Fig. 4

Stromal SPINK1 significantly modifies prostate cancer cell phenotypes in vitro. a Immunoblot analysis of EGFR-associated pathways in PCa cells treated by PSC27 CM alone, or with the EGFR inhibitor AG-1478 (2 μM). Total protein per molecule and GAPDH were used as loading control. b Immunoprecipitation (IP) followed by immunoblot assay of EGFR and SPINK1 in the whole cell lysates of PC3 treated by the CM of PSC27 sublines for 3 days. S, SPINK1; GAPDH, loading control. c Scramble or SPINK1-specific shRNA-transduced PSC27 cells were treated with either DMSO or bleomycin (BLEO) and subject to SA-β-Gal assay. Upper, comparative statistics. Lower, representative images of SA-β-Gal staining. C, scramble. d Proliferation measurement of PCa cells treated with the CM of PSC27 sublines for 3 days. e Migration assay of PCa cells seeded within transwells in 6-well plates, with cells cultured for 3 days in PSC27 subline CM as indicated in (c). Bottom, representative images of PC3 cell migration measured via wound healing assay at 72 h. Scale bars, 100 μm. f Invasiveness appraisal of PCa cells across the transwell membrane upon culture with PSC27 subline CM. Bottom, representative images of PC3 cell invasion across the transwell measured at 72 h. Scale bars, 20 μm. g Chemoresistance assay of PCa cells cultured with PSC27 subline CM. MIT (mitoxantrone) was applied at the concentration of IC50 value pre-determined per cell line. AG-1478 (2 μM), cetuximab (50 μg/ml), or SPINK1 mAb (1 μg/ml) were applied alongside with PSC27 CM. h Dose–response curves (non-linear regression/curve fit) of PC3 cells cultured with PSC27 CM and concurrently treated by a wide range of concentrations MIT. AG-1478 (2 μM), cetuximab (50 μg/ml), and/or SPINK1 mAb (1 μg/ml) were applied with PSC27 CM. Data are mean ± SD and representative of 3 independent experiments, with 3 technical replicates run per cell-based experiment. P values were calculated by Student’s t-test (d–h) and one-way ANOVA (c) (^P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001)