Phenotypic Basis for Matrix Stiffness-Dependent Chemoresistance of Breast Cancer Cells to Doxorubicin

Abstract

The persistence of drug resistant cell populations following chemotherapeutic treatment is a significant challenge in the clinical management of cancer. Resistant subpopulations arise via both cell intrinsic and extrinsic mechanisms. Extrinsic factors in the microenvironment, including neighboring cells, glycosaminoglycans, and fibrous proteins impact therapy response. Elevated levels of extracellular fibrous proteins are associated with tumor progression and cause the surrounding tissue to stiffen through changes in structure and composition of the extracellular matrix (ECM). We sought to determine how this progressively stiffening microenvironment affects the sensitivity of breast cancer cells to chemotherapeutic treatment. MDA-MB-231 triple negative breast carcinoma cells cultured in a 3D alginate-based hydrogel system displayed a stiffness-dependent response to the chemotherapeutic doxorubicin. MCF7 breast carcinoma cells cultured in the same conditions did not exhibit this stiffness-dependent resistance to the drug. This differential therapeutic response was coordinated with nuclear translocation of YAP, a marker of mesenchymal differentiation. The stiffness-dependent response was lost when cells were transferred from 3D to monolayer cultures, suggesting that endpoint ECM conditions largely govern the response to doxorubicin. To further examine this response, we utilized a platform capable of dynamic ECM stiffness modulation to allow for a change in matrix stiffness over time. We found that MDA-MB-231 cells have a stiffness-dependent resistance to doxorubicin and that duration of exposure to ECM stiffness is sufficient to modulate this response. These results indicate the need for additional tools to integrate mechanical stiffness with therapeutic response and inform decisions for more effective use of chemotherapeutics in the clinic.

Keywords: 3D cell culture; chemotherapy; extracellular matrix; resistance; tumor microenvironment.

Figure 1

Cells were cultured in hydrogels of varying stiffness before treatment with doxorubicin. The experimental protocol is outlined in (A). Briefly, cells were seeded onto tissue culture plastic or into hydrogels that ranged in stiffness from 200 to 2,000 Pa. After 6 days in culture, samples were exposed to doxorubicin for 48 h and cell viability was determined using AOPI staining. Hydrogel stiffness was determined by calculating Young's modulus from frequency sweep measurements obtained from a rheometer (B).

Figure 2

MDA-MB-231 cells have a stiffness-dependent resistance to doxorubicin. Dose response curves of MDA-MB-231 and MCF7 cells cultured in (A,B) 200 Pa hydrogel, (C,D) 2,000 Pa hydrogel, and (E,F) 2D monolayer following 48 h exposure to doxorubicin. Percent cell death was determined by staining samples with AOPI and counting live cells using a Nexcelom Cellometer.

Figure 3

An alginate hydrogel platform was used to dynamically stiffen hydrogels to mimic progressive ECM stiffening. (A) Cells were seeded into hydrogels and cultured for 3 days before dynamic stiffening with NIR light. After stiffening, cultures were given 1–5 days to acclimate to the new stiffness of the hydrogel before being exposed to doxorubicin for 2 days (48 h). Following treatment with doxorubicin, viability assays were performed to determine doxorubicin resistance. (B) 200 Pa hydrogels were exposed to NIR light for 45 s to achieve ECM stiffness similar to 2,000 Pa static hydrogels. The same technique was used to stiffen 2,000 Pa hydrogels to 3,000 Pa. (C) NIR light induces surface plasmon resonance in encapsulated gold nanorods (gold) to heat liposomes (pink) close to their gel-to-liquid transition temperature. This causes calcium (green) to leak from the liposomes and form additional alginate cross-links, thereby stiffening the hydrogel. The above figure was adapted from Joyce et al. (35).

Figure 4

MDA-MB-231 cultures have an acclimation-dependent increase in resistance to doxorubicin. (A) MDA-MB-231 and (B) MCF7 cells were cultured in hydrogels with an initial stiffness of 200 Pa or 2,000 Pa for 3 days. On day 3, hydrogels either remained static (200 or 2,000 Pa) or were stiffened (200 –>1,600 Pa or 2,000 –>3,000 Pa) using NIR light. Samples were then given 24–120 h to acclimate to the hydrogel stiffness before 48 h treatment with doxorubicin. MDA-MB-231 cells showed a higher resistance to doxorubicin as hydrogel stiffness increased and this stiffness-dependent resistance was found to be partially dependent on duration of exposure to hydrogel stiffness. Comparable MCF7 samples did not show any significant change in resistance to doxorubicin across hydrogel stiffness or acclimation time. *p < 0.01; **p < 0.02; ***p < 0.03; NS, not significant, p > 0.05.

Figure 5

Stiffer ECM increases nuclear localization of YAP and decreases expression of E-Cadherin. (A) MDA-MB-231 and (B) MCF7 cells were cultured on 200 or 2,000 Pa hydrogels for 3 days before fixation and staining with YAP (red) antibodies and DAPI (blue). Images were captured using confocal microscopy and analysis was done in ImageJ to determine nuclear localization of YAP (n = 123 for MDA-MB-231 cells cultured in 200 Pa hydrogels, n = 119 for MDA-MB-231 cells cultured in 2,000 Pa hydrogels, n = 72 for MCF7 cells cultured in 200 Pa hydrogels, n = 90 for MCF7 cells cultured in 2,000 Pa hydrogels). There is a stiffness-dependent increase in nuclear localization of YAP for both 231 (p = 1.38E-22) and MCF7 (p = 0.02) cultures, though the increase in MCF7 cultures is small. MDA-MB-231 cultures showed significantly higher expression of nuclear YAP compared to similar MCF7 cultures (p = 2.37E-37 for 200 Pa and p = 4.44E-46 for 2,000 Pa). Quantitative PCR indicates that cells cultured in stiffer hydrogels have decreased expression of E-Cadherin for both MDA-MB-231 (p = 0.001) and MCF7 (p = 0.014) cultures. Scale bar = 50 μm. *p < 0.05; **p = 0.01; ***p = 0.001; ****p << 0.001.