Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver

Abstract

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Figure 1. Pancreatic ductal adenocarcinoma-derived exosomes target and activate Kupffer cells, induce liver fibrosis pathways, and increase liver metastasis

(a) Evaluation of liver metastasis by liver weight (grams) in mice injected intra- splenically with PAN02 cells (TU, for tumor bearing) following pre-education with phosphate-buffered saline (PBS) or PAN02 exosomes (Exo). Percentage of mCherry⁺ PAN02 cells in the liver was measured by immunofluorescence (left panels; n = 4 mice were pooled from two independent experiments) and livers were weighed (right panel; n = 9 (CTL) and n = 8 (TU and Exo+TU) mice were pooled from three independent experiments) at day 21 post-PAN02 cell injection. Representative liver images are shown. **P < 0.01 by two-tailed t-test (middle panel), ***P < 0.05 by ANOVA (right panel). Scale bars, immunofluorescence: 200μm, whole organ images: 1cm. (b) Evaluation of liver metastasis by liver weight (grams) in mice pre-educated with PBS, exosomes from PAN02 cells, or normal pancreas (NP) prior to intra-splenic injection of PAN02 cells; n = 4 (TU) and n = 5 (PAN02exo + TU and NPexo + TU) mice from one experiment. **P < 0.01 by ANOVA. Scale bar, 1cm. (c) Fluorescence microscopy analysis of PKH67-labeled exosome incorporation (green) by F4/80⁺ Kupffer cells (red). Exosomes were isolated from NP, PAN02, and PKCY cells. Scale bars, 50μm. (d) Representative flow cytometric profiles of CD11b and F4/80 expression in liver cells treated with unlabeled exosomes or with PKH67-labeled NP or PDAC-derived exosomes (PAN02 and PKCY). Quantification of PKH67-positive liver cells (upper panel) and of PKH67- positive liver cells expressing CD11b and F4/80 (lower panel). For NP, n = 4 mice from one experiment; n = 7 (PAN02) and n = 8 (PKCY) mice pooled from two experiments. ***P < 0.01, **P < 0.01, *P < 0.05 by ANOVA. (e) Analysis of canonical pathways enriched in genes upregulated by Kupffer cells following in vitro education with PAN02 or NP exosomes. The list is comprised of genes related to liver fibrosis. Data were obtained from one experiment assessing three biologically independent samples. Statistical source data are presented in Supplementary Table 1. All data are represented as mean±s.e.m.

Figure 2. Role of pancreatic ductal adenocarcinoma-derived exosome education in extracellular matrix component expression and liver pre-metastatic niche formation

(a) Immunofluorescence quantification in arbitrary units (A.U.) of vitronectin, tenascin C, collagen I and fibronectin (FN) expression in liver after education with PAN02 exosomes (Exo) or PBS (CTL); n = 4 mice pooled from two experiments. Statistical source data are presented in Supplementary Table 4. The data are represented as mean±s.e.m. ***P < 0.001 by two-tailed t-test. Scale bars, 200μm. (b) Fluorescence microscopy analysis of FN expression in αSMA⁺ hStCs in livers of mice educated with PAN02 exosomes. Line scan for histogram calculation is shown. Scale bars, 50μm.

Figure 3. Pancreatic ductal adenocarcinoma-derived exosomes induce fibronectin expression and migration of bone marrow-derived cells to the liver

(a) Immunofluorescence quantification of FN expression in arbitrary units (A.U.) and F4/80⁺ cell frequency in the livers of mice during the course of PAN02 exosome education; n = 4 mice from one experiment. **P < 0.01, ***P < 0.001 by ANOVA relative to control (day 0). Scale bars, 100μm. (b) Immunofluorescence quantification of the frequency of bone marrow (BM)-derived F4/80⁺ (upper) and Gr1⁺ (lower) cells in the liver following transplantation of GFP⁺ BM from mice educated with PBS (CTL) or PAN02 exosomes (Exo); n = 4 (F4/80) and n = 3 (Gr1) mice from one experiment. **P < 0.01, *P < 0.05 by two-tailed t-test. Scale bars, 50μm. All statistical source data are presented in Supplementary Table 4. All data are represented as mean±s.e.m.

