Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors (original) (raw)
Two RTKIs have distinctive efficacy profiles. Previously, we evaluated the VEGFR inhibitor SU5416 (11, 19) in the three distinctive therapeutic trials in RIPTag2 mice (12, 13); these data are illustrated here to facilitate evaluation of the comparative and combination trials involving this and other RTKI. In the regression trial, which treats end-stage mice having bulky disease, thus being analogous to the typical phase-3 clinical trial of investigational anticancer drugs (Figure 1), treatment with SU5416 produced a modest increase in life span (to a defined endpoint 2.5 weeks after sham-treated mice were euthanized due to incipient death from tumor burden), concomitant with a lower rate of tumor growth, but this drug was not capable of regressing tumor mass or producing stable disease (Figure 1; RT). As such, we found the SU5416 efficacy profile to be similar to that of endostatin, angiostatin, and the MMP inhibitors BB94 and BAY-129566 (3, 12, 13).
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.
Given that SU5416 was very efficacious against earlier-stage disease (Figure 1), phenocopying the VEGF-A gene knockout in its impairment of angiogenic switching and tumor growth, we reasoned that other regulatory molecules might become involved in controlling angiogenesis and maintaining of the tumor vasculature in well-established solid tumors. We therefore evaluated another RTKI (SU6668) with somewhat broader selectivity in the three distinctive preclinical trials in the Rip1Tag2 model.
SU6668, a small molecule kinase inhibitor with demonstrable antiangiogenic activity (20–22), inhibits phosphorylation and signal transduction of PDGFRs, VEGFRs, and FGF receptors (FGFRs). While SU6668 has significantly higher biochemical activity against PDGFR-α and -β (_K_I = 0.0008 μM) than VEGFR-2 and FGFR-1 (_K_I = 2.1 μM and 1.2 μM, respectively) (21), cell-based assays reveal functionally appreciable inhibitory activity against both VEGFR-21 and PDGFR (21, 23, 24). By contrast, SU5416 is predominantly active against VEGFR-2 (_K_I = 0.04 μM), with minimal activity against PDGFR or FGFR (11, 25).
The trials with SU6668 produced a different efficacy profile from that of SU5416. Thus, SU6668 was less effective at blocking angiogenic switching in the prevention trial and was similar at impairing tumor growth in an intervention trial (Figure 1). Surprisingly, this agent proved much more effective than SU5416 in the regression trial, producing a condition of stable disease, in that tumor burden at the culmination (a defined end point after 4 weeks of treatment) was similar to that at the 12-week-old starting point. How can these differences be explained? We suspect that the relatively poor activity of SU6668 at blocking angiogenic switching (Figure 1; PT) reflects its lower _K_I against VEGFR; by contrast, SU5416 produced similar reductions in angiogenic switching to the gene knockout of VEGF-A (14), indicative of the singular importance of VEGF signaling at this early stage. As for its significantly better effects on well-established tumors, we hypothesized that the broader specificity of SU6668, in particular against PDGFR, might underlay its benefits. We sought, therefore, to compare the histological effects of treatment with these two agents that might reflect distinctive functional effects and to investigate the expression of PDGF ligands and receptors in the islet tumors, given that SU6668 potently inhibits these receptors.
To investigate the underlying mechanisms of these stage-specific effects, we first assessed the vascular morphology in the treated versus control tumors from the regression trials of these two drugs. Perfusion of the circulatory system with a fluorescent lectin was used to assess the functional tumor vasculature, revealing (Figure 2; left panel) demonstrable decrease in vascularity of SU6668-treated tumors and modest reduction in tumors treated with SU5416. We further assessed the vascular morphology by immunostaining tissue sections with Ab’s recognizing two vascular markers: CD31 (PECAM), a cell adhesion molecule expressed on endothelial (and hematopoietic) cells, and desmin, a marker of mature periendothelial support cells (pericytes) (26, 27). We were motivated to investigate pericytes both by a report (28) and our unpublished data that pericytes were abundant in tumors of the RIP1Tag2 model. A typical intimate association was seen between endothelial cells and pericytes in the untreated tumors, as well as in the SU5416-treated tumors (Figure 2; right panel). By contrast, SU6668-treated tumors showed marked disruption of pericyte-endothelial cell association. Pericytes had become separated from the endothelial cells, and the blood vessels were enlarged and distorted (Figure 2). Thus, SU6668 affected the tumor vasculature differently than SU5416: not only was the vascularity of the tumors reduced, but the integrity of the association of endothelial cells and pericytes was markedly perturbed. Collectively, the results led us to suspect that the distinctive effects of SU6668 against solid tumors resulted from its targeting of pericytes; this was further supported by a report suggesting that SU6668 could reduce the number pericytes in a tumor transplant model (29). Further rationale for this hypothesis came from the knowledge that pericytes express PDGFRs during vessel formation in the developing embryo and that functional disruption of PDGFR-β or its ligand PDGF-B leads to a lack of pericytes, causing severe vascular defects and embryonic lethality in late gestation (30–32). Such considerations raised a question: were PDGFRs expressed in pericytes or other cell types in the islet tumors, and if so were PDGF ligands expressed?
