Exosome-mediated breast cancer chemoresistance via miR-155 transfer

Abstract

Breast cancer remains the most prevalent cause of cancer mortality in woman worldwide due to the metastatic process and therapy resistance. Resistance against cancer therapy is partially attributed to cancer stem cells (CSCs). These cells arise from epithelial cells undergoing epithelial-to-mesenchymal transition (EMT) and might be responsible for tumor recurrence. In this study, we reported the relevance of miR-155 upregulation in chemoresistant cells associated with EMT. Notably, we found miR-155 induction in exosomes isolated from CSCs and resistant cells, followed by resistant cells' exosome transfer to the recipient sensitive cells. Functionally, miR-155 mimic assay showed an enrichment in miR-155 from exosome concomitant with miR-155 exosome transfer to breast cancer cells. In parallel to these effects, we also observed EMT change in miR-155 transfected cells. The chemoresistance phenotype transfer to sensitive cells and the migration capability was analyzed by MTT and scratch assays and our results suggest that exosomes may intermediate resistance and migration capacity to sensitive cells partly through exosome transfer of miR-155. Taken together, our findings establish the significance of exosome-mediate miR-155 chemoresistance in breast cancer cells, with implications for targeting miR-155 signaling as a possible therapeutic strategy.

Figure 1

Morphological and molecular changes of breast cells in response to doxorubicin and paclitaxel treatment. (A) MCF-7 morphological changes after chemoresistance induction; (B) MDA-MB-231 morphological changes after chemoresistance induction, a – sensitive cells, b – DOX-resistant cells, c – PTX-resistant cells; (C) EMT markers expression in sensitive, DOX and PTX-resistant MCF-7 cell line; (D) EMT markers expression in sensitive, DOX and PTX-resistant MDA-MB-231 cell line. The results were analyzed using t-test. *p < 0.05 and **p < 0.01 compared with the sensitive cells group.

Figure 2

Breast CSC population characteristics. (A) 3D morphological characteristic of mammospheres generated from MCF-7 cell line; (B) Breast CSCs markers in mammospheres (CSCs) and chemoresistant breast cells; (C) miR-155 expression in mammospheres (CSCs) and chemoresistant breast cells; (D) Mammosphere number following miR-155 transfection or control and co-culture with exosomes from MCF-7 cells transfected with miR-155 or control; The results were analyzed using t-test. *p < 0.05 and **p < 0.01 compared with the sensitive cells or control group; (E) Mammosphere formation from miR-155 transfection or control using the ELDA platform; (F) Mammosphere formation from cells co-cultured with exosomes from MCF-7 cells transfected with miR-155 or control using the ELDA platform. The slope of the line is the log-active cell fraction. Solid lines depict the mean, the dotted lines give the 95% confidence interval, and circles indicate the values obtained in each cell dilution.

Figure 3

Characterization of exosomes. (A) Western blot for the exosome-related proteins CD-63 and CD-9, S – exosomes from sensitive cells/R – exosomes from resistant cells; In the figure are reported the cropped blots, and full-length blots are presented in Supplementary Figure S1; (B) Representative fluorescence microscopy showing uptake of PKH26-labeled exosomes into recipient MCF-7 cells, blue – nuclei, red - PKH26-labeled exosomes; (C) Exosome protein concentrations from mammospheres (CSCs), sensitive, DOX and PTX resistant MCF-7 and MDA-MB-231 breast cells; (D) miR-155 level into exosomes from sensitive, DOX-resistant, PTX-resistant and CSCs MCF-7 cells; (E) miR-155 level into exosomes from sensitive, DOX-resistant, and PTX-resistant MDA-MB-231 cells. The results were analyzed using t-test. *p < 0.05 and **p < 0.01 compared with the sensitive cells group.

Figure 4

Effects of exosome co-cultures. (A) miR-155 level in MCF-7 cells following co-culture with exosomes from sensitive, DOX-resitant, PTX-resistant and CSCs MCF-7 cells, and miR-155 level in MDA-MB-231 following co-culture with exosomes from sensitive, DOX-resistant and PTX-resistant MDA-MB-231 cells; (B) mRNA levels of EMT markers following co-culture with exosomes from DOX-resitant, PTX-resistant and CSCs breast cells; (C) miR-155 targets expression following co-culture with exosomes from DOX-resitant, PTX-resistant and CSCs breast cells in MCF-7 and MDA-MB-231 cells; (D) Chemosensitivity to doxorubicin and paclitaxel following co-culture with exosomes from sensitive, DOX and PTX resistant cells, and mammospheres (CSCs) MCF-7 cells; (E) Migration assay following co-culture with exosomes from sensitive, DOX and PTX resistant MCF-7 cells; (F) Chemosensitivity to doxorubicin and paclitaxel following co-culture with exosomes from sensitive, DOX and PTX-resistant MDA-MB-231 cells; (G) Migration assay following co-culture with exosomes from sensitive, DOX and PTX resistant MDA-MB-231 cells. The results were analyzed using t-test. *p < 0.05 and **p < 0.01 compared with the EXO/S group.

Figure 5

miR-155 transfection. (A) miR-155 level into exosomes from MCF-7 cells transfected with miR-155 (mimic) or negative control; (B) miR-155 expression in MCF-7 cells transfected with miR-155 or control and MCF-7 cells co-cultured with exosomes from cells transfected with miR-155 or control; (C) miR-155 targets expression in MCF-7 cells transfected with miR-155 or control and MCF-7 cells co-cultured with exosomes from cells transfected with miR-155 or control; (D) EMT mRNA markers expression in MCF-7 cells transfected with miR-155 or control and MCF-7 cells co-cultured with exosomes from cells transfected with miR-155 or control. The results were analyzed using t-test. *p < 0.05 and **p < 0.01 compared with the control group. (E) E-cadherin immunofluorescence in MCF-7 cells transfected with miR-155 or control and MCF-7 cells co-cultured with exosomes from cells transfected with miR-155 or control.

Figure 6

Chemosensitivity and migration capacity of cells co-cultured with exosomes from cells transfected with miR-155. (A) Chemosensitivity to doxorubicin and paclitaxel following co-culture with exosomes from MCF-7 cells transfected with miR-155; (B) Migration assay of MCF-7 cells transfected with miR-155 or control; (C) Migration assay of MCF-7 cells co-cultured with exosomes from cells transfected with miR-155 or control. The results were analyzed using t-test. *p < 0.05 and **p < 0.01 compared with the EXO/C group.

Figure 7

Putative mechanism of cell-to-cell communication exosome-mediated resistance transmission.