CAFs secreted exosomes promote metastasis and chemotherapy resistance by enhancing cell stemness and epithelial-mesenchymal transition in colorectal cancer

Abstract

Background: Cancer associated fibroblasts (CAFs) are key stroma cells that play dominant roles in tumor progression. However, the CAFs-derived molecular determinants that regulate colorectal cancer (CRC) metastasis and chemoresistance have not been fully characterized.

Methods: CAFs and NFs were obtained from fresh CRC and adjacent normal tissues. Exosomes were isolated from conditioned medium and serum of CRC patients using ultracentrifugation method and ExoQuick Exosome Precipitation Solution kit, and characterized by transmission electronic microscopy, nanosight and western blot. MicroRNA microarray was employed to identify differentially expressed miRNAs in exosomes secreted by CAFs or NFs. The internalization of exosomes, transfer of miR-92a-3p was observed by immunofluorescence. Boyden chamber migration and invasion, cell counting kit-8, flow cytometry, plate colony formation, sphere formation assays, tail vein injection and primary colon cancer liver metastasis assays were employed to explore the effect of NFs, CAFs and exosomes secreted by them on epithelial-mesenchymal transition, stemness, metastasis and chemotherapy resistance of CRC. Luciferase report assay, real-time qPCR, western blot, immunofluorescence, and immunohistochemistry staining were employed to explore the regulation of CRC metastasis and chemotherapy resistance by miR-92a-3p, FBXW7 and MOAP1.

Results: CAFs promote the stemness, epithelial-mesenchymal transition (EMT), metastasis and chemotherapy resistance of CRC cells. Importantly, CAFs exert their roles by directly transferring exosomes to CRC cells, leading to a significant increase of miR-92a-3p level in CRC cells. Mechanically, increased expression of miR-92a-3p activates Wnt/β-catenin pathway and inhibits mitochondrial apoptosis by directly inhibiting FBXW7 and MOAP1, contributing to cell stemness, EMT, metastasis and 5-FU/L-OHP resistance in CRC. Clinically, miR-92a-3p expression is significantly increased in CRC tissues and negatively correlated with the levels of FBXW7 and MOAP1 in CRC specimens, and high expression of exosomal miR-92a-3p in serum was highly linked with metastasis and chemotherapy resistance in CRC patients.

Conclusions: CAFs secreted exosomes promote metastasis and chemotherapy resistance of CRC. Inhibiting exosomal miR-92a-3p provides an alternative modality for the prediction and treatment of metastasis and chemotherapy resistance in CRC.

Keywords: Chemotherapy resistance; Colorectal cancer; Exosomes; Metastasis; Stemness; miR-92a-3p.

Fig. 1

CAFs-derived exosomes promote invasion, metastasis, and chemotherapy resistance in CRC. a Transmission electron micrograph of NFs-derived exosomes (NFs-exos) and CAFs-derived exosomes (CAFs-exos). White arrow head points at exosomes. b Exosomes markers CD63, CD81 and TSG101 proteins were detected by western blot assay in NFs-exos, CAFs-exos, and the corresponding supernatant of NFs-CM and CAFs-CM obtained through ultracentrifugation. c Internalization of exosomes by SW480 cells examined by laser scanning confocal microscope. d Effect of NFs-CM, CAFs-CM, CAFs-exos, and CAFs-exos depleted of exosomes on migration and invasion of SW480, SW620 and LOVO cells by Boyden chamber assay. e Effect of NFs-CM, CAFs-CM, CAFs-exos, and CAFs-CM depleted of exos on the formation of metastasis nodules in lung. f-h Effect of NFs-CM, CAFs-CM, CAFs-exos, CAFs-CM depleted of exosomes on abilities of cell survival f, colony formation g and apoptosis h of SW480, SW620 and LOVO cells by CCK-8, colony formation and flow cytometry assays. i Effect of NFs-exos and CAFs-exos on percentage of CD133⁺CD44⁺ in SW480, SW620 and LOVO cells by flow cytometry assay. j-l Effect of NFs-exos and CAFs-exos on the expressions of stemness markers CD133, CD44, OCT4 j, k and EMT markers (l in SW480, SW620 and LOVO cells by real-time PCR and western blot assays

