Fig 2: Hsp27-modified surface marker expression on myeloid–prostate cancer cell co-cultures. (a) representative flow cytometry overlay histograms of CD163 fluorescence in THP-1 cells co-cultured with PC-3 cells; (b–h) normalized mean fluorescence intensities of THP-1 cells obtained from monoculture (mc) or prostate cancer co-cultures (cc) 96 h after Hsp27 exposure, determined using multicolor flow cytometry; (i) representative flow cytometry overlay histograms of CD71 fluorescence in HL-60 cells co-cultured with LNCaP cells; (j–o) normalized mean fluorescence intensities of HL-60 cells obtained from monoculture (mc) or prostate cancer co-cultures (cc) 96 h after Hsp27 exposure, determined using multicolor flow cytometry. Data are representative of at least three experiments; statistical analysis was performed using one-way ANOVA with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Fig 3: High-content imaging of THP-1 cells co-cultured with prostate cancer cells under Hsp27 treatment. (a) representative overlay images of untreated (control) and Hsp27-treated co-cultures (cc) of CellTrace Violet-labeled prostate cancer cells (blue) and CellTrace Far Red-labeled THP-1 cells (red), with SYTOX Green (green) as dead cell marker at 96 h; (b) PCAs of several morphology properties quantified by algorithm-based image analysis of THP-1 cells; (c–l) algorithm-based image quantification of THP-1 total cell count (c,d), the total aggregate area in µm2 (e,f), number of cells interacting with prostate cancer cells (g,h), distance to the nearest prostate cancer cell in µm (i,j), and mean fluorescence intensity (MFI) of dead prostate cancer cell material internalized by THP-1 cells (k,l). Data are presented as mean + SE. Statistical analysis was performed using unpaired, two-tailed t-test with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). ns = not significant. Scale bar is 100 µm.

Fig 4: High-content imaging of HL-60 cells co-cultured with prostate cancer cells under Hsp27 treatment. (a) representative overlay images of untreated (control) and Hsp27-treated co-cultures (cc) of CellTrace Violet-labeled prostate cancer cells (blue) and CellTrace Far Red-labeled HL-60 cells (red), with SYTOX Green (green) as dead cell marker at 96 h; (b) PCAs of several morphology properties quantified by algorithm-based image analysis of HL-60 cells; (c–l) algorithm-based image quantification of HL-60 total cell count (c,d), the total aggregate area in µm2 (e,f), number of cells interacting with the prostate cancer cells (g,h), distance to the nearest prostate cancer cell in µm (i,j), and mean fluorescence intensity (MFI) of dead prostate cancer cell material internalized by HL-60 cells (k,l). Data are presented as mean + SE. Statistical analysis was performed using unpaired, two-tailed t-test with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). ns = not significant. Scale bar is 100 µm.

Fig 5: Oxidative stress-induced Hsp27 and its effects in individual cell lines. (a) fold-change increase in oxidative stress-induced Hsp27 release after 48 h, 72 h, and 96 h in LNCaP and PC-3 cells; (b) representative photograph of the resazurin-based metabolic activity assay; (c,d) normalized metabolic activity of prostate cancer cells 96 h (c) and myeloid cells directly (4 h) and 24 h (d) following exposure to Hsp27; (e) representative flow cytometry dot plot of Hsp27-treated HL-60 cells; (f) normalized number of viable myeloid cells determined via flow cytometry directly and 24 h after Hsp27 exposure; (g) representative flow cytometry overlay histograms of mitotracker orange fluorescence in HL-60 cells; (h) normalized mean fluorescence intensity of dichlorofluorescein (DCF), aminophenyl fluorescein (APF), and mitotracker orange (MTO) in myeloid cells determined by flow cytometry; (i) normalized chemokine, cytokine, and growth-factor secretion profile 96 h after Hsp27 exposure in myeloid cells (additional soluble mediators investigated were below the limit of detection, including IL-1β, TNF-α, IFN-γ, IL-12p70, and IL-10). Data are representative of at least three experiments; statistical analysis was performed using one-way (c) or two-way ANOVA (i) with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).