Contrasting effects of VEGF inhibition on growth and invasion of RIP-Tag2 tumors
RIP-Tag2 mice treated with neutralizing anti-mouse VEGF antibody or sunitinib from age 14 to 17 weeks had significantly smaller tumors (). The sectional area of tumors was 75% less after the antibody and 78% less after sunitinib, compared to age-matched controls treated with vehicle ().
Inhibition of VEGF signaling: Effect on aggressiveness of RIP-Tag2 tumors
Despite their smaller size, tumors treated with anti-VEGF antibody or sunitinib appeared to be more invasive, as judged by the irregularity of the tumor border and the abundance of clusters of amylase-positive acinar cells of the exocrine pancreas trapped inside tumors (). Quantitative measures of the tortuosity of the tumor border (Invasion index, see Methods) and the number of trapped acinar cells were significantly greater (). The relevance of amylase-positive cells within tumors, as an indicator of invasion, was assessed by comparing amylase staining to the basement membrane protein type IV collagen and to type I collagen, a known constituent of the capsule of RIP-Tag2 tumors (4
). The three approaches gave complementary results (Supplemental Figure 1
). Tumors with abundant amylase cells inside had strong staining for type IV collagen around the trapped exocrine cells, as in normal pancreatic acini, but the border had little or no type IV collagen or type I collagen (Supplemental Figure 1, A-C, G-I
). Tumors that had few or no amylase-stained cells inside had type IV collagen around blood vessels, and the border had a layer of type IV collagen and a capsule of type I collagen (Supplemental Figure 1, D-F, J-L
Tumors of 14-week old RIP-Tag2 mice treated with normal goat IgG for 1 or 3 weeks resembled those of mice treated with vehicle (data not shown).
Tumor cell changes in RIP-Tag2 tumors after VEGF inhibition
Proliferating cells marked by phosphohistone H3 immunoreactivity were abundant throughout vehicle-treated tumors (Supplemental Figure 2A
). After treatment with anti-VEGF antibody for 3 days, proliferating cells were still abundant at the tumor border (area density: 14.7% vs. 14.3% for vehicle) but were half the control value at the tumor center (6.8% vs. 13.3% for vehicle, P
< 0.05) (Supplemental Figure 2B
). Abundant phosphohistone H3-positive cells in finger-like projections of tumor contrasted with rare dividing cells in the surrounding exocrine pancreas (Supplemental Figure 2C
Apoptotic cells identified by activated caspase-3 immunoreactivity were more abundant after anti-VEGF antibody for 3 days, but were less numerous than proliferating cells under all conditions (Supplemental Figure 2, D-F
). Apoptotic cells were no more frequent in finger-like projections than elsewhere in tumors.
Snail1, N-cadherin, and vimentin as markers of mesenchymal phenotype had stronger bands in western blots of tumors after treatment with anti-VEGF antibody or sunitinib than in corresponding mice treated with vehicle from age 14 to 15 weeks (). Densitometry values for Snail1, N-cadherin, and vimentin were 3, 5, and 10 times greater, respectively, after anti-VEGF antibody (P < 0.05) and 3, 10, and 5 times greater after sunitinib (P < 0.05).
E-cadherin, as a marker of epithelial phenotype, was weaker in tumors of RIP-Tag2 mice at age 17 weeks () than at age 10 weeks (data not shown), but was even less in tumors treated with anti-VEGF antibody (age 14 to 17 weeks), where tumor cell identity was verified by insulin staining (). E-cadherin staining was inversely related to staining for vimentin () and c-Met (Supplemental Figure 2, G-H
). E-cadherin was stronger in vehicle treated mice, and vimentin and c-Met were stronger after anti-VEGF antibody (Supplemental Figure 2, G-J
Hypoxia and c-Met in RIP-Tag2 tumors after VEGF inhibition
Tumors in RIP-Tag2 mice treated with anti-VEGF antibody or sunitinib from age 14 to 17 weeks had fewer blood vessels than in corresponding vehicle-treated tumors (), as found previously after inhibition of VEGF signaling (19
). The reduced vascularity was accompanied by greater hypoxia, reflected by staining for pimonidazole, carbonic anhydrase IX (CA-IX), or glucose transporter 1 (Glut1) (, Supplemental Figure 3, A-B, D-E
). The staining patterns for the three markers was similar: staining was patchy in control tumors and was widespread and strongest in regions of vascular pruning in VEGF inhibitor-treated tumors. Measurements confirmed an inverse relationship between tumor vascularity and amount of pimonidazole staining (). The amount of HIF-1α protein, assessed by western blot, was also greater after anti-VEGF antibody or sunitinib ().
