Tunable synthetic extracellular matrices to investigate breast cancer response to biophysical and biochemical cues

Abstract

The extracellular matrix (ECM) is thought to play a critical role in the progression of breast cancer. In this work, we have designed a photopolymerizable, biomimetic synthetic matrix for the controlled, 3D culture of breast cancer cells and, in combination with imaging and bioinformatics tools, utilized this system to investigate the breast cancer cell response to different matrix cues. Specifically, hydrogel-based matrices of different densities and modified with receptor-binding peptides derived from ECM proteins [fibronectin/vitronectin (RGDS), collagen (GFOGER), and laminin (IKVAV)] were synthesized to mimic key aspects of the ECM of different soft tissue sites. To assess the breast cancer cell response, the morphology and growth of breast cancer cells (MDA-MB-231 and T47D) were monitored in three dimensions over time, and differences in their transcriptome were assayed using next generation sequencing. We observed increased growth in response to GFOGER and RGDS, whether individually or in combination with IKVAV, where binding of integrin β1 was key. Importantly, in matrices with GFOGER, increased growth was observed with increasing matrix density for MDA-MB-231s. Further, transcriptomic analyses revealed increased gene expression and enrichment of biological processes associated with cell-matrix interactions, proliferation, and motility in matrices rich in GFOGER relative to IKVAV. In sum, a new approach for investigating breast cancer cell-matrix interactions was established with insights into how microenvironments rich in collagen promote breast cancer growth, a hallmark of disease progression in vivo , with opportunities for future investigations that harness the multidimensional property control afforded by this photopolymerizable system.

Understanding ER+ Breast Cancer Dormancy Using Bioinspired Synthetic Matrices for Long‐Term 3D Culture and Insights into Late Recurrence

“…These observations at both short and long times in culture were consistent with previous reports that TN MDA-MB-231 is particularly responsive to the integrin-binding biochemical content of the matrix. [29,31,33] Quantifying viability over time, increased variance was observed for TN MDA-MB-231 cells relative to what had been observed with ER+ cells, with differential responses to matrix composition ( Figure 3B). In the low wt% matrix with collagen cue GFOGER, viability was initially high (>90%) and then decreased to ≈80-90% over days 10-40.…”

“…Formation and Rheological Characterization of PEG Hydrogels: Hydrogels were polymerized and mechanical and biochemical properties characterized using established methods. [31] 20 kDa 4-arm PEG-SH was either synthesized by established protocols [38] or purchased (JenKem Technology, Plano, TX), breaking any disulfides prior to use by overnight treatment with tris(2-carboxyethyl)phosphine (350 mg per 1 g of PEG-SH in ≈30 mL) followed by dialysis (molecular weight cutoff of 1 kDa, Spectrum Laboratories, for 24 hours against deionized water at pH 4) and lyophilization. 20 kDa 4-arm PEG-tetrathiol (PEG-4SH) and crosslinking and pendant peptides (IKVAV or GFOGER) were dissolved in phosphate buffered saline (PBS, Invitrogen) supplemented with 1% penicillin streptomycin (PS, Invitrogen) and 0.5 µg mL −1 fungizone (FZ, Invitrogen).…”

“…growth and metastasis, [26][27][28] including breast cancer cells. [29][30][31] Few studies have applied synthetic biomaterials to the study of breast cancer dormancy in 3D culture. [10] These include examination of breast cancer cell morphology and proliferation when seeded on non-bioactive stiff electrospun poly(capralactone) fibers [32] or entrapped within crosslinked poly(siloxanes).…”

“…[37] Hydrogels were formed by crosslinking a four-arm PEG-thiol (PEG-4-SH, M n ≈ 20 kDa) with a cell-degradable bis-alkene peptide crosslinker derived from collagen I (K(alloc) GGPQG↓IWGQGK(alloc)), which undergoes a rapid step growth polymerization reaction in the presence of cytocompatible doses of long wavelength UV light (10 mW cm −2 at 365 nm for 1 min). [31] Pendant peptides (2 mm of collagen cue GFOGER or laminin cue IKVAV at preparation) were functionalized with a K(alloc) group for facile incorporation as pendant groups during hydrogel formation. This approach for matrix formation was selected based on our prior work that shows i) consistent peptide incorporation, ii) independent control of hydrogel mechanical properties and biochemical cues for mimicking different soft tissues like those of interest here, and iii) a uniform encapsulation of single cancer cells within a thin hydrogel layer for quantitative analysis of cell responses in three dimensions using imaging-based techniques.…”

“…This approach for matrix formation was selected based on our prior work that shows i) consistent peptide incorporation, ii) independent control of hydrogel mechanical properties and biochemical cues for mimicking different soft tissues like those of interest here, and iii) a uniform encapsulation of single cancer cells within a thin hydrogel layer for quantitative analysis of cell responses in three dimensions using imaging-based techniques. [31,38,39] We previously have established the relevance of this materials system for shortterm studies of the growth and morphology of mesenchymal stem cells or breast cancer cells and now exploit the property control it affords for studying the effects of specific bioinspired matrix compositions on breast cancer dormancy in long-term 3D cultures.…”