The enrichment of CD44 in exosomes from breast cancer cells treated with doxorubicin promotes chemoresistance [post]

2020 unpublished
Exosomes have been shown to be associated with chemotherapy resistance transmission between cancer cells. However, the cargo and function of exosomes changed in response to doxorubicin remains unclear. Methods: We compared proteome profiles of exosomes extracted from the supernatant of MCF-7(S/Exo) and MCF-7/ADR(A/Exo) cells. We confirmed the differential expression of the candidate target-exosomic-CD44 by immune gold staining and western blot. We further studied the changes of chemosensitivity
more » ... of chemosensitivity and CD44 expression in MCF-7 cells co-incubated with A/Exo. We analyzed the levels of exosomal CD44 from patient plasma, and compared the sensitivity and specificity of exosomic CD44 and plasma CD44 on diagnosis of chemoresistance. We modified the MCF-7-derived exosomes loaded with siRNA against CD44 to observe the effects of targeting reduced CD44 expression in lumimal A breast cancer cells. Results: DOX increased the exosomes release from MCF-7/ADR cells and the exosomes mediated proteins intercellular transfer in breast cancer chemoresistance regulation. The candidate target of CD44 in A/Exo was much higher than in S/Exo and the increase levels of exosomic CD44 (21.65-fold) was much higher than cellular CD44 (6.55-fold). The same results were obtained in clinical samples. Exosome-siRNA targeted CD44 (Exos-siCD44) could efficiently targeted to silence its expression. When co-cultured on Exos-siCD44, breast cancer cells exhibited reduced cell proliferation and enhanced susceptibility to DOX and the same phenomenon was observed in mice. Conclusion Drug-resistant breast cancer cells spread resistance capacity to sensitive ones by releasing exosomes to transfer proteins in intercellular. Background Breast cancer is the most prevalent cancer among women and doxorubicin (DOX) is still the first-line clinical chemotherapy in breast cancer. However, doxorubicin therapy has a very low response rate (25-40%) owing to acquired drug resistance [1] . Thus, to analyze the crucial factors in the development of resistance of tumor cells is of great importance. Proteomics comparing analyses between drug-resistant and drug-sensitive MCF-7 cells identified that several proteins involved in apoptosis, the cell cycle, glucose metabolism, and fatty acid oxidation contribute to drug resistance[2-5] which could provides a good foundation for understanding the development of drug resistance in cell lines. Although the resistance-related proteins have been investigated and several metabolic pathways involved in MCF-7/ADR have been found [6] , little is known about the global metabolic pattern and shift between the sensitive and resistant breast tumor cells. Exosomes as membranous nanovesicles (30-150 nm size) of endocytic origin secreted by cells and carry along a wealth of cell-of-origin cargo and the cargo changes with cellular physiology and represents diagnostic opportunities for both tumor physiology mapping and cancer progression without accessing parent cells directly, which makes exosomes an attractive alternative as potential prognostic biomarkers [7] . In recent years, exosome-induced chemoresistance is emerging as a novel mechanism[8]. Exosome signaling creates 'therapeutic tumor heterogeneity' and favorably condition tumor microenvironment [9, 10] . In the previous study, we also have found that the levels of exosomes released from doxorubicin-resistant breast cancer cells (MCF-7/ADR) are higher than MCF-7 cells and their transcriptome changes in response to doxorubicin [11] . Mechanistically, exosomes impart resistance by direct drug export, transport of drug efflux pumps and miRNAs exchange among cells [12] . However, the protein cargo and function of exosomes transmitted in response to doxorubicin remains unclear. Through the discovery of candidate biomarkers, mass spectrometrybased proteomics may provide a better understanding of the role of exosomes in intercellular chemoresistance transfer. So in this study, we analyzed the proteome profile of the breast cancer line MCF-7 in dependence on doxorubicin resistance by LC-MS/MS. The finding of this study may serve to develop a potential therapy for chemotherapy resistance in breast cancer. Materials And Methods Cell culture The human breast cancer cell lines MCF-7 was purchased from the Nanjing KeyGen. MCF-7 cells were induced by different concentrations of doxorubicin to establish resistance cell lines MCF-7/ADR. MCF-7 cells were cultured in RPMI-1640 medium supplemented with 10% exosome-depleted FBS. MCF-7/ADR 5 were cultured in the