Lineage-specific mechanisms and drivers of breast cancer chemoresistance revealed by 3D biomimetic culture

Abstract

To improve the success rate of current preclinical drug trials, there is a growing need for more complex and relevant models that can help predict clinical resistance to anticancer agents. Here, we present a three-dimensional (3D) technology, based on biomimetic collagen scaffolds, that enables the modeling of the tumor hypoxic state and the prediction of in vivo chemotherapy responses in terms of efficacy, molecular alterations, and emergence of resistance mechanisms. The human breast cancer cell lines MDA-MB-231 (triple negative) and MCF-7 (luminal A) were treated with scaling doses of doxorubicin in monolayer cultures, 3D collagen scaffolds, or orthotopically transplanted murine models. Lineage-specific resistance mechanisms were revealed by the 3D tumor model. Reduced drug uptake, increased drug efflux, and drug lysosomal confinement were observed in triple-negative MDA-MB-231 cells. In luminal A MCF-7 cells, the selection of a drug-resistant subline from parental cells with deregulation of p53 pathways occurred. These cells were demonstrated to be insensitive to DNA damage. Transcriptome analysis was carried out to identify differentially expressed genes (DEGs) in treated cells. DEG evaluation in breast cancer patients demonstrated their potential role as predictive biomarkers. High expression of the transporter associated with antigen processing 1 (TAP1) and the tumor protein p53-inducible protein 3 (TP53I3) was associated with shorter relapse in patients affected by ER⁺ breast tumor. Likewise, the same clinical outcome was associated with high expression of the lysosomal-associated membrane protein 1 LAMP1 in triple-negative breast cancer. Hypoxia inhibition by resveratrol treatment was found to partially re-sensitize cells to doxorubicin treatment. Our model might improve preclinical in vitro analysis for the translation of anticancer compounds as it provides: (a) more accurate data on drug efficacy and (b) enhanced understanding of resistance mechanisms and molecular drivers.

Keywords: 3D models; DNA repair; breast cancer; doxorubicin; drug resistance; lysosomes.

Fig. 1

Characterization and drug sensitivity of breast cancer cells in 3D collagen scaffold. (A, D) SEM micrograph of collagen scaffolds showing the porous surface of the material cellularized with MCF‐7 (A) and MDA‐MB‐231 (B) (n = 3). (B, E) Hematoxylin‐and‐eosin–stained histological sections of MCF‐7 (B) and MDA‐MB‐231 (E) within the scaffolds at day 7 (n = 3). (C, F) Confocal microscopy images of MCF‐7 (C) and MDA‐MB‐231 (F) within the scaffold at day 7 (n = 3). Cells are stained with DAPI (blue) and phalloidin (red). Scale bars for all pictures: 100 µm. (G) Percentages of survival of MCF‐7 and MDA‐MB‐231 after 72 h of treatment with different concentrations of doxorubicin in monolayer culture (2D), within the scaffold (3D), or orthotopically implanted into a murine model (in vivo). Data represent mean ± S.D. (n = 3 for in vitro data, n = 6 for in vivo data). (H) Nonlinear fit of log–dose responses curves and IC₅₀ calculation.

Fig. 2

Transcriptomic data analysis of breast cancer cells treated with doxorubicin. (A, D) Gene count of significantly altered pathways identified in MCF‐7 (A) or MDA‐MB‐231 (D) treated with doxorubicin within 3D collagen scaffolds. (B, E) Log fold change of DEGs in each identified pathway for MCF‐7 (B) or MDA‐MB‐231 (E). (C, F) Relative expression levels from qPCR data of candidate DEGs belonging to the identified pathway for MCF‐7 (C) and MDA‐MB‐231 (F) treated with doxo. The values are relative to untreated control samples. Data represent mean ± S.D. (n = 3). *P < 0.05, two‐tailed Student's t‐test.

Fig. 3

Mechanism of drug resistance in MCF‐7 cultured within the scaffold. (A) Western blot for caspase 3 in MCF‐7 untreated or treated with different concentrations of doxorubicin for 72 h in monolayer culture (2D) or within the scaffold (3D). (B) Percentages of apoptotic MCF‐7 after 72 h of treatment with different concentrations of doxorubicin in 2D or 3D cultures. Data represent mean ± S.D. (n = 3). *P < 0.05, two‐tailed Student's t‐test. (C) Immunofluorescence staining of γH2AX in MCF‐7 cells untreated (ctr) or treated with doxorubicin for 2, 6, 24, and 48 h in 2D or 3D cultures; quantification of the percentages of γH2AX‐positive cells and of the average number of γH2AX foci per cell. Data represent mean ± S.E.M (n = 5). *P < 0.05, two‐tailed Student's t‐test. Scale bars for all pictures: 20 µm. Arrows indicate apoptotic cells. (D) Schematic representation of doxorubicin effects in MCF‐7 cell line cultured within the scaffold. The most significantly altered pathway implicated in drug resistance with the list of relative DEGs are reported in the box. Green is indicative of upregulation.