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.
Expression in tumor vasculature of PDGF ligands and PDGFRs. To assess the expression of PDGF ligands and PDGFRs, RNA was isolated from whole tumors and analyzed by RT-PCR, revealing expression of the PDGF-A, -B, and -D ligands, as well as both PDGFR-α and -β (Figure 3a). To identify the cell type that expressed PDGF ligands and receptors, we developed a protocol for fractionating primary tumors into constituent cell types by flow cytometry. Endothelial cells were sorted as CD31+, Gr1–, Mac1–; inflammatory cells were collected as Gr1+, Mac1+ (not shown); and tumor cells were gated by size and collected as unlabeled with these three Ab’s. RNA was collected from unsorted and sorted populations and analyzed by RT-PCR. As illustrated in Figure 3a, the endothelial cells were found to exclusively express the genes for PDGF ligands A, B, and D. None of the sorted cell populations, tumor cells, inflammatory cells (not shown), or endothelial cells, expressed the two PDGFR genes, despite demonstrable expression in whole-tumor RNA. We therefore stained tumor tissue sections with Ab’s specific for PDGFR-β to ask whether the receptor was expressed, and if so, in which cell type. The tissue sections were costained with anti-CD31/PECAM to mark the endothelial cells. The data shown in Figure 3b clearly reveal expression of PDGFR-β in perivascular cells that are in close contact with endothelial cells, but not in the endothelial cells or tumor cells, consistent with the analysis of the sorted cell types. (PDGFR-α expression is very low in the pancreatic tumors, and the mouse protein could not be detected with available Ab’s in tissue sections.) We went on to collect the PDGFR+ cells by FACS using an anti–PDGFR-β Ab; this cell population proved to have a small size that was gated out in the unlabeled tumor cell fraction shown in Figure 3a and furthermore expressed known markers of pericytes (e.g., desmin and smooth-muscle actin [SMA], as shown in Figure 3c) consistent with identification of the PDGFR-β+ cells in these tumors as a class of pericyte. The data indicate that PDGF ligands are expressed in the tumor endothelial cells and that PDGFR-β is expressed in cells associated with the angiogenic vasculature that morphologically score as perivascular cells (pericytes). The data are consistent with the hypothesis that SU6668 is targeting PDGFR+ pericytes, causing their dissociation from the tumor vasculature, leading to vascular dysfunction. It is formally possible that the VEGFR activity of SU6668 is contributing to the observed effects on tumor growth and tumor vascularity. The VEGFR selective inhibitor SU5416 did not induce dissociation of pericytes from tumor blood vessels (Figure 2), however, suggesting that SU6668’s modest activity against the VEGFRs is not the primary basis for its effect on tumor pericytes. We cannot at present exclude possible contributions to the observed effects of SU6668’s inhibition of FGFRs, given that there is evidence supporting involvement of FGF signaling in this model (33). Data to be presented below, however, suggest that the primary activity of SU6668 in this model of pancreatic islet carcinogenesis involves its inhibition of PDGFR signaling.
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.
