The overall goal of this study was to determine the cellular effects of two small molecule RTK inhibitors, XL880 and XL184, that block key signaling pathways involved in angiogenesis and tumor invasiveness. Both inhibitors target VEGFR and c-Met at low nanomolar or subnanomolar concentrations together with several other RTKs, and both had rapid, robust, profound, and largely similar cellular effects on blood vessels and tumor cells of RIP-Tag2 tumors. These effects differed qualitatively and quantitatively from those found after treatment with XL999, an RTK inhibitor that does not target c-Met but does block VEGFR plus multiple other receptors blocked by XL880 and XL184.
Treatment with XL880 or XL184 led to rapid and severe changes in tumor blood vessels, which were greater than after XL999 over a similar dosage range or after other VEGF inhibitors (4
). VEGFR-2 and VEGFR-3 proteins and their respective mRNA expression were reduced even more than corresponding values for CD31, consistent with dual effects of vessel regression and downregulation of expression on the remaining tumor vessels (4
Selective inhibition of VEGF signaling causes regression of some tumor vessels, but those that remain tend to be more normal (3
), which were confirmed after XL999. However, surviving tumor vessels were qualitatively different after XL880 or XL184, which were narrow, fragmented, and not stained by intravenous FITC-LEA lectin, suggestive of continuing regression (4
). The tumor vascularity continued to decrease during the second week of XL880 treatment. This progressive change differs from vascular regression due to many selective VEGF inhibitors, which tends to plateau after a week or so as tumor vessels normalize (3
Basement membrane sleeves and pericytes were decreased after treatment with XL880 or XL184. Some reduction was also found with XL999, as has been reported for other VEGF inhibitors (4
), but the decrease was less. Basement membrane sleeves depend in part on the viability of the associated endothelial cells and/or pericytes, but they were not lost proportionally: endothelial cells were reduced 80%, pericytes 71%, and basement membrane only 52%.
Tumor vessels rapidly regrow after withdrawal of VEGF inhibitors (36
). Tumor vessels also regrew after withdrawal of XL880 or XL184, but the regrowth was slowed. Although the mechanism is uncertain, one factor for the reduced vascular regrowth after XL880 or XL184 may be the greater loss of pericytes and basement membrane sleeves that provide a scaffold for regrowing blood vessels (36
To better understand the robust effects of XL880 and XL184 on blood vessels in RIP-Tag2 tumors, we examined the amount and distribution of c-Met immunoreactivity. Strong staining for c-Met was consistently found in some tumor vessels at baseline. Colocalization with CD31 indicated the vascular staining of c-Met was restricted to endothelial cells. In addition, some tumor cells marked by SV40 T-antigen had faint c-Met immunoreactivity. Vascular staining for c-Met was conspicuously less after XL880 or XL184 but was unchanged after XL999. Consistent with this finding, total c-Met and phosphorylated c-Met, assessed by immunoprecipitation, were both reduced after XL184, but neither was significantly reduced after XL999. Based on intensity of c-Met immunoreactivity, vascular endothelial cells probably made a significant contribution to the signal in the immunoblots and were targets of XL880 and XL184, but the presence of weak staining in tumor cells indicates that c-Met blockade is also likely to have direct effects on tumor cells in the RIP-Tag2 model.
The robust effects of XL880 and XL184 on RIP-Tag2 tumors were not limited to tumor vessels. Intratumoral hypoxia and tumor cell apoptosis were also widespread. Induction of hypoxia and apoptosis was probably due to the extensive and progressive pruning of tumor vessels without vessel normalization. The association of pimonidazole staining with regions of tumor that had few or no blood vessels is consistent with this interpretation.
Invasiveness, which is a typical feature of aggressive tumors (42
), increases in some preclinical models after inhibition of VEGF (5
). Treatment with XL880 resulted in smaller and less invasive tumors in RIP-Tag2 mice, and importantly, recovery of tumor invasiveness was slowed and did not rebound at an exaggerated rate during the first two weeks after XL880 was withdrawn.
The involvement of c-Met in the anti-invasive action of XL880 and XL184 is consistent with their potent inhibitory actions on c-Met, the presence of c-Met immunoreactivity in RIP-Tag2 tumors under baseline conditions, and the reduction in c-Met and invasiveness after treatment. XL880 and XL184 had IC50
values for c-Met of 0.4 and 1.3 nM, compared to 463 nM for XL999. Involvement of c-Met also fits with the reduction in phospho-c-Met protein in the tumors after treatment with XL184. XL999 lacked the robust effect of XL880 and XL184 on apoptosis of blood vessels and tumor cells in RIP-Tag2 tumors. Although inhibition of AXL could contribute to the anti-invasive action, as reported in other models (46
), this is unlikely to be the main target because XL880, XL184, and XL999 block AXL about equally, with IC50
values in the range of 4.6–11 nM.
The kinase profiles of XL880, XL184, and XL999 had differences in addition to c-Met, and drug efficacy is also influenced by in vivo absorption, stability, clearance, and other chemical properties that influence pharmacokinetics and pharmacodynamics. Therefore, confirmation of essential or exclusive roles of inhibition of c-Met and VEGFR in the anti-invasive effects of XL880 and XL184 will require the use of more selective inhibitors, comparison of drug actions after genetic deletion of c-Met, or other strategies that target these signaling pathways without impacting other receptors.
In conclusion, we found that XL880 and XL184 caused rapid and extensive regression of endothelial cells and pericytes of blood vessels in RIP-Tag2 tumors. Regression of the tumor vasculature was widespread, progressive, and accompanied by extensive intratumoral hypoxia and apoptosis of tumor cells. The treated tumors were smaller, less invasive, and accompanied by fewer liver metastases. The findings are consistent with the distinctive anti-tumoral actions of XL880 and XL184 resulting from inhibition of VEGFR and c-Met together. However, inhibition of other receptors and chemical properties that influence pharmacokinetics and pharmacodynamics could also contribute. Careful assessment using strategies for selective inhibition or knockdown of individual receptors or pathways will be necessary to determine whether inhibition of VEGFR and c-Met is necessary and sufficient to reproduce all of the robust effects of XL880 and XL184 on tumors.