Physicochemical properties of microfabricated hydrogel scaffolds modulate tumor dormancy and chemotherapy resistance in a hepatic all-human microphysiologic model of metastasis

Abstract

Event Abstract Back to Event Physicochemical properties of microfabricated hydrogel scaffolds modulate tumor dormancy and chemotherapy resistance in a hepatic all-human microphysiologic model of metastasis Alex J. Wang1, Marianna Sofman1, Jorge Valdez1, Sarah E. Wheeler2, Amanda M. Clark2, Venkateswaran C. Pillai3, Raman Venkataramanan2, 3, Carissa L. Young1, Douglas A. Lauffenburger1, Donna B. Stoltz2, Alan Wells2 and Linda G. Griffith1 1 MIT, Department of Biological Engineering, United States 2 University of Pittsburgh, Department of Pathology, United States 3 University of Pittsburgh, Department of Pharmaceutical Sciences, United States Introduction: Micrometastases are clinically chemoresistant, particularly during presumed dormancy, which prevents clearance of these cells that often emerge later to kill patients. Liver is a common site of metastasis for many cancers, including the particularly fatal “triple negative” breast cancer (TNBC). We have previously developed a microscale reactor for long-term highly functional culture of 3D liver-like tissue for use in drug metabolism and toxicity studies [1],[2]. In previous work, we used a thin polystyrene scaffold that fostered formation of 3D tissue structures when seeded with primary human liver cells comprising a mixture of hepatocytes and non-parenchymal cells (NPCs). Adaptation of this reactor system to study TNBC micrometastases in the liver microenvironment requires attention to environmental factors, such as scaffold biophysical properties, that may influence the phenotype of the liver cells. Polystyrene scaffolds are stiff and induce a mild inflammatory phenotype in the liver cells, and this inflammatory response may stimulate growth of the micrometastases. To eliminate this deleterious effect, we developed an entirely new approach based on microfabrication of functionalized PEG hydrogels. We hypothesized that providing a more physiological environment for liver cell seeding and subsequent introduction of cancer cells would promote breast cancer cell dormancy and provide increased protection from chemotherapy as seen in patients, enabling the study of this elusive but critical stage in cancer progression. Materials and Methods: Hydrogel scaffolds with defined channel geometries were formed by micromolding of polyethylene glycol macromers (700 MW linear PEG-diacrylate or 20K MW 8-arm PEG-norbornene) with thiol crosslinkers using UV-initiated polymerization of macromer solutions. Hydrogels were functionalized with fibronectin-derived cell adhesive domains [3]. Physiochemical properties, including swelling, stiffness, and peptide composition, were systematically varied [4],[5]. Hydrogel crosslinking strategies included the use of peptides susceptible to proteolytic degradation and/or controlled dissolution of gel upon addition of a bacterial transpeptidase that releases the trapped cells for further characterization. 3D liver co-cultures employing primary human liver cells were initiated in microperfused bioreactors and seeded with fluorescently-labelled MDA-MB-231 or MCF-7 breast cancer cells (500-1000 cells/well) as described previously [1]. Functional behavior of liver cells, inflammatory cytokines, and growth of tumor cells were monitored over 1-3 weeks of culture in either homeostatic or simulated inflammatory (addition of LPS) conditions. Results: Hydrogel scaffolds seeded with human hepatocytes and NPCs were comparable in efficiency and overall markers of hepatocellular health as assessed using injury markers, functional markers, and tissue morphology when compared to traditional polystyrene scaffolds. Interestingly, the LPS-induced inflammatory response was enhanced in hydrogel compared to standard scaffolds, presumably due to reduced basal activation. Cancer cell numbers and markers for proliferation in hydrogel were decreased on day 15 in hydrogel compared to rigid scaffolds, indicating a dormant phenotype. Treatment of cultures with chemotherapy showed a decline in cancer cell numbers in rigid scaffolds, which contained more proliferating cells. Conversely, cancer cell numbers in hydrogel scaffolds were unaffected, which implies increased dormancy. Discussion: The PEG hydrogel microenvironment more closely reflects physiologically relevant properties and appears to better maintain NPC survival and health, as well as increasing hepatic tissue density within each channel. Cancer cell proliferation is attenuated in hydrogels, possibly due to decreased cellular stress and inflammatory response by NPCs. The use of hydrogels in this system allows for the first model of metastasis escape from chemotherapy in an all-human system. Ongoing work is elucidating the signaling pathways involved in this response, as well as continuing to design more robust and modular hydrogel scaffolds for the liver bioreactor. We thank Linda Stockdale for assistance with micromolding of hydrogels; We thank Kasper Renggli for insightful discussions