Synergistic effect of combining sunitinib with a peptide-based vaccine in cancer treatment after microenvironment remodeling

Figures & data

Figure 1. SVX vaccine modulates tumor angiogenesis with a transient decrease in VEGF levels in the tumor.

As represented in (a), mice were s.c. inoculated with CT26 tumor cells and immunized with SVX vaccine (s.c.) plus adjuvant on day 4 and on day 11 (without adjuvant) or injected with PBS for the control group. Tumors were harvested on days 7, 14, and 21 (n = 10–12 mice/group/day) to perform tumor lysate or immunofluorescence staining on frozen tissue sections. (b) Histograms represent tumor volume (mm³) at sacrifice for each group. (c) VEGF-A levels measured by enzyme-linked immunosorbent assay in total tumor lysate per mg of tumor from SVX-treated or control mice at different time points. (d-g) Mice were sacrificed one hour after hypoxyprobe injection (i.p.). (d) Tissues were stained for pimonidazole adducts (Hypoxyprobe, yellow) and hypoxic areas were determined using Inform® tissue segmentation analysis and quantified as mean percentage of hypoxic area per field (right). (e) Tumors were also stained for endothelial cells (CD31, green), pericytes (a-sma, red) and nucleus (DAPI, blue) and Inform® tissue segmentation algorithms were also applied. (f) Microvascular density was assessed as the mean percentage of CD31⁺ area per field. (g) Pericyte coverage was calculated as [α-sma⁺area/CD31⁺ are × 100]. Data are shown as means ± SEM and are the pool of two independent experiments. Statistical analysis was performed using two-way Anova with Sidak’s multiple comparisons test (b, d, f, g) and with Tukey’s multiple comparisons test (c). * P < .05; ** P < .01; **** P < .0001.

Figure 2. Strong therapeutic efficacy of the combination when the vaccination is initiated at the end of sunitinib treatment.

Mice (n = 8–10/group) were s.c. inoculated with CT26 tumor cells. In a first experimental setting (a/b), mice were immunized on day 4 (with adjuvants) and on day 11 (without adjuvant) (SVX). Sunitinib treatment was started, daily by oral gavage either on day 7 (a) or on day 14 (b) for a period of 14 consecutive days at a dosing equivalent to 40 mg/kg. In a second experimental setting (c/d), sunitinib treatment was started 5 days after tumor inoculation and mice were immunized for the first time either on day 12 (c) or on day 19 (d) with a second immunization one week after. In every setting, the combination group was compared to a sunitinib-treated group and a control group (DMSO). Data are presented as mean tumor volume (mm³) calculated as [length.(width²)/2] ± SEM and as tumor weight at sacrifice (gram) ± SEM with the number of complete regressions indicated. Colored stars represent statistical significance between the control group and either the sunitinib-treated group (blue) or combined sunitinib + vaccine group (orange). Black stars represent statistical significance between the sunitinib-treated group and combined sunitinib + vaccine group. Statistical analysis was performed using two-way Anova with Sidak’s multiple comparisons for differences in tumor volume and with the Mann–Whitney t-test for differences in tumor mass (d, right). * P < .05; ** P < .01.

Figure 3. Pericyte coverage and CD8⁺ T cell infiltration increase one week after the interruption of sunitinib.

As represented in (a), mice were s.c. inoculated with CT26 tumor cells and daily treated with 40 mg/kg of sunitinib by oral gavage starting on day 5 for a period of 14 days. Tumors and spleens were harvested either after one week of sunitinib treatment (d12), at the end of sunitinib treatment (d19) or one week after stopping the treatment (d26). Tumors were frozen to perform immunofluorescence in situ analysis of tumor vasculature (n = 8–10 mice/group/day). (b) Tumor mass (gram) of treated or control mice at different time points of sacrifice. (c) Representative fields of control and treated tumors at d12, d19, and d26 stained for endothelial cells (CD31, green), pericytes (NG2, red), CD8⁺ T cells (CD8, white) and nucleus (DAPI, blue). (d) InForm tissue segmentation algorithms were used to determine the CD31⁺ and NG2⁺ areas representative of vascular density (left) and pericyte density (right) respectively. (e) Pericyte coverage was calculated as [NG2⁺area/CD31⁺ are × 100]. (f) InForm cell segmentation and phenotyping algorithm was used to determine the number of CD8⁺ cells per field. (g) Correlation between the number of CD8⁺ cells and pericyte coverage per field at day 26 independently of treatment. Spearman correlation was used with a simple linear regression represented with the 95% confidence bands of the best-fit line. Data are shown as means ± SEM. Statistical analysis was performed using two-way Anova with Sidak’s multiple comparisons test (b, d, e, f). * P < .05; ** P < .01; *** P < .001; **** P < .0001.