Inhibition of VEGF signaling: Effect on tumor vascularity, hypoxia, and c-Met
Comparison of c-Met immunoreactivity in RIP-Tag2 tumors treated with vehicle, anti-VEGF antibody, or sunitinib confirmed the known relationship between hypoxia and c-Metexpression (8
). Staining for c-Met was weak or absent in tumor cells but was strong in tumor vessels of control 15-week old mice (, upper
). After anti-VEGF antibody or sunitinib for one week, c-Met staining was strong and widespread in tumor cells (, middle and lower
). Tumor vessel staining was present but less conspicuous because of the vascular pruning.
Expression of c-Met mRNA was 3-fold the control value after anti-VEGF antibody and 6-fold the control value after sunitinib (). The amount of c-Met mRNA in RIP-Tag2 tumors treated with normal goat IgG was similar to that of vehicle-treated controls. The amount and distribution of phospho-c-Met was strongly influenced by treatment. Total c-Met and phospho-c-Met assessed by immunoprecipitation were 3- and 5-fold greater, respectively, in RIP-Tag2 tumors after anti-VEGF antibody for one week (). Phospho-c-Met immunoreactivity was mainly in blood vessels in vehicle-treated tumors, but was strong and widespread in tumor cells after anti-VEGF antibody or sunitinib for one week (). The two inhibitors of VEGF signaling had similar effects on phospho-c-Met staining ().
Neither HGF mRNA expression nor HGF immunoreactivity changed detectably in tumors of RIP-Tag2 mice treated with anti-VEGF antibody for 1 week (Supplemental Figure 3, I-K
To determine whether hypoxia had a direct effect on c-Met activation in tumor cells, we isolated tumor cells from 14-week old RIP-Tag2 mice and exposed them to 21% oxygen (normoxia) or 1% oxygen (hypoxia) for 4 hours in vitro. Phospho-c-Met assessed by immunoprecipitation was greater in tumor cells exposed to hypoxia ().
We addressed the question of whether tumor cells, like blood vessels, are direct targets of inhibitors of VEGF signaling in RIP-Tag2 tumors. Blood vessels in all tumors had strong staining for VEGFR-2 and VEGFR-3 (Supplemental Figure 4
), as reported previously (34
). In addition, 5-10% of tumor cells in RIP-Tag2 mice at age 17 weeks had moderate VEGFR-2 immunoreactivity (Supplemental Figure 4, A-C
). This was not changed by inhibition of VEGF signaling. VEGFR-3 immunoreactivity was not detected in tumor cells under any of the treatment conditions (Supplemental Figure 4, D-F
Treatment of 14-week old RIP-Tag2 mice with anti-VEGF antibody for 1 week did not result in significant changes in expression of angiopoietin-1 (ANG1), angiopoietin-2 (ANG2), basic fibroblast growth factor (FGF2), insulin-like growth factor 1 receptor (IGF-1R), macrophage stimulating 1 receptor (RON/MST1R), RET, SKY, or MER in tumors, but increases were found in AXL (2.8 times) and TGF-β (15 times) (Supplemental Figure 5A
Reduction in invasion of RIP-Tag2 tumors after inhibition of c-Met
To determine whether tumor invasiveness promoted by inhibition of VEGF signaling was dependent on c-Met activity, we treated RIP-Tag2 mice with PF-04217903, a selective inhibitor of c-Met signaling (21
), together with anti-VEGF antibody or sunitinib from age 14 to 17 weeks. Tumors in mice treated with the drug combination had a smoother contour, fewer projections into the exocrine pancreas, and fewer intratumoral acinar cells than after anti-VEGF antibody or sunitinib alone (). Measurements of intratumoral acinar cells () and Invasion index (Supplemental Figure 5B
) showed significant reductions after treatment with PF-04317903 alone or in combination with anti-VEGF antibody or sunitinib. Tumors treated with PF-04317903 in combination with sunitinib had 38% fewer amylase-positive cells than the 14-week old onset controls (3.0 ± 0.4% vs. 4.8 ± 0.5%, P
< 0.05), indicative of reversal of this measure of invasion.
