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. 2015 Feb;17(2):215-24.
doi: 10.1016/j.neo.2014.12.011.

Successful combination of sunitinib and girentuximab in two renal cell carcinoma animal models: a rationale for combination treatment of patients with advanced RCC

Jeannette C Oosterwijk-Wakka  1 , Mirjam C A de Weijert  2 , Gerben M Franssen  3 , William P J Leenders  4 , Jeroen A W M van der Laak  4 , Otto C Boerman  3 , Peter F A Mulders  5 , Egbert Oosterwijk  2
Affiliations

Successful combination of sunitinib and girentuximab in two renal cell carcinoma animal models: a rationale for combination treatment of patients with advanced RCC

Jeannette C Oosterwijk-Wakka et al. Neoplasia. 2015 Feb.

Abstract

Anti-angiogenic treatment with tyrosine kinase inhibitors (TKI) has lead to an impressive increase in progression-free survival for patients with metastatic RCC (mRCC), but mRCC remains largely incurable. We combined sunitinib, targeting the endothelial cells with Girentuximab (monoclonal antibody cG250, recognizing carbonic anhydrase IX (CAIX) targeting the tumor cells to study the effect of sunitinib on the biodistribution of Girentuximab because combination of modalities targeting tumor vasculature and tumor cells might result in improved effect. Nude mice with human RCC xenografts (NU12, SK-RC-52) were treated orally with 0.8 mg/day sunitinib, or vehicle for 7 to 14 days. Three days before start or cessation of treatment mice were injected i.v. with 0.4 MBq/5 μg (111)In-Girentuximab followed by biodistribution studies. Immunohistochemical analyses were performed to study the tumor vasculature and CAIX expression and to confirm Girentuximab uptake. NU12 appeared to represent a sunitinib sensitive tumor: sunitinib treatment resulted in extensive necrosis and decreased microvessel density (MVD). Accumulation of Girentuximab was significantly decreased when sunitinib treatment preceded the antibody injection but remained unchanged when sunitinib followed Girentuximab injection. Cessation of therapy led to a rapid neovascularization, reminiscent of a tumor flare. SK-RC-52 appeared to represent a sunitinib-resistant tumor: (central) tumor necrosis was minimal and MVD was not affected. Sunitinib treatment resulted in increased Girentuximab uptake, regardless of the sequence of treatment. These data indicate that sunitinib can be combined with Girentuximab. Since these two modalities have different modes of action, this combination might lead to enhanced therapeutic efficacy.