Figure 4. TGFβ signaling induces fibronectin upregulation and macrophage recruitment to the liver pre-metastatic niche

Immunofluorescence quantification of FN and αSMA expression in arbitrary units (A.U.) and F4/80⁺ cells in livers of mice educated with PBS (CTL), PAN02 exosomes alone (Exo), or in combination with the TGFβ receptor inhibitor A83-01 (Exo+A83-01); n = 4 (CTL) and n = 7 (Exo and Exo+A83-01) mice pooled from two experiments. The data are represented as mean±s.e.m. ***P < 0.001, N.S. stands for not significant by ANOVA. Scale bars, 200μm.

Figure 5. Fibronectin and macrophages play major roles in PAN02 exosome- mediated liver pre-metastatic niche formation

(a) Immunofluorescence quantification of FN and αSMA expression and F4/80⁺ cell frequency in livers of FN-conditional knockout mice. Cre^{−/ −}Fn^fl/fl (CRE^-) and Cre^+/−Fn^fl/fl (CRE⁺) mice were tamoxifen-treated (TMX) and educated with PAN02 exosomes (Exo). Control PBS-educated livers (CTL): n = 3 (CTL F4/80), n = 4 (CTL αSMA, FN), n = 6 (CRE⁺ F4/80), n = 7 (CRE⁻ αSMA, F4/80), and n = 8 (CRE⁻ FN; CRE⁺ αSMA, FN) mice from two experiments. ***P < 0.001, **P < 0.01 by ANOVA. (b) Evaluation of liver metastasis by liver weight (grams) in tumor-free mice (CTL), PBS-educated mice injected intra-splenically with PAN02 cells (TU), and tumor-bearing CRE⁻ and CRE⁺ mice TMX-treated during Exo education; n = 4 (CTL), n = 6 (CRE⁻ and CRE⁺), and n = 10 (TU) mice from three experiments. ***P < 0.001, *P < 0.05 by ANOVA. (c) Evaluation of liver metastasis in TMX-treated CRE⁻ versus CRE⁺ mice injected intra-splenically with PAN02 cells (TU); n = 4 (CRE⁺) and n = 5 (CRE^-) mice from one experiment. (d) Quantification of FN and αSMA expression and F4/80⁺ cell frequency in mice transiently depleted of CD11b⁺ cells using a diphtheria toxin (DT)- inducible system during PAN02 exosomes (Exo) education. Control PBS-educated livers (CTL): n = 3 (CTL F4/80), n = 4 (CTL αSMA, FN; DTR⁻ αSMA), n = 7 (DTR⁻ FN, F4/80; DTR⁺ αSMA, FN), and n = 9 (DTR⁺ F4/80) mice from two experiments. ***P < 0.001, **P < 0.01 by ANOVA. (e) Evaluation of liver metastasis in tumor-free mice (CTL), PBS-educated mice injected intra-splenically with PAN02 cells (TU), and tumor-bearing CD11b-DTR⁻ or -DTR⁺ mice DT-treated during Exo education; n = 5 (CTL), n = 6 (DTR^-), n = 9 (DTR⁺), and n = 10 (TU) mice pooled from three experiments. ***P < 0.001, **P < 0.01 by ANOVA. (f) Evaluation of liver metastasis in mice transiently depleted of CD11b⁺ cells and injected intra-splenically with PAN02 cells; n = 4 (DTR^-) and n = 5 (DTR⁺) mice from one experiment. All data are represented as mean±s.e.m. N.S. for not significant by two-tailed t-test. Scale bars, immunofluorescence: 200μm, whole organ: 1cm.

