGF production has been previously described in the ovary pericytes (48
) and pericytes isolated from RIP-Tag2 tumors have high VEGF transcription level (25
). In addition, previous studies have shown that pericytes facilitate the maintenance of endothelial cells by secreting growth factors (10
) as VEGF (16
). Macrophages have been described to be important source of VEGF (42
). In LLC tumors double stain with VEGF and macrophage markers showed that only few macrophages were positive for VEGF. Similarly VEGF immunoreactivity was not observed in LLC tumor cells. We cannot rule out that VEGF was not expressed it these cells but the fact that the amount was too little to be detectable by immunohistochemistry suggests that in LLC tumor pericytes are the major source of VEGF.
Interestingly when we analyzed the effects of VEGF and PDGF inhibition in a LLC tumor line expressing high amount of VEGF, LLCx tumors, we found that combination therapy had a greater effect compared to single treatment. Moreover PDGF blockage induced only a small reduction of the blood vessel density. These findings supported our hypothesis that the amount and distribution of VEGF determine the effects of PDGF inhibition on tumor blood vessels.
Endothelial cells in RIP-Tag2 tumors express genes for PDGF-A and PDGF-B (24
). In our experiments, both tumor cells and endothelial cells produced PDGF in RIP-Tag2 tumors while only endothelial cells produced PDGF in LLC tumors.
These findings suggest that VEGF and PDGF inhibitors affect blood vessels differently depending on the amount and cellular distribution of VEGF and PDGF within the tumor. Our model is that in LLC tumors, PDGF inhibition affects blood vessels by first eliminating pericytes, the major source of VEGF; similarly, VEGF inhibition reduces the number of pericytes by targeting eliminating endothelial cells, the major source of PDGF. This model is consistent with our results showing that in LLC tumors, inhibition of VEGF strongly reduced PDGF immunoreactivity and inhibition of PDGF strongly reduced VEGF immunoreactivity.
In RIP-Tag2 tumors, where tumor cells produce both VEGF and PDGF, targeting endothelial cells via VEGF inhibition did not lead to a reduction in pericytes because PDGF was unaffected. Similarly, targeting pericytes, via PDGF inhibition, did not impact endothelial cells because VEGF was unaffected. Our results illustrated that PDGF inhibitors can have effects on tumor vessels similar to those of VEGF inhibitors but only in tumors where pericytes are the main source of VEGF. Future experiments in other tumor models will broaden our findings.
In conclusion, the present study highlights the importance of VEGF and PDGF signaling in sustaining the tumor vasculature. Our results also illustrate that tumor vasculature can respond to VEGF and/or PDGF inhibition differently depending on the cellular source and amount of the growth factors within the tumor. These findings emphasize the importance of the tumor phenotype in the responsiveness to inhibitors of VEGF and PDGF. A better understanding of the interaction of factors from endothelial cells, pericytes, and other tumor compartments is required to design the most effective anti-tumor therapy.