IL-23 secreted by myeloid cells drives castration-resistant prostate cancer

Abstract

Patients with prostate cancer frequently show resistance to androgen-deprivation therapy, a condition known as castration-resistant prostate cancer (CRPC). Acquiring a better understanding of the mechanisms that control the development of CRPC remains an unmet clinical need. The well-established dependency of cancer cells on the tumour microenvironment indicates that the microenvironment might control the emergence of CRPC. Here we identify IL-23 produced by myeloid-derived suppressor cells (MDSCs) as a driver of CRPC in mice and patients with CRPC. Mechanistically, IL-23 secreted by MDSCs can activate the androgen receptor pathway in prostate tumour cells, promoting cell survival and proliferation in androgen-deprived conditions. Intra-tumour MDSC infiltration and IL-23 concentration are increased in blood and tumour samples from patients with CRPC. Antibody-mediated inactivation of IL-23 restored sensitivity to androgen-deprivation therapy in mice. Taken together, these results reveal that MDSCs promote CRPC by acting in a non-cell autonomous manner. Treatments that block IL-23 can oppose MDSC-mediated resistance to castration in prostate cancer and synergize with standard therapies.

Extended Data 1. Multispectral images of PMN-MDSCs in human biopsies and set up of the different CRPC mouse models.

a, Multispectral microscopy images (EpCAM yellow, CD15 green, CD33 red; CD11b pink) of castration sensitive vs castration resistant prostate cancers. n=3 biological independent patients. Scale Bar 20μM. b, Quantification of the number of CD11b⁺ CD15^- cells within the tumor of castration sensitive vs castration resistant prostate cancers (CSPCs, n=51 vs CRPCs, n=51 biological independent patients). Cells were counterstained with the nuclear marker DAPI (blue). Statistical analyses (negative binomial regression model): P=0.062. c, MRIs of one representative Sham-operated (Sham) or surgical castrated (CTX) Pten^pc-/- mouse of the three analyzed at different time points. d, Waterfall plot depicting proportional change in tumor response for Sham (n=3) and CTX (n=3) Pten^pc-/- mice. e, Prostate PMN-MDSCs frequencies determined by flow cytometry in Sham (n=3) and CTX (n=3) Pten^pc+/+ mice (12 weeks after castration). Statistical analyses (two sided unpaired t test): P=0.85. f, Schematic representation of the experiment. Six-week-old C57BL/6 males were challenged s.c. with TRAMP-C1 cells. When tumors reached ≈ 100 mm³, mice were sham-operated (Sham, n=9) or surgically castrated (CTX, n=5). g, Tumor PMN-MDSCs frequencies determined by flow cytometry during CS and CR phase. Sham CS n=5, CTX CS n=4, Sham CR n=8, CTX CR n=6. h, Schematic representation of the experiment. Six-week-old FVB males were challenged s.c. with MyC-CaP cells. When tumors reached ≈ 100 mm³, mice were sham-operated (Sham, n=3) or surgically castrated (CTX, n=3). i, Tumor PMN-MDSCs frequencies determined by flow cytometry during CS and CR phase. Sham CS n=3, CTX CS n=4, Sham CR n=4, CTX CR n=3. b,d,e,g,i, Data are reported as mean ± SEM. d,g,i, Statistical analyses (Unpaired two-sided t-test): ns, not significant; *P <0.05; **P <0.01; ***P <0.001. f,h, Statistical analyses (two sided unpaired t test followed by Wilcoxon post-test): *P <0.05.

Extended Data 2. Multiparametric flow cytometry analysis of myeloid populations in the tumors of different CRPC mouse models.

a, Gating strategy relative to the quantification of PMN-MDSCs in the tumor of Sham and CTX-operated mice for Pten^pc-/-, TRAMP-C1 and MyC-CaP models. b, Representative dot plot reporting the Ly6G and Ly6C positive cells in the tumor of Sham and CTX-operated mice for Pten^pc-/-, TRAMP-C1 and MyC-CaP models. Data were validated in at least three biological independent animals. c, Representative dot plot and quantification of CD11b⁺ F4/80⁺ cells and CD11c⁺ CD206^- (M1-like) or CD11c^- CD206⁺ (M2-like) macrophages in the tumor of Sham and CTX-operated mice for Pten^pc-/- (Sham n=5, CTX n=9), TRAMP-C1 (Sham n=7, CTX n=6), and MyC-CaP (Sham n=4, CTX n=4), models. Data were reported as mean ± SEM. Statistical analyses (Unpaired two-sided t-test): **P <0.01.

