Hypoxia-inducing cryogels uncover key cancer-immune cell interactions in an oxygen-deficient tumor microenvironment.

Hypoxia, an important feature of solid tumors, is a major factor shaping the immune landscape, and several cancer models have been developed to emulate hypoxic tumors. However, to date, they still have several limitations, such as the lack of reproducibility, inadequate biophysical cues, limited immune cell infiltration, and poor oxygen (O ₂ ) control, leading to non-pathophysiological tumor responses. As a result, it is essential to develop new and improved cancer models that mimic key features of the tumor extracellular matrix and recreate tumor-associated hypoxia while allowing cell infiltration and cancer-immune cell interactions. Herein, hypoxia-inducing cryogels (HICs) have been engineered using hyaluronic acid (HA) as macroporous scaffolds to fabricate three-dimensional microtissues and model a hypoxic tumor microenvironment. Specifically, tumor cell-laden HICs have been designed to deplete O ₂ locally and induce long-standing hypoxia. This state of low oxygen tension, leading to HIF-1α stabilization in tumor cells, resulted in changes in hypoxia-responsive gene expression and phenotype, a metabolic adaptation to anaerobic glycolysis, and chemotherapy resistance. Additionally, HIC-supported tumor models induced dendritic cell (DC) inhibition, revealing a phenotypic change in plasmacytoid B220 ⁺ DC (pDC) subset and an impaired conventional B220 ^- DC (cDC) response in hypoxia. Lastly, our HIC-based melanoma model induced CD8+ T cell inhibition, a condition associated with the downregulation of pro-inflammatory cytokine secretion, increased expression of immunomodulatory factors, and decreased degranulation and cytotoxic capacity of T cells. Overall, these data suggest that HICs can be used as a tool to model solid-like tumor microenvironments and identify a phenotypic transition from cDC to pDC in hypoxia and the key contribution of HA in retaining cDC phenotype and inducing their hypoxia-mediated immunosuppression. This technology has great potential to deepen our understanding of the complex relationships between cancer and immune cells in low O ₂ conditions and may pave the way for developing more effective therapies.