(C-104) Targeting EZH2 in a Microfluidic Tumor-Spheroid Inducing Microgel System: A Novel Treatment for Triple-Negative Breast Cancer

Introduction:: Introduction: Triple-negative breast cancer (TNBC) cells are aggressive, difficult to target, and form metastasis in 46% of the patients, leading to high mortality rates. Although patients with TNBC undergo chemotherapy as the most effective treatment, novel therapeutics targeting the intrinsic biology of TNBC cells are being developed to personalize therapeutics. Among them is targeting the epigenetic pathway by inhibiting the catalytic activity of enhancer of zeste homologue 2 (EZH2), a methyltransferase targeting H3K27. Despite the association between EZH2 and the TNBC phenotype, EZH2 inhibitors are inadequate at treating TNBC. While effective at treating liquid tumors, in solid tumors, EZH2 demonstrated pleiotropic effects outside its methylating capabilities, such as tumor proliferation. Therefore, targeting the catalytic activity of EZH2 does not suffice. Recently, the activation of the dopamine 1 receptor (D1R) has been shown to affect TNBC cells. Therefore, we sought to inhibit EZH2 and activate D1R in hopes of a new therapeutic option. Most therapeutic investigations utilize 2D monolayer culture to measure the potential of novel therapies. However, 2D methods oversimplify the cancer microenvironment, subjecting the cancer cells to homogeneous growth conditions and doses of therapeutics, which is atypical in solid tumors. Hydrogels mimic the viscoelastic properties of decellularized tissue, isotropic conditions, and allow for the growth of tumor spheroids. Therefore, apart from testing a novel therapy, we engineered the microfluidic high-throughput generation of homogeneous microgels without binding sites to induce tumor spheroid formation and model avascular tumor cytotoxicity, compatible with immunofluorescence for an in-situ understanding of the action mechanism.


Materials and Methods:: Materials and Methods: In silico docking investigations were performed on the affinity of small-molecule EZH2 inhibitors and D1R agonists to determine the feasibility of the combination therapy. The cytotoxic capabilities of the combination therapy were first validated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The tumor-spheroid microgel inducing system (TSIMS) was generated via a flow-focusing microfluidic device containing three streams: the first stream, the continuous phase, consisting of a fluorosurfactant diluted in a hydrofluoroether, and the second and third streams, the dispersed phases, consisting of calcium-carbonate nanoparticles diluted in sodium alginate and TNBC cells diluted in sodium alginate, respectively (Fig. 1A). The microgels were crosslinked with a two-step method, internally with acetic acid and externally with calcium chloride. The microgels were demulsified with a short-chain fluorosurfactant and transferred to media for subsequent therapeutic screenings. The TNBC cells were allowed to aggregate before treatment with EZH2 inhibitors and D1R agonists. The tumor spheroids were monitored over the treatment to determine therapeutic kinetics. CRISPR-Cas9 was performed to knockout (KO) EZH2. Yo-Pro-1 iodide and ethidium homodimer-1 (EthD-1) were utilized to assess cell death as apoptosis or necrosis, respectively. Immunofluorescence was visualized with epifluorescence and confocal microscopy to understand the downstream targets of the combination therapy. Furthermore, the effects of the combination therapy on the stability of the polycomb repressive complex 2 (PCR2) with which EZH2 is associated was determined with immunoprecipitation.


Results, Conclusions, and Discussions:: Results: The in-silico data revealed two pockets in the catalytic site of EZH2, one hydrophobic and the other hydrophilic. EZH2 inhibitors bound to both pockets, whereas D1R agonists bound to the hydrophilic pockets (Fig. 1B). Furthermore, the MTT assay on a TNBC cell monolayer reduced viability with a combination index of 0.24, demonstrating synergy between EZH2 inhibitors and D1R agonists. To better understand the cytotoxicity of the combination therapy in solid tumors, we engineered the TSIMS. The microfluidic approach yielded a coefficient of variation of microgel diameters of 1.73 ± 0.46 %, indicating highly homogeneous and reproducible conditions for cell growth. The combination treatment utilizing the TSIMS enhanced the reduction of tumor spheroid diameter to 37.85 ± 2.18 % that of the empty vehicle, with a coefficient of drug interaction of 0.68, further demonstrating synergy. The kinetics of tumor spheroid growth further indicated spheroid regression over treatment (Fig. 1C). Furthermore, the combination therapy induced necrosis but not apoptosis (Fig. 1D). Utilizing immunofluorescence, we observed a similar downregulation of H3K27me3 with the EZH2 inhibitor and the combination treatment (Fig. 1E). The combination caused complete inhibition of EZH2 expression (Fig. 1F). This may suggest the possible abrogation of both the canonical and non-canonical functions of EZH2 by the combination treatment. Immunoprecipitation further validated the results and demonstrated the dissociation of PCR2. Therefore, we developed EZH2-KO cells and subjected them to the TSIMS. The KO cells were unable to form tumor spheroids. Furthermore, the cellular aggregates of the KO cells in TSIMS were visualized with confocal microscopy, revealing the destabilization of actin and tubulin.


Conclusions and Discussions: The TSIMS allowed for a physiologically relevant perspective of the combination therapy, which revealed regression of tumor spheroids. Furthermore, the compatibility of the TSIMS with biomarker characterization allowed for a mechanistic approach to understanding the effect of the combination therapy. We found that the combination therapy degraded EZH2, of which the complete degradation revealed cytoskeletal disorganization. Therefore, combination therapy provides a novel treatment for TNBC that not only targets the primary tumor via necrosis but may also disrupt micrometastatic formation in distant sites.


Acknowledgements (Optional): : This work is supported by DOD: W81XWH2010065, for Eswar Shankar; this work is supported by NIH: UG3/UH3 TR002884 and U18TR003807 for Eduardo Reátegui.


References (Optional): : Lab Chip, 2022, 22, 2502-2518