Figure 4. Sunitinib decreases myeloid populations in the spleen over time but transiently increases PMN-MDSCs infiltrating the tumor. In the same experimental settings as described before, CT26-bearing mice were treated or not with sunitinib (d5 to d18). Tumors and spleens were harvested at d12, d19 or d26 to perform phenotyping analysis by flow cytometry (n = 14–18 mice/group/day).

(a) Number of infiltrating CD45⁺ immune cells represented per gram of tumor. (b) Representative flow plots showing polymorphonuclear (Ly6G ^high Ly6C ^int, violet) and monocytic (Ly6C ^high Ly6G ^neg, blue-green) MDSC populations among CD45⁺ cells in the spleen at d12, d19, and d26 in treated or control animals. (c-f) Percentage of different myeloid population among CD45⁺ cells (see gating strategy in Suppl. S4, A) in the spleen (c, d) and the tumor (e, f) with (c, e) polymorphonuclear MDSCs (Ly6G ^high Ly6C ^int, left) and monocytic MDSCs (Ly6C ^high Ly6G ^neg, right) and (d, f) Macrophages (CD11b ^highF4/80⁺). Data are shown as means ± SEM and are the pool of two independent experiments. Statistical analysis was performed using two-way Anova with Sidak’s multiple comparisons test (a, d, f) and with Tukey’s multiple comparisons test (c, e). * P < .05; ** P < .01; *** P < .001; **** P < .0001.

Figure 5. NK populations are decreased during sunitinib treatment and increased after the cessation of treatment in both tumor and spleen.

Briefly, tumors and spleens of CT26-bearing mice treated or not with sunitinib (d5 to d18) were harvested at d12, d19, or d26 (n = 14–18 mice/group/day for a and d; n = 6–8mice/group/day for b) to perform phenotypic analysis by flow cytometry. (a) Percentages of NK cells (NKp46⁺CD3⁻) among CD45⁺ immune cells in the spleen (left) and in the tumor (right). (B) Percentage of proliferating (Ki67⁺) NK cells in the tumor. (c) Representative dot plots showing CD11b expression by infiltrating NK cells in the tumor of control or sunitinib-treated mice at d12, d19 or d26. (d) Percentage of CD11b expressing NK cells. Data are shown as means ± SEM and are the pool of two independent experiments (for a and d). Statistical analysis was performed using two-way Anova with Sidak’s multiple comparisons test (a) and with Tukey’s multiple comparisons test (b, d). * P < .05; ** P < .01; **** P < .0001.

Figure 6. Sunitinib treatment decreases Treg in the spleen and strongly increases CD8⁺ T cells population and function in the tumor.

(a-d) Briefly, spleens and tumors of CT26-bearing mice treated or not with sunitinib (d5 to d18) were harvested at d12, d19, or d26 (n = 14–18 mice/group/day). (a-b, d) Lymphocyte populations were assessed by flow cytometry in the spleen (top) and the tumor (bottom) as the percentage of total T cells among CD45⁺ cells (a), regulatory T cells (CD4⁺FoxP3⁺) among T lymphocytes (b), and CD8⁺ T cells among T lymphocytes (d). (c) Percentage of proliferating (Ki67⁺) regulatory T cells in the spleen (top) and the tumor (bottom). (e-g) Tumors of CT26-bearing mice treated or not with sunitinib (d5 to d18) were harvested at d26. (e) Percentage of PD-1⁺ cells among CD8⁺ infiltrating T lymphocytes (n = 5 mice/group). (f) Percentage of Granzyme B⁺ CD8⁺ cells among infiltrating T lymphocytes (left) and Geometric Mean of Granzyme B in CD8⁺ infiltrating T cells (right) (n = 8-9 mice/group). (g) CD8⁺ infiltrating lymphocytes were enriched by magnetic beads. 5 × 10⁴ cells (representing a pool of 2 tumors) were in vitro restimulated with anti-CD3/anti-CD28 beads. Cytokine production was measured by Luminex assay performed on the supernatant after 24 h of culture (n = 4 pool of 2 mice/group). Data are shown as means ± SEM and (a-d) are the pool of two independent experiments. Statistical analysis was performed using two-way Anova with Sidak’s multiple comparisons test (a-d) and Mann–Whitney t-test (e-g). * P < .05; ** P < .01; **** P < .0001.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, CT, upon reasonable request.