Engineering the Hypoxic Immunologically Cold High-grade Serous Ovarian Carcinoma Microenvironment as a therapeutic target

Introduction:: High-Grade Serous Ovarian Carcinoma (HGSOC) is the most common and lethal subtype of ovarian cancer with a 5-year survival rate lower than 20% in the advanced stage, characterized by recurrence and resistance to chemotherapy and immunotherapy. Ovarian cancer was one of the first cancers where the presence of tumor-infiltrated lymphocytes (TILs) was correlated with higher patient survival and better immunotherapeutic response. Paradoxically, HGSOC is one of the most hypoxic (low oxygen levels) tumor types, dictating an immunologically cold microenvironment with unbalanced immunosuppressive cell types and signals and activated cancer-associated fibroblasts (CAFs) through TGF-beta signaling leading to dysregulated collagen secretion increasing the stiffness of the extracellular matrix (ECM) that acts as a physical barrier to the infiltration of TILs and immunotherapy. Unfortunately, current studies compare the hypoxic HGSOC environment (1.5% O₂) with hyperoxic culture models (21% O₂) even though the physiologically relevant oxygen levels in the healthy ovary are about 7% O₂. Therefore, there is a need to establish tissue biomimetic models that allow for the recapitulation of the malignant hypoxic and healthy physoxic ovarian environments. Our overall objective is to develop and validate a physiologically relevant biomimetic immuno-engineered system to study immune evasion in HGSOC and validate novel therapeutic targets. In the absence of these models, the development of effective targeting strategies turning hypoxic immunologically “cold” tumors into “hot” ones will likely remain difficult.


Materials and Methods:: We have bioengineered a patient-derived physiologically relevant 3D model to recapitulate the hypoxic O₂ levels of the ovarian cancer microenvironment (3D-Hypoxia) and the physoxic levels of the healthy ovary (3D-Physoxia) by manipulating 3D crosslinking, matrix height, and exposure to external controlled oxygen levels (Fig. A). The 3D model is based on crosslinking of plasma fibrinogen, generating a personalized 3D matrix used for multi-culturing matching peripheral blood mononuclear cells (PBMCs), HGSOC cells and CAFs from HGSOC patient biopsies recapitulating the HGSOC tumor microenvironment (TME), absence of HGSOC and CAFs was used as a control (Blank). Mechanical characterization of the 3D model consisted of validation of oxygen levels by optical sensor and confocal imaging of hypoxia green ImageiT dye, as well as determination of 3D model stiffness and collagen I composition by atomic force microscopy (AFM) and immunohistochemistry staining (IHC), respectively. PBMCs were either added on top of the 3D model to study immune infiltration, or co-cultured inside the matrix to study evasion mechanisms once they penetrate into the tumor in either 3D-Physoxia or 3D-Hypoxia models. Immunophenotyping studies were performed to identify changes to the different immune populations including T cells, B, cells, NK cells, macrophages and myeloid-derived suppressor cells (MDSCs), as well as activation, exhaustion and intracellular cytokine expression was measured to validate their functionality by flow cytometry. Cytokine multiplex array identified secreted immune cytokines. A TGF-β1 receptor inhibitor (Galunisertib, 10 μM) was used to overcome CAF activation and the dysregulated ECM remodeling in order to prevent the hypoxia-induced immunosuppressive environment.


Results, Conclusions, and Discussions:: The 3D matrix recapitulated the physiologically relevant physoxic healthy ovary oxygen level (7% O₂, 3D-Physoxia) when incubated in 1.5% O₂ or the hypoxic HGSOC oxygen level (0.7% O₂, 3D-Hypoxia) when incubated in 21% O₂ incubator at predefined crosslinking and height parameters (Fig B). We confirmed hypoxic status (green), higher secretion of collagen I and significantly higher stiffness in multi-culture of PBMCs, HGSOC and CAFs when in 3D-Hypoxia compared to 3D-Physoxia (Fig C&D). Infiltration studies demonstrated a significant impaired infiltration of CD8+ T cytotoxic cells and NK cells in 3D-Hypoxia/ECM^high compared to 3D-Physoxia/ECM^low driven by the TME presence (Fig E). Functionality and phenotyping of CD8+ cells revealed significantly enhanced expression of activation markers CD69, intracellular IL-2, and secreted IFNγ in 3D-Hypoxia/ECM^high compared to 3D-Physoxia/ECM^low (Fig F). Moreover, CD8+ cells expressed higher levels of cytolytic enzyme Granzyme B and had higher cytotoxic effect on HGSOC cells when cultured in 3D-Hypoxia/ECM^high compared to 3D-Physoxia/ECM^low (Fig G). Pretreatment with galunisertib (Gal) reversed CD8+ T cells infiltration into the matrix with TME cells, confirming the implication of CAFs in ECM remodeling and immune infiltration through TGF-β signaling (Fig H). Immunophenotyping in multi-culture conditions further revealed an increased immunosuppressive Treg population but activated CD8+ cells with higher Granzyme B and IL-2 secretion in the hypoxic HGSOC TME (Fig I&J). Critically, these results suggest that hypoxia acts as a friend and foe in the HGSOC-TME by increasing CD8 activation and cytotoxicity to kill the cancer cells but conversely also enhancing an immunosuppressive TME with ECM remodeling impairing immune penetration into the tumor. In conclusion, we have engineered and functionally characterized a physiologically relevant 3D model to investigate the influence 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 enhance immunotherapy efficacy and how to activate the immune system to destroy cancer cells by turning immunologically “cold” tumors into “hot” ones, possible targeting the dysregulated ECM remodeling via TGF-β signaling.


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


References (Optional): :