Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro

Abstract

Extracellular vesicles (EV) secreted in the tumour microenvironment (TME) are emerging as major antagonists of anticancer therapies by orchestrating the therapeutic outcome through altering the behaviour of recipient cells. Recent evidence suggested that chemotherapeutic drugs could be responsible for the EV-mediated tumour-stroma crosstalk associated with cancer cell drug resistance. Here, we investigated the capacity of tumour EV (TEV) secreted by normoxic and hypoxic (1% oxygen) C26 cancer cells after doxorubicin (DOX) treatment to alter the response of naïve C26 cells and RAW 264.7 macrophages to DOX. We observed that C26 cells were less responsive to DOX treatment under normoxia compared to hypoxia, and a minimally cytotoxic DOX concentration that mounted distinct effects on cell viability was selected for TEV harvesting. Homotypic and heterotypic pretreatment of naïve hypoxic cancer and macrophage-like cells with normoxic DOX-elicited TEV rendered these cells slightly less responsive to DOX treatment. The observed effects were associated with strong hypoxia-inducible factor 1-alpha (HIF-1α) induction and B-cell lymphoma-extra-large anti-apoptotic protein (Bcl-xL)-mediated anti-apoptotic response in normoxic DOX-treated TEV donor cells, being also tightly connected to the DOX-TEV-mediated HIF-1α induction, as well as Bcl-xL levels increasing in recipient cells. Altogether, our results could open new perspectives for investigating the role of chemotherapy-elicited TEV in the colorectal cancer TME and their modulatory actions on promoting drug resistance.

Keywords: colon cancer; doxorubicin; extracellular vesicles; hypoxia; macrophages; normoxia.

Figure 1

The effects of doxorubicin (DOX) on C26 murine colon carcinoma cells under normoxic and hypoxic conditions. (A) Percentage of cell viability reduction compared to the viability of control untreated cells after 12 h incubation of C26 cells with increasing concentrations of DOX ranging from 0.1 µM to 1.25 µM under either normoxia (C26 N 12 h) or hypoxia (C26 H 12 h). (B) Percentage of cell viability reduction compared to the viability of control cells after 24 h incubation of C26 cells with increasing concentrations of DOX ranging from 0.1 µM to 1.25 µM under either normoxia (C26 N 24 h) or hypoxia (C26 H 24 h). Data are shown as mean ± SD of triplicate measurements of two independent experiments; ns: not significant, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2

Fluorescence microscopy displaying DOX uptake pattern by C26 murine colon carcinoma cells after 12 h incubation with 0.3 µM DOX under normoxic and hypoxic conditions. (A) Fluorescence microscopy images acquired with different filters. DIC: differential interference contrast images of C26 cells after 12 h incubation with the drug; DAPI: fluorescence images of C26 cells subjected to 4′,6-diamidino-2-phenylindole (DAPI) staining after 12 h incubation with DOX to highlight the nuclei (excitation 365 nm, emission > 397 nm); DOX: fluorescence images of DOX uptake by C26 cells after 12 h incubation with the drug (excitation 470 nm, emission 581–679 nm); MERGE: overlays of fluorescence and DIC images. The same settings were applied for each photo taken from every experimental condition; magnification = 40×; scale bar = 10 µm; Control = untreated C26 cells cultured under normoxia. (B) Mean absolute intracellular DOX fluorescence was measured from several images using ImageJ software and the results were expressed as mean ± SD. Unpaired t-test was used for the statistical comparison between the DOX fluorescence under normoxia compared to hypoxia; ***, p < 0.001.

Figure 3

Size distribution and concentration of TEV. Nanoparticle tracking analysis was used to determine TEV concentration (A) and their size (B) under normoxia and hypoxia after 12 h incubation with 0.3 µM DOX. The production (C) and size (D) of extracellular vesicles (EVs) secreted by C26 cells after 24 h incubation with 0.3 µM DOX under normoxia and hypoxia are also shown. EV production was expressed as particle concentration/mL ± SD of triplicate measurements and data were normalised for the protein concentration obtained from cell lysates at the time of EV harvesting; ns: not significant, p > 0.05; *, p < 0.05; ***, p < 0.001.

Figure 4

C26 cell viability reduction under normoxia and hypoxia after pretreatment with EVs from C26 cells and exposure to DOX treatment. The effects of TEV secreted by untreated normoxic C26 cells (TEV N) or by 0.3 µM DOX-treated normoxic cells (DOX-TEV N) on hypoxic C26 cells (C26 H) treated with different DOX concentrations for 12 h are shown in panel (A) and for 24 h are shown in panel (C). The effects of TEV secreted by untreated hypoxic C26 cells (TEV H) or by 0.3 µM DOX-treated hypoxic cells (DOX-TEV H) on normoxic C26 cells (C26 N) treated with different DOX concentrations for 12 h are shown in panel (B) and for 24 h are shown in panel (D). Data from interexperimental duplicates are represented as mean ± SD and expressed as a percentage compared to the corresponding controls (untreated C26 cells, C26 cells treated with 0.5 µM DOX that were not pretreated with TEV, and C26 cells treated with 0.75 µM DOX that were not pretreated with TEV, respectively); ns: not significant, p > 0.05; *, p < 0.05; **, p < 0.01.