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Figures

Figure 1
Figure 1
Biodistribution of cG250 in nude mice with NU12 and SK-RC-52 tumors. 111In-Girentuximab biodistribution in BALB/c nu/nu mice shows decreased uptake with sunitinib in NU12 tumors and increased uptake with sunitinib in SK-RC-52 tumors. Treatment schedules shown on top of graphs. Groups of 4–5 mice (in F: 8–9 mice) were treated with sunitinib every day for 1 week (A,D,F) or until they were euthanized (B,E). Three days before start or 3d (in one experiment also 7 or 10 days) after stop of treatment, mice were injected with 111In-Girentuximab (0.4 MBq, 5 μg) and mice were euthanized at various timepoints. The activity in the samples was expressed as % injected dose per gram tissue (%ID/g). A: Biodistribution of NU12 mice with sunitinib treatment preceding 111In-Girentuximab injection, B: Biodistribution of NU12 mice injected with 111In-Girentuximab before sunitinib treatment, C: G250 antibody uptake was plotted for individual tumors from experiment. Red open circles: control tumors; black closed circles: sunitinib treated tumors A, D: Biodistribution of SK-RC-52 mice treated with sunitinib preceding 111In-Girentuximab injection, E: Biodistribution of SK-RC-52 mice treated with sunitinib followed by 111In-Girentuximab, F: Biodistribution of SK-RC-52 mice with 111In-Girentuximab injection 3 days, 7 days or 10 days after cessation of sunitinib, G: G250 antibody uptake was plotted for individual tumors from experiment F. *P < .05, **P < .01, ***P < .005. P-values shown for the biodistribution are based on all sunitinib treated animals (N = 15, 9, 28,18, 25 for Figure 1, A, B, D–F, respectively) compared to all control animals (N = 13, 16, 30, 18, 27 for Figure 1, A, B, D–F, respectively).
Figure 2
Figure 2
Phenotypic analysis of NU12 and SK-RC-52 tumors. Phenotypic analysis of NU12 and SK-RC-52 tumors of mice treated with sunitinib shows necrosis, decreased accumulated cG250 and decreased number of microvessels in NU12 tumors and very limited necrosis, increased accumulated cG250 and unchanged number of microvessels in SK-RC-52 tumors. A-D, NU12 control tumors; E-H, NU12 sunitinib treated tumors; I-L, SK-RC-52 control tumors; M-P, SK-RC-52 sunitinib treated tumors. HE staining in A, E, I and M. Sunitinib treatment did not modify CAIX expression in either NU12 or SK-RC-52 (B,F and J,N). In NU12 control tumors (A-D), homogeneous accumulation of cG250 (C) was observed. D: tumor vasculature visualized by staining with 9 F1. In sunitinib treated NU12 tumors (E-H), extensive necrosis was present as judged by HE (E) and accumulated cG250 (G) and microvessels (H) were only observed in the tumor rim. SK-RC-52 control tumors (I-L), revealed focal accumulation of injected cG250 (J) and moderate microvessel density (MVD) as visualized by staining with 9 F1 (L). Accumulation of cG250 was increased in sunitinib treated SKRC52 tumors (O vs. K) and MVD appeared to be increased (P vs. L). Necrosis was limited regardless of treatment (I, M). N: necrosis. Original magnification × 25 and × 200.
Figure 3
Figure 3
Microvessel density analysis. Density of microvascular profiles was assessed automatically as described previously using an AxioCam MRc connected to an AxioPhot microscope (Zeiss). Microvessel density (MVD) was defined as the percentage of microvessel area/total tumor area. All image acquisition and processing was performed using custom written macros in KS400 image analysis software (Zeiss). Sunitinib treatment resulted in significant decrease in % of microvessels in NU12 tumors (A) but no change in % of microvessels in SK-RC-52 tumors (B). Triple immunofluorescence staining of NU12 control (C) and sunitinib treated tumor (D) and SK-RC-52 control (E) and sunitinib treated tumor (F). Low proliferation of NU12 tumor cells (nuclei in blue as visualized by DRAQ5) as visualized by Ki67 staining (red) is observed, both in controls (C) as well as in sunitinib treated tumors (D). Please note decrease of endothelium (9 F1 staining) in sunitinib treated NU12 tumor (green). In SK-RC-52, Triple immunofluorescence staining revealed high proliferation of SK-RC-52 tumor cells, both in controls (E) as well as in sunitinib treated tumors (F) Necrosis was minimal. ****P < .001.
Figure 4
Figure 4
SPECT/CT imaging of mice with SK-RC-52 tumors. SPECT/CT analysis was performed to visualize the biodistribution and the intra-tumoral distribution of the radiolabeled antibody. Sixteen mice bearing SK-RC-52 were treated with sunitinib for 7 days and injected with 111In-Girentuximab with a specific activity of 22,5 MBq/5 μg, 3 days before start or 3 days after stop of treatment. Micro-SPECT images of mouse bearing SK-RC-52 tumor on right flank (arrow) at 7 d after injection of 111In-girentuximab show that in addition to tumor uptake, minimal uptake in other organs was observed. More radiolabel was observed in the sunitinib treated tumors than in vehicle (A).This is in concordance with the biodistribution data. In all groups radiolabel was distributed throughout the tumor and no difference in radiolabel distribution was observed in the various treatment groups (B).
Figure 5
Figure 5
Expression of VEGF-A isoforms in NU12 and SK-RC-52 tumors. VEGF-A/RT-PCR was performed on NU12 and SK-RC-52 cells as well as on harvested xenografts of both sunitinib-treated (from 2 days up to 7 days) as untreated animals. After correction for loading differences (as judged by GAPDH), no differences in VEGF-A expression was observed between tumor models, regardless of sunitinib treatment.
Figure S1
Figure S1
Tumor growth of NU12. Mean % of growth of NU12 tumors during treatment with sunitinib. Treatment with sunitinib was started when tumors had reached a tumor volume of 100 to 200 mm3 (Day 10) and continued until Day 3. Three days later mice were injected with 111In-cG250. Percentage tumor growth was set at 100% at start of treatment with sunitinib. Tumor growth is strongly inhibited by sunitinib treatment (black line) in comparison to control (red line). Figure S2 Tumor growth of SK-RC-52. Mean % of growth of SK-RC-52 tumors during treatment with sunitinib. Treatment with sunitinib was started when tumors had reached a tumor volume of 100 to 200 mm3 (Day 10) and continued until Day 3. Three days later mice were injected with 111In-cG250. Percentage tumor growth was set at 100% at start of treatment with sunitinib. Tumor growth is stabilized by sunitinib treatment (black line) in comparison to control (red line).
Figure S1
Figure S1
Tumor growth of NU12. Mean % of growth of NU12 tumors during treatment with sunitinib. Treatment with sunitinib was started when tumors had reached a tumor volume of 100 to 200 mm3 (Day 10) and continued until Day 3. Three days later mice were injected with 111In-cG250. Percentage tumor growth was set at 100% at start of treatment with sunitinib. Tumor growth is strongly inhibited by sunitinib treatment (black line) in comparison to control (red line). Figure S2 Tumor growth of SK-RC-52. Mean % of growth of SK-RC-52 tumors during treatment with sunitinib. Treatment with sunitinib was started when tumors had reached a tumor volume of 100 to 200 mm3 (Day 10) and continued until Day 3. Three days later mice were injected with 111In-cG250. Percentage tumor growth was set at 100% at start of treatment with sunitinib. Tumor growth is stabilized by sunitinib treatment (black line) in comparison to control (red line).
Figure S3
Figure S3
Phenotypic analysis of NU12 tumors. Phenotypic analysis of NU12 tumors of mice treated with sunitinib. Mice were injected intravenously with 111In-Girentuximab (0.4 MBq, 5 μg) 3 days pre-sunitinib treatment. Accumulation of Girentuximab remained unchanged when sunitinib followed Girentuximab injection. Tumors shown were harvested at Day 7 (4 days after start of sunitinib treatment). A, C: HE staining of untreated and sunitinib treated tumor respectively; B, D: Girentuximab accumulation. Original magnification X25 and X200.
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