Extended Data 3. Factors secreted by MDSCs promote insensitivity to androgen deprivation in androgen-dependent murine and human prostate cancer cell lines and CXCR2 antagonist impairs MDSCs tumor recruitment in Pten^pc-/- mice.

a, Representative dot plot reporting the BM-MDSCs upon in vitro differentiation. Data were validated in two biological independent experiments. b, Experimental scheme. c, Cell proliferation of MyC-CaP cells (None n=5, C.M. BM-MDSCs n=3, F.A.D. n= 13, F.A.D. + BM-MDSCs n= 13 biological independent samples). d, Percentage of AnnexinV and 7AAD-negative MyC-CaP cells. e, Percentage of AnnexinV and 7AAD-positive MyC-CaP cells. f, qRT-PCR analyses of the indicated genes in MyC-CaP cells. g, h, i, Cell proliferation of VCaP (None n=4, C.M. hBM-MDSCs n=4, F.A.D. n=7, F.A.D. + hBM-MDSCs n=8 biological independent samples), 22Rv1 (None n=4, C.M. hBM-MDSCs n=8, F.A.D. n=8, F.A.D. + hBM-MDSCs n=4 biological independent samples), and PC3 (None n=4, C.M. hBM-MDSCs n=8, F.A.D. n=4, F.A.D. + hBM-MDSCs n=8 biological independent samples) prostate cancer cells. j, Tumor MDSCs frequencies determined by flow cytometry of prostate tumors of CTX Pten^pc-/- mice treated or not with CXCR2 antagonist (αCXCR2) at completion of the study (12-weeks upon CTX). CTX CS n=3, CTX+ αCXCR2 CS n=3, CTX CR n=4, CTX+ αCXCR2 CR n=7 biological independent animals. k, Cell proliferation of TRAMP-C1 cells after 72h of treatment with CXCR2 antagonist (αCXCR2) l, qRT-PCR analyses of the indicated genes in TRAMP-C1 cells after 24h of treatment (fold change compared with F. A. D. condition). k, l, Aggregated data from three independent experiments are reported, fold change compared with F. A. D. condition. c-l, Data were reported as mean ± SEM. c-f, n=3 biological independent samples d,e,g-j, Statistical analyses (Unpaired two-sided t-test): ns, not significant; *P <0.05; **P <0.01; ***P <0.001. c,f, Statistical analyses (two sided unpaired t test followed by Wilcoxon post-test): *P <0.05.

Extended Data 4. Impaired tumor recruitment of MDSCs enhances response to surgical castration in different allograft models of prostate cancers.

a, Schematic representation of the experiment. Six-week-old C57BL/6 males were challenged s.c. with TRAMP-C1 cells. When tumors reached ≈ 100 mm³, mice were surgically castrated and left untreated (CTX, n=8) or treated with CXCR2 antagonist (CTX + αCXCR2, n=9). Representative flow cytometry plots of PMN-MDSCs (CD11b⁺ Ly6G⁺ cells, gated on CD45⁺ cells) in tumors for each experimental condition. b, qRT-PCR analyses of the indicated genes in the prostate tumors of CTX and CTX + αCXCR2 (n=3 per group). Data were reported as mean ± SEM. Statistical analyses (Unpaired two-sided t-test):*P <0.05; ***P <0.001. c, Average tumor volume (±SE) for each experimental group. Statistical analyses (Unpaired two sided t test followed by Wilcoxon post-test): ***P <0.001. d, Survival curves are reported in Kaplan-Meier plot. Statistical analyses (two sided Log rank test): ***P <0.001. e, Schematic representation of the experiment. Six-week-old FVB males were challenged s.c. with MyC-CaP cells. When tumors reached ≈ 100 mm³, mice were surgically castrated and left untreated (CTX, n=5) or treated with CXCR2 antagonist (CTX + αCXCR2, n=5). Representative flow cytometry plots of PMN-MDSCs (CD11b⁺ Ly6G⁺ cells, gated on CD45⁺ cells) in tumors for each experimental condition. f, qRT-PCR analyses of the indicated genes in the prostate tumors of CTX and CTX + αCXCR2 (n=3 per group). Data were reported as mean ± SEM. Statistical analyses (Unpaired two-sided t-test):**P <0.01; ***P <0.001. g, Average tumor volume (±SEM) for each experimental group. Statistical analyses (two sided unpaired t test followed by Wilcoxon post-test): *P <0.05. h, Survival curves reported as Kaplan-Meier plot. Statistical analyses (two sided Log-rank test): **P <0.01. i, Schematic representation of the experiment. Six-week-old NOD SCID males were challenged s.c. with LNCaP cells or with LNCaP cells and hBM-MDSCs. When tumors reached ≈ 70 mm³, mice were sham-operated (Sham, n=5) or sham-operated and injected every three days i.p. with 3x10⁶ hBM-MDSCs (Sham + hBM-MDSCs, n=5) or surgically castrated and left untreated (CTX, n=8) or treated with hBM-MDSCs (CTX + hBM-MDSCs, n=5). j, Average tumor volume (±SEM) for each experimental group. Statistical analyses (Unpaired two sided t test followed by Wilcoxon post-test): **P <0.01.

Extended Data 5. IL23 pathway is the most up-regulated in the tumor upon castration.

a, Gene expression of selected genes determined by NanoString nCounter gene expression assay in Sham Pten^pc-/- and CTX Pten^pc-/- tumors. Data are shown as pool of n=5. b, Analyses of the conditioned media of BM-derived MDSCs tested for the indicated soluble molecules by Mouse CytokineMAP B version 1.0. The graph reports the concentration of the indicated soluble molecules as Log10 of the concentration found in the conditioned medium of BM-MDSCs, the values were subtracted of the background (culture medium). Data are shown as pool of n=10. c, qRT-PCR analyses of the indicated genes in Sham (n=6) and CTX (n=6) Pten^pc-/- tumors. Data were reported as mean ± SEM of biological independent animals. Statistical analyses (Unpaired two sided t test): *P <0.05. d, Protein level of CXCL1, CXCL2 and CXCL5 in CTX Pten^pc-/- tumors. Data are analyzed as ratio between CTX (pool of 3 samples) and Sham (pool of 3 samples) Pten^pc-/- tumors and reported as fold increase in protein level. e-f, IL23R protein level analyzed by flow cytometry and western blot on TRAMP-C1 cells under normal culture condition (FBS) or androgen deprivation culture condition (CS-FBS). n=4 biological independent samples per group. f, Numbers indicate fold change in protein level. Loading control: anti–β–actin antibody. The WB was validated at twice. g, Protein profile of the plasma of CSPC and CRPC patients. Data are analyzed as ratio between CRPC (pool of 18 samples) and CSPC (pool of 17 samples) and reported as fold increase in protein level.

Extended Data 6. Characterization of IL23⁺ cells in the tumor of CTX Pten^pc-/- mice and CRPC patients.

