Fig 2: NRP1 expression is associated with in vivo tumor progression, cancer stemness and spheroid-initiating potential. a Western blot analysis of NRP1 expression in non-targeting control (shNT) and NRP1 shRNA-silenced (shNRP1 [1]) SUM159 cells inoculated into mice. b Post-inoculation tumor volumes, c tumor volume at week 8 post-inoculation and d Kaplan–Meier analysis of overall survival in SUM159 shNT and NRP1 knockdown groups. e Representative images of NRP1 immunohistochemistry in shNT and shNRP1 tumors and f quantification of NRP1 IHC staining across shNT and NRP1 knockdown groups. g NRP1 expression across CL1, CL2 and CL3 claudin-low subtypes as well PAM50 classifiers in the METABRIC dataset obtained via cBioportal [11, 25]. h qPCR (left and center panel; n = 3) and Western blot (right panel) analysis of ZEB1 expression in HS578T cells after 72 h NRP1 knockdown versus NT control. i qPCR analysis of ITGA6 mRNA expression in HS578T cells (leftmost panel; NRP1 expression shown in h) and SUM159 cells (center and right panel) after 72 h NRP1 knockdown versus NT control (n = 3). j Western blot showing ITGA6 expression in HS578T, SUM159 and MDA-MB-231 cells after 72 h NRP1 knockdown versus NT control. k Western blot showing expression of ZEB1 and NRP1 in FACS sorted CD44+/CD24lo and CD44+/CD24hi populations of SUM159 cells. l Number of spheroids (> 50 µM) formed by day 6 following seeding of single cell SUM159 and Hs578T cell cultures containing 1,200 cells in the presence of 50 µg/ml Vesencumab (red lines) or IgG control (black lines). n = 5, along with m representative images of (mi) SUM159 and (mii) Hs578T spheroid cultures at days 4 and 6 post-seeding. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, ****p ≤ 0.0001, error bars represent SEM

Fig 3: NRP1 inhibition suppresses EGFR and PDGFRα signaling in claudin-low cells. ai Receptor tyrosine kinase array showing EGFR and PDGFRα expression in SUM159 cells after 60 min treatment with 50 μg/mL IgG or Vesencumab (top panel), or 72 h after transfection with NRP1 targeting siRNA (siNRP1 [1] or siNRP1 [2]) versus non-targeting (NT) control (bottom panel), with aii corresponding densitometry. Ref1 and Ref2 represent positive controls. b Western blot showing NRP1, total EGFR, phospho-EGFR (pEGFR Y1068), total PDGFRα and phospho-PDGFRα (Y1018) expression in SUM159, MDA-MB-231 and HS578T cells after 72 h NRP1 knockdown versus NT control. c Correlation analysis between NRP1 and EGFR (right panel; Pearson: 0.24, p = 2.7e−26) and PDGFRα (left panel; Pearson: 0.52, p = 3.47e−130) mRNA expression in the METABRIC dataset (n = 1904) [25]. Data was obtained from cBioportal. d Immunohistochemical analysis of phosphorylated (T202/Y204) p42/44 levels in endpoint (7 weeks post-tumor inoculation) Vesencumab or IgG control treated tumors, with e representative images (right panel); scale bars = 50 µm, n = 5–6. Error bars represent SEM, *p ≤ 0.05; **p ≤ 0.01 versus control

Fig 4: NRP1 is over-expressed in the claudin-low molecular subtype of breast cancer. a Heatmap showing NRP1 expression association with PAM50, claudin-low, core claudin-low (CoreCL), ER and HER2 tumor status, as well as core claudin-low signature genes. b NRP1 mRNA expression (log2 signal) in intrinsic breast cancer subtypes and claudin-low tumors (CLDNlow) in the METABRIC patient dataset (n = 1904), obtained through cBioPortal [25]. c NRP1 mRNA expression across intrinsic subtypes subdivided into claudin-low (CL) and non-claudin-low tumors. Correlation of claudin-low di up-gene (CLDNlow UP GES) and dii down-gene (CLDNlow DN GES) GSVA-derived signature scores with NRP1 expression. Claudin-low gene signature scores were obtained via GSVA. Sample subtype is represented according to color scheme used in A-C. e NRP1 mRNA expression in METABRIC core claudin-low (CoreCL), non-core claudin-low (OtherCL) and non-claudin-low tumors [10]. Error bars represent SEM, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001

Fig 5: NRP1 is associated with the claudin-low signature in breast cancer cell lines and promotes proliferation. a Heatmap showing NRP1 mRNA expression across luminal, basal A, basal B and claudin-low human breast cancer cell lines, as well as association with core claudin-low signature genes [10, 28]. NRP1 mRNA expression in b luminal, basal A and basal B breast cancer cell lines, C claudin-low versus non-claudin-low cell lines, and association with key claudin-low markers di claudin 3 (CLDN3) and dii vimentin across breast cancer cell lines. E Flow cytometry analysis of NRP1 expression across breast cancer cell lines representing different intrinsic subtypes and the claudin-low (CLDNlow) subtype. f Western blot analysis of NRP1 expression in BrCa cell lines including luminal A (LumA; T47D and MCF-7), luminal B (LumB; MDA-MB-361), HER2+ (BT-474, HCC1569 and SKBR3), basal (BT-20 and MDA-MB-468) and claudin-low (SUM159, MDA-MB-231 and HS578T) cells, with GAPDH loading control. g Western blot showing NRP1 expression in claudin-low cell lines (MDA-MB-231, BT549, SUM159 and HS578T) at day 3 post-transfection with NRP1 siRNA (siNRP1 [1] or siNRP1 [2]) or non-targeting control siRNA (siNT). h Cell viability of claudin-low cell lines (MDA-MB-231, BT549, SUM159 and HS578T) at 0, 1, 2, 4 and 7 days after transfection with NRP1 siRNA (siNRP1 [1] or siNRP1 [2]) relative to siNT as measured by CyQuant™ DNA quantification assay; n = 3. Error bars represent SEM, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, ****p ≤ 0.0001