Tumor-Derived Exosomal Protein Tyrosine Phosphatase Receptor Type O Polarizes Macrophage to Suppress Breast Tumor Cell Invasion and Migration

Abstract

Tumor-derived exosomes, containing multiple nucleic acids and proteins, have been implicated to participate in the interaction between tumor cells and microenvironment. However, the functional involvement of phosphatases in tumor-derived exosomes is not fully understood. We and others previously demonstrated that protein tyrosine phosphatase receptor type O (PTPRO) acts as a tumor suppressor in multiple cancer types. In addition, its role in tumor immune microenvironment remains elusive. Bioinformatical analyses revealed that PTPRO was closely associated with immune infiltration, and positively correlated to M1-like macrophages, but negatively correlated to M2-like macrophages in breast cancer tissues. Co-cultured with PTPRO-overexpressing breast cancer cells increased the proportion of M1-like tumor-associated macrophages (TAMs) while decreased that of M2-like TAMs. Further, we observed that tumor-derived exosomal PTPRO induced M1-like macrophage polarization, and regulated the corresponding functional phenotypes. Moreover, tumor cell-derived exosomal PTPRO inhibited breast cancer cell invasion and migration, and inactivated STAT signaling in macrophages. Our data suggested that exosomal PTPRO inhibited breast cancer invasion and migration by modulating macrophage polarization. Anti-tumoral effect of exosomal PTPRO was mediated by inactivating STAT family in macrophages. These findings highlight a novel mechanism of tumor invasion regulated by tumor-derived exosomal tyrosine phosphatase, which is of translational potential for the therapeutic strategy against breast cancer.

Keywords: breast cancer; invasion and migration; macrophage polarization; protein tyrosine phosphatase receptor type O; tumor-derived exosomes.

FIGURE 1

PTPRO expression was associated with TAM infiltration in breast cancer. (A) Box and scatter plots showing the immune scores of PTPRO high expression group and PTPRO low expression group in the GEO dataset GSE36774. (B) Correlation between PTPRO expression and immune scores in the GEO dataset GSE36774. (C) GSEA showing the positive correlation between PTPRO expression and adaptive immune response signature in the GEO dataset GSE36774. (D) GSEA showing the positive correlation between PTPRO expression and macrophage differentiation-related gene signature in the GEO dataset GSE36774. (E) The correlation between PTPRO expression and the infiltration level of macrophages was presented by Tumor Immune Estimation Resource (TIMER; cistrome.shinyapps.io/timer). (F) Box and scatter plots showing M1/M2 ratio difference between PTPRO high expression group and PTPRO low expression group according to GEO dataset GSE36774. FDR q, false-discovery rate q-value; NES, normalized enrichment score; **P < 0.01, ***P < 0.001 by Student’s t-test.

FIGURE 2

Tumor-derived PTPRO reeducated macrophages toward a M1-like phenotype. (A) THP-1 cells were treated with PMA for 24 h. Representative images of THP-1 derived macrophages were shown. Scale bars = 50 μm. (B) qRT-PCR was used to detect the expression of CD68 (macrophage marker) in THP-1 cells. (C) Representative images of immunofluorescence staining of iNOS and CD206 in THP-1 derived macrophages after incubating with ZR-75-1-vector or ZR-75-1-PTPRO cells for 48 h. Scale bars = 50 μm. (D) Representative images of immunofluorescence staining of iNOS and CD206 in THP-1 derived macrophages after incubating with ZR-75-1-shCtrl or ZR-75-1-shPTPRO cells for 48 h. Scale bars = 50 μm. Error bars, SEM. **P < 0.001 by Student’s t-test.

FIGURE 3

Tumor cell-derived PTPRO inflected macrophage polarization via exosome carrier. (A) Electron microscopy images of exosomes isolated from ZR-75-1-PTPRO cells. (B) Nanoparticle tracking analysis (NTA) of exosomes isolated from ZR-75-1-PTPRO cells. (C) The expressions of Alix, TSG101, CD63, CD9, Calnexin, and PTPRO were measured by immunoblotting in exosomes isolated from ZR-75-1-PTPRO cells. Relative protein expression was quantified. (D) Representative images of immunofluorescence staining of iNOS and CD206 in THP1-derived macrophages after treating with ZR-75-1-vector-exo or ZR-75-1-PTPRO-exo for 48 h. Scale bars = 50 μm. (E) qRT-PCR was applied to detect the relative expression of IL-1β, TNF-α, IL-10, and TGF-β in THP-1 derived macrophage after treating with ZR-75-1-vector-exo or ZR-75-1-PTPRO-exo for 48 h. (F) Representative images of immunofluorescence staining of iNOS and CD206 in THP1-derived macrophages after treating with ZR-75-1-shCtrl-exo or ZR-75-1-shPTPRO-exo for 48 h. Scale bars = 50 μm. (G) qRT-PCR was applied to detect the relative expression of IL-1β, TNF-α, IL-10, and TGF-β in THP-1 derived macrophage after treating with ZR-75-1-shCtrl-exo or ZR-75-1-shPTPRO-exo for 48 h. Error bars, SEM. ***P < 0.001 by Student’s t-test.

FIGURE 4

Tumor cell-derived exosomal PTPRO inhibited cancer cell invasion and migration. (A) Schematic illustration of the in vitro transwell co-culture system. (B) Cell migration and invasion assays were used to study the migration and invasion ability of ZR-75-1 cells. THP-1 cells prestimulated with PMA were treated with ZR-75-1-vector-exo or ZR-75-1-PTPRO-exo and ZR-75-1-shCtrl-exo or ZR-75-1-shPTPRO-exo at a concentration of 25 μg/mL or PBS; then, the upper chamber was obtained and incubated with ZR-75-1 cells for 24 h. Original magnification: 200×. Error bars, SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by a one-way ANOVA with post hoc intergroup comparisons.

FIGURE 5

Tumor cell-derived exosomal PTPRO polarized the M1 macrophage via dephosphorylated STAT3/STAT6 signaling. (A) GSEA showing inverse correlation between STAT3 expression and M1-gene signature (MONOCYTE_VS_CLASSICAL_M1_MACROPHAGE_UP) and positive correlation between STAT6 expression and M2-gene signature (MONOCYTE_VS_ALTERNATIVE_M2_MACROPHAGE_DN) in a published cohort of breast cancer. (B) Immunoblotting revealed that PTPRO was efficiently over-expressed in ZR-75-1 cells and the mutation of PTPRO did not affect it’s protein expression level. Relative protein expression was normalized to β-actin. (C) The expressions of STAT3/STAT6 protein and corresponding phosphorylated protein in THP-1 derived macrophage after treating with ZR-75-1-vector-exo, ZR-75-1-PTPRO-exo or ZR-75-1-PTPRO-CS-exo. Relative protein expression was normalized to β-actin. (D) The expressions of STAT3/STAT6 protein and corresponding phosphorylated protein in THP-1 derived macrophage after treating with ZR-75-1-shCtrl-exo or ZR-75-1-shPTPRO-exo. Relative protein expression was normalized to β-actin. FDR q, false-discovery rate q-value; NES, normalized enrichment score.