Neuropilin 1 and Neuropilin 2 gene invalidation or pharmacological inhibition reveals their relevance for the treatment of metastatic renal cell carcinoma

Figures

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  Fig. 1. NRP1 or NRP2 gene invalidation results in inhibition of cell proliferation and migration. a The locus of the NRP1 gene was sequenced in control (NRP1 Ctrl) and in two independent clones (#NRP1 2.2 and #NRP1 2.7) KO for NRP1. b The locus of NRP2 was sequenced in control (NRP2 Ctrl) and in two independent clones (#NRP2 2.3 and #NRP1 2.28) KO for NRP2. c NRP1 and NRP2 mRNA levels were tested by qPCR in control (786O), in two independent clones (#NRP1 2.2 and 2.7) KO for NRP1, and in two independent clones (#NRP2 2.3 and 2.28) KO for NRP2. d NRP1 and NRP2 protein levels were evaluated by flow cytometry in control (786O), in two independent clones (#NRP1 2.2 and 2.7) KO for NRP1, and in two independent clones (#NRP2 2.3 and 2.28) KO for NRP2. e The proliferation of NRP1 and NRP2 KO cells were tested by counting the cells at the indicated time points. f The migration of NRPs KO cells was determined in scratch assays by measuring the time of wound closure. g VEGFA and VEGFC expression was tested in control (Ctrl) and KO clones by ELISA. *p < 0.05; **p < 0.01; *** p < 0.001

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  Fig. 2. NRPs KO inhibits experimental RCC growth in immunocompetent and immunodeficient mice. a Experimental tumors in nude mice were obtained after injection of 3 × 105 control (Ctrl, 10 mice) or NRPs KO RENCA cells (5 mice for each condition). One NRP1 KO clone (4.1 7) and one NRP2 KO clone (5.1 8) were injected. Tumor incidence (percentage of mice with tumors) at the indicated times is presented. b Experimental tumors in immuno-competent Balb-C mice (5 mice per condition) were obtained after subcutaneous injection of 3 × 105 control (Ctrl) or the above-mentioned NRPs KO RENCA cells. The tumor volume is represented for the indicated time

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  Fig. 3. The NRP inhibitor NRPa-308 inhibits ccRCC cell proliferation more efficiently than sunitinib and EG00229. The effects of NRPa-308, sunitinib and the commercially available NRP inhibitor (EG00229) measured by MTT assays, were tested in (a) 786-O cells, (b) on A498 cells and (c) on HDF cells. d Determination of the IC50 for each treatment in the different cell lines and their selectivity index. *p < 0.05; **p < 0.01; *** p < 0.001

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  Fig. 4. NRPa-308 exerts a wide range of anti-proliferative effect on primary ccRCC cells. a The effects of NRPa-308 on cell viability were tested on 786-O (786-O) and 786-O cells resistant to sunitinib (786R). b The relative expression of NRP1 and NRP2 mRNA in 786-O and 786R cells was evaluated in a RNA seq analysis and confirmed by RT qPCR. c The sensitivity of NRPa-308 was tested on already described primary cells [15] by MTT assays. d The relative mRNA levels were evaluated in 786-O cells that served as reference values (100%) and in the different primary cells

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  Fig. 5. NRPa-308-dependent inhibition of cell proliferation relies mainly on NRP2 in 786-O cells. a Effects of NRPa-308 on cell viability of 786-O cells, of two independent NRP1 (#NRP1 2.2 and #NRP1 2.7) KO clones and of two independent NRP2 (#NRP2 2.3 and #NRP2 2.28) KO clones, measured by MTT assays, are represented to determine NRPa-308 specificity to NRP1 and/or to NRP2. b Clonogenic tests were performed on control 786-O, NRP1- and NRP2-KO cells in the presence of NRPa-308 (0.2 μM) for 6 days or following 6 days treatment then a 29 days’ period without NRPa-308 (0.2 μM). c The percentage of inhibition of VEGFA binding to NRP1 and NRP2 and of VEGFC binding to NRP2 at different concentration of NRPa-308 is presented. *p < 0.05; **p < 0.01

