Cancer Research | Multimodal γδ T-cell Cytotoxicity Overcomes Cancer Cell–Mediated Immunomodulation

This study systematically evaluates the interactions between γδ T cells and patient-derived organoids at the single-cell level through high-throughput analysis, revealing that multimodal cytotoxicity (AIC and ADCC) can overcome cancer cell-mediated immunomodulation. It provides new therapeutic strategies for colorectal cancer, particularly the microsatellite-stable (MSS) subtype.

 

Literature Overview
The article, titled 'Phenoscaping Reveals Multimodal γδ T-cell Cytotoxicity as a Strategy to Overcome Cancer Cell–Mediated Immunomodulation', published in the journal Cancer Research, reviews and summarizes the cytotoxic mechanisms of γδ T cells in various colorectal cancer organoid models and how their activity is affected by cancer cell immunomodulation. The study uses systematic single-cell analysis to reveal the functional heterogeneity of γδ T cells across different patient-derived organoids and explores how engineering can enhance their anti-tumor activity.

Background Knowledge
Colorectal cancer is a highly heterogeneous solid tumor, and its tumor microenvironment (TME) typically exhibits immunosuppressive properties, particularly in the microsatellite-stable (MSS) subtype, which shows poor response to immune checkpoint inhibitors. γδ T cells are innate immune cells capable of MHC-independent cytotoxicity and can also kill tumor cells through Fcγ receptor-mediated ADCC mechanisms. However, the functional regulatory mechanisms of γδ T cells across different donors and tumor models remain incompletely understood. Moreover, colorectal cancer stem cells (CSCs) are highly plastic, transitioning from chemotherapy-sensitive proCSCs to chemotherapy-resistant revCSCs, increasing therapeutic challenges. This study systematically maps the interaction landscape between γδ T cells and colorectal cancer organoids using high-throughput single-cell analysis (TOBis MC) and organoid co-culture systems, providing a theoretical foundation for developing more effective γδ T cell-based immunotherapies.

 

 

Research Methods and Experiments
The research team employed a novel high-dimensional single-cell analysis technique called TOBis MC (Thiol-Reactive Organoid Barcoding in situ Mass Cytometry) to analyze over 1,000 co-cultures of γδ T cells and colorectal cancer patient-derived organoids (PDOs). This method enabled quantification of γδ T cell signaling pathway activation, cell cycle status, apoptosis levels, and immune phenotype changes. CRISPR technology was also used to generate B7-H3 knockout PDO models to assess whether γδ T cell ADCC function depends on B7-H3 expression. Furthermore, flow cytometry and single-cell RNA sequencing (scRNA-seq) were employed to validate the immunomodulatory mechanisms of γδ T cells.

Key Conclusions and Perspectives

• Unmodified γδ T cells exhibited limited cytotoxicity in co-culture, while engineered γδ T cells expressing an IL15Rα–IL15 fusion protein (stIL15) effectively killed PDO organoids without requiring exogenous cytokine support.
• When relying solely on AIC-mediated killing, PDO organoids specifically suppressed γδ T cell cytotoxicity by reprogramming their PTM signaling network, demonstrating that cancer cells can negatively modulate γδ T cell function.
• Adding anti-B7-H3 monoclonal antibodies enabled γδ T cells to initiate ADCC mechanisms, effectively overcoming cancer cell immunomodulation and restoring cytotoxicity.
• The dual-mode (AIC + ADCC) killing strategy efficiently eliminated chemotherapy-resistant revCSCs, highlighting its potential for treating refractory colorectal cancers.
• Different PDO models exhibited patient-specific immunomodulation of γδ T cells, indicating that cancer cell heterogeneity (ITH) significantly impacts γδ T cell function.
• Engineered γδ T cells showed enhanced survival and proliferation in vivo, and B7-H3 knockout experiments confirmed the antigen-specific nature of the ADCC mechanism.

Research Significance and Prospects
This study reveals the multimodal cytotoxicity mechanisms of γδ T cells in colorectal cancer treatment and demonstrates that engineered γδ T cells can effectively overcome cancer cell immunomodulation. Future research could further optimize γδ T cell engineering, explore their applicability in other solid tumors, and combine them with personalized medicine strategies to improve anti-tumor efficacy.

 

 

Conclusion
Through high-throughput single-cell phenotypic analysis, this study systematically uncovered the complex interaction network between γδ T cells and colorectal cancer organoids. It was found that engineered γδ T cells expressing stIL15 can autonomously maintain cytotoxicity and overcome cancer cell immunosuppression through B7-H3-targeted ADCC mechanisms. Moreover, the multimodal (AIC + ADCC) killing strategy effectively eliminates chemotherapy-resistant cancer stem cells, offering a novel immunotherapeutic approach for colorectal cancer, particularly the MSS subtype. These findings not only deepen our understanding of γδ T cell anti-tumor mechanisms but also provide experimental support for optimizing γδ T cell therapy in solid tumors.

 

Callum Baird Nattress, Rhianna O’Sullivan, Daniel Fowler, Jonathan PH Fisher, and Christopher J Tape. Phenoscaping Reveals Multimodal γδ T-cell Cytotoxicity as a Strategy to Overcome Cancer Cell–Mediated Immunomodulation. Cancer Research.