Fig 1: Repression of Tsp-1 by PRSS2 creates and immunosuppressive TME.A Immunofluorescence staining of CD3 (green) and FoxP3 (red) in tumors formed by Pan02 (n = 4) and Pan02shPRSS2 (n = 3) cells in WT C57Bl6/J mice and Tsp1-/- C57Bl6/J mice (scale bar = 1 mm). B Plot of the percentage of CD3 + /FoxP3 + T cells out of total CD3 + T cells in tumors formed by Pan02 (n = 4) and Pan02shPRSS2 (n = 3) cells in WT C57Bl6/J mice and thbs1-/- C57Bl6/J mice (P value calculated via two-sided Mann–Whitney U test). C Immunofluorescence staining of CD3 (green), CD4 (red), and CD8 (white) in tumors formed by Pan02 (n = 4) and Pan02shPRSS2 (n = 3) cells in WT C57Bl6/J mice and Tsp1-/- C57Bl6/J mice (scale bar = 1 mm). D Plot of percentage of CD3 + /CD8 + T cells out of total CD3 + T cells in tumors formed by Pan02 (n = 4) and Pan02shPRSS2 (n = 3) cells in WT C57Bl6/J mice and thbs1-/- C57Bl6/J mice (P value calculated via two-sided Mann–Whitney U test). E Plot of ratio of CD3 + /CD8 + T cells to CD3 + /FoxP3 + T cells in tumors formed by (n = 4) and Pan02shPRSS2 (n = 3) cells in WT C57Bl6/J mice and thbs1-/- C57Bl6/J mice (WT/WT minimum = 0.099; maximum = 0.58; median = 0.37; percentile = 25–75; P value calculated via two-sided Mann–Whitney U test). F Representative pictures of immunohistochemistry of CD8 + and FoxP3 + T cells in prostate tumors (n = 458 patients) with high or weak levels of PRSS2 (scale bar = 50 µm). G Kaplan–Meier curves of: upper left: correlation of high PRSS2 and low CD8 levels with survival after radical prostatectomy. Upper right: correlation of high PRSS2 and high FoxP3 levels with survival after radical prostatectomy; and lower left: correlation of low PRSS2 and high CD8 levels with survival after developing castration resistance; and lower right: correlation of high PRSS2 and high FoxP3 levels with survival after developing castration resistance (P values calculated via two-sided Mann–Whitney U test). H Schematic diagram of PRSS2 regulation of tumor immune microenvironment.
Fig 2: PRSS2 represses Tsp-1 by activating Rac downstream of LRP1.A Dot plot of GTP-bound Rac and Rho in WI38 cells that were untreated (control), treated with CM from 293T cells transfected with empty vector (pCMV) or PRSS2 (PRSS2) (n = 3; P value calculated via two-sided Student’s t test). B Western blot of Tsp-1 and actin expression in WI38 cells that were untreated (–) or treated with PRSS2 (+) in the absence (–) and presence (+) of a small molecule inhibitor of Rac1 NSC23766 (n = 3). C Dot plot of quantitation of Tsp-1 western blots from all replicates of WI38 that were untreated (–) or treated with PRSS2 (+) in the absence (–) and presence (+) of NSC23766 (n = 3; P value calculated via two-sided Student’s t test). D Western blot of phospho c-Jun, total c-Jun and actin in WI38 cells that were treated with saline (–), PRSS2, Rac1 inhibitor NSC23766, or PRSS2 and NSC23766 in combination (n = 3). E Dot plot of quantitation of phospho c-Jun, total c-Jun, and actin in WI38 cells that were treated with saline (–), PRSS2, Rac1 inhibitor NSC23766, or PRSS2 and NSC23766 in combination (P values calculated by two-sided ANOVA; n = 3). F Western blot of phosphor-JNK, total JNK and actin in WI38 cells that were treated with saline (–), PRSS2, Rac1 inhibitor NSC23766, or PRSS2 and NSC23766 in combination (n = 5). G Dot plot of quantitation of phospho-JNK, total JNK and actin in WI38 cells that were treated with saline (–), PRSS2, Rac1 inhibitor NSC23766, or PRSS2 and NSC23766 in combination (P values calculated by two-sided ANOVA; n = 5). H Western blot of Tsp-1 and actin in WI38 cells that were treated with saline (–), PRSS2, JNK1 inhibitor III, or PRSS2 and JNK1 inhibitor III in combination (n = 3). I Schematic diagram of PRSS2 repression of Tsp-1 via LRP1-Rac signaling.
Fig 3: Breast and prostate cancer cells repress Tsp-1 in a paracrine manner via PRSS2.A Immunohistochemistry (IHC) of thrombospondin-1 (Tsp-1) expression in tumors formed in the prostate gland of SCID mice by PC3 and PC3M-LN4 prostate cancer cells (scale bar = 100 µm) (n = 8 per group). B Schematic diagram of the proteomic screening and identification of Tsp-1 repressing protein in PC3M-LN4 conditioned media. C ELISA of Tsp-1 expression (normalized to total protein levels) in PC3M-LN4 fractions eluted from a Heparin-sepharose-Cu2+ column (n = 3). D Western blot of PRSS2 and actin protein levels in PC3 and PC3M-LN4 prostate cancer cells (n = 3). E Western blot of PRSS2 and actin protein levels in MDA-MB-231 (231), MDA-MB-231-LM2 (LM2), MCF7 and SUM159 breast cancer cells (n = 3). F Tsp-1 and actin levels in MRC5 fibroblasts and primary human peripheral blood mononuclear cells (PBMCs) that were untreated (–) or treated with recombinant human PRSS2 ( + ) (n = 3). G Plot of Tsp-1 mRNA fold change, measured by real-time quantitative RT-PCR in WI38 cells treated with conditioned media of 293T cells engineered to overexpress PRSS2 (n = 3; P value calculated via two-sided Student’s t test). H Upper panel: Western blot of PRSS2 in SUM159 cells that were untransfected (C), transfected with empty pLKO.1 vector (V), or pLKO.1 vector expressing three independent shRNA sequences directed against PRSS2 (sh1, sh2, sh3). Lower panel: Western blot of Tsp-1 and actin in WI38 cells that were untreated (–) or treated with conditioned media from SUM159 cells with empty vector (V) or PRSS2 shRNA (n = 3). I Western blot of Myc, PRSS2 and actin in SUM159 cells transfected with vector control (V) or three shRNA sequences directed against c-Myc (sh1, sh2 and sh3) (n = 3). J Dot plot of fold change of Myc and PRSS2 in SUM159 cells transfected with vector control (V) or three shRNA sequences directed against c-Myc (sh1, sh2, and sh3 (n = 3). K Dot plot of fold change of Myc and PRSS2 in PC3M-LN4 cells transfected with vector control (V) or three shRNA sequences directed against c-Myc (sh1, sh2, and sh3) (n = 3).
