Fig 2: ADT Induces NRG1 Expression in the Stroma of Prostate Cancer Patients(A) Representative images showing H&E (left, middle) and immunohistochemistry (right) analysis of stromal NRG1 staining in radical prostatectomy specimens from high-grade primary PCa patients. Asterisk denotes areas of intraductal carcinoma (no. 1) or invasive cancer (no. 2).(B) Top: pie chart showing percentage of NRG1 positivity in hormone-intact or neoadjuvant ADT-treated groups, and table showing number of patients with NRG1-positive biopsies in hormone-intact or neoadjuvant ADT-treated group.(C) Western blot analysis of NRG1 and CAF markers (PDFGRa, FAP, vimentin, and a-SMA) in five independent patient-derived primary PCa CAFs (pCAFs). pCAF no. 1 is from a neoadjuvant ADT-treated patient and pCAFs no. 2–5 are from hormone-intact patients. HSP90 serves as loading control. Bands were quantified using ImageJ and normalized to mean of HSP90 intensity in each group.(D) Western blot analysis of HER3-AKT activation in 22Pc-EP cells stimulated with conditioned media from patient-derived primary CAFs (pCAFCM). Total AKT serves as loading control. Bands were quantified using ImageJ and normalized to mean of total AKT intensity in each group.(E) Growth of 22Pc-EP cells in CSS media supplemented with pCAFCM. CellTiter-Glo reading on day 4. Media Ctrl, serum-free media.(F) Growth of 22Pc-EP cells in FBS media supplemented with pCAFCM and treated with Enz (0.1 µM). The Enz group was normalized to the Veh group. CellTiter-Glo reading on day 7. Media Ctrl, serum-free media.(G) Growth of 22Pc-EP cells in CSS media supplemented with pCAFCM treated with YW538.24.71 (10 µg/mL) or AMG888 (10 µg/mL). CellTiter-Glo reading on day 4. Media Ctrl, serum-free media.(H) Growth of VCaP or VCaP + pCAF no. 1 co-injected tumor xenografts in castrated mice treated with YW538.24.71 (25 mg/kg), neratinib (20 mg/kg), or vehicle. Treatment started when tumors reached 200 mm3 (n = 5 mice per group).(I) qRT-PCR analysis of NRG1 mRNA expression in pCAF no. 1 treated with CSS, Enz (10 µM), or Veh (DMSO) on day 7. NRG1 expression is normalized to ACTB.(J) Western blot analysis of NRG1 protein in pCAFs no. 2 and 3 treated with CSS, Enz (10 µM), or Veh (DMSO) on day 7. Cyclophilin B serves as loading control. Assays were performed with three biological replicates. ****p < 0.0001, *p < 0.05; n.s., not significant. (E–G) One-way ANOVA compared with Ctrl/Veh/IgG group, (H) one-way ANOVA compared with VCaP alone + Vehicle group, (I) Student’s t test compared with Ctrl group, (E–G and I) data are represented as mean ± SD, (H) data are represented as mean ± SEM. Ctrl, serum-free media. See also Figure S7 and Tables S4–S7.

Fig 3: NRG1 Promotes Antiandrogen Resistance in Androgen-Dependent PCa Models(A) Western blot analysis of NRG1 expression in 22Pc-CAF in which Nrg1 was deleted using CRISPR/Cas9. sgNT, non-targeting guide control; sgNrg1 (no. 1–4), four independent guides targeting Nrg1. ß-Actin serves as loading control.(B) Western blot analysis of HER3-AKT activation in 22Pc-EP after stimulation with either sgNT- or sgNrg1-22Pc-CAFCM. Total AKT serves as loading control.(C) Growth of 22Pc-EP cells in CSS media supplemented with either sgNT- or sgNrg1-22Pc-CAFCM. CellTiter-Glo reading on day 4. Media Ctrl, serum-free media.(D) Growth of 22Pc-EP cells in FBS media supplemented with either sgNT- or sgNrg1-22Pc-CAFCM treated with Enz (0.1 µM) or Veh (DMSO). The Enz group was normalized to the Veh group. CellTiter-Glo reading on day 7. Media Ctrl, serum-free media.(E) Western blot analysis of HER3-AKT activation in 22Pc-EP cells after stimulation with 22Pc-CAFCM in the presence of either NRG1 neutralizing antibody YW538.24.71 (1, 10, or 100 µg/mL) or IgG (100 µg/mL). Total AKT serves as loading control.(F) Growth of 22Pc-EP cells in CSS media supplemented with 22Pc-CAFCM treated with YW538.24.71 (1, 10, or 20 µg/mL) or IgG (20 µg/mL). CellTiter-Glo reading on day 4. Media Ctrl, serum-free media.(G) Growth of 22Pc-EP cells in Enz (0.1 µM) or Veh (DMSO) containing FBS media supplemented with 22Pc-CAFCM treated with YW538.24.71 (1, 10, or 20 µg/mL) or IgG (20 µg/mL). The Enz group was normalized to the Veh group. CellTiter-Glo reading on day 7. Media Ctrl, serum-free media.(H) Western blot analysis of HER3-AKT activation in 22Pc-EP after stimulation with 22Pc-CAFCM in the presence of an HER3-blocking antibody AMG888 (1 or 10 µg/mL) or IgG (10 µg/mL). Total AKT serves as loading control.(I) Growth of 22Pc-EP cells in CSS media supplemented with 22Pc-CAFCM treated with AMG888 (1 or 10 µg/mL) or IgG (10 µg/mL). CellTiter-Glo reading on day 4. Media Ctrl, serum-free media.(J) Growth of 22Pc-EP cells in Enz (0.1 µM) or Veh (DMSO) containing FBS media supplemented with 22Pc-CAFCM treated with AMG888 (1 or 10 µg/mL) or IgG (10 µg/mL). The Enz group was normalized to the Veh group. CellTiter-Glo reading on day 7. Media Ctrl: serum-free media.(K) Western blot analysis of HER3-AKT activation in 22Pc-EP after stimulation with either recombinant NRG1 or EGF (10 or 50 ng/mL). Total AKT serves as loading control.(L) Growth of 22Pc-EP in CSS media supplemented with either recombinant NRG1 or EGF (10 or 50 ng/mL). CellTiter-Glo reading on day 4. Media Ctrl, serum-free media.(M) Growth of 22Pc-EP cells in Enz (0.1 µM) or Veh (DMSO) containing FBS media treated with recombinant NRG1 or EGF (10 or 50 ng/mL). The Enz group was normalized to the Veh group. CellTiter-Glo reading on day 7. Media Ctrl, serum-free media.Assays were performed with three biological replicates. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; n.s., not significant, Student’s t test. Data are represented as mean ± SD. See also Figure S4.