Inhibition of VEGF and/or c-Met: Effect on tumor invasion, vascularity, hypoxia, and cell markers
The addition of PF-04217903 to anti-VEGF antibody or sunitinib was not accompanied by greater vascular pruning (CD31 immunoreactivity, ), reduction in tumor size (data not shown), or intratumoral hypoxia (pimonidazole staining, ) than was found with the single agents. In the assessment of intratumoral hypoxia under these conditions, CA-IX and Glut1 had patterns similar to pimonidazole staining (Supplemental Figure 3, A-F, G
). Glut1 had a stronger signal than CA-IX and was more intense in tumors treated with PF-04217903 in combination with anti-VEGF antibody or sunitinib (Supplemental Figure 3, G and H
). HIF-1α was not increased in western blots from baseline after treatment with PF-04217903 alone. HIF-1α was increased by anti-VEGF antibody or sunitinib alone () or by either one combined with PF-04217903 (data not shown).
PF-04217903 given in combination with anti-VEGF antibody or with sunitinib prevented the increase in c-Met and phospho-c-Met immunoreactivities produced by anti-VEGF antibody or sunitinib given alone (Supplemental Figure 5, D and E
Tumors of RIP-Tag2 mice treated with PF-04217903 alone had a smoother border and fewer trapped acinar cells than after vehicle (). However, tumors treated with PF-04217903 alone for 3 weeks were similar in size to age-matched, vehicle-treated controls (data not shown). PF-04217903-treated tumors had no reduction in overall vascularity (), vascular patency assessed by injection of Lycopersicon esculentum
), or intratumoral hypoxia (), and had no apparent change in vessel branching or in pericyte coverage assessed by NG2 and α-SMA immunoreactivities (data not shown).
To test whether the anti-invasive effects of PF-04217903 could be reproduced by another c-Met inhibitor, we performed similar experiments using the c-Met inhibitor, PF-02341066 (21
), in RIP-Tag2 mice treated from age 14 to 17 weeks. As with PF-04217903, treatment with PF-02341066 reduced invasiveness, as reflected by trapped acinar cells and Invasion index, but did not change tumor vascularity or hypoxia (Supplemental Figure 6, A-B, E-F, G-H, K-L
). Importantly, treatment with PF-02341066 in combination with sunitinib resulted in changes similar to those found after PF-04217903 plus sunitinib: tumors were less invasive than after vehicle or sunitinib alone (Supplemental Figure 6, C-F
), and tumor vascularity and hypoxia were about the same as after sunitinib alone (Supplemental Figure 6, I-L
To determine whether inhibition of c-Met suppressed the increase in mesenchymal markers after inhibition of VEGF, we compared Snail1, N-cadherin, and vimentin in western blots prepared from tumors of RIP-Tag2 mice treated with anti-VEGF antibody, PF-04217903, or the combination from age 14 to 15 weeks. The bands for these markers were strong after anti-VEGF antibody but were weaker after the antibody plus PF-04217903 or after PF-04217903 alone (). Similarly, Snail1 was less in tumors treated with sunitinib plus PF-04217903 than with sunitinib alone, but N-cadherin and vimentin showed less difference (Supplemental Figure 5C
). The reduction in mesenchymal markers after PF-04217903 fits with the reversal of the mesenchymal phenotype by c-Met blockade.
Reduction in liver metastasis after inhibition of c-Met in RIP-Tag2 mice
The liver is a common site of metastasis in RIP-Tag2 mice. Metastases were rarely visible grossly in the liver at autopsy of untreated 14-week old RIP-Tag2 mice (onset controls), but micrometastases, consisting of single or small groups of cells, were found by microscopy after staining for SV40 T-antigen (0.05 ± 0.01 per mm2 sectional area). Liver metastases were 14 times more numerous in control mice at age 17 weeks (0.7 ± 0.2 per mm2), and were much more abundant and larger after anti-VEGF antibody or sunitinib from age 14 to 17 weeks (). Measurements showed 5 times as many liver metastases after anti-VEGF antibody and twice as many after sunitinib, compared to the 17-week old vehicle group (). Compared to the 90 ± 9 μm mean diameter of metastases in the vehicle group, metastases were nearly twice as large after anti-VEGF antibody (mean diameter, 172 ± 29 μm) and 4 times as large after sunitinib (mean diameter, 369 ± 267 μm). The incidence of liver metastases in RIP-Tag2 mice treated with normal goat IgG from age 14 to 17 weeks was similar to mice treated with vehicle (data not shown).