a, Multispectral microscopy images (EpCAM yellow, CD15 green, IL23 red) of three human castration resistant prostate cancers. b, Correlation analyses of the numbers of CD15^- CD11b⁺ cells in the tumor and the IL23 levels in the plasma of CRPC patients (n=28). Statistical analyses (negative binomial regression model) p=0.63. c, d, IL23 and IL23R mRNA expression in the tumor of HSPCs (n=549) and mCRPCs (n=116). e, f, Expression of IL23 in PMN-MDSC marker (CD11b, CD33, CD15) positive HSPC or mCRPC patients. c-f, Statistical analyses (Unpaired two sided t test) are reported. g, Representative plots of IL23⁺, CD45⁺ and CD45^-, Ly6G^bright CD11b⁺ and Ly6Gint CD11bint, CD11b⁺ F4/80⁺ cells pre-gated on the reported population in the tumor of CTX Pten^pc-/- mice. IL23 gate was decided based on Isotype control panel (insert). Pie reports the percentage of the different subsets gated on IL23⁺ cells in the tumor of Pten^pc-/- mice (Mean, n=9). h, qRT-PCR analyses of IL23 in the prostate tumors of castrated (CTX; n=6) or castrated and treated with CXCR2 antagonist (CTX + αCXCR2; n=7) Pten^pc-/- mice. Mean ± SEM. i, PMN-MDSCs and TAMs frequencies determined by flow cytometry in the tumor of castrated NSG™ TRAMP-C1 allografts upon treatment with Isotype, αCSF1R, αCXCR2. Data are reported as mean ± SE (n=3 per group). j, qRT-PCR analyses of IL23 in the tumor of castrated NSG™ TRAMP-C1 allografts upon treatment with Isotype (n=4), αCSF1R (n=5), αCXCR2 (n=5). Data are reported as mean ± SEM. Each dot represents a biological independent animal. h, i, j, Statistical analyses (Unpaired two sided t test): *P <0.05; **P <0.01; ***P <0.01.

Extended Data 7. BM-MDSCs^IL23wt and BM-MDSCs^IL23ko have equal secretome and equal immunosuppressive capability.

a, Correlation analyses of the gene expression of selected genes determined by NanoString nCounter gene expression assay in BM-MDSCs^IL23wt and BM-MDSCs^IL23ko reported by scatter plot analyses. Data are shown as pool of n=10. R² values were determined using a linear regression analyses. b, Representative flow cytometry plots of CFSE dilution (gated on CD8⁺ cells) and quantification of proliferating CD8⁺ T cells exposed to C. M. from BM-MDSCs^IL23wt or BM-MDSCs^IL23ko. Mean ± SE of three independent biological samples. Statistical analyses (Unpaired two sided t test): **P <0.01; ***P <0.01.

Extended Data 8. Genetic inhibition of IL23 limits castration resistance in Pten^pc-/- mice.

a, Experimental set-up. Sham-operated (Sham) or Castrated (CTX) Pten^pc-/- mice were lethally irradiated and transplanted with BM precursors depleted by T, B, and NK cells from IL23^wt and IL23^ko mice. Then, monitored by MRI for tumor progression. b, Representative dot plot of BM precursors pre- and post-depletion of T, B, and NK cells. Data were validated in two biological independent experiments. c, Quantification of the tumor size of Sham-operated Pten^pc-/-;IL23^wt (n=4) and Pten^pc-/-;IL23^ko (n=4), and castrated Pten^pc-/-;IL23^wt (n=4) and Pten^pc-/-;IL23^ko (n=7) mice at completion of the study is reported as fold increase of the prostate anterior lobe (AL) volume (fold change compared with CTX Pten^pc-/-;IL23^wt group). Data are reported as mean ± SE. Statistical analyses (Unpaired two sided t test): **P <0.01, ***P <0.001. d, H&E, Ki-67, and pSTAT3^Y705 immunohistochemical staining (Ki-67 and pSTAT3^Y705 brown; nuclei, blue) of one representative Pten^pc-/-;IL23^wt and Pten^pc-/-;IL23^ko mouse of at least the three analyzed at completion of the study. Scale Bar 50 or 25 μM.

