Tunable Poly(ethylene glycol)-based Hydrogels for Reductionist Models of the Tumor Microenvironment

Tumor growth, survival, and metastasis depend upon interactions with the matrix and cells which compose the tumor microenvironment (TME). Tumor cell interactions with transformed extracellular matrix (ECM) and immune cells, such as tumor associated neutrophils (TANs) have been shown to affect tumor progression both clinically and in animal models. Unfortunately, while the complexity of the TME is difficult to recapitulate in standard cell culture, it is also difficult to analyze and to influence in vivo. Researchers have sought to circumvent these challenges by developing 3D models in naturally derived matrices like collagen and Matrigel, but these scaffolds often sacrifice biochemical tunability and fail to meet the stiffness regimes often seen in malignant ECM. Thus, there is a need for highly controlled 3D culture systems which mimic the biophysical properties and cell-cell interactions of the TME to better investigate how these interactions direct cancer development and progression. To meet this goal, researchers have employed synthetic hydrogel systems for 3D in vitro cell culture. Our lab has previously engineered a synthetic scaffold to serve as an ECM by incorporating a matrix metalloproteinase cleavable peptide into a biocompatible poly(ethylene glycol) (PEG) backbone and grafting in an integrin-binding peptide. This system allows independent tuning of adhesivity and matrix stiffness and supports tumor cell growth and spheroid formation. Here, we present two applications of this system to model cell-matrix interactions in the TME. First, we orthogonally tuned the adhesion ligand concentration and stiffness of our PEG-based hydrogels to investigate the individual and interactive impact of these matrix properties in a physiologically relevant regime. We assessed the tumor progression of a fibrosarcoma cell line (derived from mesenchymal cells) and a triple-negative breast carcinoma cell line (TNBC, derived from epithelial cells) cultured in and on these hydrogels. We observed that the cell proliferation, invasion, and focal complex formation in the fibrosarcoma cells responded to changes in matrix stiffness, while the same behaviors in the TNBC cell line occurred in response to changes in matrix adhesivity. to discern the differential behavior of these broad classes of tumors. We found no interactive effect between the two matrix properties within the conditions tested. These results helped to reiterate the importance of independently tunable systems in assessing cell response to specific matrix properties and established our system’s ability to discern differential tumorigenic behavior across diseases. Next, we incorporated neutrophil extracellular traps (NETs) in our system to study their impact on tumorigenesis in TNBC cells in a highly controlled environment. We observed that NETs helped to increase cell survival, proliferation, and pro-metastatic morphological phenotype. We also showed that the presence of NETs influenced the secretion of IL-8, a pro-NETosis factor, and that conditioned media from cells cultured in these gels influenced NETosis in an IL-8 dependent manner. The results observed in this system correlate with murine models and clinical studies in the literature and help to provide additional insight of the individual factors at play in the NET-mediated promotion of TNBC progression and metastasis. To expand upon current models of cell-matrix interactions within the TME, we applied an existing model to assess the differences in behavior between a fibrosarcoma and TNBC cell line and developed a new model for assessing NETs as a matrix biomolecule in TME models. These two reductionist models help to advance our understanding of the roles specific aspects of the TME play in tumor progression, ultimately helping to drive better rational design of TME-targeted therapies to improve clinical outcomes of cancer patients.