Extracellular vesicles of carcinoma-associated fibroblasts creates a pre-metastatic niche in the lung through activating fibroblasts

Abstract

Objectives: Carcinoma-associated fibroblasts (CAFs) have been known to promote cancer progression by modifying the primary tumor microenvironment. We aimed to elucidate the intercellular communication between CAFs and secondary organs in salivary adenoid cystic carcinoma (SACC) metastasis.

Methods: Pre-metastatic and metastatic animal models of SACC were established using extracellular vesicles (EVs) from CAFs and SACC cells. Lung fibroblasts (LFs) were treated with EVs and their transcriptomic alterations were identified by RNA sequencing. ITRAQ were performed to analyze EV cargos. TC I-15 was used to inhibit EV uptake by LFs and SACC lung metastasis in vivo.

Results: Here, we show that CAF EVs induced lung pre-metastatic niche formation in mice and consequently increased SACC lung metastasis. The pre-metastatic niche induced by CAF EVs was different from that induced by SACC EVs. CAF EVs presented a great ability for matrix remodeling and periostin is a potential biomarker characterizing the CAF EV-induced pre-metastatic niche. We found that lung fibroblast activation promoted by CAF EVs was a critical event at the pre-metastatic niche. Integrin α2β1 mediated CAF EV uptake by lung fibroblasts, and its blockage by TC I-15 prevented lung pre-metastatic niche formation and subsequent metastasis. Plasma EV integrin β1 was considerably upregulated in the mice bearing xenografts with high risk of lung metastasis.

Conclusions: We demonstrated that CAF EVs participated in the pre-metastatic niche formation in the lung. Plasma EV integrin β1 might be a promising biomarker to predict SACC metastasis at an early stage. An integrated strategy targeting both tumor and stromal cells is necessary to prevent SACC metastasis.

Keywords: Carcinoma-associated fibroblast; Extracellular vesicles; Lung metastasis; Pre-metastatic niche.

Fig. 1

CAF EVs promoted SACC lung metastasis and pre-metastatic niche formation. a FAP expression and quantification in the stroma of human SACC with or without lung metastasis. Scale bar = 50 μm. b Metastatic SACC-LM cells were detected by immunofluorescent staining with anti-human Pan CK antibody. C57BL/6 J mice were pre-treated with SACC-LM EVs (n = 5), CAF-A1 EVs (n = 4) and CAF-A2 EVs (n = 5). Scale bar = 50 μm. c Immunofluorescence staining and quantification of FN, LOX and MMP9 in lung sections from tumor-free mice injected every other day for 3 days with the indicated EVs (n = 5 per group). Immunofluorescence staining and quantification of VEGFR1+ cells in lung sections from tumor-free mice injected every other day for 14 days with the indicated EVs (n = 5 per group). Scale bar = 50 μm. * P < 0.05, **

Fig. 2

CAF EVs activated LFs via TGF-β signaling pathway. a CAF-A1/A2 EVs were labeled with PKH67 (green) and the lung tissue sections were stained with Collagen I antibody (red). Scale bar = 50 μm. b Collagen gel contract assay using EVs from CAF-A1 and CAF-A2. EV concentrations tested were 0, 5, 15, 25 μg/well. c Heatmap of RNA sequencing. Enriched Biological Processes with GO term using differentially expressed genes were listed. POSTN was the top one among the enriched gene in both ECM Organization and Cellular Response to TGF-β Stimulus Biological Processes. d Expression of p-Smad2 and p-Smad3 in LFs with or without CAF EV pre-treatment assessed by western blot and immunofluorescent staining. Scale bar = 100 μm. e POSTN expression in LFs in vitro and in the lung tissues of mice (n = 5 per group) treated with CAF-A1/A2 EVs for 3 days. Scale bar = 100 μm. f Images and quantization of adhesion and proliferation of SACC-LM-GFP cells (green) on LFs treated with EVs from SACC-LM, CAF-A1 and CAF-A2. Scale bar = 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 3

Comparison of proteins between CAF-A1/A2 EVs and SACC-LM EVs by ITRAQ. a Overexpressed proteins in the two comparison groups (CAF-A1 EVs vs. SACC-LM EVs, CAF-A2 EVs vs. SACC-LM EVs). b Heat map of the 66 proteins overexpressed in both CAF-A1 and CAF-A2 EVs, compared with SACC-LM EVs. c Biological Process enrichment of the 66 overexpressed proteins. d TSP1, LOXL2, and MMP2 expression in the EVs from SACC-LM, CAF-A1 and CAF-A2 cells by western blot. e TSP1 was downregulated in CAF-A1/A2 EVs by TSP1-specific siRNA transfection. Expression of p-Smad3 in LFs treated with CAF-A1/A2 EVs-NC, CAF-A1/A2 EVs-siTSP1–1, and CAF-A1/A2 EVs-siTSP1–2 assessed by western blot and immunofluorescent staining. Scale bar = 100 μm. ** P < 0.01

Fig. 4

Integrin α2β1 mediated CAF EV uptake by LFs. a Western blot analysis of integrin expression in CAF-A1/A2 EVs. b Inhibition assay of CAF EV uptake by LFs in vitro. The TC I-15 concentrations tested were 0, 0.5, 1 and 2 μM. c TC I-15 inhibitory effects on the expression of p-Smad3 in LFs induced by CAF-A1/A2 EVs. d TC I-15 inhibitory effects on the expression of FAP, α-SMA and POSTN in LFs induced by CAF-A1/A2 EVs. e TC I-15 inhibitory effects on collagen gel contract promoted by CAF-A1/A2 EVs. Scale bar = 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 5

TC I-15 inhibited CAF EV-induced pre-metastatic niche formation and metastasis in vivo. a TC I-15 inhibitory effects on the expression of FN, LOX, MMP9, and POSTN induced by CAF-A1/A2 EVs in the lungs (n = 5 per group). b TC I-15 inhibitory effects on lung metastasis promoted by CAF-A1/A2 EVs (n = 5 per group). Scale bar = 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 6

Plasma EV integrin β1 predicted SACC lung metastasis in mice. a Schematic of the animal experiments using BALB/c nude mice. b POSTN expression in the lungs of mice at week 3. Metastatic colony number and area in the lungs of mice at week 5. Survival analysis for the mice raised for up to 12 weeks. c Western blot analysis of plasma EV integrin β1 expression in the mice at the pre-metastatic stage. d Western blot analysis of plasma EV integrin β1 expression in the mice at the metastatic stage. e Illustration of the CAF EVs constructed pre-metastatic niche in the lungs. Scale bar = 50 μm. * P < 0.05, ** P < 0.01