Upregulation of exosome secretion from tumor-associated macrophages plays a key role in the suppression of anti-tumor immunity

Abstract

Macrophages play a pivotal role in tumor immunity. We report that reprogramming of macrophages to tumor-associated macrophages (TAMs) promotes the secretion of exosomes. Mechanistically, increased exosome secretion is driven by MADD, which is phosphorylated by Akt upon TAM induction and activates Rab27a. TAM exosomes carry high levels of programmed death-ligand 1 (PD-L1) and potently suppress the proliferation and function of CD8⁺ T cells. Analysis of patient melanoma tissues indicates that TAM exosomes contribute significantly to CD8⁺ T cell suppression. Single-cell RNA sequencing analysis showed that exosome-related genes are highly expressed in macrophages in melanoma; TAM-specific RAB27A expression inversely correlates with CD8⁺ T cell infiltration. In a murine melanoma model, lipid nanoparticle delivery of small interfering RNAs (siRNAs) targeting macrophage RAB27A led to better T cell activation and sensitized tumors to anti-programmed cell death protein 1 (PD-1) treatment. Our study demonstrates tumors use TAM exosomes to combat CD8 T cells and suggests targeting TAM exosomes as a potential strategy to improve immunotherapies.

Keywords: CP: Cancer; CP: Immunology; exosome; immunotherapy; programmed cell death ligand 1; tumor-associated macrophages.

Figure 1.. Transition of macrophages to TAMs leads to increased exosome secretion

(A) NTA of sEVs purified from human monocyte-derived macrophages (Mφ) and TAMs induced by WM9 cell CM. The x axis represents the diameter and the y axis represents the concentration (particles/mL) of the sEVs. Quantification of the sEVs released from these cells is shown at the right. (B) NTA of sEVs purified from murine bone marrow-derived macrophages, and TAMs induced by YUMM1.7 cell CM (mTAM). Quantification of the sEVs is shown at the right. (C) A representative TEM image of sEVs purified from TAMs and mTAMs, respectively. Scale bar, 100 nm. (D) Western blot analysis showing the expression levels of exosomal marker proteins (CD63, TSG101, and CD9) in whole-cell lysate (Wcl) and exosomes from human Mφ and TAMs. The sEVs from the same number of cells were loaded for the western blot analysis. (E) Western blot analysis showing the expression levels of CD63, TSG101, and CD9 in whole-cell lysate (Wcl) and exosomes from murine Mφ and mTAMs. The sEVs from the same number of cells were loaded for western blot analysis. Data represent mean ± SD (n = 3). Statistical analysis was performed using two-sided unpaired t test (A, B, D, E).

Figure 2.. Akt phosphorylation of MADD mediates Rab27a activation and exosome secretion in TAMs