Fig. 2

Direct transfer of CAFs secreted exosomal miR-92a-3p to CRC cells. a Hierarchical clustering analysis of differentially expressing miRNAs among NFs-exos, CAFs-exos, SW480^NFs-exos and SW480^CAFs-exos using microRNA microarray. b Relative expression of miR-92a-3p, miR-181d-5p, miR-221–3p, miR-125b-5p, miR-185-5p and miR-625-3p in NF-exos, CAFs-exos, SW480^NFs-exos and SW480^CAFs-exos cells by real-time PCR assay. U6 was used as internal control. c Relative expression of miR-92a-3p in normal colorectal cell NCM460, CRC cell line cells, matched CRC tissues and normal colorectal mucosa, CAFs and NFs, CAFs-exos and NFs-exos from a same CRC patient by real-time PCR analysis. U6 was used as internal control. d Relative expression of miR-92a-3p in Blank, SW480^CAFs-exos, SW480^NFs-exos, SW620^CAFs-exos, SW620^NFs-exos, LOVO^CAFs-exos and LOVO^NFs-exos cells by real-time PCR assay. U6 was used as internal control. e & f Relative expression of miR-92a-3p e and pre-miR-92a-3p f in SW480^CAFs-exos, SW480^NFs-exos, SW620^CAFs-exos, SW620^NFs-exos, LOVO^CAFs-exos and LOVO^NFs-exos cells at indicated time by real-time PCR assay. U6 was used as internal control. g Relative expression of miR-92a-3p in SW480, SW620 and LOVO cells treated with antimiR-NC, antimiR-92a-3p, antimiR-92a-3p + NFs-exos, antimiR-92a-3p + CAFs-exos by real-time PCR analysis. U6 was used as internal control. h Transwell co-culture of SW480 cells with CAFs and CAFs/miR-92a-3p-FAM cells. Cultured SW480 cells were harvested and observed using laser scanning confocal microscope. i Incubation of SW480 cells with CAFs-exos and CAFs/miR-92a-3p-FAM secreted exosomes. Cells were observed using laser scanning confocal microscope

Fig. 3

CAFs secreted exosomal miR-92a-3p promoted metastasis and chemotherapy resistance of CRC. a Effects of NFs-exos, CAFs-exos, CAFs-exos/antimiR-92a-3p, CAFs-exos/antimiR-92a-3p + miR-92a-3p mimics treatment on cell migration and invasion of SW480, SW620 and LOVO cells. b Gross and microscopy observations of primary colon tumors and liver metastases in mice injected with SW620^NFs-exos, SW620^CAFs-exos, SW620^CAFs-exos/antimiR-92a-3p n = 8). The number of metastatic nodules in individual mice was counted under the microscope. The liver sections were stained with H&E. (C&D) Effects of NFs-exos, CAFs-exos, CAFs-exos/antimiR-92a-3p, CAFs-exos/antimiR-92a-3p + miR-92a-3p mimics treatment on abilities of cell survival c, colony formation and apoptosis d of SW480, SW620 and LOVO cells. e SW480 cells were injected into the flank of mice to establish xenografts. 5-FU/L-OHP (5 mg/kg) or same volume of PBS every 3 days were injected subsequently. When tumors formed, NFs-exos, CAFs-exos, and CAFs-exos/antimiR-92a-3p were injected into the vicinity of the subcutaneous tumors every 3 days. Tumor volume was calculated using the formula V = length × width²/2. f Effect of PBS, 5-FU/L-OHP, NFs-exos/5-FU/L-OHP, CAFs-exos/5-FU/L-OHP, and CAFs-exos/antimiR-92a-3p + 5-FU/L-OHP treatment on SW480 cells derived tumor apoptosis assessed by TUNEL assay. Red arrows pointed out the apoptotic cells in indicated group of tumors. g-k Effect of NFs-exos, CAFs-exos, CAFs-exos/antimiR-92a-3p and CAFs-exos/antimiR-92a-3p + miR-92a-3p mimics treatment on sphere formation ability g, percentage of CD133⁺CD44⁺ cells h, CD133, CD44, OCT4 i & j and EMT markers expression k in SW480, SW620 and LOVO cells using sphere formation, flow cytometry, real-time PCR and western blot assays. GAPDH was used as internal control

