(275d) Microgel-Based Screening of Triple-Negative Breast Cancer Therapeutic Synergy

Problem: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer due to its lack of common biomarkers that can be targeted for therapeutics or diagnostics. Currently, the most effective treatment is chemotherapy, which has several drawbacks, including reduced immune response and damage to healthy cells. Therefore, it is critical to utilize the intrinsic biology of TNBC to develop personalized therapeutics. TNBCs are known to undergo epigenetic changes mediated by the catalytic activity of enhancer of zeste homolog 2 (EZH2), a methyltransferase that targets H3K27. Conversely, activation of the dopamine D1 receptor (D1R) disrupts TNBC progression, leading to our strategy to target D1R and EZH2 simultaneously. The FDA Modernization Acts 2.0 and 3.0 have eliminated the requirement for animal models before human clinical trials and now encourage the development of bioengineered assays for preclinical testing. Most TNBC research is conducted in murine models, which offer physiological relevance but lack simplicity, and two-dimensional culture models, which offer simplicity but compromise physiological relevance. Three-dimensional culturing methods more accurately replicate in vivo responses but may reduce control and viability. Hydrogels, water-retaining polymers, mimic the viscoelastic properties of decellularized tissue, creating isotropic culture conditions. Microgels, which are miniaturized hydrogels, provide similar technical replicates and can be cultured as colloids, enabling high-throughput testing. Therefore, we developed a microfluidic approach to generate TNBC-cell-laden microgels that simulate tumor growth, allowing us to assess the efficacy of EZH2 inhibition and D1R agonism as a novel therapeutic strategy for TNBC.

Methods: Microgels were generated using a flow-focusing microfluidic device with fluorinated oil and a fluorosurfactant as the continuous phase, and an alginate pre-polymer mixture containing sodium alginate and calcium carbonate nanoparticles as the dispersed phase. The TNBC cell line, MDA-MB-231, was included in the dispersed phase to simulate the growth of TNBC tumors. The microgels were crosslinked by exchanging sodium ions with calcium ions, which was achieved internally by decreasing the pH of the fluorinated oil stoichiometrically with acetic acid, allowing the dissociation of the calcium carbonate nanoparticles. Following crosslinking, the microgels were demulsified with a short-chain fluorosurfactant and incubated with calcium chloride to further crosslink the microgels externally. The cell-laden microgels were cultured for two days, allowing tumor spheroids to form. Various concentrations and combinations of multiple EZH2 inhibitors (GSK126 and Tazemetostat) and D1R agonists (A77636 and SKF38393) were introduced into the assay and quantified via live-cell microscopy. Cell death was measured with Yo-Pro-1 iodide and ethidium homodimer-1 (EthD-1) to quantify apoptosis and necrosis, respectively. Immunofluorescence and standard molecular techniques were employed to elucidate the molecular mechanisms underlying the combination therapy. Lastly, EZH2 knockout cells were generated using CRISPR-Cas9 and encapsulated within the microgels.

Results: The microfluidic approach generated homogeneous microgels with a coefficient of variation of 1.73 ± 0.46%. The size of the microgels was tuned linearly with respect to the flow rate ratio. Similarly, the cellular occupancy of the microgels was tuned linearly with respect to the initial cell concentration. Furthermore, by combining an internal and external crosslinking mechanism, crosslinking times and reagent concentrations were reduced, resulting in a viability of 94.31 ± 9.85%. Therefore, the microgel-based method provided a homogeneous, tunable, and highly viable platform for effectively screening TNBC therapeutics. Tumor spheroids that were allowed to form after two days were subjected to single and combined therapies of GSK126/Tazemetostat and A77636/SKF38393 over a four-day period. Single doses demonstrated a dose-dependent decrease in the area of tumor spheroids. In contrast, the combination treatment showed a regression in spheroidal growth and a synergistic reduction in area (coefficient of drug interaction < 0.6), particularly for GSK126 and A77636. Interestingly, the observed regression of spheroidal growth was induced by necrosis, but not apoptosis. To further understand the mechanisms of action, the overall expression of EZH2 and its downstream target, H3K27, was analyzed. While the decrease in H3K27me3/H3K27 ratio upon treatment with GSK126 and the combination suggests that catalytic inhibition of EZH2 was primarily driven by the EZH2 inhibitor, a notable downregulation of EZH2 expression was observed only with the combination treatment. Immunoprecipitation analyses also demonstrated instability of the polycomb repressive complex 2 (PRC2), with which EZH2 is associated, further strengthening these results. Given the loss of EZH2 after the combination treatment, EZH2 knockout cells were generated and encapsulated. The EZH2 knockout cells were unable to form compact tumor spheroids and exhibited chaotic cytoskeletal formation. In contrast, wild-type cells had organized actin and tubulin structures. Therefore, the combination treatment targets the intrinsic expression of EZH2 in TNBC cells, thereby disrupting cellular function and viability.

Implications: The microgel-based system not only enabled a rapid method for screening therapeutic combinations but also provided a facile interface for investigating the mechanism of action of the promising combination of EZH2 inhibitors and D1R agonists. We demonstrate that the combination leads to tumor regression, due to necrotic cell death, decreased expression of EZH2, and cytoskeletal disorganization. Altogether, the treatment yields a promising new therapeutic option for TNBC.