Exosomes Produced by Mesenchymal Stem Cells Drive Differentiation of Myeloid Cells into Immunosuppressive M2-Polarized Macrophages in Breast Cancer

Abstract

Tumor-associated macrophages are major contributors to malignant progression and resistance to immunotherapy, but the mechanisms governing their differentiation from immature myeloid precursors remain incompletely understood. In this study, we demonstrate that exosomes secreted by human and mouse tumor-educated mesenchymal stem cells (MSCs) drive accelerated breast cancer progression by inducing differentiation of monocytic myeloid-derived suppressor cells into highly immunosuppressive M2-polarized macrophages at tumor beds. Mechanistically, MSC-derived exosomes but not exosomes from tumor cells contain TGF-β, C1q, and semaphorins, which promote myeloid tolerogenic activity by driving PD-L1 overexpression in both immature myelomonocytic precursors and committed CD206⁺ macrophages and by inducing differentiation of MHC class II⁺ macrophages with enhanced l-Arginase activity and IL-10 secretion at tumor beds. Accordingly, administration of tumor-associated murine MSC-derived exosomes accelerates tumor growth by dampening antitumor immunity, and macrophage depletion eliminates exosome-dependent differences in malignant progression. Our results unveil a new role for MSC-derived exosomes in the differentiation of myeloid-derived suppressor cells into macrophages, which governs malignant growth.

Figure 1.. MSC-derived exosomes promote conversion of M-MDSCs into immunosuppressive macrophages.

(A) BM-derived GM-CSF + IL-6-induced myeloid cells treated with or without exosomes from EpCAM⁺ tumor cells or 3T3 or MSCs for 48 hr. Bar graphs showing number (percentage) of M-MDSCs (CD11b⁺F4/80⁺Ly6G⁻Ly6C⁺IA/IE⁻) and macrophages (CD11b⁺ F4/80⁺Ly6G⁻Ly6C⁺IA/IE⁺) among the total myeloid population analyzed by flow cytometry. Flow cytometry dot plots of IA/IE in untreated and MSC-derived exosome treated cells gated for viable CD11b⁺F4/80⁺Ly6G⁻Ly6C⁺ cells. Bar graphs showing percentages of IA/IE⁺ cells in different treatment groups. Experiments were performed twice. (B) Dot plots showing CD206 expression in M-MDSCs (upper) and macrophages (lower) in untreated and MSC-derived exosome treated cells. Bar graphs showing percentages of CD206⁺ M-MDSCs and MFI of CD206 in M-MDSCs (upper); and CD206⁺ macrophages and MFI of CD206 in macrophages (lower), in different treatment groups. Experiments were performed twice. (C) Dot plots showing PD-L1 expression in M-MDSCs (upper) and macrophages (lower) in untreated and MSC-derived exosome treated cells. Bar graphs showing percentages of PD-L1⁺ M-MDSCs (upper), and PD-L1⁺ macrophages (lower) in different treatment groups. Experiments were performed twice. (D) Bar graph showing arginase activity of GM-CSF + IL-6-induced BM-derived myeloid cells upon treatment with or without EpCAM⁺ tumor cells or 3T3 or MSCs-derived exosomes for 48 hr. Experiments were performed three times. (E) Bar graphs showing IL-10 concentration in conditioned media from BM-derived myeloid cells upon treatment with or without EpCAM⁺ tumor cells or 3T3 or MSCs-derived exosomes for 48 hr. Experiments were performed three times and ELISA was performed with 4 replicates from each. (F) Heat map showing (left) differences in Z-scores for indicated genes in BM-derived myeloid cells upon treatment with or without EpCAM⁺ tumor cells or 3T3 or MSCs-derived exosomes for 48 hr. Graph (right) showing major upregulated pathways exclusively observed MSC-exosome treated BM-derived myeloid cells, calculated and represented as −log₁₀ values of False Discovery Rate (FDR). Experiments were performed in triplicate. (G) Dilution of Cell Trace Violet in labeled T cells activated with anti-CD3/CD28 antibodies and co-cultured with increasing ratios of BM-derived myeloid cells, treated with or without EpCAM⁺ tumor cells or MSCs-exosomes for 48 hr. Bar graph showing percentage of T cells proliferated after incubation with BM-derived myeloid cells for 3 days. Experiments were performed three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant.