Inhibition of VEGF and/or c-Met: Effect on number and size of liver metastases
Liver metastases assessed by SV40 T-antigen staining were significantly less numerous in mice treated with PF-04217903 or PF-02341066 (). The baseline number of liver metastases in 17-week old RIP-Tag2 mice (vehicle treatment) was reduced 98% by administration of PF-04217903 and reduced 73% by PF-02341066 (). Metastases in mice treated with sunitinib were reduced 99% by PF-04217903 and reduced 53% by PF-02341066 (). Metastases in mice treated with anti-VEGF antibody were reduced 92% by co-administration of PF-04217903 (). Metastases were visible microscopically in the liver of all 17-week old mice treated with vehicle, anti-VEGF antibody, or sunitinib alone, but were found in only 1 of 7 mice given vehicle plus PF-04217903, 3 of 6 mice given anti-VEGF antibody plus PF-04217903, and 1 of 5 mice given sunitinib plus PF-04217903.
Liver metastases were smaller in all groups that received PF-04217903 (). The mean diameter of metastases was reduced 40% when PF-04217903 was combined with vehicle, 50% when combined with anti-VEGF antibody, and 81% when combined with sunitinib (). These reductions reflect average decreases in volume (metastatic tumor burden) of 78%, 88%, and 99%. Metastases with diameters larger than 100 μm were 3 times more numerous after anti-VEGF antibody or sunitinib than after vehicle, but were rare in the groups receiving PF-04217903 ().
Survival of RIP-Tag2 mice during the 3-week treatment from age 14 to 17 weeks was strongly influenced by the treatment: 42% survival with vehicle, 50% with anti-VEGF antibody, 70% with anti-VEGF antibody plus PF-04217903, and 80% with sunitinib alone or in combination with PF-04217903.
Reduction in invasion of Panc-1 tumors after inhibition of c-Met
To compare the effects of inhibition of VEGF signaling alone and with concurrent blockade of c-Met signaling in a second tumor model, we performed similar experiments on implanted Panc-1 pancreatic adenocarcinomas (26
), which invade locally and can metastasize (36
). Growth of luciferase expressing Panc-1 tumors in the pancreas was monitored by bioluminescence imaging (). Tumors in mice treated with vehicle grew steadily and invaded the exocrine pancreas during 3-week treatment (). Tumors treated with sunitinib grew slower, and those treated with sunitinib plus PF-04217903 grew even less ().
Inhibition of VEGF and/or c-Met: Effect on Panc-1 tumor growth, c-Met, and invasion
Treatment with sunitinib was accompanied by 75% reduction in tumor vascularity and doubling of intratumoral hypoxia, assessed by pimonidazole staining (). Vascular pruning and hypoxia were similar after treatment with sunitinib plus PF-04217903 (). Treatment with PF-04217903 alone had little effect on vascular pruning or intratumoral hypoxia ().
c-Met immunoreactivity was moderate in some blood vessels and faint in others and was faint in most tumor cells of vehicle-treated Panc-1 tumors. However, c-Met was stronger in both locations after treatment with sunitinib (). Invasiveness of Panc-1 tumors, reflected by intratumoral acinar cells stained for amylase, was exaggerated by sunitinib () and was suppressed by co-administration of PF-04217903 ().
Panc-1 tumors metastasize to the liver when the tumors become aggressive (36
). At the early stage we studied, micrometastases were found in the liver of 4 of 6 mice treated with sunitinib but in none of 6 mice treated with sunitinib plus PF-04217903 (data not shown).
Reduction in invasion of RIP-Tag2 tumors after treatment with XL184
The effects of agents that block c-Met or VEGF signaling, administered alone or in combination, were compared to those of XL184, which blocks both receptors simultaneously (19
). XL184 has potent inhibitory activity against c-Met, VEGFR-2, and multiple other RTKs tested in biochemical and cell-based assays (Supplemental Figure 7A
). The IC50
for c-Met (1.3 nM) and VEGFR-2 (0.035 nM) were both in the low nanomolar or sub-nanomolar range and were the lowest of the kinases assayed.
Comparison of tumor size in RIP-Tag2 mice treated from age 14 to 17 weeks revealed that XL184-treated tumors were much smaller (mean sectional area 0.7 ± 0.1 mm2
) than those treated with vehicle (17.3 ± 6.5 mm2
), anti-VEGF antibody (4.3 ± 0.8 mm2
), or sunitinib (3.7 ± 0.8 mm2
). XL184-treated tumors of 17-week old mice were also smaller than tumors in 14-week old onset controls (2.8 ± 0.5 mm2
). Treatment with XL184 for 3 weeks resulted in widespread vascular pruning and intratumoral hypoxia (Supplemental Figure 8
): the 79% reduction in tumor vascularity was greater than found after any of the other agents studied.
