| Objective:The aim of this study was to discuss whether colorectal cancer(CRC)-derived small extracellular vesicles(s EVs)induce M2 polarization in macrophages.By performing proteomic analysis of CRC tissue-derived s EVs to find the differential protein solute carrier family 38member 1 Gene(SLC38A1),and then the role of SLC38A1 in the induction of macrophage polarization was investigated to clarify the effect of CRC-derived s EVs on macrophage polarization through SLC38A1.Methods:The s EVs were purified from CRC and paracancerous tissue-derived s EVs by differential ultracentrifugation combined with size-exclusion columns.s EVs were identified and characterized by transmission electron microscopy,nano-flow detector and western blot.s EVs were enzymatically digested after total protein quantification by BCA quantification kit,and the enzymatically digested peptides were analyzed by mass spectrometry by applying free-labeled quantification.The screened differential proteins were subjected to cluster analysis,and the differential proteins were functionally annotated according to databases such as GO,KEGG and NR.SLC38A1 was selected as the target molecule based on the functional annotation results,and the expression of SLC38A1 in CRC tissues,cells and s EVs of tissue and cell culture supernatant origin was verified by western blot.Immunohistochemistry was used to detect the enrichment of SLC38A1 and macrophages in CRC and paraneoplastic tissues.THP-1differentiation into M0 macrophages was induced with 0.75 ng/ml of PMA.Colorectal cancer cells HCT116-derived s EVs labeled with PKH67 were co-cultured with M0 macrophages,and whether M0 macrophages took up HCT116 cell-derived s EVs was observed by confocal microscopy.HCT116 cells were transfected with lentivirus to obtain HCT116 cell lines with stable knockdown of SLC38A1.Induction of macrophage polarization by CRC-derived SLC38A1~+s EVs was analyzed by real-time fluorescence quantitative PCR(q-PCR)and flow cytometry.Results:1.Transmission electron microscopy,nano-flow detector and immunoblotting results showed that s EVs are vesicles with a diameter of about 50-200 nm,vesicular shape and intact cytosol,and expressed characteristic markers such as HSP70,CD63,CD81,ALIX and TSG101on the surface.2.Proteomic analysis of CRC and paraneoplastic tissue interstitial-derived s EVs revealed that SLC38A1 was significantly highly expressed in CRC tissue interstitial-derived s EVs.3.Immunohistochemical detection of colorectal cancer and paracancerous tissues revealed that SLC38A1,M0 macrophage marker CD68 and M2 macrophage marker CD206 were significantly highly expressed in CRC tissues,while M1 macrophage marker CD86 expression was not significantly elevated.4.Immunoblotting experiments verified the upregulation of SLC38A1 expression in colorectal cancer tissues,cells,tissue interstitial and cell culture supernatant-derived s EVs.5.Confocal microscopy observed the uptake of PKH67-labeled HCT116 cell-derived s EVs in THP-1 cells after PMA induction.6.Flow cytometry analysis showed that in the SLC38A1~+s EVs treatment group the proportion of CD11b~+CD206~+cells is significantly higher than that in the KD-SLC38A1-s EVs-treated group,whereas the proportion of CD11b~+CD86~+positive cells did not change significantly.Meanwhile,q-PCR results showed that the M2 macrophage markers CD206 and Arg-1 were significantly elevated after SLC38A1~+s EVs treatment,whereas CD86 did not change significantly.Conclusion:1.SLC38A1 and macrophage infiltration were significantly higher in colorectal cancer tissues compared with paracancerous tissues.2.SLC38A1 was significantly highly expressed in colorectal cancer tissues,cells,tissue-derived s EVs and cell culture supernatant-derived s EVs.3.Small extracellular vesicles derived from colorectal cancer cells HCT116 with high expression of SLC38A1 promoted the expression of M2 macrophage markers CD206 and Arg-1,thus demonstrating that colorectal cancer-derived small extracellular vesicles can induce macrophage M2 polarization via SLC38A1. |
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