CARTIA: a functional screening platform for engineering the next generation of CAR T-cells against hypoxic solid tumors

Introduction:: Chimeric Antigen Receptor T-cell (CAR T) therapy has shown tremendous success against hematological cancers; however, this clinical efficacy has fallen short in the context of solid tumors. The immunosuppressive solid tumor microenvironment (TME), characterized by nutrient deficiency, hypoxia, and metabolic rewiring has been shown to exacerbate T-cell dysfunction. While recent advances in immunoprofiling have identified drivers of this dysfunctionality, there is still a need to elucidate the combinatorial role of TME-associated signals in impeding tumor infiltrating lymphocytes in solid tumors. A more comprehensive understanding of mechanisms in which T-cells mitigate TME-driven dysfunction will be critical in developing clinically viable CAR Ts for solid tumors.

In vitro models that recapitulate key features of the TME can be an effective strategy for studying TME regulation of T-cells. A recent study demonstrated that hypoxia and continuous antigen stimulation can drive T-cells to an exhaustive state in vitro; however, there remain significant differences in transcriptome profiles of these in vitro exhausted T-cells compared to those seen in tumor infiltrating lymphocytes of hypoxic tumors. This indicates the role of additional properties of the solid tumor that drive a distinct dysfunctional phenotype and highlights the need for a holistic interrogation of the TME-associated roadblocks to CAR T therapy.

To address these issues, we developed a 3D-CAR T Infiltration Assay (CARTIA), a biomimetic in vitro platform that combines a hypoxic 3D solid tumor model with single-cell sequencing and screening to identify CAR Ts with superior functionality in the hypoxic, nutrient deficient, and metabolically altered solid TME.


Materials and Methods:: U251 glioblastoma cells expressing a hypoxia-driven biosensor, UnaG/mOrg, were cultured in a Gelatin-Methacrylate (GelMA) hydrogel for 24-96 hours. Oxygen levels that achieve a viable and hypoxic cancer cell-embedded gel were modeled via COMSOL, and biologically validated by UnaG expression. A gel compatible cryosectioning and immunohistochemistry (IHC) protocol was developed to characterize the spatial expression of hypoxia-related proteins/genes in the gel.


Results, Conclusions, and Discussions:: COMSOL simulations show that a 2 million cell/ml embedded gel can achieve an oxygen profile along its vertical axis (Fig. 1A). Several media and gel heights were tested to ensure an oxygen gradient is formed (Fig. 1B) while cell viability within the gel is maintained up to 96 hours (Fig. 1C-E). Extended cancer cell survival within the model is critical as 1) 24-48 hours is needed to first rewire cancer cells to form a intrinsically-driven hypoxic and metabolically disrupted tumor model and 2) the T-cell:cancer cell co-culture period must be maintained for 72-120 hours to achieve TME-driven CAR T-cell dysfunction.

We further developed a cryosectioning protocol to process the hydrogels for IHC. A 50-um cryosection of a hydrogel tumor model embedded with UnaG/mOrg+ U251 cells was stained with a nuclear dye, DAPI, demonstrating uniform cell distribution within the 3D gel and maintenance of UnaG signal after hydrogel processing (Fig. 1B).

These results serve as a proof of concept for our hypoxic, cell embedded GelMA hydrogel to model the solid TME, which forms the foundation of the CARTIA screening platform. Next, we will characterize the biological response of cancer cells to hypoxia in this model through IHC and qPCR analysis. In parallel, we will introduce CAR Ts into CARTIA, to characterize and improve their functions with an overarching goal of implementing a screening library to determine CAR T variants with improved oxidative stress management, infiltrative properties, cytotoxicity, and differentiation status in the solid TME.


Acknowledgements (Optional): : This work has been supported by an NIH NCI grant R01CA220012, STOP CANCER Marni Levine Memorial Research Career Development Award, and USC Viterbi School of Engineering.

References (Optional): : [1] Chen et al. Nat. Rev. Drug Discov. (2021); [2] Albelda et al. Annu. Rev. Med. (2017); [3] Scharping et al. Nat. Immunology (2021); [4] Wang D. et al. Cancer Discov. 2021