Tumors in RIP-Tag2 mice treated with XL184 had well-defined borders, smooth contours, few projections into the acinar pancreas, and few or no islands of trapped acinar cells, and were distinctly unlike tumors treated with anti-VEGF antibody (). SV40 T-antigen-positive cells were scattered outside the tumor border in mice treated with anti-VEGF antibody () but not after XL184 (). Many tumor cells in mice treated with vehicle or anti-VEGF antibody were irregular in shape and loosely associated with one another (), but tumor cells treated with XL184 were rounder and tightly clustered (). Consistent with these differences, values for Invasion index and trapped acinar cells were significantly less after XL184 than after vehicle or anti-VEGF antibody (). Area density values for trapped acinar cells in tumors treated from age 14 to 17 weeks with XL184 (2.6 ± 0.4%) were 46% lower than for untreated mice at 14 weeks of age (4.8 ± 0.5%). This significant difference indicates that XL184 partially reversed the invasion present at the beginning of treatment.
XL184 effects on RIP-Tag2 tumor invasion and epithelial/mesenchymal markers
RIP-Tag2 tumors treated with XL184 had fainter c-Met immunoreactivity in tumor cells and blood vessels and less phospho-c-Met assessed by immunoprecipitation than corresponding vehicle-treated controls (Supplemental Figure 7B-F
). By comparison, c-Met immunoreactivity of the tumors treated with PF-04217903 together with anti-VEGF antibody or sunitinib was still strong in tumor cells and blood vessels (data not shown). Western blots of XL184-treated tumors also showed more E-cadherin protein and less Snail1, N-cadherin, and vimentin than tumors treated with anti-VEGF antibody (). Similarly, tumors treated with XL184 had stronger E-cadherin immunoreactivity than tumors treated with vehicle or anti-VEGF antibody (). Taken together, these results indicate that tumor cells treated with XL184 had a more epithelial phenotype.
Reduction in metastasis and prolonged survival after treatment with XL184
Microscopic liver metastases were consistently found in 17-week old RIP-Tag2 mice after treatment for 3 weeks with vehicle, were more numerous and larger after anti-VEGF antibody, but were not found after treatment with XL184 ().
XL184 effects on liver metastasis and overall survival of older RIP-Tag2 mice
Overall survival of RIP-Tag2 mice treated from 14 weeks of age for up to 6 weeks was strongly influenced by the treatment. Median survival of vehicle-treated mice was 14.7 weeks (). Survival was prolonged to 16.1 weeks in the PF-04217903 group, 16.4 weeks in the anti-VEGF antibody group, 17.3 weeks in the group receiving anti-VEGF antibody plus PF-04217903, and more than 20 weeks in the XL184 group (). Strikingly, all mice treated with XL184 survived until the experiment ended at age 20 weeks. One of 6 mice treated with the combination of anti-VEGF antibody and PF-04217903 survived this long, but none of the mice in the other groups reached 20 weeks of age ().
The 20-week old RIP-Tag2 mice treated with XL184 for 6 weeks had small, round, compact tumors. The mean Invasion index of these tumors (5.8), compared to tumors in mice treated from age 14 to 17 weeks, was lower than for vehicle (12.7, P < 0.05) or anti-VEGF antibody (18.9, P < 0.05) and about the same as for XL184 (4.5) ().
None of 5 mice treated with XL184 from age 14 to 17 weeks had detectable liver metastases. Small liver metastases were present in 5 of 6 mice treated with XL184 from age 14 to 20 weeks (0.3 ± 0.2 metastases/mm2), but these were much less numerous than in 17-week old mice treated with anti-VEGF antibody (3.4 ± 1.5 metastases/mm2) or sunitinib (1.3 ± 1.2 metastases/mm2) for 3 weeks ().
To determine whether XL184 had similar effects on tumors in younger RIP-Tag2 mice, we treated mice from age 10 to 14 weeks. As in the older mice, tumors in younger mice treated with XL184 were rounder and less invasive (Supplemental Figure 9, A-E
). Grossly visible liver metastases were not found in any of the mice at age 14 weeks, but micrometastases were visible microscopically after staining for SV40 T-antigen (Supplemental Figure 9, F-H
). Four times as many micrometastases were found in mice treated with anti-VEGF antibody as with vehicle, but none was found in 5 mice treated with XL184 (Supplemental Figure 9I
). Mice treated with XL184 had better overall survival than age-matched mice treated with vehicle or anti-VEGF antibody (Supplemental Figure 9J