The BEHAV3D Platform Demonstrates Cellular Immunotherapy Modes of Action

Patient-derived organoids (PDO) can recapitulate critical aspects of the original tumor, including response to immunotherapy treatments. Imaging of these PDOs could be used to determine the dynamics of cellular immunotherapy as well as potentially improve therapy design, but this has not yet been accomplished. Dekkers, Alieva, and colleagues developed the BEHAV3D system, which uses both 3D imaging and transcriptomics to allow for live tracking after cellular immunotherapy and applied this platform to evaluate approximately 60 human breast cancer PDOs treated with cancer metabolome–sensing engineered T cells (TEG). A high variation in TEG killing ability was observed, and RNA sequencing of the breast cancer PDOs revealed cytokine signaling and extracellular matrix organization gene signatures being enriched in PDOs highly sensitive to TEG therapy, with the highest association to TEG killing being found with IFN-I signaling genes. BEHAV3D also identified nine unique subpopulations of TEGs after exposure to breast cancer PDOs that exhibited different behavioral patterns, and these were also observed after exposure to other cancer types or after treatment with other engineered T cell therapies, suggesting broad applicability of this platform. Additionally, investigation into the link between tumor-targeting behavior and population phenotypes demonstrated that TEG-mediated killing was dependent on prolonged organoid contact, a key feature of super engagers, with CD8⁺, but not CD4⁺, TEGs falling into this classification. These CD8⁺ TEGs also were capable of consecutive killing of multiple cells, suggesting that CD8⁺ TEGs are more potent tumor-targeting cells. To gain additional insight into transcriptional programs that underlie BEHAV3D-identified tumor-targeting dynamics, single-cell transcriptomic profiling was performed and showed dynamic gene changes to TEG subsets during tumor targeting, with the specific gene signature related to the serial killing super engager TEGs revealing 27 new genes with no previously described T-cell function. Moreover, studies also showed that IFNβ treatment can prime resistant breast cancer PDOs for TEG-mediated killing. In summary, this study provides the organoid-based, 3D imaging–transcriptomic platform BEHAV3D, which allows for greater understanding of the mode of action behind cellular antitumor immunotherapies and supports its application in guiding patient-specific treatments moving forward.

Dekkers JF, Alieva M, Cleven A, Keramati F, Wezenaar AKL, van Vliet EJ, et al. Uncovering the mode of action of engineered T cells in patient cancer organoids. Nat Biotechnol 2022 Jul 25 [Epub ahead of print].

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