Fig. 4

FBXW7 and MOAP1 attenuate miR-92a-3p-mediated promotion of aggressiveness and chemotherapy resistance of CRC. a Sequences of miR-92a-3p and the potential miR-92a-3p-binding sites at the 3’UTR of FBXW7 and MOAP1. Also shown are nucleotides mutated in FBXW7–3′-UTR mutant and MOAP1–3′-UTR mutant. Seed sequences are marked. b & c Effect of Blank, Mock and ectopic miR-92a-3p expression on the luciferase activity of FBXW7 3’UTR wild type b, FBXW7 3’UTR mutation c, MOAP1 3’UTR wild type b, and MOAP1 3’UTR mutation c in HEK293A, SW480, SW620 and LOVO cells by dual-luciferase reported assay. d&e Expression of FBXW7 and MOAP1 in SW480, SW620 and LOVO cells transfected with Mock, miR-92a-3p, miR-92a-3p/FBXW7, and miR-92a-3p/MOAP1 by real-time PCR d and western blot e assays. GAPDH was used as internal control. f Expression of FBXW7 and MOAP1 in SW480, SW620 and LOVO cells treated with NFs-exos, CAFs-exos, CAFs-exos/FBXW7, CAFs-exos/MOAP1 by western blot. GAPDH was used as internal control. g&h Effect of Mock, miR-92a-3p, miR-92a-3p/FBXW7 treatment on migration g and invasion h of SW480, SW620 and LOVO cells by Boyden chamber. i Effect of Mock, miR-92a-3p, miR-92a-3p/FBXW7, and miR-92a-3p/MOAP1 treatment on colony formation ability of SW480, SW620 and LOVO cells by plate colony formation assay. j & k Effect of Mock, miR-92a-3p, miR-92a-3p/FBXW7 j, miR-92a-3p/MOAP1 k treatment on survival of SW480, SW620 and LOVO cells by CCK-8 assay. l Effect of Mock, miR-92a-3p, miR-92a-3p/FBXW7 treatment on sphere formation of SW480, SW620 and LOVO cells by spheres formation assay. m Effect of Mock, miR-92a-3p, miR-92a-3p/FBXW7 transfection on the expression of CD133, CD44, OCT4, CyclinD1, C-myc, E-cad and N-cad expression in SW480, SW620 and LOVO cells by real-time PCR. GAPDH was used as internal control. n Effect of Mock, miR-92a-3p, miR-92a-3p/FBXW7 treatment on the expression of FBXW7, CD133, CD44, OCT4, E-cad and N-cad expression in SW480, SW620 and LOVO cells by western blot. GAPDH was used as internal control. o Effect of Mock, miR-92a-3p, miR-92a-3p/MOAP1 transfection on the expression of MOAP1, BAX and cleaved caspase3 in SW480, SW620 and LOVO cells by western blot. GAPDH was used as internal control

Fig. 5

FBXW7 and MOAP1 attenuate miR-92a-3p-mediated promotion of aggressiveness and chemotherapy resistance of CRC in vivo. a The formation of metastatic nodules in lung derived from SW620/Mock, SW620/miR-92a-3p, and SW620/miR-92a-3p/FBXW7 cells by tail vein injection method (n = 8 in each group). b The formation of metastatic nodules in liver derived from SW620/Mock, SW620/miR-92a-3p, and SW620/miR-92a-3p/FBXW7 cells by subcapsular injection of the spleen method (n = 8 in each group). c The formation of subcutaneous tumors derived from SW480/Mock, SW480/miR-92a-3p, and SW480/miR-92a-3p/FBXW7 cells under 5-FU/L-OHP therapy by subcutaneous injection into the flank of mice n = 6 in each group). d-f Detection of proliferation and apoptosis by Ki-67 d, TUNEL e, and caspase3 f expression in tumor tissues derived from SW480/Mock, SW480/miR-92a-3p, and SW480/miR-92a-3p/FBXW7 cells by IHC and TUNEL assays. g The formation of subcutaneous tumors derived from SW480/Mock, SW480/miR-92a-3p, and SW480/miR-92a-3p/MOAP1 cells under 5-FU/L-OHP therapy by subcutaneous injection into the flank of mice (n = 6 in each group). h-j Detection of proliferation and apoptosis by Ki-67 (H), TUNEL i, and caspase3 j expression in tumor tissues derived from SW480/Mock, SW480/miR-92a-3p, and SW480/miR-92a-3p/MOAP1 cells by IHC and TUNEL assays