Figure 2.. Mesenchymal stem cell-exosomes drive increased tumor growth, upregulation of CD206 in tumor associated M-MDSCs and macrophages, EMT of cancer cells with superior invasive ability.

(A) In vivo luciferase analysis showing tumor growth in the right axillary flank after 14 days from intratumoral administration of MSC-derived exosomes or PBS into Brpkp110 breast tumors. Mouse MSC-derived exosomes (Exo^MSC) or PBS (Vehicle) was administered after 5 days of tumor challenge. Data are representative of the two independent experiments (n=5/group; one representative experiment of two). (B) Comparison of tumor weight between Exo^MSC and Vehicle group after 14 days of PBS or exosome administration (n=5/group; two experiments). Representative tumors from an individual experiment are depicted. (C) Scattered plot showing number of M-MDSCs (CD45⁺CD11b⁺F4/80⁺Ly6G⁻Ly6C⁺IA/IE⁻) among total tumor-infiltrated leukocytes (viable CD45⁺) in Vehicle and Exo^MSC tumors (n=5/group; two experiments). (D) Scattered plot showing number of macrophages (CD45⁺CD11b⁺F4/80⁺Ly6G⁻Ly6C⁺IA/IE⁺) among total tumor-infiltrated leukocytes (viable CD45⁺) in Vehicle and Exo^MSC tumors (n=5/group; two experiments). (E) Representative flow cytometry plots showing percentage of CD206⁺ cells in gated viable M-MDSCs (CD45⁺CD11b⁺F4/80⁺Ly6G⁻Ly6C⁺IA/IE⁻), and quantification of CD206⁺ M-MDSCs among total tumor-infiltrated leukocytes in Exo^MSC and Vehicle tumors are represented as scattered pots (n=5/group; two experiments). (F) Representative flow cytometry plots showing percentage of CD206⁺ cells in gated viable macrophages (CD45⁺CD11b⁺F4/80⁺Ly6G⁻Ly6C⁺IA/IE⁺), and quantification of CD206⁺ macrophages among total tumor-infiltrated leukocytes in Exo^MSC and Vehicle tumors are represented as scattered pots (n=5/group; two experiments). (G) Western blots of Slug, Snail and E-cadherin in tumors from Exo^MSC and Vehicle group, performed three times from 3 different tumors of each group. β-actin used as loading control. Bar graphs showing western blot intensities of Slug, Snail and E-cadherin, relative to β-actin. (H) Phase contrast microscopy analysis and quantification, by absorbance measurement at 560 nm, of extracellular matrix invasion by CD45⁻EpCAM⁺ cancer cells from Exo^MSC and Vehicle group, performed three times from 3 different tumors of each group. *, p < 0.05; **, p < 0.01.

Figure 3.. MSC-derived exosomes dampens anti-tumor T cell responses.