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  Fig. 6. NRPa-308 binding mode is different between NRP1 and NRP2. NRPa-308 (colored in orange) predicted binding mode into the NRP1 (a and b in cyan, left panels) and NRP2 (a and b in blue, right panels) binding sites. Hydrogen bonds are depicted as yellow dashed lines and π-stacking are depicted as magenta dashed lines

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  Fig. 7. NRPa-308 inhibits 786-O cell migration velocity more efficiently than sunitinib. a Photographs of scratch assay on cell monolayers in different experimental conditions; untreated, treated by NRPa-308 and by sunitinib. b The effects of NRPa-308 and sunitinib on 786-O cell migration at different concentrations by quantifying the above-mentioned experiments. c NRPa-308 (2 μM) inhibited the invasion of collagen-coated Boyden chambers (Control (Ctrl) and NRPa-308-treated duplicates are shown). d Quantification of the results shown in (c). **p < 0.01; *** p < 0.001

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  Fig. 8. High NRPa-308 concentration stimulates the production of NRPs ligands and of pro-angiogenic/pro-inflammatory cytokines. The effects of NRPa-308 and sunitinib on the production of different cytokines were evaluated by ELISA; a VEGFA, b VEGFC, production (c) CXCL8, d CXCL5. *p < 0.05; **p < 0.01; *** p < 0.001

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  Fig. 9. NRPa-308 decreases experimental ccRCC growth in a reverse dose-dependent manner. a Experimental tumors in nude mice (5 mice per condition) were obtained after injection of 3 × 106 786-O cells. Three concentrations of NRPa-308 (5 μg/kg, 500 μg/kg and 50 mg/kg) diluted in carboxymethyl cellulose, were given trice a week by oral gavage. The control group (Ctrl) received carboxymethyl cellulose. Tumor volume represented as a fold increase from the beginning of the treatment is presented. b Experimental tumors in immunocompetent mice (Balb-C, 5 mice per condition) were obtained after injection of 3 × 105 RENCA cells. Treatment (NRPa-308) was given trice a week by oral gavage. Two concentrations of NRPa-308- (5 μg/kg and 50 mg/kg) diluted in carboxymethyl cellulose were administered. The control group (Ctrl) received carboxymethyl cellulose. Tumor volume represented as the fold increase from the beginning of the treatment is shown. The tumor vasculature in each experimental group was detected by immuno-staining for CD31 (endothelial cells, green) and α-SMA (pericytes, cancer associated fibroblasts), red). Tumor sections were counterstained with 40,6-diamidino-2-phenylindole (DAPI)…

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  Fig. 10. Efficient NRPa-308 dose decreases the expression of pro-tumoral factors. Detection by qPCR of pro-tumoral genes in tumors generated in immunodeficient and immunocompetent mice with wild-type and with NRPs knock-out cells. *p < 0.05; **p < 0.01; *** p < 0.001

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  Fig. 11. The NRP2 associated pathway is more determinant for the aggressiveness of mccRCC but not for triple negative breast cancers. Analysis of cbioportal database highlighted the relative levels of VEGFA (a), VEGFC (b), NRP1 (c) and NRP2 (d) mRNA in a panel of RCC (769 (769P), 786-O (786), ACHN (A), Caki1 (C1), Caki2 (C2), RCC10 (R10)) and TNBC (BT474 (BT), MDAMB231 (231), MDAMB134 (134), MDAMB436 (436), MDAMB468 (468)). Correlation between genes of the NRP1 and NRP2 pathways and survival (DFS/PFS/OS) in M0 and M1 RCC patients (e) and TNBC (f) patients. The tested genes of the NRP1 pathway were the following: NRP1 (N1), VEGFA, VEGFR1 (R1), VEGFR2 (R2), Semaphorin 3A (Sema3A), Plexin A1 (PLXNA1). The tested genes of the NRP2 pathway were the following: NRP2 (N1), VEGFC, VEGFR3 (R3), Semaphorin 3F (Sema3F), Plexin A2 (PLXNA1) and PROX1. The p-values of genes associated with shorter DFS/PFS/OS appear white on a black background; the p-values of genes associated with a longer DFS/PFS/OS appear black on a gray background. Significant p-values are given; a trend to significance is indicated by a “T”. Specific cut-off are indicated (First, second or third quartile (1°, 2°, 3° Q). A score was…