Characterization of Lung Cancer Cell Growth in Hypoxia Inducing Cryogels (HICs)

Nahis Cruz-Delgado^1,2, Suzanne Lightsey³, Zachary Rogers⁴, Sidi Bencherif⁴ , and Blanka Sharma³

1. Department of Chemical Engineering, University of Puerto Rico, Mayaguez, Puerto Rico
2. Department of Chemical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, Florida
3. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, Florida
4. Department of Chemical Engineering, Northeastern University, Boston, Massachusetts

Cancer is the second leading cause of death globally. Novel immunological treatments have been developed to treat this disease, but these therapies are often less effective against solid tumors. Hypoxia, defined as low levels of oxygen (<5%O₂), is a cancer hallmark that impedes the migration of immune cells in solid tumors. Conventional 2-D cultures and tumor spheroids fail to preserve the low levels of oxygen that characterizes solid tumors. As such, a 3D platform that replicates tumor hypoxia is needed to bridge the gap between 2D and in vivo models. The objective of this study was to investigate the feasibility of using a hypoxia-inducing scaffold to model the lung tumor microenvironment, as a first step towards using this system to study immune cell interactions.

Lung cancer cells were seeded into hypoxia-inducing cryogels (HICs), which comprise methacrylated-hyaluronic acid that is cross-linked and freeze-thawed to obtain a macro-porous and interconnected 3D network. The cryogels contain glucose oxidase, which converts oxygen into hydrogen peroxide, creating a hypoxic environment. Two different lung cancer cell lines, A549 and H1299, were seeded on the HICs and cryogels without the glucose oxidase enzyme as a control. Different seeding times were examined, and cell growth was quantified over 7 days using double-stranded DNA and metabolic activity assays. To confirm the induction of cellular hypoxia, staining for hypoxia-inducible factor alpha (HIF-1α) was performed. The cultures were also characterized for TGF-β production, a major immunosuppressive cytokine.

We found that 2 hours was an optimal time of adhesion where we observed similar number of cells adhering to the hypoxia and control cryogels. When cultured over 7 days, no significant differences were observed in growth between the HICs and control group until day 7 of culture. HIF-1α expression was increased in H1299 cells in HICs on day 5, indicating that the cells are responding to hypoxia. For the TGF-β analysis we did not observe significant differences between HIC’s and the control cryogels; therefore, this interaction will need to be studied further.

Together this data indicates that HICs are capable of replicating some of the hypoxia-related processes observed in vivo. Through my work, we can predict the number of cancer cells in the cryogels and use this to estimate the effector-to-target cells ratio (E:T), which is necessary for the quantification of immune cells cytotoxicity. Future efforts will include investigating hypoxia-induced immunosuppression of natural killer (NK) cells, the major effector cells of the innate immune system.