(A) ELISpot analysis of CD8⁺ T cells isolated from draining lymph nodes of Brpkp110-tumor-bearing mice, stimulated with irradiated tumor cell-pulsed BM-derived dendritic cells. (B) Representative flow cytometry analyses of IFN-γ production by CD8⁺ and CD4⁺ T cells from Exo^MSC or Vehicle tumors, pre-induced with PMA (20 ng/mL), Ionomycin (Sigma, 1 μg/mL) and Golgi stop (0.8 μL/10⁶ cells) for 4 hr. An isotype control was utilized to set the gate for intracellular IFN-γ signal. Scattered graphs showing percentages of CD8⁺ and CD4⁺ cells from Exo^MSC or Vehicle tumors expressing IFN-γ; bar graphs showing IFN-γ MFI (n=5/group; two experiments). (C) Dilution of Cell Trace Violet in labeled T cells activated with anti-CD3/CD28 antibodies and co-cultured with increasing ratios of IA/IE⁻ M-MDSCs or IA/IE⁺ macrophages. Experiments were performed twice. (D) Representative flow cytometry analyses showing PD-L1 expression by CD206⁺ M-MDSCs and CD206⁺ M2 macrophages. Scattered graphs showing percentages of CD206⁺ M-MDSCs, and CD206⁺ M2 macrophages from Exo^MSC or Vehicle tumors expressing PD-L1; bar graphs showing PD-L1 MFI (n=5/group; two experiments). (E) Representative quantification of PD-1⁺ T cells (n=5/group; two experiments) and intensity of PD-1 expression on T cells (n=5/group; one representative experiment of two) in tumors from Exo^MSC and Vehicle group. (F) Scattered plot of log2 PD-1 mRNA expression from TCGA dataset (n=1,100) comparing breast tumors with the strongest CD271 (n=250) and weakest CD271 (n=250) expression. (G) Tgfb mRNA expression in tumor-sorted M-MDSCs (left), tumor-associated macrophages (TAMs, middle), and CD45⁻EpCAM⁺ cancer cells (right). Experiment was performed twice. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant.

Figure 4.. MSC-derived exosomes drive breast cancer progression through PD-L1-PD-1 axis, and contain M-MDSC to M2 differentiating factors.

(A) Vehicle and Exo^MSC group mice administered with anti-PD-L1 neutralizing antibodies or anti-isotype control antibodies. Volume comparison at different time points (n=5/group, one representative experiment of two) starting from day of intra-tumor exosome or PBS administration (Day 0). (B) Comparison of tumor weight between anti-PD-L1 or anti-isotype control antibody injected mice groups (n=5/group, two experiments) after resection on day 21 post Brpkp110-challenge. Representative tumors from an individual experiment are depicted. (C) Comparison of intratumoral CD11b⁺ myeloid population in mice administered with Chlodronate liposomes or Control liposomes. (D) Both Vehicle and Exo^MSC groups administered with Chlodronate liposomes or Control liposomes. Volume comparison (n=5/group, one representative experiment of two) at different time points starting from Day 0. (E) Comparison of tumor weight between Chlodronate liposomes or Control liposomes treated mice groups (n=5/group, two experiments) after resection on day 21 after Brpkp110-challenge. Representative tumors from an individual experiment are depicted. (F) List of important molecules contained exclusively or superiorly in MSC-derived exosomes. CD63 is listed as exosome quality control. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant.

Figure 5.. Infiltration of MSCs and M2 macrophages in human breast tumors shows a strong positive correlation.

(A) Surgically operated fresh IDC breast tumors were collected and RNA was isolated (n=51). Scattered plat showing correlations between mRNA expressions of CD206 and CD271 with a Spearman’s correlation co-efficient value (r) 0.896. (B) Representative dot plots of flow cytometry with dissociated tumors (n=15), showing tumors with higher percentage of CD45⁻CD271⁺MSCs has a higher percentage of CD45⁺CD3⁻CD11b⁺CD206⁺CD163⁺ M2 macrophages (left). Graph (right) showing correlation (Spearman’s correlation, r=0.83) between intratumoral percentages of MSCs and myeloid cells with features of M2 macrophage. (C) From TCGA dataset of 1,100 primary breast tumors, CD271 and CD206 mRNA expression were analyzed, and log values are represented in Y-axis and X-axis, respectively in the scattered correlation plot, showing a positive correlation (Spearman’s correlation, r=0.26) (D) Graph on left showing log CD206 mRNA expression from TCGA dataset (n=1,100) comparing breast tumors with the strongest CD271 (n=250) and weakest CD206 (n=250) expression. Graph on right showing log CD271 mRNA expression from TCGA dataset (n=1,100) comparing breast tumors with the strongest CD206 (n=250) and weakest CD206 (n=250) expression. (E) Breast TMA (n=19) stained for CD271 (Alexa Fluor 568) and CD206 (Alexa Fluor 647). Nuclei were stained with DAPI. Representative immunofluorescence images showing tumors with both high and both low CD271 and CD206. Scatter graph (upper) showing percentage of CD271⁺ cells in CD206 high (>0.4%; n=9) vs. CD206 low (<0.4%; n=10) tumors. Scattered plot (bottom) showing positive (Spearman’s correlation, r=0.3) correlation between MFI of CD271and MFI of CD206 per unit area of tumor (n=19). *, p < 0.05.