Improved efficacy by combining kinase inhibitors with distinct specificity. SU5416 was most efficacious at blocking initial angiogenic switching and similarly as effective as SU6668 at repressing growth of small, nascent, solid tumors, whereas SU6668 was more effective against end-stage bulky disease (Figure 1). Therefore, we reasoned that combining the two kinase inhibitors might improve efficacy, given their distinctive efficacy profiles and target selectiveness. To investigate this hypothesis, combination trials were performed. The data are provocative. The combination of SU5416 and SU6668 improved efficacy in each of the three trials, variously targeting angiogenic dysplasias, small tumors, or end-stage tumors (Figure 4). Thus, the combination was better than either of the single agents at all stages of carcinogenesis, producing a broad efficacy profile. Mice treated with the combination did not develop morphologically identifiable angiogenic islets in the prevention trial, in contrast with the control mice. Histological analysis revealed that treated islets did not have hemorrhages and appeared more benign, like early hyperplastic islets in which the cells had a larger cytoplasm-to-nucleus ratio (Figure 5, a and b). Most exciting was the clear and convincing reduction in tumor mass seen in the regression trial. Not only did the combination produce severe reductions in tumor mass (Figures 4 and 5, c and d), but it also elicited evident necrosis and limited the characteristic microhemorrhaging that produces blood-filled tumors (Figure 5, e and f). The blood vessel network regressed and predominantly disappeared in larger tumors, leaving the perivascular cells stretched out and distorted (Figure 5, g and h). Concomitantly, apoptosis was increased 6.7-fold (Figure 5, i and j) in the combination therapy, whereas single treatment with SU5416 or SU6668 increased apoptosis 2.7- and 3.5-fold, respectively. The data presented above suggested that these dramatic effects are the result of simultaneous targeting of VEGFRs in endothelial cells and PDGFRs in perivascular cells. The modest inhibitory profile of SU6668 against FGFRs and its demonstrable VEGFR activity, however, cannot absolutely exclude other interpretations. Therefore, we tested another tyrosine kinase inhibitor, Gleevec (STI571), which has high activity against three kinases: Bcr/Abl, c-Kit, and PDGFR (17, 34, 35). Among known kinases tested, Gleevec overlaps with SU6668 only in its inhibition of PDGFR. We performed a regression trial with Gleevec, treating near end-stage mice with Gleevec alone or in combination with SU5416. Gleevec, which has very poor pharmacokinetic properties in mice (R. van Etten, Harvard Medical School, Boston, Massachusetts, USA, personal communication; see ref. 34), was not effectual as a single agent (not shown). Nevertheless, the combination of Gleevec and SU5416 produced significant reductions in tumor burden (Figure 4) comparable to SU6668 plus SU5416 treatment. Furthermore, the Gleevec combination produced similar morphological disruptions in tumors (increased apoptosis, decreased abundance and detachment of pericytes, and reduced vascularity; Figure 5l). Interestingly, the degree of vascular disruption varied among independent tumors, apparently as a function of the size of tumors, being most apparent in larger tumors. Importantly, neither of the combinatorial treatments caused dissociation of perivascular cells from blood vessels of normal tissue (lung, liver; data not shown) or otherwise disrupted normal tissue vasculature, including that of the immediately adjacent exocrine pancreas to the islet tumors (shown for Gleevec plus SU5416 in Figure 5k), indicating that the tumor vasculature is differentially sensitive to these kinase inhibitors.
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.
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.
The dramatic effect both kinase inhibitor combinations had on tumors in a month-long trial raised the question of whether they could improve survival over a longer time period. We sought to treat end-stage mice in a multimonth trial, but found that SU5416 was poorly tolerated by older mice, requiring most mice to be removed from study (see Methods). These side effects precluded statistically significant survival studies. Nevertheless, a few mice were able to stay in trial for 2 months with SU6668 plus SU5416 in the regression trial (i.e., 6 weeks past the incipient death and obligate euthanasia of the untreated controls); these mice still had a smaller tumor burden than mice at the starting point of the trial (12 weeks) (data not shown), indicating that the combination therapies were not only regressing well-established tumors in end-stage mice, but also limiting subsequent regrowth of these tumors or other new tumors forming from the abundant angiogenic progenitors characteristic of this challenging multifocal model of carcinogenesis. We hope to address long-term survival in future studies using less toxic VEGFR inhibitors in conjunction with Gleevec and SU6668, as well as other PDGFR inhibitors.