(A) The levels of Rab27a, total Akt (t-Akt), and phospho-Akt (p-Akt) in the whole-cell lysates (Wcl) of WM9-TAMs and parental Mφ cells (left), YUMM1.7-TAMs (mTAM), and parental mouse Mφ cells (right). The levels of GTP-Rab27a bound to GST-JFC1 in these cells is shown in the lower panel. GST-JFC1 was stained with Ponceau S. (B) Quantification of the levels of GTP-Rab27a in TAM and Mφ. (C) Quantification of the levels of GTP-Rab27a in mTAM and Mφ. (D) Western blot analysis showing the levels of GTP-Rab27a in control (CTL) and MADD knockdown (KD) TAMs. Two short hairpin RNA (shRNA) constructs were used in the KD. (E) Quantification of the level of GTP-Rab27a in TAMs with or without MADD KD. (F) Western blot analysis showing the levels of GTP-Rab27a in mTAMs with or without MADD KD. (G) Quantification of the level of GTP-Rab27a in mTAMs with or without MADD KD. (H) Quantification of the exosomes secreted by TAMs using NTA. (I) Quantification of the exosomes secreted by mTAMs using NTA. (J and K) Quantification of p-Akt levels in human (J) and mouse (K) macrophages based on western blot data (A). (L) Pull-down assay for the levels of GTP-Rab27a in WM9-TAMs and YUMM1.7-TAMs (mTAM) with or without Akt inhibitor treatment. (M) Quantification of the levels of GTP-Rab27a in TAMs with or without Akt inhibitor treatment. (N) Quantification of the levels of GTP-Rab27a in mTAM with or without Akt inhibitor. (O) Quantification of exosome released from TAMs with or without Akt inhibitor. (P) Quantification of exosome released from mTAM with or without Akt inhibitor treatment. (Q) Cells expressing the wild-type MADD (WT), the phospho-deficient mutant MADD (S70A), or phospho-mimetic MADD mutant (S70D) were lysed for GST-JFC1 RBD pull-down assay to assess the levels of GTP-Rab27a. (R) Quantification of the levels of GTP-Rab27a in mTAMs expressing WT, S70A, or S70D MADD. (S) Quantification of the exosomes released from mTAMs expressing WT, S70A, or S70D MADD. Data represent mean ± SD (n = 3). Statistical analysis is performed using two-sided unpaired t test (B, C, J, K, and M–P), one-way ANOVA with Dunnett’s multiple comparison tests (E, G, H, and I), or one-way ANOVA with Sidak’s multiple comparison tests (R and S).

Figure 3.. Exosomes released from TAMs carry more PD-L1 compared with Mφ

(A) Volcano plots analysis of the levels of proteins based on RPPA comparing WM9 cell CM-induced TAM-derived exosomes (TAM Exo) or YUMM1.7 cell CM-induced murine TAM-derived exosomes (mTAM Exo) with their matching Mφ-derived exosomes (Mφ Exo). Each point represents the difference in the expression of individual proteins in the indicated exosomes. Dotted vertical lines represent expression differences of ±30%, while the dotted horizontal line represents a significance level of p < 0.05. Proteins indicated in blue are different by at least ±30% fold change with a statistically significant level of p < 0.05. PD-L1 (shown in red) is expressed at significantly higher levels in both TAM Exo and mTAM Exo. (B) A representative TEM image of macrophage-derived exosomes. Arrowheads indicate individual PD-L1 proteins labeled with 5-nm gold particles. Scale bar, 100 nm. (C) Western blot analysis of PD-L1 and exosome marker proteins (CD63, TSG101, and CD9) in the whole-cell lysate (W) and exosomes (E) purified from WM9-TAMs (TAM) and YUMM1.7-TAMs (mTAM). All lanes were loaded with equal amounts of proteins. (D) PD-L1 co-fractionated with CD63, TSG101, CD9, and CD81 on iodixanol density gradients. (E) Western blot analysis of the exosomes from human macrophages. All lanes were loaded with equal amounts of proteins. PD-L1 was upregulated in exosomes on TAM. (F) Western blot analysis of the exosomes from murine macrophages. All lanes were loaded with the same amounts of proteins. (G and H) Immunofluorescence staining of PD-L1 and CD63 in Mφ and TAMs. Scale bar, 20 μm. Quantification of the levels of co-localization of PD-L1 with CD63 in TAMs compared to their matching Mφ is shown to the right. Fifty cells from each group were used in the quantification. Data represent mean ± SD (n = 3). Statistical analysis is performed using two-sided unpaired multiple t test (A) or two-sided unpaired t test (G and H).