Figure 6. MIF-expressing PAN02 exosomes induce liver pre-metastatic niche formation

(a) Representative images and quantification of pre-metastatic niche markers in livers educated with PAN02 exosomes (Exo), PAN02shCTL exosomes (shCTLexo), PAN02shMIF exosomes (shMIFexo and shMIF(2)exo) or PBS control (CTL). Immunofluorescence analysis shows frequency of TGFβ-expressing F4/80⁺ cells, αSMA and FN expression as well as F4/80⁺ cell frequency. Inset shows TGFβ⁺/F4/80⁺ cell; n = 3 (CTL F4/80), n = 4 (CTL TGFβ, αSMA, FN; Exo TGFβ; all shCTL; shMIF TGFβ; all shMIF(2), n = 6 (Exo F4/80; shMIF F4/80), n = 7 (Exo αSMA, FN; shMIF αSMA, FN) mice pooled from two experiments. ***P < 0.001, **P < 0.001 by ANOVA. Scale bars, 100μm. (b) Evaluation of liver metastasis by liver weight (grams) in tumor-free mice (CTL), mice injected intra-splenically with PAN02 cells either pre-educated with PBS (TU), with PAN02 exosomes (Exo+TU), with PAN02 exosomes in combination with A83-01 (Exo+A83-01+TU), or with PAN02shMIF exosomes (shMIFexo+TU); n = 4 (Exo+A83-01+TU), n = 5 (CTL, TU, Exo+TU, and shMIFexo+TU) mice pooled from two experiments. **P < 0.01, *P < 0.05 by ANOVA. Scale bar, 1cm. (c) Evaluation of liver metastasis in mice injected intra-splenically with PAN02 cells either pre-educated with PBS (TU), with exosomes isolated from PAN02 cells infected with control shRNA (shCTLexo+TU) or shMIF(2) (shMIF(2)exo+TU) lentiviral vectors; n = 4 (shCTLexo+TU), n = 5 (shMIF(2)exo+TU) and n = 6 (TU) mice from one experiment. ***P < 0.01 by ANOVA. Scale bar, 1cm. (d) Enzyme-linked immune assay (ELISA) reveals increased levels of MIF (picogram per 10⁸ exosomes) in exosomes isolated from patients with pancreatic ductal adenocarcinoma (PDAC) with progression of disease post-diagnosis (POD) compared to PDAC patients with no evidence of disease 5 years post-diagnosis (NED) and to healthy controls (CTL), but not PDAC patients with liver metastasis (LM); n = 10 (NED), n = 12 (POD), n = 15 (CTL) and n = 18 (LM) patients. All patient samples were analyzed once as part of three independent ELISA assays. **P < 0.01 by ANOVA. All data are represented as mean±s.e.m

Figure 7. Liver pre-metastatic niche formation and increased exosomal MIF levels precede pancreatic ductal adenocarcinoma lesion development in PKCY mice

(a) Immunofluorescence quantification of αSMA and FN expression in arbitrary units (A.U.) and F4/80⁺ cell frequency in livers of control CY mice at 16 weeks (wks) (CTL), PKCY mice at early and late PanIN phases (4–6 and 8–11 wks, respectively), and PDAC livers (16–20 wks). Upregulation of αSMA, FN, and increased frequency of F4/80⁺ cells can be observed starting at the PanIN stages; n = 4 (all CTL; all early PanIN; all late PanIN; PDAC TGFβ and αSMA) and n = 5 (PDAC FN and F4/80) mice pooled from three experiments. Statistical source data can be found in Supplementary Table 4. ***P < 0.001, **P < 0.01, *P < 0.05 by ANOVA when compared to CTL. Scale bars, 200μm. (b) Levels of MIF in exosomes isolated from plasma of CY and PKCY mice, at PanIN (both early and late) and PDAC stages measured by ELISA; n = 5 (PanIN), n = 6 (CTL), and n = 8 (PDAC) mice plasma samples pooled from two experiments. *P < 0.05 by ANOVA when compared to CTL. All data are represented as mean±s.e.m.

Figure 8. Model for the sequential steps in liver pre-metastatic niche formation induced by pancreatic ductal adenocarcinoma-derived exosomes

Education with MIF⁺ PDAC-derived exosomes, which bind predominantly to Kupffer cells in the liver, induces TGFβ production by these cells. TGFβ activates hStCs, which in turn upregulate FN. Bone marrow-derived cells (i.e. macrophages) bind to FN-enriched hepatic sites, ultimately leading to liver pre-metastatic niche formation.