Fig 2: Fucoidan activates the JAK-STAT pathway and promotes T cell proliferation and activation. (A) IFNγ and TNFα expression in primary CD8+ T cells cultured with anti-CD3/CD28 beads, with or without fucoidan (25 μg/ml) treatments. Numbers adjacent to the outlined areas indicate the frequency of cells expressing IFNγ and TNFα. All data are quantified on the right. (B) IFNγ expression levels in CD8+ T cells treated by indicated concentrations of fucoidan. (C) CFSE assays quantifying the proliferation of CD8+ T cells stimulated by indicated concentrations of fucoidan. (D) Gene set enrichment analysis (GSEA) of the RNAseq data of fucoidan-treated (10 μg/ml) and control CD8+ T cells. (E) Heatmap of fucoidan-regulated genes involved in the JAK-STAT pathway. Ctrl, control. A1, A2, two biological replicates of fucoidan A-treated samples. F1, F2, two biological replicates of fucoidan F-treated samples. (F) Volcano plots of differentially expressed genes from RNAseq data of fucoidan-treated and control CD8+ T cells. The x axis represents log2 of fold changes (FC) of gene expression levels in fucoidan-treated cells relative to control cells, and the y axis represents log10 of corresponding P values. Genes within the JAK-STAT pathway were highlighted. (G) qRT-PCR analysis of the mRNA expression of indicated genes in fucoidan-treated and control CD8+ T cells. (H,I) IFNγ expression in CD8+ T cells activated by anti-CD3/CD28 beads, treated with fucoidan (10 μg/ml) and/or AZD1480 (50 nM). Data are representative of three independent experiments. Error bars, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: Expression of gelsolin is associated with poor clinical outcomes in patients with PCa(A) Representative examples of gelsolin (red crimson precipitates) expression in prostate cancer (PCa) cells, which was detected by immunohistochemistry, and the slide was counterstained with hematoxylin. In non-tumor prostate epithelial cells, secreted gelsolin was detected in the lumens (original magnification × 100). (B) Compared to hypertrophic region, expression of gelsolin was detected in tumor nests (Gleason score 6) (original magnification × 100). (C) In pathological tissue of PCa specimens with higher Gleason score, overexpression of gelsolin was detected in all the PCa cells (Gleason score 9) (original magnification × 100). (D) In PCa tissue with metastatic lesions, overexpression of gelsolin was detected in tumor nest and the metastatic PCa cells (Gleason score 9) (original magnification × 100). (E) In bone marrow section of PCa specimens, gelsolin was mainly detected in the invasive PCa cells (original magnification × 100). (F) Around tumor nest, in which gelsolin was minimally expressed, a swarm of lymphocytes without evident gelsolin signal were surrounding the tumor nest (original magnification × 100). (G) In some areas, many cells with darkly stained nuclei in the tumor nest were positive for gelsolin. However, nuclear morphologies of these cells were different from the neighboring PCa cells (original magnification × 400). (H) Similar to cells, which had darkly stained nuclei in PCa tumor nest, the cells were CD3-positive (original magnification × 400). (I) Expression of gelsolin in PCa and benign hypertrophic prostate epithelia (BHPE) cells was confirmed by Western blotting analysis. (J) Kaplan-Meier plots of overall survival in PCa patients with high (GSNHigh) or low gelsolin (GSNLow) expression. (K) Survival analysis of PCa patients in whom lymphocytes were positive (GSN+LYM+) or negative (GSN-LYM-) for gelsolin staining. Detection of gelsolin staining correlated with poor prognosis.

Fig 4: Secreted gelsolin desensitizes CD3-activated CD4+ T cells and induces the apoptosis of CD8+ T cells in the presence of PCa cells(A) Following the incubation of CD3 antibody-activated CD4+ and CD8+ T cells with conditioned media from LNCaP cells for 30 minutes, both cell types were positive for gelsolin, indicating that gelsolin could indeed bind to the surfaces of T cells (upper panel). (B) After being coated with gelsolin for 48 hours, both cell types shrank from about 11.12 ± 0.08 μm to 6.85 ± 0.35 μm in size (p < 0.007). (C) Gelsolin-coated lymphocytes were co-incubated with PCa cells, (D) Apoptosis of CD8+ T cells increased about 50% following gelsolin coating, suggesting that gelsolin might be involved in the inactivation and apoptosis of lymphocytes. The cytotoxic effect of PCa cells on CD4+ T cells, however, was not as evident. The white bar indicates 5 μm.

Fig 5: Splenic Th17 cells and pro-inflammatory cytokine expression. The lymphocytes were isolated from mice subjected to various treatments. (A) Flow cytometry was used to evaluate RORγt+CD3+CD4+ Th17 cells among splenic CD4+ cells obtained from mice. Quantification of (B) RORγ and (C) IL-17 from the flow cytometry results. (D) The mRNA levels of the cytokines IL-6, IFN-γ, TNF-α, IL-1β, and MCP-1β in the spleen tissue samples were assessed. (E) IL-17 mRNA and protein levels were evaluated by reverse transcription-quantitative polymerase chain reaction, western blot and ELISA. Values are expressed as the mean ± standard error of the mean (n=10 in each group). ***P<0.001 vs. the Con group; +P<0.05, ++P<0.01 and +++P<0.001 vs. the PRI group. Con, control; PRI, pristine; F25/50/100, treatment with 25/50/100 mg/kg fisetin; Th17, T-helper type 17; IL, interleukin; TNF, tumor necrosis factor; MCP, monocyte chemoattractant protein; IFN, interferon; ROR, RAR-related orphan receptor.