Figure 5

Cell viability of RAW 264.7 cells cultured under normoxia and hypoxia after pretreatment with EVs from C26 cells and exposure to DOX treatment. The effects of TEV secreted by untreated normoxic C26 cells (TEV N) or by 0.3 µM DOX-treated normoxic cells (DOX-TEV N) on hypoxic RAW 264.7 cells (RAW H) treated with different DOX concentrations for 12 h are shown in panel (A) and for 24 h are shown in panel (C). The effects of TEV secreted by untreated hypoxic C26 cells (TEV H) or by 0.3 µM DOX-treated hypoxic cells (DOX-TEV H) on normoxic RAW 264.7 cells (RAW N) treated with different DOX concentrations for 12 h are shown in panel (B) and for 24 h are shown in panel (D). Data from interexperimental duplicates are represented as mean ± SD and expressed as percentages compared to the corresponding controls (untreated RAW 264.7 cells, RAW 264.7 cells treated with 0.5 µM DOX that were not pretreated with TEV, and RAW 264.7 cells treated with 0.75 µM DOX that were not pretreated with TEV, respectively); ns: not significant, p > 0.05; *, p < 0.05; **, p < 0.01.

Figure 6

Overview on the molecular changes related to the resistance inducing mechanisms in C26 cells to DOX treatment under normoxia. Cropped Western blot images and their representative graphs displaying the percentage of protein levels after 24 h treatment with 0.3 µM DOX under normoxia compared to controls (the levels of the same proteins in untreated cell lysates) are shown for phosphatidylinositol-3 kinase (PI3K) in panel (A); for protein kinase B (Akt) in panel (B); for proto-oncogene tyrosine-protein kinase Src (c-Src) in panel (C); for the c-Jun subunit of AP-1 transcription factor (AP-1 c-Jun) in panel (D); for p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) in panel (E); for c-master regulator of cell cycle entry and proliferative metabolism (c-Myc) in panel (F); for B-cell lymphoma–extra-large anti-apoptotic protein (Bcl-xL) in panel (G), for Bcl-2-associated X protein (BAX) in panel (H); and for hypoxia-inducible factor 1-alpha (HIF-1α) in panel (I). β-actin was used as loading control. Data were expressed mean ± SD of duplicate measurements from two independent experiments. Unpaired t-test was used for statistical analysis of the data; ns: not significant, p > 0.05; *, p < 0.05; **, p < 0.01, ***, p < 0.001.

Figure 7

Overview on the molecular changes in hypoxic C26 cells to DOX treatment. Cropped Western blot images and their representative graphs displaying the percentage of protein levels after 24 h treatment with 0.3 µM DOX under hypoxia compared to controls (the levels of the same proteins in untreated cell lysates) are shown for PI3K in panel (A); for Akt in panel (B); for c-Src in panel (C); for AP-1 c-Jun (the c-Jun subunit of AP-1 transcription factor) in panel (D); for NF-κB p65 (the p65 subunit of the NF-κB transcription factor) in panel (E); for c-Myc in panel (F); for Bcl-xL in panel (G), for BAX in panel (H); and for HIF-1α in panel (I). β-actin was used as loading control. Data were expressed mean ± SD of duplicate measurements from two independent experiments. Unpaired t-test was used for statistical analysis of the data; ns: not significant, p > 0.05; *, p < 0.05.

Figure 8

Overview on the molecular changes determined by TEV and DOX-TEV pretreatment on the recipient hypoxic C26 and RAW 264.6 cells exposed to DOX treatment. Cropped Western blot images and their representative graphs displaying the percentage of protein levels after hypoxic C26 and RAW 264.7 cell pretreatment for 24 h with C26 normoxic TEV (N TEV) or DOX-TEV (N DOX TEV) under hypoxic conditions, followed by treatment with 0.75 µM DOX under hypoxia for 24 h, as compared to controls (the levels of the same proteins in hypoxic cells treated with 0.75 µM, but no TEV pretreatment), which are shown for HIF-1α (A,D), for BAX (B,E), and for Bcl-xL (C,F). Data were expressed mean ± SD of triplicate measurements from two independent experiments. Unpaired t-test was used for statistical analysis of the data; ns: not significant, p > 0.05; *, p < 0.05, **, p < 0.01, ****, p < 0.0001.