Extracellular vesicles from human breast cancer-resistant cells promote acquired drug resistance and pro-inflammatory macrophage response

Abstract

Introduction: Breast cancer is a significant public health problem around the world, ranking first in deaths due to cancer in females. The therapy to fight breast cancer involves different methods, including conventional chemotherapy. However, the acquired resistance that tumors develop during the treatment is still a central cause of cancer-associated deaths. One mechanism that induces drug resistance is cell communication via extracellular vesicles (EVs), which can carry efflux transporters and miRNA that increase sensitive cells' survivability to chemotherapy.

Methods: Our study investigates the transcription changes modulated by EVs from tamoxifen- and doxorubicin-resistant breast cancer cells in sensitive cells and how these changes may induce acquired drug resistance, inhibit apoptosis, and increase survivability in the sensitive cells. Additionally, we exposed human macrophages to resistant EVs to understand the influence of EVs on immune responses.

Results: Our results suggest that the acquired drug resistance is associated with the ability of resistant EVs to upregulate several transporter classes, which are directly related to the increase of cell viability and survival of sensitive cells exposed to EVs before a low-dose drug treatment. In addition, we show evidence that resistant EVs may downregulate immune system factors to evade detection and block cell death by apoptosis in sensitive breast cancer cells. Our data also reveals that human macrophages in contact with resistant EVs trigger a pro-inflammatory cytokine secretion profile, an effect that may be helpful for future immunotherapy studies.

Discussion: These findings are the first transcriptome-wide analysis of cells exposed to resistant EVs, supporting that resistant EVs are associated with the acquired drug resistance process during chemotherapy by modulating different aspects of sensitive cancer cells that coffer the chemoresistance.

Keywords: chemoresistance; doxorubicin; immunomodulation; membrane transporters; tamoxifen.

Figure 1

Process of generation of resistant cancer cells. Initial cell viability assay by resazurin reduction and half maximal inhibitory concentration (IC50) of sensitive MCF-7 cells treated with (A) tamoxifen (TAM) and (B) doxorubicin (DOX) and of MDA-MB-231 treated with (C) TAM and (D) DOX. Sensitive cells of MCF-7 and MDA-MB-231 exposed to (E) TAM and (F) DOX over time to generate resistant cell lines. The resistance fold calculated for each resistant cell line (G), indicating the level of resistance for each drug. The IC50s of (H) TAM- and (I) DOX-resistant MCF-7 and (J) TAM- and (K) DOX-resistant MDA-MB-231 cells calculated (red line) and compared to sensitive cells (blue line). The IC50s calculated by a nonlinear regression with an adjusted confidence level of 95%. The visualization of TAM- and DOX-resistant (L) MCF-7 and (M) MDA-MB-231 cells in a two-dimensional in vitro setup, and (N) in three-dimensional spheroids of sensitive and resistant MCF-7 cells.

Figure 2

Evaluation of the production and characterization of extracellular vesicles derived from breast cancer cells. (A) Nanoparticle-tracking analysis showing the size distribution of extracellular vesicles (EVs) produced by sensitive and resistant MCF-7 and MDA-MB-231 cells treated with tamoxifen (TAM) and doxorubicin (DOX). (B) Additional information about the average size and concentration of EVs per mL. (C) Histogram showing the EV production of each cancer cell per hour. (D) Transmission electron microscopy images of sensitive and resistant EVs from breast cancer cell lines. TAM-R, tamoxifen-resistant; DOX-R, doxorubicin-resistant; ns, non-significant. The bars represent the mean with the standard deviation from three independent experiments.

Figure 3

Sensitive breast cancer cells uptake resistant extracellular vesicles (EVs) and show increased cell viability. (A) Sensitive MCF-7 cells (Bright field) internalizing the EVs (Vybrant dil) after 24 h of exposure. (B) Sensitive MDA-MB-231 cells (Bright field) internalizing the EVs (Vybrant dil) after 24 h of exposure. TAM-R: tamoxifen-resistant; DOX-R: doxorubicin-resistant; ns: non-significant. A resazurin reduction assay evaluated the cell viability of sensitive (C) MCF-7 and (D) MDA-MB-231 cells exposed to resistant EVs. (E) Spheroids of sensitive MCF-7 cells were previously exposed to sensitive, TAM- and DOX-resistant EVs for 24 h and treated with TAM or DOX for 72h. The asterisks indicate a significant difference (p < 0.05) between tamoxifen or doxorubicin treatments when compared to no treatment (unpaired t-test).

