Identification of a mechanogenetic link between substrate stiffness and chemotherapeutic response in breast cancer

Abstract

Mechanical feedback from the tumor microenvironment regulates an array of processes underlying cancer biology. For example, increased stiffness of mammary extracellular matrix (ECM) drives malignancy and alters the phenotypes of breast cancer cells. Despite this link, the role of substrate stiffness in chemotherapeutic response in breast cancer remains unclear. This is complicated by routine culture and adaptation of cancer cell lines to unnaturally rigid plastic or glass substrates, leading to profound changes in their growth, metastatic potential and, as we show here, chemotherapeutic response. We demonstrate that primary breast cancer cells undergo dramatic phenotypic changes when removed from the host microenvironment and cultured on rigid surfaces, and that drug responses are profoundly altered by the mechanical feedback cells receive from the culture substrate. Conversely, primary breast cancer cells cultured on substrates mimicking the mechanics of their host tumor ECM have a similar genetic profile to the in situ cells with respect to drug activity and resistance pathways. These results suggest substrate stiffness plays a significant role in susceptibility of breast cancer to clinically-approved chemotherapeutics, and presents an opportunity to improve drug discovery efforts by integrating mechanical rigidity as a parameter in screening campaigns.

Keywords: Chemotherapy; Drug screening; Genotyping; Hydrogel; Mechanobiology.

Fig. 1.

Mechanical profiling of breast tumor ECM. (a) Top Left: Stiffness profile of primary mammary tumor tissue, as measured by AFM analysis and reported as the elastic modulus (units of kPa). Top right: Mechanical profile of decellularized tumor sections. Inset: Cellularized (blue) stiffness data overlaid onto decellularized (red) results between 5 to 25 kPa (n ≥ 10). Bottom: H&E staining reveal tumor sections are composed of multi-focal regions of cancer cells (purple; cell nuclei) encapsulated by a dense fibrotic stroma (pink; collagen), while decellularized tissues are primarily composed of tumor ECM (scale bar = 400 μm; n = 3). (b) Fraction of stiffness measurements collected from primary tumor tissue (blue) or decellularized sections (red), binned as distinct regimes of elastic moduli. (c) Average stiffness values recorded at ten positions along the central axis of the cellularized tumor sections; error bars represent one standard deviation from the mean (n ≥ 10).

Fig. 2.

Breast tumor ECM mimetic stiffness array. (a) 96 well PA hydrogel stiffness arrays encumbering the mechanical profile of primary tumor ECM. Here, stiffness is systematically increased across the plate, with untreated glass included as a rigid control (moduli ≈10⁵ MPa). To prepare the multi-well array, glass bottom plates are chemically treated (red) to covalently attach PA gels formed in each well (purple). Functionalization of the gel surface with fibronectin (orange) allows for attachment of seeded cells. (b) Stiffness array gels were prepared with elastic moduli values that matched the rigidity profile of primary mammary tumor ECM (5 to 60 kPa) by controlling the weight percent of bis-acrylamide (bis) crosslinker added to the polymer solution (n = 3). (c) Representative immnofluorescent image demonstrating homogenous display of covalently linked fibronectin (red) on the gel surface (scale bar = 400 m). Associated z-stacks confirm protein coating is limited to the top of the gels. (d) Proliferation of primary mammary tumor cells isolated from MMTV-PYMT mice on 5 to 30 kPa PA gels, as measured by cell nuclei counting (n = 3). (e) Cell spreading as a function of substrate stiffness for primary tumor cells (1° tumor) or the human breast cancer cell li ne MCF-7 (n = 30). Error bars represent one standard deviation

Fig. 3.

Stiffness-dependent chemotherapeutic response of breast cancer cells. Left: Plots demonstrating the change in activity of selected small molecule chemotherapeutics after a 72 hour incubation with primary tumor cells, or the human breast cancer cell line MDA-MB-453, as a function of the culture substrate’s elastic modulus. PTX = Paclitaxel; DOX = Doxorubicin; MTX = Methotrexate; 5FU = 5-Fluorouracil; TAM = Tamoxifen. Dashed red line represents highest concentration tested, with the symbol ‘~’ indicating that an IC50 could not be reached at this concentration for the specified condition. Error bars represent one standard deviation from the mean (n = 9). Right: Table of IC50 values for each drug towards the panel of breast cancer cell lines tested following their culture on substrates of varying elastic modulus (E). Molecular sub-type status (ER/PR/HER2) is shown below the name of the tested cell line. Fold change (FC) in IC50 of cells cultured on each of the gels versus the glass control surface is tabulated to the right of the IC50 values, and color coded to visually symbolize order of magnitude.

Fig. 4.

Substrate stiffness alters the expression of drug resistance genes. (a) Gross changes in MDA-MB-453 gene expression when cultured on 5 kPa or 30 kPa PA gels compared to cells grown on glass, in units of log₂ (p<1×10⁻⁶). Cutoffs were set at ≥2 folds up-regulated (green) or ≤−2 folds down-regulated (red). (b-d) Fold change in the expression of genes associated with resistance (orange) or sensitivity (purple) of cells towards (b) PTX, (c) DOX or (d) MTX/5FU, following culture of MDA-MB-453 on 5 kPa gels versus glass. (e) Fold change in selected oncogene/proto-oncogenes (yellow) and tumor suppressor genes (blue). P value cutoff for panels b–e set at p<0.05. Data is averaged from n = 4 biologic replicates.

Fig. 5.

Cancer cells seeded onto gels that mimic tumor ECM stiffness recapitulate in situ gene expression. (a-e) Z-normalized expression levels of selected mouse genes whose human orthologs are involved in (a) apoptosis, (b) cell signaling, (c) cell cycle and growth, (d) response to drug and (e) small molecule metabolism and transport pathways. Gene expression of primary mammary tumor cells is shown for the in situ cells (isolate) immediately after removal, or following their culture for 5 days on soft gels (5 kPa), intermediate stiffness gels (30 kPa) or the highly rigid glass control surface. (f) Folds change (units of log₂; p<1×10⁻⁶) in the expression of genes associated with resistance (orange) or sensitivity (purple) of cells towards PTX and DOX between cells seeded onto soft (5 kPa) gels versus glass. Data is averaged from n ≥ 3 biologic replicates.