Poster T4 - Collagen Fiber Architecture Promotes Metastatic Ovarian Cancer Cell Adhesion and Spheroid Disaggregation

Introduction: High grade serous ovarian cancer (HGSOC) is the most common and deadly gynecologic cancer, with a 5-year survival rate of < 50%. In HGSOC, cells metastasize as single cells and spheroids in a transcoelomic fashion from the primary tumor to distal metastatic sites within the peritoneal cavity [1]. These distal sites undergo extracellular matrix alterations with disease progression; specifically, the density of fibrillar collagen is increased in metastatic tissues [2]. Collagen I (Col I) is known to direct cell behaviors such as adhesion and movement, but the influence of the contribution of fibers has not been extensively investigated in the context of HGSOC metastasis. Therefore, we sought to investigate the influence of increased Col I fibers on HGSOC behaviors that are directly related to metastasis – i.e., single-cell adhesion and spheroid spreading – using biomimetic in vitro scaffolds. Furthermore, we sought to model and independently modulate the biochemical adhesive sites and biophysical structure unique to fibrillar collagen and elucidate which fiber-specific features were responsible for guiding cell outcomes. Using these representative model systems, we will be able to interrogate the effect of fibrillar collagen presentation on incoming HGSOC cell adhesion and spheroid spreading and understand what aspects of fibrillar collagen drives HGSOC progression.

Materials and

Methods: To model the presentation of Col I fibers representative of healthy vs metastatic omentum we used an in vitro semi-interpenetrating methacrylated gelatin (GelMa) network to evaluate the influence of Col I fibers independent of changes in protein density or stiffness [3]. In this material, stiffness was kept at 12 kPa by tuning GelMA cross-linking, and we added equal concentrations of either fibrillar Col I (GelMa/Col I) or denatured Col I (commonly called gelatin, GelMa/Gel). To further probe the independent contributions of Col I biochemistry and architecture to cell outcomes, two different synthetic-based scaffold systems were employed. Specifically, polyethylene glycol (PEG) was functionalized with adhesive peptides found in gelatin (RGD) or unique to fibrillar Col I (GFOGER), while polycaprolactone (PCL) was either electrospun into fibrillar mats with Col-mimetic fiber dimensions or presented as a flat, non-fibrillar sheet. Single-cell adhesion and spheroid spreading were evaluated for HGSOC cells/spheroids seeded atop gels made of GelMa/Gel, GelMa/Col, PEG-RGD, or PEG-GFOGER, and flat or fibrous PCL constructs. HGSOC single-cell adhesion was evaluated after 3 hours by washing with PBS and quantifying for number of cells adhered per 10x frame, while spheroid spreading was quantified by calculating fold change in spheroid area after 48 hours.

Results, Conclusions, and Discussions: Single-cell adhesion and spheroid spreading were significantly increased on GelMa/Col I substrates relative to GelMa/Gel, suggesting a pro-adhesive role for Col I (Fig. 1 A). HGSOC single-cell morphology at 24 hours also displayed significantly more spread morphology on GelMa/Col I as compared to GelMa/Gel. These behaviors do not appear to be dependent upon the biochemical presentation of Col I-specific adhesive sequences, as adhesion and spheroid spreading were not promoted on mimetic PEG-GFOGER substrates compared to PEG-RGD. (Fig. 1 B). In contrast, single-cell adhesion and spheroid spreading were significantly higher on fibrous compared to non-fibrous PCL constructs, suggesting that Col I architecture plays a dominant role in these processes (Fig. 1 C). These data suggest that Col I fiber presentation could help to promote metastatic progression by supporting adhesion through a mechanism that is independent of adhesive ligand identity and instead may be driven by the biophysical architecture of Col I. In our future work we hope to interrogate the mechanism driving these promoted HGSOC behaviors by investigating how fiber morphology/architecture influences adhesion initiation and strengthening. Specifically, we will evaluate how cell actin polymerization and myosin II contractility is altered as a result of interactions with Col I fibers and potentially linked with the promoted HGSOC cell outcomes.

Acknowledgements (Optional): Funding for this study was provided by NIH 5R01CA232517. UW Soft Materials Characterization Lab and UW Optical Imaging Core were used to help analyze samples.

References: (1) Tan, D. S., (2006). The Lancet. Oncology, 7(11), 925–934 (2) Pearce, O. M. T., (2018). Cancer discovery, 8(3) (3) Berger, A. J., (2017). Biomaterials, 141, 125–135.