Figure 4

Transcriptome-wide analysis showing the differential expression in sensitive breast cancer cell lines exposed to resistant extracellular vesicles (EVs) for 24 hours. (A) Histogram of the total number of differentially expressed genes (DEGs) in MCF-7 and MDA-MB-231. Distribution of gene ontology (GO) slim terms associated with MCF-7 transcripts after tamoxifen-resistant EVs (TAM-R EVs) exposure (B) and doxorubicin-resistant EVs (DOX-R EVs) exposure (C). For MDA-MB-231, the distributions of GO-slim enriched terms after TAM-R and DOX-R EV exposure are shown in (D, E), respectively. (F) Heatmap showing each condition’s top 10 up- and downregulated genes based on their log2 fold change.

Figure 5

Deep over-representation analysis of the differentially expressed genes (DEGs) in breast cancer cells exposed to extracellular vesicles (EVs) released by tamoxifen-resistant (TAM-R) and doxorubicin (DOX-R) cell lines. The gene ontology (GO) analysis enriched more specific terms considering the up- and downregulated DEGs in sensitive MCF-7 cells exposed to (A) TAM-R EVs and (B) DOX-R EVs for 24 h For sensitive MDA-MB-231 cells, the enriched terms for up- and downregulated DEGs after exposure to TAM-R and DOX-R EVs are displayed in (C, D), respectively. The number in front of each bar represents the total of enriched DEGs for each term. The x-axis of A to D represents the adjusted p-value (q-value) with a false discovery rate of 1%. Protein-protein interaction networks were generated using the DEG lists as references. (E, F) display the networks created by the MCF-7 DEGs after exposure to TAM-R and DOX-R EVs, respectively. For MDA-MB-231 cells, the exposure of TAM-R and DOX-R EVs generated the networks represented in (G, H), respectively.

Figure 6

Resistant extracellular vesicles (EVs) induce the upregulation of genes associated with acquired drug resistance and increase the sensitive cells’ survivability. The quantification of several genes by quantitative polymerase chain reaction (qPCR) of (A) sensitive MCF-7 exposed to tamoxifen-resistant (TAM-R) and doxorubicin-resistant (DOX-R) EVs. (B) The gene expression level of sensitive MDA-MB-231 cells exposed to TAM-R and DOX-R. (C) The apoptotic rate by annexin V/propidium iodide labeling in sensitive MCF-7 and sensitive MDA-MB-231 previously exposed to resistant EVs and treated with TAM and DOX. (D) Representative images and the clonogenic surviving fraction of MCF-7 cells previously exposed to TAM-R and (E) DOX-R EVs treated with the respective drug. (F) Representative images and the clonogenic surviving fraction of MDA-MB-231 cells previously exposed to TAM-R and (G) DOX-R EVs treated with the respective drug. Values are displayed as the mean ± standard deviation from three independent experiments. The asterisks indicate a significant difference between TAM-R EVs or DOX-R EVs exposure compared to sensitive EV effects (unpaired t-test). *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

Figure 7

Extracellular vesicles (EVs) released by tamoxifen-resistant (TAM-R) and doxorubicin-resistant (DOX-R) breast cancer cell lines modulate the cytokine production and release of human macrophages. Human CD14+/CD16- peripheral blood mononuclear cells (PBMC)-derived macrophages were exposed to EVs released by resistant MCF-7 and MDA-MB-231, and the quantification level of (A) tumor necrosis factor (TNF)-α, (B) interleukin (IL)-1β, (C) IL-10, and (D) interferon (IFN)-γ were evaluated. Additionally, THP-1-macrophages were exposed to resistant EVs, and the secretion levels of (E) TNF-α, (F) IL-1β, (G) IL-10, and (H) IFN-γ measured by the ELISA assay. Values are displayed as the mean ± standard deviation from three independent experiments. The asterisks indicate a significant difference between TAM-R EVs or DOX-R EVs exposure compared to sensitive EV effects (unpaired t-test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; media, RPMI 1640; LPS, lipopolysaccharide (positive control).