Introduction: Hypoxia, defined as low oxygen tension, is a characteristic feature of solid tumors and a hallmark of aggressive cancers. The rapid growth of tumors often results in the development of a hypoxic microenvironment, leading to tumor cell growth and invasion, resistance to apoptosis, and multidrug resistance. Multicellular tumor spheroids (MCTS) have been used to model solid tumors and emulate key aspects of tumor biology such as hypoxia. However, challenges with MCTS formation and reproducibility, inadequate biomechanical cues provided to cells, and uncontrolled oxygen depletion among other limitations led to nonphysiologic tumor cell responses. As a result, predicting clinical responses to anticancer drugs with MCTS remains a major challenge in cancer treatment. To model solid tumors more accurately, our team has recently developed an innovative approach using a cryogel scaffold inducing rapid oxygen depletion while enabling cell rearrangement into 3D spherical cellular aggregates. Our main objectives were: (1) to engineer hypoxia-inducing cryogels (HIC) to generate cellular hypoxia; (2) to provide a biophysical support enabling tumor cell attachment, proliferation, and remodeling; and (3) to evaluate acquired resistance of hypoxic B16-F10 melanoma cells to a common anticancer drug.
Methods: HIC were fabricated via a cryogelation process using methacrylated hyaluronic acid (HAGM). RGD peptide, catalase, and glucose oxidase (GO) were incorporated into the gel to promote cell attachment and viability while depleting oxygen. Oxygen depletion was monitored using needle-type oxygen microprobes in the presence or absence of glucose. B16-F10 cell viability was evaluated by confocal imaging. Cellular hypoxia was monitored using a fluorescent hypoxyprobe. Finally, alamarblue assay was performed to evaluate drug resistance of B16-F10 cells to various concentrations of doxorubicin (DOX).
Results: We demonstrated that HICs induce a controlled and sustained depletion of oxygen leading to hypoxia (~1% O2) up to 48h. Hypoxia induction is dependent on both GO and glucose contents. Unlike regular cryogels (control), HIC promoted cell remodeling into MCTS-like structure with high cell viability (95 ± 2%), while inducing a substantial level of cellular hypoxia (94 ± 3%) after 24h. Finally, HIC significantly induced melanoma resistance to DOX. With our highest DOX concentration (2μM), 100% and 77% of drug-resistant B16-F10 cells in HIC remained alive after 24h and 48h treatment, respectively. In comparison, all B16-F10 cells died within 48h in our control condition (blank cryogels).
Conclusions: We have engineered advanced cryogels, namely HIC, capable of inducing hypoxia while promoting tumor cell remodeling and anticancer drug resistance. This suggests that tumor-laden HIC may mimic key aspects of aggressive tumor microenvironments, making them a promising platform for drug screening and potentially improving preclinical drug discovery and testing.
Citation Format: Thibault Colombani, James Sinoimeri, Sidi A. Bencherif. Engineering cryogels to modulate oxygen tension in reconstructed tumor microenvironments [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B106.