Co-targeting of VEGFR2 and PD-L1 promotes survival and vasculature normalization in pleural mesothelioma

Abstract

Pleural mesothelioma (PM) is an aggressive cancer caused by asbestos exposure, with limited treatment options and poor prognosis, highlighting the need for more effective therapies. Combining immune checkpoint blockade with anti-angiogenic therapy has shown potential in other cancers. Our study investigated the combined inhibition of PD-L1 and VEGFR2 in a mouse model of PM. Using C57BL/6 mice with subcutaneous AE17 mesothelioma tumors, we assessed the effects of anti-PD-L1 therapy with induction, concomitant, or consolidation anti-VEGFR2 treatment. Mice received intraperitoneal doses every three days for three treatments. Tumor growth, survival, tumor-infiltrating immune cells and intra-tumoral vasculature were analyzed. Results demonstrated that combining anti-PD-L1 with induction or concomitant anti-VEGFR2 significantly delayed tumor growth, improved survival, and promoted vascular maturation. Flow cytometry suggested T cell exhaustion in monotherapy groups, while no significant changes were seen with concomitant treatment. Depleting CD4⁺ T cells reversed the positive effects of concomitant treatment. These findings suggest that dual inhibition of PD-L1 and VEGFR2 is a promising therapeutic approach for PM, with CD4⁺ T cells playing a critical role in the immune response. This dual targeting of immune checkpoints and angiogenesis offers a potential new avenue for improving outcomes in PM treatment and warrants further clinical exploration.

Keywords: Anti-angiogenesis; cancer immunotherapy; combination therapy; immune checkpoint blockade; pleural mesothelioma.

Graphical abstract

Figure 1.

Anti-VEGFR2 monoclonal antibody delays tumour growth and extends survival of the AE17 C57BL/6 PM mouse model. (a-b) Kaplan-Meier curves showing (a) the progression-free survival (PFS) and (b) the disease-specific survival (DSS) of 87 pleural mesothelioma patients in function of their VEGFR2 protein expression level. (c) bubble plot showing the correlations between CD274 (PD-L1), KDR (VEGFR2), PDCD1 (PD-1), and VEGFA gene expression, VEGFR2 protein expression, and tumour inflammation signature (TIS) scores. (d) correlation analysis between PDCD1 mRNA expression and TIS within VEGFR2-low (left) and VEGFR2-high (right) subgroups. (e) Kaplan-Meier curve showing the survival of mice treated with increasing doses of anti-VEGFR2 mAb or PBS control. Pooled data from three independent experiments (n = 15 mice per group). Statistical significance was determined using a log-rank test. (f) tumour growth curves from mice treated with increasing doses of anti-VEGFR2 mAb or PBS control. Tumour size (mm³) as measured over time from the start of treatment (day 0) until the first mouse of each group was euthanised. Mice were treated every third day for a total of three doses (q3dx3). Statistical significance was determined using a mixed model ANOVA. Error bars represent the mean ± SEM. Data representative of three independent experiments (n = 5 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Combined treatment with anti-VEGFR2 and anti-PD-L1 slows tumour growth and conveys a survival benefit in the AE17 C57BL/6 mesothelioma mouse model. (a) schematic of the in vivo experimental set-up and treatment timelines. (b) Kaplan-Meier curves showing the post-treatment survival of mice treated with the indicated therapies. Pooled data from three independent experiments (n = 8–14 mice per group). Statistical significance was determined using a log-rank test. (c) tumour growth curves from mice treated as indicated. Tumour size (mm³) was measured over time from tumour inoculation and is shown here from the day of start of treatment until the first mouse of each group was euthanised. Statistical significance was determined using a mixed model ANOVA. Error bars represent the mean ± SEM. Data representative of at least three independent experiments (n = 3–5 mice per group). (d) spaghetti plots showing tumour kinetics data, with each line representing an individual mouse within the different treatment groups. The grey dotted line at day 9 post-treatment represents the timepoint at which 50% of the control animals had reached their humane endpoint; the coloured dotted lines represent the timepoints at which 50% of animals of each individual group reached their humane endpoint. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Created with BioRender.com.

