(68b) Elucidating Dormancy in Invasive Lobular Carcinoma through Multidimensional Bioengineering

Introduction: Metastasis is the dissemination of tumor cells to vital organs and the leading cause of cancer-associated deaths. In breast cancer, the 5-year survival rate decreases from ~99% to ~31% once the cancer spreads from the primary tumor site to secondary locations. This indicates that the disseminated tumor cells (DTCs) acquire distinct survival mechanisms that circumvent traditional therapies and inflammatory responses. Metastasis can occur after long-term remission, providing evidence for DTCs that persist in the secondary locations and contradict the rapid growth kinetics of cancer. Further comprehending these dormant DTCs can inform therapeutic interventions. Late recurrences are prevalent in hormone-receptor-positive breast cancers of which the most common are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC). Despite accounting for 10-15% of breast cancers, ILC is understudied and treated on evidence from IDC research. Although ILC has overall lower rates of survival and increased numbers of DTCs compared to IDC, indicating dormancy as an underlying mechanism, research on ILC dormancy is minimal. ILC cells are slow-growing and require months for murine models to develop distant metastases. Therefore, there is an unmet need to develop novel in-vitro methods that expedite the dormancy timeline, while providing physiological relevance specific to ILC.

Methods: To model ILC dormancy, we developed three micro-compartmentalization approaches that span various cell culture dimensions. To investigate the role of the extracellular matrix (ECM), we developed large-scale arrays of protein micropatterns. The large-scale arrays were generated through surface chemistry via the covalent binding of poly(ethylene glycol) (PEG) to the coverslip surface. Digital-micromirror device (DMD)-based ultraviolet (UV) illumination degraded the PEG coating and promoted the adsorption of ECM into specific surface locations. To remove the interaction with the ECM, we developed large-scale non-adhesive microcuvettes through the in-situ radical polymerization of acrylated PEG. Briefly, oxygen inhibition was repurposed to produce topographical structures by tuning the photon flux of UV light. Lastly, we developed an isotropic culturing method by producing a microgel colloid via microfluidic generation of agarose microdroplets, which provided a non-adhesive and mechanically confining matrix for the cells to self-assemble. ILC cells were made drug-resistant to an anti-estrogen, tamoxifen, via long-term culture with low-dose tamoxifen. Furthermore, the ILC cells were engineered to express a biosensor that monitors the cell cycle in real time. Dormancy was induced by growth factor and oxygen deprivation and validated through immunofluorescence.

Results: Utilizing the three bioengineering approaches, we recapitulated the ductal and lobular morphologies of ILC and IDC, where the former were incapable of forming complex structures via the absence of E-cadherin, a cell-cell junction protein. While the drug-resistant cells were similar to the drug-sensitive cells in the microcuvettes and microgels, the cells demonstrated striking differences on the ECM, where the drug-resistant cells spread throughout the ECM surface and the drug-sensitive cells remained stagnant. By inducing dormancy on the micropatterns, the drug-resistant cells expressed a dormant phenotype to a greater extent than the drug-sensitive cells as described by p27^Kip1 positivity, which depended on the ECM that was micropatterned. To further understand the relationship between drug resistance and dormancy, we screened for various biomarkers and determined the epigenetic upregulation of histone methylation and downregulation of drug-resistance-associated miRNA as drivers of p27^Kip1 positivity. Furthermore, the drug-resistant cells were stem-cell-like under dormancy-inducing conditions with elongated morphologies and participated in mechanosensing as measured with nuclear YAP. Therefore, we removed the cellular association with the ECM by utilizing the microcuvettes and the microgels. By removing the ECM, the drug-resistant cells exhibited a deeper state of dormancy that was less pronounced for the drug-sensitive cells.

Conclusions: This investigation provides evidence for the benefits of utilizing chemical engineering approaches to overcome challenges in human health and in vivo research. Herein, various cell culturing technologies provided distinct aspects of cellular biology in high throughput. With the United States Food and Drug Administration (FDA) Modernization Act 2.0, bioengineering assays are receiving public attention and esteem as replacements for animal models. Therefore, we hope that this investigation further catalyzes the field of dormancy for an underrepresented subtype of breast cancer that deserves the spotlight.