Figure 6.. Human mesenchymal stem cells boost CD206^high PD-L1^high M2 macrophage polarization.

(A) Flow cytometry histograms showing expression of CD11b in untreated or PMA-induced THP-1 cells (M0 THP-1). (B) Western blot analysis showing expressions of PD-L1, CD206, CD163 and β-actin in M0 THP-1 cells, with different treatment combinations, representative of two experiments. M0 THP-1 cells treated with IL-4 and IL-13 as a positive control of M2 polarization. In the remaining combinations, M0 THP-1 cells cultured in conditioned media (CM) from either MCF10A or MDA-MB-231 or T47D and co-cultured with or without human MSCs. (C) Densitometry analysis of western blots showing intensities of CD206, PD-L1, CD163 bands relative to respective β-actin bands (D) Flow cytometry dot plots showing percentages of CD206⁺ and PD-L1⁺ and histograms showing MFI of CD206 and PD-L1 in M0 THP-1 cells in different treatment combinations as mentioned, representative of two experiments. (E) Bar graphs showing mean±SEM percentages of CD206⁺, PD-L1⁺ cells. *, p < 0.05; **, p < 0.01; NS, not significant.

Figure 7.. Human mesenchymal stem cells-derived exosomes drive elevated PD-L1 and CD206 expression by macrophages.

(A) Western blot analysis of PD-L1 (left) in M0 THP-1 cells, grown in CM from breast cancer cell lines treated with or without 20 μM GW4869, and co-cultured with mesenchymal stem cells treated with or without GW4869. β-actin used as loading control. Experiments were performed twice. Bar graphs (right) showing mean±SEM intensities of PD-L1 western blot bands relative to β-actin. (B-D) M0 THP-1 cells treated with different combinations (a,b,c and d) of purified exosomes from breast cancer cell lines and mesenchymal stem cells. (B) Western blot analysis of PD-L1, representative of two experiments (left). Bar graphs (right) showing mean±SEM intensities of PD-L1 western blot bands relative to β-actin. (C) Flow cytometry analysis of CD206 and PD-L1, representative of two experiments. (D) Bar graphs showing mean±SEM percentages of CD206⁺, PD-L1⁺ cells. (E) PD-L1 mRNA expression in human breast tumors (n=51) relative to healthy breast tissues, quantified by Q-PCR, and grouped according to CD271 mRNA fold change less or more than 6. (F) Graph showing level of mRNA of PD-L1 in 1,100 breast tumors (showed with color intensity gradient) analyzed from TCGA data where each dot represents individual tumors with X-axis value for log2 CD206 expression, Y-axis value for log2 CD271 expression. Spearman’s Rank correlation co-efficient between CD271 and PD-L1 is 0.06483444; and between CD206 and PD-L1 is 0.497973717. Scattered plot of log2 PD-L1 mRNA expression from TCGA dataset (n=1,100) comparing breast tumors with the strongest CD271-CD206 co-expression (n=250) and weakest CD271-CD206 expressions (n=250). *, p < 0.05; **, p < 0.01; ***, p < 0.001.