Figure 3.

Intratumoural vasculature in AE17 C57BL/6 mice is normalised by combined anti-VEGFR2 and anti-PD-L1 treatment. (a) Representative immunofluorescence image of tumour tissue stained for CD31 (red), αSMA (green), and DAPI (blue), 40x magnification. (b) heatmap of VECTASHIELD® and ImageJ analysis results expressed as the Fold change normalised to the PBS control group. (c) VECTASHIELD® quantification of: percentage of CD31 expression (top left); number of branching points (top middle); total tube length expressed in pixels (px) (top right); number of tubes (bottom left); number of loops (bottom middle). Quantification of CD31-αSMA co-localisation, expressed as the percentage of CD31-positive cells covered in αSMA-positive cells (yellow signal) (bottom right). Error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4.

The complex tumour immune microenvironment of the AE17 C57BL/6 mesothelioma mouse model treated with anti-VEGFR2 and/or anti-PD-L1 therapy. (a) schematic detailing the experimental setup. Experiment repeated twice with a total of n = 9–12 mice per group. (b) tumour weights in milligram (mg) with mean ± SEM. Dots represent individual tumours. (c-k) quantification of CD45.2⁺ cells (leukocytes) (c), CD45.2⁺ CD3⁻ NKp46⁻ CD11c⁺ MHCII⁺ dendritic cells (d), CD45.2⁺ CD3⁻ NKp46⁺ natural killer cells (e), CD45.2⁺ CD3⁺ T cells (f), CD45.2⁺ CD3⁺ CD4⁻ CD8⁻ double-negative T cells (g), CD45.2⁺ CD3⁺ CD8⁺ T cells (h), CD45.2⁺ CD3⁺ CD4⁺ FOXP3⁻ CD25⁻ Th cells (i), CD45.2⁺ CD3⁺ CD4⁺ FOXP3⁺ CD25⁺ Tregs (j), and CD45.2⁺ CD3⁺ CD4⁺ FOXP3⁺ CD25⁻ Tregs, expressed as the absolute number of cells per mg of tumour tissue. Error bars represent the mean ± SEM and dots represent individual tumours. (l) heatmaps of the percentage of PD-1- (left), PD-L1- (middle), and ICOS-expressing (right) immune cells expressed as the Fold change normalised to the PBS control group. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Created with BioRender.com.

Figure 5.

Depletion of CD4⁺ T cells reverses therapeutic effects of combined anti-VEGFR2 and anti-PD-L1 treatment and abrogates intratumoural vasculature. (a) schematic of the experimental design and timeline. (b) Kaplan-Meier curves showing the post-treatment survival of mice treated with concomitant anti-VEGFR2 and anti-PD-L1 treatment (combo) as well as CD8⁺ (left), CD4⁺ (middle), and CD25⁺ (right) immune cell depleting antibodies (n = 5–9 mice per group). Statistical significance was determined using a log-rank test. (c) Tumour growth curves from mice treated as indicated. Tumour size (mm³) was measured over time from tumour inoculation and is shown here from the day of start of treatment until the first mouse of each group was euthanised. Statistical significance was determined using a mixed model ANOVA. Error bars represent the mean ± SEM (n = 5–9 mice per group). (d) Representative immunofluorescence images of tumour tissue from a mouse treated with concomitant anti-VEGFR2 and anti-PD-L1 treatment (top) and a mouse treated with combo + CD4⁺ immune cell depletion (bottom), stained for CD31 (red) and DAPI (blue). 10x magnification. (e) heatmap of VECTASHIELD® analysis results expressed as the Fold change normalised to the concomitant treatment group. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Created with BioRender.com.