Tumor-promoting effects of pancreatic cancer cell exosomes on THP-1-derived macrophages

Abstract

Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages (TAMs), yet the mechanisms by which tumor cells and TAMs communicate are not fully understood. Here we show that exosomes secreted by PDAC cell lines differed in their surface proteins, lipid composition, and efficiency of fusing with THP-1-derived macrophages in vitro. Exosomes from AsPC-1, an ascites-derived human PDAC cell line, were enriched in ICAM-1, which mediated their docking to macrophages through interactions with surface-exposed CD11c on macrophages. AsPC-1 exosomes also contained much higher levels of arachidonic acid (AA), and they fused at a higher rate with THP-1-derived macrophages than did exosomes from other PDAC cell lines or from an immortalized normal pancreatic ductal epithelial cell line (HPDE) H6c7. Phospholipase A2 enzymatic cleavage of arachidonic acid from AsPC-1 exosomes reduced fusion efficiency. PGE2 secretion was elevated in macrophages treated with AsPC-1 exosomes but not in macrophages treated with exosomes from other cell lines, suggesting a functional role for the AsPC-1 exosome-delivered arachidonic acid in macrophages. Non-polarized (M0) macrophages treated with AsPC-1 exosomes had increased levels of surface markers indicative of polarization to an immunosuppressive M2-like phenotype (CD14hi CD163hi CD206hi). Furthermore, macrophages treated with AsPC-1 exosomes had significantly increased secretion of pro-tumoral, bioactive molecules including VEGF, MCP-1, IL-6, IL-1β, MMP-9, and TNFα. Together, these results demonstrate that compared to exosomes from other primary tumor-derived PDAC cell lines, AsPC-1 exosomes alter THP-1-derived macrophage phenotype and function. AsPC-1 exosomes mediate communication between tumor cells and TAMs that contributes to tumor progression.

Fig 1. Exosomes secreted by pancreatic cancer cell lines have similar physical properties.

(A) Exosomes sized by dynamic light scattering demonstrate similar average size, **p<0.005. Data from three independent experiments were combined and are represented as mean ± SEM. (B) BxPC-3 exosomes (1) and PANC-1 exosomes (2) imaged by transmission electron microscopy display size heterogeneity and characteristic cup-shaped morphology (arrows).

Fig 2. ICAM-1 and CD11c co-localize during exosome-macrophage interactions.

(A) Immunoblotting analysis reveals exosomes from the pancreatic cancer cell lines AsPC-1 and BxPC-3 are enriched in the surface-exposed proteins ICAM-1 and EpCAM, while CD9 is more broadly expressed. The pan-exosomal marker flotillin-1 is used as a loading control. (B) Co-localization of exosome proteins and macrophage proteins is demonstrated by immunostaining for the exosome marker ICAM-1 (green, panel 1) or the macrophage marker CD11c (red, panel 2) after mixing AsPC-1 exosomes with THP-1-derived, non-polarized macrophages. Panel 3 is a merged image showing co-localization of ICAM-1 and CD11c staining (yellow, indicated by an arrow), and is suggestive of ICAM-1 and CD11c protein-protein interaction. THP-1-derived macrophages not mixed with exosomes (cells only) show little ICAM-1 staining (green, panel 4), suggesting exosomes were the main source of the ICAM-1 signal. In the absence of exosomes, more CD11c is associated with the THP-1 cell surface (red, panel 5), and merged images of THP-1 only staining show few areas of signal co-localization (panel 6, yellow). Scale bar = 3.0 μm (C) Quantitation of ICAM-1:CD11c co-localized signal in THP-1 cells mixed with AsPC-1 exosomes or in THP-1 cells alone. ***p<0.005.

Fig 3. AsPC-1 exosomes are enriched in arachidonic acid and exosomal arachidonic acid contributes to the efficiency of AsPC-1 exosome-macrophage fusion.

(A) Compared to exosomes from other PDAC cell lines and from HPDE cells, AsPC-1 exosomes are enriched in arachidonic acid (C20:4) species. Comparison of the percent of arachidonic acid in each glycerophospholipid class showing that the percentage of AA is highest in the PI and PE fractions. Phosphatidylcholine = PC, phosphatidylserine = PS, phosphatidylethanolamine = PE, phosphatidic acid = PA, phosphatidylinositol = PI. (B) Representative fusion curves of exosomes with non-polarized THP-1 macrophages. (C) Compared with exosomes from other pancreatic cell lines, AsPC-1 exosomes fuse at the highest rate with THP-1-derived macrophages. Relative arachidonic acid (AA) levels shown with plus signs. (D, E) Pre-treatment of AsPC-1 exosomes with phospholipase A₂ (PLA₂), which removes AA from phospholipids, decreased the exosome fusion rate. Data from three independent experiments are represented as mean ± SEM. *****p<0.0005 by ANOVA.

Fig 4. AsPC-1 exosomes polarize THP-1-derived macrophages towards an immunosuppressive M2 phenotype.

(A) Workflow of in vitro THP-1 monocyte differentiation and positive controls for macrophage polarization. LPS + IFNɣ are M1 macrophage polarization controls, while IL-4 + IL-13 are controls for M2 macrophage polarization. (B) PMA-induced differentiation of THP-1 monocytes into non-polarized macrophages significantly increased CD14 protein levels; data from three independent experiments are represented as mean ± SEM, ****p<0.0005 by unpaired two-tailed t-test. Non-polarized CD14^hi macrophages treated with AsPC-1 exosomes show no change in markers of M1 polarization, HLA-DR, CD80, or CD11c (C, D, E), or in the level of the immunoregulatory protein PD-L1 (F). In contrast, CD14^hi macrophages treated with AsPC-1 exosomes show significantly increased levels of the M2 markers CD163 (G) and CD206 (H), and Panc-1 exosomes also increased CD163 expression. Data from at least two independent experiments are represented as mean ± SEM. *****p<0.0005, ***p<0.005, *p<0.05.

Fig 5. THP-1-derived macrophages treated with AsPC-1 exosomes secrete bioactive molecules associated with tumor progression, angiogenesis, invasion, and metastasis.

THP-1 macrophages treated with AsPC-1 exosomes secrete elevated amounts of prostaglandin E2 (PGE₂) (A), angiogenic factor VEGF (C), cytokines and chemokines MCP-1, IL-6, IL-1β, and TNFα (B, D, F, G) and the metalloprotease MMP-9 (E). Exosomes from BxPC-3 also stimulated macrophage secretion of MCP-1, and exosomes from both BxPC-3 and HPDE cells significantly enhanced the secretion of MMP-9. Data from at least three independent experiments are represented as mean ± SEM. *****p<0.0005, ***p<0.005, *p<0.05.

Fig 6. Proposed model for the immunomodulatory and pro-tumoral effects of AsPC-1 exosomes.

While lipids form the basis of exosome membrane structure, exosomes may also shuttle bioactive lipid mediators, such as AA, between tumor cells and macrophages to promote a local, pro-tumor microenvironment.