Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors

Abstract

Functions of receptor tyrosine kinases implicated in angiogenesis were pharmacologically impaired in a mouse model of pancreatic islet cancer. An inhibitor targeting VEGFRs in endothelial cells (SU5416) is effective against early-stage angiogenic lesions, but not large, well-vascularized tumors. In contrast, a kinase inhibitor incorporating selectivity for PDGFRs (SU6668) is shown to block further growth of end-stage tumors, eliciting detachment of pericytes and disruption of tumor vascularity. Importantly, PDGFRs were expressed only in perivascular cells of this tumor type, suggesting that PDGFR(+) pericytes in tumors present a complimentary target to endothelial cells for efficacious antiangiogenic therapy. Therapeutic regimes combining the two kinase inhibitors (SU5416 and SU6668) were more efficacious against all stages of islet carcinogenesis than either single agent. Combination of the VEGFR inhibitor with another distinctive kinase inhibitor targeting PDGFR activity (Gleevec) was also able to regress late-stage tumors. Thus, combinatorial targeting of receptor tyrosine kinases shows promise for treating multiple stages in tumorigenesis, most notably the often-intractable late-stage solid tumor.

Figure 1

Different stage-specific efficacy profiles for the VEGFR inhibitor SU5416 and the PDGF (+VEGF/FGF) receptor inhibitor SU6668 in the three distinct stages of pancreatic islet carcinogenesis in RIP1Tag2 transgenic mice. Mice were either treated with SU5416 or SU6668 as described in Methods. The average number of angiogenic islets ± SEM at 10.5 weeks in control and treated mice, the average tumor burden ± SEM in PBS/vehicle–treated mice (at 10, 12, 13.5 weeks), and SU5416- and SU6668-treated mice (at 13.5 and 16 weeks) are shown. The prevention trial (PT) started at 5 weeks, when mice harbor hyperplastic/dysplastic islets, and ended at 10.5 weeks, when the first small tumors appear. Islets that have switched on angiogenesis are scored by their reddish color (resulting from microhemorrhage and leakiness associated with VEGF-induced angiogenesis). In the intervention trial (IT), mice with a small tumor burden (10 weeks) are treated until the end stage (13.5 weeks), while in the regression trial (RT), 12-week-old mice with substantial tumor burden and a life expectancy of less than 2 weeks are treated until 16 weeks, when control mice are already dead. Statistical analysis was performed with a two-tailed, unpaired Mann-Whitney test comparing experimental groups to PBS-injected control mice. Tumor burdens of experimental groups in the Regression Trial were compared to that of 12-week-old Rip1Tag2 mice. Cohorts of 6–21 animals were used. P values less than 0.1 are considered statistically significant. P values of SU5416 PT = 2.26 × 10^–5, SU6668 PT = 0.0002, SU5416 IT = 0.0009, SU6668 IT = 0.0001, SU5416 RT = 0.1827, and SU6668 RT = 0.3228.

Figure 2

Comparison of vascular morphology (left panels) and association of endothelial cells and perivascular cells (right panels) in treated versus control tumors. Tumor-bearing pancreata were taken from end-stage 13.5-week-old control Rip1Tag2 mice and from 16-week-old Rip1Tag2 mice treated with SU5416 or SU6668 (Regression Trial). To visualize the functional blood vessels in tumors, mice were first anesthetized and injected intravenously with 0.05 mg FITC-labeled tomato lectin (Lycopersicon esculentum) and then heart perfused with 4% PFA. Pancreata were frozen in OCT medium and sectioned at 50 μm. To visualize endothelial cells (green in right panels) and pericytes (red in right panels) by immunohistochemical analysis, mice were anesthetized, heart perfused with PFA, and pancreata collected, frozen in OCT medium, and 15-μm sections prepared. Endothelial cells were detected with FITC-labeled lectin; pericytes were identified with CY3-labeled anti-desmin (1:3,000), a marker of mature pericytes.