Fig. 4

Mechanism of drug resistance in MDA‐MB‐231 cultured within the scaffold. (A) Doxorubicin median fluorescence intensity detected by flow cytometry in MDA‐MB‐231 after 6, 24, 48, and 72 h of treatment with different doxo concentrations in monolayer culture (2D) or within the scaffold (3D). Data represent mean ± S.D. (n = 3). (B) Histogram plot of MDA‐MB‐231 stained with lysotracker (LT) green in 2D or 3D cultures and median fluorescence intensity of LT green in control cells or cells treated with 4 µg·mL⁻¹ doxorubicin after 24, 48, and 72 h. Data represent mean ± S.D. (n = 3). (C) Confocal microscopy images of MDA‐MB‐231 treated with doxorubicin within the scaffold. Red is doxorubicin autofluorescence and green is LT green signal. Scale bar is 10 µm (D) Schematic representation of doxorubicin effects in MDA‐MB‐231 cell line cultured within the scaffold. The most significantly altered pathways implicated in drug resistance with the list of relative DEGs are reported in the box. Green is indicative of upregulation. Red is indicative of downregulation.

Fig. 5

Role of hypoxia in 3D‐induced drug resistance. (A) Survival percentages (day 7) of MCF‐7 and MDA‐MB‐231 cultured in monolayer (2D) of within the scaffold (3D) in the absence (CTR) or presence of a hypoxia inhibitor (HI). Data represent mean ± S.D. (n = 5). (B) HIF‐1α expression in histological sections of MCF‐7 and MDA‐MB‐231 cultured within the scaffold in control conditions (CTR) or in the presence of an hypoxia inhibitor (HI). Scale bars: 50 µm. (C) Percentage of survival of MCF‐7 and MDA‐MB‐231 after 72 h of treatment with different concentrations of doxorubicin in monolayer culture (2D), within the scaffold (3D) and in the presence (3D HI) of a hypoxia inhibitor. Data represent mean ± S.D. (n = 3). (D) Relative expression levels from qPCR data of candidate DEGs in MCF‐7 untreated or treated with 4 µg·mL⁻¹ doxorubicin under control conditions (ctr) or in the presence of hypoxia inhibition (HI). Data represent mean ± S.D. (n = 3). *P < 0.05, two‐tailed Student's t‐test. (E) Relative expression levels from qPCR data of candidate DEGs in MDA‐MB‐231 untreated or treated with 4 µg·mL⁻¹ doxorubicin under control conditions (ctr) or in the presence of hypoxia inhibition (HI). Data represent mean ± S.D. (n = 3). *P < 0.05, two‐tailed Student's t‐test.

Fig. 6

Expression levels of the identified biomarkers in vivo and in breast cancer patients. (A) Relative expression levels from qPCR data of candidate DEGs in MCF‐7 untreated or treated with 4 µg·mL⁻¹ doxorubicin in an orthotopic murine model. Data represent mean ± S.D. (n = 3). *P < 0.05, two‐tailed Student's t‐test. (B) Relative expression levels from qPCR data of candidate DEGs in MDA‐MB‐231 untreated or treated with 4 µg·mL⁻¹ doxorubicin in an orthotopic murine model. Data represent mean ± S.D. (n = 3). *P < 0.05, two‐tailed Student's t‐test. (C) Expression levels of TAP1 and TP53I3 in ER‐positive breast cancer patients and LAMP1 and LAMP2 in triple‐negative breast cancer patients in relation to response to anthracycline treatment. Patients were classified as responder or nonresponder according to the 5‐year relapse‐free survival. ROC curves of TAP1 and TP53I3 as predictor of response to anthracycline treatment in ER‐positive breast cancer patients, and LAMP1 and LAMP2 as predictor of response to anthracycline treatment in triple‐negative breast cancer patients. *P < 0.05, two‐tailed Student's t‐test, Mann–Whitney test.