Extended Data 9. Genetic inhibition of IL23 limits castration resistance in TRAMP-C1 allograft model in vivo.

a, Schematic representation of the experiment. Six-week-old C57BL/6 males were lethally irradiated and transplanted with BM precursors from IL23^wt and IL23^ko mice. After the BM engraftment, the animals were challenged s.c. with TRAMP-C1 cells. When tumors reached ≈ 100 mm³, mice were surgically castrated and monitored for tumor progression. b, H&E, Ki-67 and pSTAT3^Y705 immunohistochemical staining (Ki-67 and pSTAT3^Y705 brown; nuclei, blue) of representative TRAMP-C1 IL23^wt and TRAMP-C1 IL23^ko bearing mice. Scale Bar 25 μM. c, Quantification of Ki-67 positive cells is reported as a percentage of total. TRAMP-C1 IL23^wt (n=8) and TRAMP-C1 IL23^ko (n=4), one tumor per mouse, mean of three sections per mouse, ≥ 3 fields per section. Biological mean ± SE. Statistical analyses (Unpaired two-sided t test): **P <0.01. d-e, PMN-MDSCs frequencies determined by flow cytometry in the tumor and in the spleen of TRAMP-C1 IL23^wt (n=3) and TRAMP-C1 IL23^ko (n=3) bearing mice 10 days after castration. Data are reported as mean ± SE. f, Quantification of pSTAT3^Y705 reported as a percentage of total. TRAMP-C1 IL23^wt (n=8) and TRAMP-C1 IL23^ko (n=4), one tumor per mouse, mean of three sections per mouse, ≥ 3 fields per section. Statistical analyses (Unpaired two sided t test): ***P <0.001. g, Western blot for RORγ, pSTAT3^Y705 and total STAT3 levels of prostate tumors of TRAMP-C1 IL23^wt and TRAMP-C1 IL23^ko bearing mice. Loading control: HSP90 antibody or total ERK antibody. The WB was validated al least two times. h, Quantification is reported as mean ± SEM of biological independent experiments: TRAMP-C1 IL23^wt RORγ n=9; and TRAMP-C1 IL23^ko RORγ n=9; TRAMP-C1 IL23^wt pSTAT3^Y705 n=4; and TRAMP-C1 IL23^ko pSTAT3^Y705 n=3. Statistical analyses (Unpaired two sided t test): *P <0.05.

Extended Data 10. Pharmacological inhibition of IL23 in association with ADT delays disease progression in prostate cancer.

a, H&E and Ki-67 immunohistochemical staining (Ki-67 brown; nuclei, blue) of representative castrated-Pten^pc-/- mice treated with αIL23, ENZA or both. Scale Bar 50 μM b, Quantification of Ki-67 reported as a percentage of total within the glands. One tumor per mouse, three sections per mouse, ≥ 3 fields per section. Mean ± SEM of a biological independent animals. Untreated n=6, αIL23 n=6, ENZA n=5 or both n=6. Statistical analyses (Unpaired two sided t test): **P <0.01; ***P <0.001. c, Upon castration, PMN-MDSCs progressively infiltrate the tumor bed mainly recruited by CXCL5. Within the tumor, PMN-MDSCs start to produce higher amount of IL23, thus establishing a positive-feedback loop that induce the over-expression of IL23R on the tumor epithelial cells and confer castration resistance by activating the STAT3/RORγ pathway. Enzalutamide treatment can block androgen receptor inducing sensitiveness of prostate cancer cells to androgen deprivation, but the persistent presence of PMN-MDSCs-derived IL23 rescues the drug sensitiveness leading to androgen deprivation resistance. Anti-IL23 treatment reverts castration resistance in prostate cancer enhancing the efficacy of enzalutamide.

Figure 1. MDSCs infiltrate CRPC paralleling the activation of AR pathway and conferring castration resistance.