medium containing 1ug/ml doxorubicin in order to maintain the drug resistant phenotype, and were then maintained in drug-free medium for at least two days prior to use. Cells tested negative for mycoplasma. All cells were maintained in 5% CO 2 at 37℃. Exosomes isolation, identification and peptide extraction To gauge the participation of exosomes in doxorubicin-induced transmission of drug-resistant, we used sequential ultracentrifugation to isolate exosomes from MCF-7 and MCF-7/ADR cells that were cultured in exosome-depleted medium for 48h. Exosome-depleted medium was prepared as follows: medium was ultracentrifuged for 16h, and then filtered using a 200-nm filter. Exosomes were identified by transmission electron microscopy (TEM) and ZetaView Nanoparticle-tracking analysis (NTA) instrumentation. To detect exosome-specific markers on isolated exosomes, purified exosome pellets were lysed with RIPA and quantified using NanoDrop (Thermo Scientific), and proteins were analyzed with an SDS-PAGE gel and detected using Odyssey® Infrared Imaging System(LI-COR). Samples were incubated with primary anti-CD63 (proteintech, 1:100) and anti-Calnexin antibodies (proteintech, 1:100), followed by IRDye® 800CW IgG (H+L) and IRDye® 680RD IgG (H+L) antibody (LI-COR, 1:15,000). The exosomes extracted from the supernatant of MCF-7 and MCF-7/ADR cells were named S/exo and ADR/exo, respectively. Protein separation, digestion and peptide extraction for LC-MS/MS were prepared according to the previous research methods [13] . The obtained peptides were labeled by TMT (Thermo Fisher Scientific, MA, USA) and cleaned, desalted and vacuum-dried. Quantitative proteomic analysis and bioinformatic analysis The obtained peptides from S/exo and A/exo were subsequently analyzed with an on-line twodimensional nano LC/MS/MS by BioNovoGene. Scatter-plot matrices and density plots were created with the R programming language to verify the correlations between the data samples. The differently expressed proteins (DEPs) between S/exo and ADR/exo were selected by the difference multiples (log2|FoldChange|≧1.0) and significant levels (q-value<0.001). Then we performed GO enrichment analysis and KEGG pathway enrichment analysis as we described before [14] . Flow cytometry analysis of the differently expressed proteins Given the low amount of exosomal protein, we chose flow cytometry analysis instead of western blotting to further verify the differently expressed proteins(log2|FoldChange|≧1.5). The collected exosomes were suspended by 100 μl PBS and attached to 10 μl aldehyde/sulfate latex beads (4 μm, A37304, Invitrogen, Carlsbad, CA, USA) for 2h continuous rotation at room temperature. This suspension was diluted to 1 ml PBS and rotated for 2h at RT. Centrifuge the suspension at 580 g for 5 min at RT and discard the supernatant. Resuspend the pellet with 1 mL of 100 mM glycine solution and incubate for 30 min at RT. Exosomes-bound beads were washed 1 time in 1× PBS/2% BSA and centrifuge for 1 min at 14,800 g, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1× PBS/2% BSA and centrifuged for 1 min at 14,800 g and incubated with fluorescent antibodies ( BioLegend 6 μl of antibody in 100 μl of 2% BSA/1× PBS) during 30 min rotating at 4°C. The percent positive beads were calculated relative to the CD63+ beads analyzed per sample and the log2|FoldChange| was calculated by the positive percentage. Exosomal CD44 of breast cancer cells were detected by western blotting and immunogold labeling To further validate the different level of CD44 in S/exo and ADR/exo, we performed immunogold labeling and western blot analysis which were conducted according to the previous protocol. Exosomes were fixed in 1% paraformaldehyde with 0.05 glutaraldehyde; The grids were immediately placed into the primary antibody of CD44 (Proteintech) at a 1:30 dilution and an isotype-matched negative control antibody for 36 h at 4℃. Then all of the grids were floated on drops of 6 nm gold particles (1:30 dilution) for 1 h at room temperature. Samples were then negatively stained with filtered aqueous 2% uranyl acetate for 1 min. Excess uranyl acetate was drained with filter paper and samples were examined on transmission electron microscope at an accelerating voltage of 120 Kv. The effect on MCF-7 after incubation with ADR/Exo To evaluate the changes of CD44 in MCF-7 after incubation with ADR/Exo, 1×10 6 MCF-7 cells were cultured in 6-well plates with different concentration (1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 and 1×10 9 particles/mL harvested at 7th day) and different time (1×10 8 particles/mL ADR/Exo harvested at 0,1 st ,
doi:10.21203/rs.2.22928/v1 fatcat:6j6vnilfgzhhxevkvfbkxl4p44