Spindle pole body component 25 and platelet-derived growth factor mediate crosstalk between tumor-associated macrophages and prostate cancer cells

Abstract

Tumor-associated macrophages (TAMs) are involved in the growth of prostate cancer (PrC), while the molecular mechanisms underlying the interactive crosstalk between TAM and PrC cells remain largely unknown. Platelet-derived growth factor (PDGF) is known to promote mesenchymal stromal cell chemotaxis to the tumor microenvironment. Recently, activation of spindle pole body component 25 (SPC25) has been shown to promote PrC cell proliferation and is associated with PrC stemness. Here, the relationship between SPC25 and PDGF in the crosstalk between TAM and PrC was investigated. Significant increases in both PDGF and SPC25 levels were detected in PrC specimens compared to paired adjacent normal prostate tissues. A significant correlation was detected between PDGF and SPC25 levels in PrC specimens and cell lines. SPC25 increased PDGF production and tumor cell growth in cultured PrC cells and in xenotransplantation. Mechanistically, SPC25 appeared to activate PDGF in PrC likely through Early Growth Response 1 (Egr1), while the secreted PDGF signaled to TAM through PDGFR on macrophages and polarized macrophages, which, in turn, induced the growth of PrC cells likely through their production and secretion of transforming growth factor β1 (TGFβ1). Thus, our data suggest that SPC25 triggers the crosstalk between TAM and PrC cells via SPC25/PDGF/PDGFR/TGFβ1 receptor signaling to enhance PrC growth.

Keywords: SPC25; crosstalk; pdgf; prostate cancer; tumor associated macrophage (TAM).

Figure 1

SPC25 increases PDGF levels in PrC (A, B) ELISA measurement of SPC25 protein level in 40 pairs of prostate cancer (PrC) and normal tissues (NT) as a group (A), and as per pair (B). (C, D) PDGF protein levels measured by ELISA in 40 pairs of PrC and NT, as a group (A), and as per pair (B). (E) Correlation between SPC25 and PDGF in PrC specimens (R = 0.84; p < 0.0001). (F) RT-qPCR for SPC25 in several human PrC cell lines. (G) mRNA level of SPC25 in human PrC cell lines DU145 and LNCap transfected by either shSPC25 or SPC25, compared to cells transfected by scrambled plasmids (scr). (H) ELISA for SPC25 in DU145 and LNCap transfected by either shSPC25 or SPC25, compared to cells transfected by scrambled plasmids (scr). *p < 0.05. n = 40 for clinical studies (A–E) and n = 5 for cell line studies (F, G). Relative values were shown. For (F, G), Scr = 1.

Figure 2

Establishment of a mouse PrC model (A) A diagram of a lenti vector carrying a luciferase and a green fluorescent protein (GFP) reporter under a CMV promoter. (B) The lenti-pCMV-LUC-GFP was used to transduce DU145 cells, sorted by flow cytometry as GFP-positive cells. (C) GFP-positive cells show green fluorescence. (D) The lenti-pCMV-LUC-GFP-transduced DU146 cells received luciferin to show the presence of luciferase expression in the cells. (E) LUC-GFP-DU145 cells were subcutaneously injected into nude mice to establish the PrC model and detected by bioluminescence (three left mice). Two control mice that did not receive tumor cell implantation were shown as controls (two right mice). Scale bar is 20 µm.

Figure 3

PrC cells express a high level of PDGF and TAMs express a high level of PDGFR (A) PrC cells from the mice model were detected by green fluorescence. (B) PDGF mRNA level of GFP-positive cells vs. GFP-negative cells. (C) Analyses of GFP-positive cells and PDGFR-positive cells from the PrC mouse model. PDGFR+ cells were divided into two populations based on CD31 and F4/80 expression. PDGFR+ F4/80+ cells were further analyzed for CD206 expression. The gating strategy for the flow cytometry was shown. (D) Staining with a PDGFR antibody or isotype control on green cells. (E) % of F4/80+, CD31+, and other cells in PDGFR+ cells measured by flow cytometry. (F) % of CD206+, CD206- cells in PDGFR+F4/80+ cells. *p < 0.05. n = 5. Scale bars are 20 µm.

Figure 4

PrC cells regulate macrophage polarization, which, in turn, mediates PrC cell growth (A) A diagram of a transwell co-culture system for DU145 cells and bone-marrow-derived macrophages (MΦ). Group 1: macrophages alone; Group 2: DU145 cells alone; Group 3: co-culture of macrophages and DU145; Group 4: co-culture of macrophages and DU145-shSPC25; Group 5: co-culture of macrophages and DU145 with presence of an anti-PDGFR antibody; Group 6: macrophages alone with presence of PDGF; Group 7: macrophages alone with presence of PDGF and the anti-PDGFR antibody. Cells were incubated for 2 days. (B, C) An MTT assay was performed in DU145 cells (B) and in macrophages (C). (D, E) DNA content determination on DU145 cells (D) and in macrophages (E). (F) Cell invasion and migration in macrophages. (G, H) Flow cytometry analysis of CD206+ cells in macrophages, shown by quantification (G) and by representative flowcharts (H). *p < 0.05. NS, non-significant. n = 5.

Figure 5

Polarized TAMs promote PrC cell growth through TGFβ1 (A) RT-qPCR for TGFβ1 mRNA level in CD206+ versus CD206- macrophages. (B) A transwell co-culture diagram of DU145 cells and bone-marrow-derived macrophages (MΦ). Group 1: MΦ. Group 2: MΦ with inhibitor SB431542 (SB). Group 3: MΦ and DU145 cells. Group 4: MΦ and DU145 cells transduced with shSPC25. Group 5: MΦ and DU145 cells with SB. Cells were incubated for 2 days. (C, D) Flow cytometry analysis of CD206+ cells in macrophages, shown by quantification (C) and by representative flowcharts (D). (E) MTT assay on DU145 cells. *p < 0.05. NS, non-significant. n = 5.

Figure 6

SPC25 activates PDGF through Egr-1 (A, B) Mithramycin A (MMA) was added at a dose of 0.1 µg/ml into the cultured LNCap cells. (A) Representative Western blots of SPC25, Egr-1, and PDGF, and (B) corresponding quantification. (C) A schematic of the model in this research: SPC25 in PrC cells enhances PDGF production likely through Egr-1 to polarize macrophages towards TAM through their surface expression of PDGFR. Polarized macrophages (TAM) produce TGFβ1 to promote growth of PrC cells. *p < 0.05. NS, non-significant. n = 5.