a, CD11b⁺ CD33⁺ CD15⁺ PMN-MDSCs within the tumors of CSPCs vs CRPCs. EpCAM yellow, CD15 green, CD33 red; CD11b pink; DAPI blue, n=51 biological independent patients per group reported as mean ± SEM. Statistical analyses (negative binomial regression model): P <0.001. b-d, Pten^pc-/- mice sham-operated (Sham) or surgically castrated (CTX) Pten^pc-/- mice at different time points. b, Tumor volume of the anterior prostate lobe. c, qRT-PCR analyses of the indicated genes in the prostate tumors at t=4 (castration sensitive phase; CS) and t=12 (castration resistance phase; CR). d, Flow cytometry for tumor PMN-MDSCs (gated on CD45⁺ cells). e, Percentages of tumor-infiltrating immune cell populations (gated on CD45⁺ cells). f, Experimental scheme. g, TRAMP-C1 cell proliferation. h, Percentage of AnnexinV and 7AAD-negative TRAMP-C1 cells. i, Percentage of AnnexinV and 7AAD-positive TRAMP-C1 cells. j, qRT-PCR analyses of the indicated genes in TRAMP-C1 cells. k, Experimental scheme. l, LNCaP cell proliferation. m, Volume of prostate tumors of CTX Pten^pc-/- mice treated or not with CXCR2 antagonist (αCXCR2) at completion of the study (12-weeks upon CTX). n, qRT-PCR analyses of the indicated genes in the prostate tumors of mice treated as in m. Specific n values of biological independent animals (b, c, d, m, n) and independent samples (g-j, l) are shown and reported as mean ± SEM. b, d, h, i, -, l, m, Statistical analyses (Unpaired two-sided t-test): ns, not significant; *P <0.05; **P <0.01; ***P <0.001. b, d, Statistical analyses (One-way ANOVA, two-sided): P <0.001. c, g, j, n, Statistical analyses (Paired two-sided t-test): *P <0.05; **P <0.01; ***P <0.001.

Figure 2. Tumor-infiltrating MDSCs produce IL23 that drives insensitivity to androgen deprivation.

a-c Prostate tumors from Pten^pc-/- mice Sham or CTX analyzed at t=12 a, Representative images of Ly6G⁺ IL23⁺ cells (nuclei, blue) and representative dot plot of Ly6G⁺ IL23⁺ cells gated on CD45⁺ cells in CTX mice. b-c, Representative histograms (left) and quantification (right; mean ± SE) showing b, Mean fluorescence intensity (MFI) of IL-23 within CD45⁺ CD11b⁺ Ly6G⁺ cells and c, MFI of IL-23R gated on CD45^- EpCAM⁺ cells. b, c, n=3 biological independent animals per group. d, Representative images of IL23, CD15, EpCAM positive cells within the tumor of CRPC patients. a, d, Data were validated in at least three experiments. e, CD33⁺ IL23⁺ CD11b⁺ CD45⁺ cells within the tumor of CSPCs vs CRPCs (n=4 biological independent patients/cohort). f, IL23 levels in the plasma of CSPCs (n=20) and CRPCs (n=120) patients. g, Correlation analyses of the numbers of tumor-infiltrating PMN-MDSCs and plasmatic IL23 levels in CRPC patients (n=28). Statistical analyses (negative binomial regression model) p <0.001. h-i, Tumor progression (t=days post-castration) of NSG™ TRAMP-C1 allografts treated with Isotype control (Untreated; n=4), anti-CSF1R antibody (αCSF1R; n=5) or αCXCR2 (n=5). h, Average tumor volume. i, Survival curves reported as Kaplan-Meier plot. Statistical analyses (Log rank (Mantel-Cox) test): **P <0.01. j, TRAMP-C1 cell proliferation. k, Percentage of AnnexinV and 7AAD-negative TRAMP-C1 cells. l, Percentage of AnnexinV and 7AAD-positive TRAMP-C1 cells. m, qRT-PCR analyses of the indicated genes in TRAMP-C1 cells. n, Cell proliferation of 3D cultures of reported organoids. rIL23 conditions were normalized to the None or F.A.D. condition respectively. e,f,h,j-n, Data are reported as mean ± SE. Specific n values of biological independent samples are shown in j-n. b, c, e, f, h, j-l, n, Statistical analyses (Unpaired two-sided t-test) and m, Statistical analyses (Paired two-sided t-test), *P <0.05; **P <0.01; ***P <0.001.