Figure 3

Identification of the cell types expressing PDGF ligands and receptors in pancreatic islet carcinomas. (a) Primary tumors were fractioned into constituent cell types by flow cytometry. RNA was isolated from unsorted and sorted populations and analyzed by RT-PCR. Pancreatic tumors of end-stage Rip1Tag2 mice were excised and enzymatically dispersed with collagenase into single cells. The cell suspension was incubated with Ab’s for CD31 and Gr1 and Mac1. Endothelial cells were collected by FACS as a CD31⁺, Gr1^–, Mac1^– population, whereas tumor cells were gated by size and collected as unlabeled with these three Ab’s. Inflammatory cells were collected as Gr1⁺, Mac1⁺; these cells did not express PDGF ligands or receptors (not shown). (b) Tumor sections (prepared as in Figure 2) were costained with anti–PDGFR-β-FITC (1:200) and anti–CD31-rhodamine to reveal PDGFR-β-expressing cells in green and endothelial cells in red. (c) PDGFR-β⁺ cells from tumors were isolated by FACS (PDGFR-β Ab, 1:50), RNA isolated, and analyzed by RT-PCR for pericyte markers. ECs, endothelial cells; TCs, tumor cells; PCs, perivascular cells.

Figure 4

Improved efficacy at all stages of islet carcinogenesis produced by combining the VEGFR-inhibitor SU5416 with SU6668 or Gleevec, two drugs that inhibit PDGFR signaling. Mice were injected subcutaneously with 50–75 mg/kg SU5416 twice a week and in addition received either daily oral administration of 200 mg/kg SU6668 or twice daily dosing of 50 mg/kg Gleevec (STI571). (The dosage of SU5416 had to be reduced from that used in the single-agent trials shown in Figure 1 due to SU5416-specific toxic side effects.) The average number of angiogenic islets ± SEM at 10.5 weeks in control and treated mice are shown in the prevention trial. The average tumor burden ± SEM of PBS/vehicle–treated mice is indicated at 10, 12, and 13.5 weeks, for comparison with SU5416 + SU6668–treated mice at 13.5 and 16 weeks, and with SU5416 + Gleevec–treated mice at 16 weeks. Tumor burdens were assessed as described in Methods. Statistical analysis was performed with a two-tailed, unpaired Mann-Whitney test comparing experimental groups with PBS-injected control mice. Tumor burdens of experimental groups in the regression trial were compared with that of 12-week-old Rip1Tag2 mice. Cohorts of 6–21 animals were used. P values less than 0.1 are considered statistically significant. (P values of SU5416 + SU6668 PT = 0.0002, SU5416 + SU6668 IT = 0.0008, SU5416 + SU6668 RT = 0.0003, and SU5416 + Gleevec RT = 0.0007.

Figure 5

Effects of the combined therapy using SU5416 + SU6668 or SU5416 + Gleevec. Hematoxylin and eosin staining of islets from untreated (a) and SU6668 + SU5416–treated transgenic mice (b) at 10.5 weeks in a prevention trial. Gross pathology of dissected pancreata from untreated (c) and SU5416 + SU6668–treated mice (d) in a 4-week regression trial targeting late-stage disease. Hematoxylin and eosin staining of tumors from untreated (e) and SU5416 + SU6668–treated mice (f). Arrows indicate hemorrhage formation, and dotted area confines necrotic region. Comparison of the functional vasculature in control (g) and SU5416 + SU6668–treated mice (h) from a regression trial. Mice were injected intravenously with FITC-labeled tomato lectin (Lycopersicon esculentum) to stain blood vessels in green, and then heart perfused with 4% PFA, followed by immunohistochemical staining with Cy3-labeled desmin Ab to label desmin-expressing perivascular cells in red. Apoptotic cells in tumors of control (i) and SU6668 + SU5416–treated mice (j) were detected by TUNEL staining with fluorescent visualization (red), and the vasculature was revealed as above by intravenous FITC-lectin perfusion before sacrifice. Mice were treated with SU5416 + Gleevec in the regression trial, and blood vessels and perivascular cells of exocrine pancreas (k) and adjacent islet tumors (l) were visualized with FITC-lectin and a Cy3-labeled desmin Ab.

Figure 6

Two distinctive vascular cell types in tumors present complementary targets for anticancer drugs. The results presented herein suggest that the conceptual notion of tumors as aberrant organs composed of both cancer cells and conscripted normal cell types, all making functional contributions to tumor phenotypes (43), be expanded both to include PDGFR-β⁺ pericytes and to recognize that tumors can have vasculature that is either immature or mature, with different responses to angiogenesis inhibitors. Combined therapy efficacy against otherwise intractable late-stage islet carcinomas is observed when VEGFRs on endothelial cells and PDGFRs on perivascular cells are targeted together. There is reason to envision that when the other constituent cell types, in particular the overt cancer cells, are also targeted, long-term therapeutic benefit can be achieved.