Figure 4.. TAM-derived exosomes inhibit CD8 T cells

(A) Confocal microscopy images showing the association of stimulated human CD8 T cells (red) with CFSE-labeled TAM-derived exosomes (green). Nuclei were stained with DAPI (blue). The association of TAM-derived exosomes with CD8 T cells is indicated by arrows. Scale bar, 10 μm. (B) Representative images of flow cytometry of human CD8 T cells with or without anti-CD3/CD28 antibody stimulation after incubation with CFSE-labeled TAM exosomes. Quantification of the exosome-bound CD8 T cells is shown at the right. (C) Representative images of flow cytometry of murine CD8 T cells with or without CD3/CD28 antibody stimulation after incubation with CFSE-labeled mTAM exosomes. Quantification of the exosome-bound CD8 T cells is shown to the right. (D) Representative images of flow cytometry of human CD8 T cells with CD3/CD28 antibody stimulation after incubation with CFSE-labeled Mφ- or TAM-derived exosomes. Quantification of the exosome-bound CD8 T cells is shown at the right. (E) Representative images from flow cytometry of murine CD8 T cells with CD3/CD28 antibody stimulation after incubation with CFSE-labeled Mφ- or mTAM-derived exosomes. Quantification of the exosome-bound CD8 T cells is shown at the right. (F) Representative histogram of human peripheral CD8 T cells examined for the expression of Ki-67 and GzmB after indicated treatments. Quantification of cells with positive GzmB and Ki-67 expression in CD8 T cells after indicated treatments is shown at the right. (G) Representative histogram of murine CD8 T cells examined for the expression of Ki-67 and GzmB after indicated treatments. Quantification of cells with positive GzmB and Ki-67 expression in CD8 T cells after indicated treatments is shown at the right. (H) Schema of isolation of melanoma patient tumor-tissue-derived exosomes (see section “experimental model and subject details”). (I) Characterization of exosomes purified from melanoma patient tumor tissues using NTA. The x axis represents the diameters of the isolated vesicles; the y axis represents the concentration of isolated vesicles. (J) Schema of CD163⁺ exosome removal from tumor-tissue-derived exosomes by magnetic beads (see section “experimental model and subject details”). (K) Western blot analysis of the total (Control), remaining (Void), and CD163⁺ exosomes purified from the tumor samples of three representative melanoma patients (MP). All lanes were loaded with equal amounts of exosome proteins. (L) Inhibition of stimulated CD8 T cells by total exosomes (Total Exo) and CD163 removed exosomes (Void Exo) from the tumor samples of three melanoma patients (MP1, MP2, and MP3), as demonstrated by the decreased proportion of cells expressing GzmB and Ki-67. (M) Quantification of CD8 T cells with positive GzmB and Ki-67 expression after indicated CD163⁺ exosomes treatments. Data represent mean ± SD (n = 3). Statistical analysis was performed using two-sided unpaired t test (B–E), Welch ANOVA with Sidak’s T3 multiple comparison tests (F, G, and M), or one-way ANOVA with Dunnett’s multiple comparison tests (L).

Figure 5.. Exosomes from TAMs induced by PD-L1-negative tumor cells inhibit CD8 T cells

(A) Western blotting of PD-L1 in exosomes from TAMs induced by WM9 cells (TAM) and PD-L1 KO WM9 cells (PD-L1 KO WM9-TAM). All lanes were loaded with equal amounts of exosomes. (B and C) Flow cytometry showing the percentage of CD8 T cells with GzmB (B) and Ki-67 (C) expression after indicated treatments. Quantification of cells with GzmB- or Ki-67-expressing CD8 T cells with indicated treatments is shown at the right. (D) Western blotting of PD-L1 in exosomes from mTAMs induced by YUMM1.7 cells (YUMM1.7-TAM) and PD-L1 KO YUMM1.7 cells (PD-L1 KO YUMM1.7-TAM). (E and F) All lanes were loaded with equal amounts of exosomes. Flow cytometry showing the percentage of CD8 T cells with GzmB (E) and Ki-67 (F) expression after indicated treatments. Quantification of cells with GzmB- or Ki-67-expressing CD8 T cells with indicated treatments is shown at the right. Data represent mean ± SD (n = 3). Statistical analysis was performed using Welch ANOVA with Sidak’s multiple comparison tests (B, C, E, and F).