Figure 3. IL23/IL23R axis regulates castration resistance in vivo and in vitro.

a, MRIs of representative Pten^pc-/-;IL23^wt and Pten^pc-/-;IL23^ko mice at the endpoint (upper panels). Waterfall plot depicting proportional change in tumor response for Pten^pc-/-;IL23^wt (n=3) and Pten^pc-/-;IL23^ko (n=3) mice. Mean ± SE. Statistical analyses (Unpaired two-sided t-test): *P <0.05, One-way ANOVA: P=0.0008. b, Representative H&E at the endpoint. Scale Bar 100 μM. Data are representative of at least three biological independent animals. c, Quantification of adenocarcinoma, prostatic intraepithelial neoplasia (PIN) or normal-like glands in Pten^pc-/-;IL23^wt (n=3) and Pten^pc-/-;IL23^ko (n=3) mice. d, Quantification of Ki-67⁺ cells in Pten^pc-/-;IL23^wt (n=4) and Pten^pc-/-;IL23^ko (n=6). e, qRT-PCR analyses in the prostate tumors. f, g, Tumor volume and survival curves of TRAMP-C1 IL23^wt and TRAMP-C1 IL23^ko bearing mice. f, Statistical analyses (Unpaired two-sided t-test followed by Wilcoxon post-test): *P <0.05. h, qRT-PCR analyses in the tumors of TRAMP-C1 IL23^wt or IL23^ko bearing mice. i-j, NSG™ males challenged with TRAMP-C1 or TRAMP-C1-IL23RKO cells, after CTX were treated with Isotype control (Untreated) or anti-IL23 antibody (αIL23) and monitored for i, tumor progression and j, survival. WB for IL23R as insert (performed at least in double). g, j, Statistical analyses (Log rank (Mantel-Cox) test): **P <0.01; ***P <0.001. k, Quantification of pSTAT3^Y705+ cells in Pten^pc-/-;IL23^wt (n=4) and Pten^pc-/-;IL23^ko mice (n=6). l-m WB and quantification for RORγ, pSTAT3^Y705 and total STAT3 levels in prostate tumors. n, TRAMP-C1 cell proliferation o, qRT-PCR analyses in TRAMP-C1 cells. c, d, k, Data are reported as mean ± SE of one tumor per mouse (mean of three sections per mouse, ≥ 3 fields per section). e-i,j,k,m-o, Data are reported as mean ± SE. Specific n values of biological independent animals (i,j,k) and biological independent samples (m-o) are reported in figure. c, d, i, k, m, n, Statistical analyses (Unpaired two-sided t-test), e, h, o, Statistical analyses (Paired two-sided t-test), *P <0.05; **P <0.01; ***P <0.001.

Figure 4. IL23 inhibition improves enzalutamide efficacy in vivo.

a, CR Pten^pc-/- mice (12 weeks after castration) were randomly enrolled in the preclinical trial. Treatments: Isotype control (Untreated), anti-IL23 antibody (αIL23; 100ng/per mouse i.p. weekly), Enzalutamide (ENZA, 30 mg/kg/day administered daily by oral gavage on a Monday to Friday schedule) and Enzalutamide in combination with anti-IL23 antibody (ENZA+αIL23). b, Histological score. n=3 biological independent animals. Statistical analyses (Two-way ANOVA): p<0.001. c, Fold increase of the prostate anterior lobe volume (fold change compared with untreated group). d, Representative H&E and Ki-67 staining in the tumors at completion of the study. Scale Bar 50 μM. e, qRT-PCR analyses of the indicated genes in the prostate tumors of CTX-Pten^pc-/- mice at completion of the preclinical trial. Statistical analyses (two-sided Paired t test): *P <0.05; ***P <0.001. f, Representative cleaved-Caspase3 staining in the tumors after one week of treatments. Scale Bar 50 μM. g, Quantification of cleaved-Casp 3 (percentage of total within the glands). d, f, Data are representative of at least in three biological independent animals. b, g, Data are reported as mean ± SEM of one tumor per mouse (mean of three sections per mouse, ≥ 3 fields per section). b,c,e,g Data are reported as mean ± SEM. Specific n values of biological independent animals are shown in c, e, g. c, g, Statistical analyses (Unpaired two-sided t-test): ns, not significant; *P <0.05; **P <0.01; ***P <0.001.