Fig 4: Biochemical Fractionation of CAF-Secreted Resistance Activity Implicates NRG1(A) Schematic diagram showing fraction purification and resistance activity in 22Pc-CAFCM.(B) Growth of 22Pc-EP cells in CSS media supplemented with purified fractions from input (22Pc-CAFCM), Q30, or Q100. CellTiter-Glo reading on day 4. Ctrl, PBS.(C) Growth of 22Pc-EP cells in CSS media supplemented with sub-purified fractions (Q3–Q17) from Q30. CellTiter-Glo reading on day 4. Input, Q30; Ctrl, PBS.(D) Western blot analysis of HER3 and FGFR activation in 22Pc-EP after stimulation with FBS media, 22Pc-CAFCM, Q30 or Q100. Ctrl, PBS. ß-Actin serves as loading control.(E) Western blot analysis of HER3-AKT activation in 22Pc-EP after stimulation with different Q fractions and analysis of secreted NRG1 in corresponding fractions. Ctrl, PBS; Input, Q30; FT, flow through. Total AKT serves as loading control.(F) Western blot analysis of HER3-AKT activation in 22Pc-EP after stimulation with 22Pc-CAFCM in the presence of a commercial HER3-blocking antibody (10 or 30 µg/mL) or immunoglobulin G (IgG) (30 µg/mL). Total AKT serves as loading control.(G) Growth of 22Pc-EP cells in CSS media supplemented with 22Pc-CAFCM treated with a commercial HER3-blocking antibody (10 or 30 µg/mL) or IgG (30 µg/mL). CellTiter-Glo reading on day 4. Media Ctrl, serum-free media.(H) Western blot analysis of NRG1 from 22Pc-EPCM or 22Pc-CAFCM (concentrated 1×, 10×, 20×, or 50×).(I) qRT-PCR analysis of Nrg1 expression in CWR22Pc (n = 8) or 22Pc-EP (n = 10) tumor xenografts using mouse-specific primers.(J) Representative images showing RNA in situ hybridization (ISH) analysis of mouse Nrg1 expression in CWR22Pc or 22Pc-EP tumor xenografts. T, tumor; S, stroma; mouse-specific Nrg1 probe, brown dots.Assays were performed with three biological replicates. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; n.s., not significant. (B and C) One-way ANOVA compared with Ctrl group, (G and I) Student’s t test. Data are represented as mean ± SD. See also Figure S3.

Fig 5: Efficacy of pertuzumab and patritumab with or without trastuzumab on BT474-HRG and JIMT-1 tumor xenograft models. A, Vehicle (PBS: 100 µL), trastuzumab (10 mg/kg), pertuzumab (25 mg/kg), patritumab (25 mg/kg), trastuzumab (10 mg/kg) + patritumab (25 mg/kg), pertuzumab (25 mg/kg) + patritumab (25 mg/kg), and trastuzumab (10 mg/kg) + pertuzumab (25 mg/kg) + patritumab (25 mg/kg) were administered via intraperitoneal injection once per week to mice bearing BT474-HRG cell xenografts. B, Vehicle (PBS: 100 µL), trastuzumab (10 mg/kg), pertuzumab (25 mg/kg), patritumab (25 mg/kg), pertuzumab (25 mg/kg) + patritumab (25 mg/kg), and trastuzumab (10 mg/kg) + pertuzumab (25 mg/kg) + patritumab (25 mg/kg) were administered via intraperitoneal injection once per week to mice bearing JIMT-1 cell xenografts. Tumor volume (a) and body weights (b) were measured twice per week. Data represent the mean ± standard error. *P < 0.05 (two-way ANOVA). C, Posttreatment expression of HER2 and HER3 in tumors from xenograft-bearing mice, as determined by immunoblotting. Cell lysates were analyzed for HER2 and HER3 expression, with ß-actin serving as a loading control.