Figure 6.. Exosomal PD-L1 from TAMs suppressed the anti-tumor immune response

(A) Exosome treatment of PD-L1^−/− C57BL/6 mouse model with PD-L1 KO YUMM1.7 tumors (see section “experimental model and subject details”). (B) Growth curves of PD-L1 KO YUMM1.7 tumors in PD-L1^−/− C57BL/6 mice injected with exosomes derived from Mφ cells (Mφ Exo), TAMs induced from mBMDMs of WT C57BL/6 mice (TAM Exo), TAMs induced from mBMDMs of PD-L1^−/− C57BL/6 mice (PD-L1^−/− TAM Exo), TAMs induced by PD-L1 KO YUMM1.7 from mBMDMs from WT C57BL/6 mice (pkoTAM Exo), or PD-L1^−/− C57BL/6 mice (PD-L1^−/− pkoTAM Exo). (C) Tumor weights for mice with indicated treatments. (D) Representative IHC images of CD8⁺ TILs in tumor tissues. Scale bar, 100 μm. (E) The number of CD8⁺ TILs per mm² was quantified from IHC analysis. (F) The number of CD8⁺ TILs per gram of tumor was quantified from flow cytometry. (G) The percentage of Ki-67⁺GzmB⁺ CD8 TILs was quantified by flow cytometry. (H and I) The percentages of Ki-67⁺ GzmB⁺ CD8 T cells from lymphatic nodes (H) and spleens (I) were quantified by flow cytometry. Data represent mean ± SD (n = 7). (J and K) Schema showing that PD-L1⁺ tumor cells not only attack CD8 T cells using their own exosomes but also reprogram macrophages to TAMs, which secrete a large number of exosomes carrying a higher level of PD-L1 to inhibit CD8⁺ T cells (J). PD-L1⁻ tumor cells can also induce TAMs to secrete PD-L1 exosomes to inhibit CD8⁺ T cells (K). Statistical analysis is performed using two-way ANOVA with Tukey’s multiple comparison tests (B), or Welch ANOVA with Dunnett’s T3 multiple comparison tests (C and E–I).

Figure 7.. Targeting Rab27a by siRNA-loaded LNPs sensitized tumors to anti-PD-1 antibody

(A) Heatmap showing scaled expression values of RAB27A, RAB27B, MADD, HGS, PDCD6IP, and TSG101 in four clusters of cells, including B cells (BCs), plasma cells (PCs), monocytes/macrophages (TAM), and dendritic cells (DCs). (B) The expression level of RAB27A in macrophages expressing different markers, including CD163, CD206, CD68, CD80, CD86, HLA-DRA, HLA-DRB, HLA-DRB5, or MSR. (C) Spearman correlation analysis showing that expression status of RAB27A in macrophages from melanoma biopsies possessed a significant correlation with the proportion of CD8 T cells in total CD45⁺ immune cells. (D) A syngeneic C57BL/6 mouse model was established using YUMM1.7 cells and treated as indicated. (E) Growth curves of YUMM1.7 tumors in mice with indicated treatments. (F) The weights of YUMM1.7 tumors in mice with indicated treatments. (G) Survival curves of mice in the indicated groups. (H) Representative IHC images of CD8⁺ TILs in tumor tissues. Scale bar, 100 μm. (I) The number of CD8⁺ TILs for each group of mice quantified from IHC analysis. (J) The number of CD8⁺ TILs per gram of tumor was quantified from flow cytometry. (K) The number of F4/80⁺ TAMs for each group quantified from flow cytometry. (L) The percentage of Ki-67⁺ GzmB⁺ CD8 T cells quantified by flow cytometry. (M) The percentage of Ki-67⁺ GzmB⁺ CD8 T cells quantified by flow cytometry. (N) The percentage of Ki-67⁺GzmB⁺ CD8 T cells quantified by flow cytometry. Data represent mean ± SD (n = 5). Statistical analysis was performed using Spearman’s correlation (B), two-way ANOVA with Tukey’s multiple comparison tests (E), Welch ANOVA with Dunnett’s T3 multiple comparison tests (F and I–N), or log rank test (G).