Reprogramming the Tumor-Immune Microenvironment: Turning Hypoxic Immunologically “Cold” Ovarian Tumors into “Hot” Tumors

Introduction: High-Grade Serous Ovarian Carcinoma (HGSOC) is the most common and deadliest subtype of ovarian cancer (OC) characterized by recurrence and resistance to chemotherapy and immunotherapy in more than 80% of patients in advanced stage. Paradoxically, OC was one of the first cancers where the presence of tumor-infiltrated lymphocytes (TILs) was correlated with better immunotherapeutic response and survival rate. One of the key hallmarks of HGSOC is hypoxia (low oxygen levels) which dictates an immunologically cold tumor microenvironment (TME) characterized by enhanced immunosuppressive signals and cell types and activation of cancer-associated fibroblasts (CAF). Activated CAF contribute to a dysregulated collagen secretion through TGF-β signaling leading to increased stiffness of the extracellular matrix (ECM) that forms a physical barrier for the infiltration of TILs and immunotherapy. Despite the physiologically relevant oxygen levels in the healthy ovary being about 7% O2, current studies compare the hypoxic TME (1.5% O2) with hyperoxic culture models (21% O2). Therefore, there is a pressing need to establish tissue biomimetic models to recapitulate malignant hypoxic and healthy physoxic ovarian environments.

Our overall objective is to develop and validate a physiologically relevant biomimetic immuno-engineered model to study immunologically cold TME in HGSOC and identification of novel therapeutic targets. In the absence of these models, the development of effective targeting strategies turning hypoxic immunologically “cold” tumors into “hot” ones would remain difficult.

Materials and

Methods: A patient-derived physiologically relevant 3D model was bioengineered to mimic the physoxic levels of the healthy ovary (3D-Physoxia) and the hypoxic O2 levels of the OC-TME (3D-Hypoxia) by modulating matrix height, crosslinking, and exposure to externally controlled oxygen levels (Fig. A). The 3D model is based on crosslinking of human plasma fibrinogen into fibrin, generating a personalized 3D scaffold used for culture of patient-matched peripheral blood mononuclear cells (PBMCs) with cancer cells and CAF from HGSOC tumor biopsies mimicking the HGSOC-TME. Biomechanical characterization of the 3D model relied on the determination of oxygen levels by optical sensor and confocal imaging of Image-iT hypoxia dye, validation of stiffness by atomic force microscopy (AFM), and collagen I secretion by immunohistochemistry. For immune infiltration assays, PBMCs were added on top of the 3D model, or they were co-cultured inside the matrix to study evasion mechanisms once they penetrated the tumor in either 3D-Physoxia or 3D-Hypoxia. Confocal fluorescent microscopy was used to detect infiltrated CD45+ (green) immune cells into 3D matrix with OC cells MUC-1+ (magenta) in monoculture or coculture with CAF CD90+ (red) and confirmed by immunophenotyping studies by flow cytometry to determine changes in different immune populations including T cells, B, cells, NK cells, macrophages, and myeloid-derived suppressor cells, as well as activation, exhaustion, and intracellular cytokine expression to validate their functionality. Secreted immune cytokines were detected by cytokine multiplex array. Galunisertib, a TGF-β1 receptor inhibitor, was used to overcome CAF activation and ECM remodeling to overcome the hypoxia-induced immunosuppressive environment.

Results, Conclusions, and Discussions: The engineered 3D platform recapitulated the physiologically relevant hypoxic HGSOC oxygen level (0.7% O2, 3D-Hypoxia) when incubated in 1.5% O2 incubator and the physoxic healthy ovary oxygen level (7% O2, 3D-Physoxia) when incubated in 21% O2 (Fig B). Hypoxic status (green), an enhanced expression of collagen I, and significantly higher stiffness in coculture of HGSOC, CAF and PBMCs were confirmed in 3D-Hypoxia compared to 3D-Physoxia (Fig C&D). Significantly impaired infiltration of CD8+ T cytotoxic cells into the coculture of HGSOC with CAF was observed by flow cytometry in 3D-Hypoxia/ECMhigh compared to 3D-Physoxia/ECMlow (Fig E) and was rescued by pre-treatment with TGF-β inhibitor galunisertib (Fig F), suggesting an implication of CAF in ECM remodeling and immune infiltration through TGF-β signaling. Similar results were observed by confocal microscopy where the 3D-Hypoxia decreased infiltration of CD45+ PBMCs was rescued by galunisertib (Fig G). Furthermore, CD8+ cells expressed higher levels of cytolytic enzyme Granzyme B and had higher cytotoxic effect on HGSOC cells when cultured in 3D-Hypoxia/ECMhigh compared to 3D-Physoxia/ECMlow (Fig H). Phenotyping and functionality of CD8+ cells showed significantly enhanced expression of activation markers CD69, intracellular IL-2, and secreted IFNγ in 3D-Hypoxia/ECMhigh compared to 3D-Physoxia/ECMlow (Fig I). Moreover, immunophenotyping in multi-culture conditions revealed an increased activated CD8+ cells with higher granzyme B and IL-2 secretion, but also immunosuppressive Treg population in the hypoxic HGSOC-TME (Fig J&K). Critically, these data suggest that hypoxia acts as a friend and foe in the HGSOC-TME by enhancing CD8 activation and cytotoxicity to kill the tumor cells but conversely also increasing an immunosuppressive TME with ECM remodeling impairing immune infiltration into the tumor.

In conclusion, we have engineered and functionally characterized a physiologically relevant 3D model to study the role of hypoxia and the dysregulated ECM remodeling on tumor-immune interactions. Our results are expected to have an important positive impact because they will shed light on how to improve immunotherapy efficacy and how to activate the immune system to kill tumor cells by turning immunologically “cold” tumors into “hot” ones by targeting the dysregulated ECM remodeling via TGF-β signaling.

Acknowledgements (Optional): This project is supported by NIH/NCI R21CA259158