Fig 4: Myeloid-specific deletion of LRP1 inhibits tumor growth by preventing the repression of Tsp-1.A Plot of percentage of LRP1 + multiple myeloid and lymphoid cells in wild-type (n = 5) and LysM-Cre/LRP1fl/fl (n = 5) mice as determined by FACS analysis. B Representative FACS plots of LRP1 expression in myeloid and lymphoid cells in wild-type and LysM-Cre/LRP1fl/fl mice (n = 5 per group). C Plot of relative abundance of CD45 + myeloid and lymphoid cells in wild-type (n = 5) and LysM-Cre/LRP1fl/fl (n = 5) mice as determined by FACS analysis. D Western blot of PRSS2 and Actin in Pan02 murine pancreatic cancer cells, SUM159 human breast cancer cells, and E0771 murine breast cancer cells (n = 3). E Plot of average tumor volume (as measured by calipers) of orthotopic mammary tumors formed by E0771 murine breast cancer cells in wild-type (red line), LysM-Cre/LRP1fl/fl (green line), and LysM-Cre/LRP1fl/fl/THBS1-/- (blue) mice (n = 7 per group) (error bars depict SEM). F Dot plot of volume of tumors formed by E0771 cells in LysM-Cre/LRP1fl/fl mice (LRP1mKO) (n = 7 mice per group). G Photographs of tumors formed by E0771 murine breast cancer cells in wild-type, LysM-Cre/LRP1fl/fl, and LysM-Cre/LRP1fl/fl/THBS1-/- mice. H H&E and Tsp-1 immunohistochemical staining of tumors formed by E0771 murine breast cancer cells in wild-type, LysM-Cre/LRP1fl/fl, and Tsp-1-/-/LysM-Cre-LRP1fl/fl mice (n = 7 mice per group) (scale bars = 100 µm).
Fig 5: PRSS2 is required for efficient tumor formation of SUM159 cells.A Immunohistochemical analysis of breast cancer patient Series 1 (n = 544 patients) for expression of: (i) PRSS2 in tumors with strong (high) PRSS2 expression in tumor cells, (ii) PRSS2 in tumors with weak (low) PRSS2 expression in tumor cells, (iii) PRSS2 in tumors with strong PRSS2 expression in TME, (iv) PRSS2 in tumors with weak PRSS2 expression in the TME; (v) proliferating microvessel density (pMVD) in tumors with strong PRSS2 expression, (vi) pMVD in tumors with weak PRSS2 expression; (vii) proliferation (Ki67) in tumors with strong PRSS2 expression; and (viii) Ki67 in tumors with weak PRSS2 expression (scale bar = 50 µm, original magnification ×400). B H&E staining and immunohistochemical analysis of prostate cancer patient series (n = 458 patients) for expression of: (i) PRSS2 in tumors with strong (high) tumor cell expression of PRSS2 in localized prostatic carcinoma; (ii): PRSS2 in tumors with weak (low) tumor cell expression of PRSS2 in localized prostatic carcinoma; (iii) PRSS2 in tumors with strong expression of PRSS2 in castration-resistant carcinoma (CR); (iv) PRSS2 in tumors with weak expression of PRSS2 in castration-resistant carcinoma; (v) VEGF-A in localized carcinoma with strong PRSS2 expression; (vi) VEGF-A in localized carcinoma with weak PRSS2 expression; (vii) Ki67 in localized carcinoma with strong PRSS2 expression; (viii) Ki67 in localized carcinoma with weak PRSS2 expression (scale bar = 50 µm, original magnification ×400). C Kaplan–Meier curve of clinical progression following radical prostatectomy of prostate cancer patients with strong and weak expression of PRSS2 (P values were determined via two-sided log-rank test). D Kaplan–Meier curve of overall survival following the acquisition of castration resistance of prostate cancer patients with strong and weak expression of PRSS2 (P values were determined via a two-sided log-rank test). E Plot of in vitro proliferation of SUM159 and SUM159shPRSS2 cells over 3 days (no significant difference was determined via two-sided ANOVA). F Plot of in vivo luciferase activity of orthotopic tumors formed by mammary gland injection of SUM159 and SUM159shPRSS2 cells (P value was determined via two-sided Mann–Whitney U test; error bars depict SEM). G In vivo luciferase imaging of mice bearing SUM159 (upper panel) and SUM159shPRSS2 (lower panel) tumors. H Photographs of tumors formed by SUM159 and SUM159shPRSS2 cells. I Plot of the volume of tumors formed by SUM159 and SUM159shPRSS2 cells (P value calculated via two-sided Mann–Whitney U test). J Immunohistochemistry of Tsp-1 in tumors formed by SUM159 and SUM159shPRSS2 cells (n = 8 mice per group) (scale bar = 200 µm).
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