Fig. 6

FBXW7 and MOAP1 attenuated miR-92a-3p mediated Wnt/β-catenin activation and mitochondrial inhibition in CRC. a Effect of Mock, miR-92a-3p, miR-92a-3p/FBXW7 treatment on Wnt/β-catenin activation, stemness and EMT markers expression in SW480, SW620 and LOVO cells by western blot assay. b Effect of Mock, miR-92a-3p, miR-92a-3p/FBXW7 transfection on Wnt/β-catenin activation in SW480, SW620 and LOVO cells by laser scanning confocal microscope. c Effect of Mock, FBXW7, NC and siFBXW7 transfection on ubiquitination and degradation of β-catenin in SW480, SW620 and LOVO cells by western blot. d Effect of Mock and FBXW7 transfection on expression of nucleus β-catenin in SW480, SW620 and LOVO cells by western blot assay. e Effect of Mock, FBXW7, NC, and siFBXW7 treatment on expression of β-catenin in SW480, SW620 and LOVO cells by laser scanning confocal microscope. f Effect of Mock, miR-92a-3p, MOAP1 on expression of MOAP1, BAX, cytochrome C, caspase9, caspase3 in in SW480, SW620 and LOVO cells CRC cells treated with 5-FU/L-OHP therapy. g Effect of Mock, miR-92a-3p, MOAP1 on expression of cytochrome C in in SW480, SW620 and LOVO cells cells treated with 5-FU/L-OHP therapy

Fig. 7

The expression of miR-92a-3p correlates negatively with FBXW7 and MOAP1 and is associated with metastasis in serum exosomes. a Real-time PCR analysis of miR-92a-3p, FBXW7 and MOAP1 in non-metastatic and metastatic fresh CRC tissues n = 20 for each group) (tumor ration to normal). The expression of miR-92a-3p was compared between CRC with metastasis and CRC without metastasis. U6 was used as internal control. b & c Real-time PCR analysis of FBXW7 b and MOAP1 c in non-metastatic and metastatic fresh CRC tissues (n = 20 for each group) (tumor ration to normal). GAPDH was used as internal control. (D&E) Pearson correlation analysis between miR-92a-3p and FBXW7 levels, miR-92a-3p and MOAP1 levels in 20 cases of non-metastatic CRC d, 20 cases of metastatic CRC e and corresponding normal colorectal mucosa. f Real-time PCR analysis of miR-92a-3p in serum-derived exosomes collected from 30 normal persons, 30 non-metastatic CRC patients and 30 metastatic CRC patients. U6 was used as internal control. g Real-time PCR analysis of miR-92a-3p in serum-derived exosomes collected from 18 cases of 5-FU/L-OHP sensitive CRC patients and 18 cases of 5-FU/L-OHP resistant CRC patients. U6 was used as internal control. h Briefly, Colorectal cancer (CRC) cells uptake cancer associated fibroblasts secreted exosomes, leading to an increase of miR-92a-3p and stemness, EMT, metastasis, and 5-FU/L-OHP resistance in CRC cells. Mechanically, miR-92a-3p promotes aggressiveness and chemotherapy resistance by directly binding to 3’UTR of FBXW7 and MOAP1 and suppressing their expressions in CRC cells. Re-expression of FBXW7 and MOAP1 attenuate the role of miR-92a-3p by inhibiting Wnt/β-catenin and mitochondrial apoptosis in CRC