Immunology

Microwave ablation (MWA) is a thermal ablation technique widely used for local tumor control that has the added potential to stimulate systemic anti-tumor immunity. Although MWA alone rarely eliminates recurrent or metastatic disease, its ability to remodel the tumor microenvironment makes it a promising partner for adoptive cell therapies such as chimeric antigen receptor (CAR)-T cells. However, reproducible protocols for combining these approaches remain limited. This protocol describes the integration of MWA with CAR-T therapy in tumor-bearing mouse models. Human hepatocellular carcinoma cell lines (Hep3B and SK-HEP-1) are inoculated subcutaneously into NOG mice to establish tumors. Localized MWA is performed at adjustable power and duration to induce partial or complete ablation. At defined intervals following MWA, CAR-T cells derived from healthy donor T cells and transduced with a lentiviral vector are injected intravenously. This experimental design uniquely separates MWA and CAR-T delivery, enabling precise evaluation of thermal preconditioning effects on the tumor microenvironment and subsequent CAR-T activity. By combining localized ablation with adoptive immunotherapy, the protocol provides a translationally relevant platform to optimize treatment timing, enhance CAR-T efficacy in solid tumors, and address key barriers in tumor immunology and cancer therapy.

Adoptive immune cell therapy, especially chimeric antigen receptor T (CAR-T) cells, has emerged as a promising strategy in solid tumor treatment, owing to its unique ability to specifically recognize and effectively eliminate tumor cells. Patient-derived organoids (PDOs) offer a robust and physiologically relevant platform for assessing the safety and efficacy of CAR-T-cell-based therapies. We now describe a detailed protocol for an in vitro evaluation system based on the co-culture of PDOs and CAR-T cells. This system encompasses the establishment of tumor organoids from patient tumor specimens, the isolation of T cells from matched peripheral blood mononuclear cells (PBMCs), and the generation of antigen-specific CAR-T cells. Through the use of fluorescent labeling to visualize different cells and apoptosis-related events post-interaction, along with quantitative analyses of T-cell proliferation, tumor organoid apoptosis, and the secretion of immune effector molecules, this system enables a robust and multifaceted evaluation of CAR-T cell cytotoxicity in vitro. Collectively, this co-culture system provides a systematic and reproducible in vitro platform for evaluating the functional activity of CAR-T cells and advancing research in tumor immunology and immunotherapy.

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector–target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality.