The Study Of Breast Cancer Cell-derived Exosome Regulating Macrophage Polarization

Macrophages are a group of highly heterogeneous cell populations.They can be polarized into classically activated M₁ macrophages and alternative activated M₂macrophages based on different physiological and pathological environmental conditions.Macrophages polarization affects the tumor outcome.Although macrophages modulating the biological behavior of tumor parenchymal cells have been deeply studied,the mechanism of macrophage polarization induced by tumor parenchymal cells remains to be elucidated.In this study,PMA-treated THP-1 cells（M₀）were treated by different malignant breast cancer cell supernatant.It was found that supernatant from different malignant breast cancer cells can induce M₀ macrophage into M₂ macrophage,and the induction is closely related to breast cancer cell-derived exosomes.These results indicate that mammary tumor parenchymal cells can induce macrophage M₂ polarization through exosomes,which provides important insights into greater understanding of the molecular mechanisms of tumor cell-derived exosome-mediated macrophage polarization.ObjectiveTo explore whether macrophages can be polarized by exosomes from different malignant phenotypic mammary tumor cell,and provide reference for the clinical diagnosis and treatment of breast cancer through targeted exosomes.Methods1.Downloading GSE52292 raw data,using R language analysis software to analyze the expression of 55 macrophage markers,to screen the molecular markers of M₀,M₁ and M₂ macrophages.Human monocyte THP-1 cells were treated by different doses of PMA and different incubation time,the changes of cell morphology were observed by microscope,and the expression of M₀ macrophage marker CD68 was detected by q RT-PCR and immunofluorescence.M₀ macrophages were treated with100ng/ml LPS and 20ng/ml IFN-γ,or 25ng/ml IL4 and IL13 25ng/ml,respectively.q RT-PCR,ELISA,immunofluorescence and flow cytometry were used to detect the expression of M₁ macrophage markers CCR7,TNF-α,IL1β,IL12,HLADR,CXCL10,and M₂ macrophage markers CD206,CCL17,CCL18,CCL22,IL10.2.Cell culture supernatants of highly malignant breast cancer cell lines BT549,MDA-MB-231（M-231）and low malignant breast cancer cell lines MCF7,T47D mixing with complete medium in a 1:1 volume to culture M₀ macrophages for 24h and 72h,the expression of M₁ macrophage marker CD68/CCR7 and M₂ macrophage marker CD68/CD206 were detected by flow cytometry.3.The exosomes in the culture supernatant of high-malignant breast cancer cell lines BT549,M-231 and low malignant breast cancer cell lines MCF7 and T47D were extracted and identified by immunoblotting,transmission electron microscopy,and nanoparticle tracking analysis.The four tumor cell-derived exosomes were added into complete medium to culture M₀ macrophages,and the expression of M₂ macrophage marker CD68/CD206 was detected by flow cytometry.Breast cancer cell lines were treated with 10μM sphingomyelinase inhibitor GW4869 for 48h,and exosomes were extracted by the same method to incubate M₀ macrophages and M₂ macrophage,and the expression of M₂ macrophage marker CD68/CD206 was detected by flow cytometry.Results1.The GSE52293 data set analysis showed that the expression of CD68 was significantly higher in the process of monocyte differentiation into macrophages,and in the M₁ treatment group,the expression of most M₁ macrophage markers was higher than that in the M₀ group,and was different from that in the M₂ group.In the M₂ treatment group,the expression of most of the M₂ type macrophage markers was not significantly different from the M₀ and M₁ groups.After PMA action,morphology of human THP-1 monocytes was transformed from suspended spherical cells into attached adherent cells;q RT-PCR and immunofluorescence showed that 20ng/ml of PMA treated THP-1 cell line for 24h,M₀ macrophage marker CD68 was stably expressed.The expression of CCR7,TNF-α,and CXCL10 in M₁ macrophage was increased notably after 24h induction of M₀ macrophages with LPS combined with IFN-γ;M₂ macrophages were induced successfully after 72h using IL-4 in combination with IL-13,the expression of M₂ macrophage markers CD206,CCL17,CCL18,CCL22 and IL10 significantly increased,and there are statistically significant differences between M₁ and M₂ macrophages.2.M₀ macrophages treated with high malignant phenotypes of breast cancer BT549 and M-231 cell supernatant,the pseudopodia became longer and the number increased;while M₀ macrophages treated with low malignant phenotypes of breast cancer MCF7 and T47D cell supernatant,the M₀macrophages morphology was still spherical and only a few pseudopodia.Compared with the control group,the expression of CCR7 in macrophages induced by the culture supernatant of breast cancer cell lines had no significant changes（p>0.05）,and the expression of CD206was significantly increased（p<0.05）.There was no significant difference in the expression of CD206 in macrophages which were induced by different malignant breast cancer supernatant（p>0.05）.3.Western blot analysis revealed CD63 expression in exosomes extracted from culture supernatants of four breast cancer cell lines;transmission electron microscopy and nanoparticle tracking analysis revealed that exosomes exhibited cupped,bilayer membrane structures and particle diameter distributions from 80nm to 200nm.After the exosomes of breast cancer cell lines were added into complete medium to culture M₀ macrophages,the expression of CD206 in macrophages was significantly increased compared with the control group（p<0.05）,but there was no significant difference between the experimental group（p>0.05）.After GW4869 was applied to breast cancer cells,the exosome-induced the expression of M₂ macrophage marker CD206 was significantly decreased compared with the control group（p<0.05）,but there was no significant difference between the experimental group（p>0.05）.Conclusion1.The time difference in the expression of M₁ and M₂ macrophage-related molecular markers exists;the flow cytometric analysis of CD68⁺/CCR7⁺and CD68⁺/CD206⁺can be used to detect M₁ and M₂ macrophage markers.2.Different malignant phenotypes of breast tumor cells can induce M₀macrophages into M₂ macrophages in vitro.This effect can be mediated by exosomes.