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 through high-throughput single-cell analysis, revealing that multimodal cytotoxicity (AIC and ADCC) can overcome cancer cell immunomodulation and offering new therapeutic strategies for colorectal cancer, particularly the microsatellite-stable (MSS) type.

 

Literature Overview
This paper, 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 influenced by cancer cell immunomodulation. Using systematic single-cell analysis, the study reveals functional heterogeneity of γδ T cells across different patient-derived organoids and explores how their anti-tumor activity can be enhanced through engineered modifications.

Background Knowledge
Colorectal cancer is a highly heterogeneous solid tumor, and its tumor microenvironment (TME) is typically immunosuppressive, especially 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 via Fcγ receptor-mediated ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity). However, the functional regulatory mechanisms of γδ T cells across different donors and tumor models are not fully understood. Moreover, colorectal cancer stem cells (CSCs) exhibit high plasticity, transitioning from chemotherapy-sensitive proCSCs to drug-resistant revCSCs, which complicates treatment. This study employs high-throughput single-cell analysis (TOBis MC) and organoid co-culture systems to systematically map the interaction landscape between γδ T cells and colorectal cancer organoids, providing a theoretical basis for developing more effective γδ T-cell immunotherapies.

 

 

Research Methods and Experiments
The research team employed a novel technology termed TOBis MC (Thiol-Reactive Organoid Barcoding in situ Mass Cytometry) to perform high-dimensional single-cell analysis on over 1,000 co-cultures of γδ T cells and patient-derived colorectal cancer organoids (PDOs). Using this approach, they quantified signal pathway activation, cell cycle status, apoptosis levels, and changes in immune phenotypes of γδ T cells. Additionally, CRISPR technology was used to generate B7-H3 knockout PDO models to assess whether ADCC functionality of γδ T cells is dependent on B7-H3 expression. Flow cytometry and single-cell RNA sequencing (scRNA-seq) were further employed to validate the immunomodulatory mechanisms of γδ T cells.

Key Conclusions and Perspectives

• Unmodified γδ T cells showed limited cytotoxicity in co-culture, whereas 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 (Antibody-Independent Cytotoxicity), PDO organoids specifically suppressed γδ T-cell cytotoxicity by reprogramming their PTM (post-translational modification) signaling network, indicating that cancer cells can inversely modulate γδ T-cell function.
• By introducing anti-B7-H3 monoclonal antibodies, γδ T cells activated the ADCC mechanism, effectively overcoming cancer cell immunomodulation and restoring cytotoxicity.
• The dual-mode (AIC + ADCC) killing strategy efficiently eliminated chemotherapy-resistant revCSCs, highlighting its potential in treating refractory colorectal cancer.
• Immune modulation of γδ T cells by different PDO models exhibited patient-specific patterns, indicating that cancer cell heterogeneity (ITH) significantly influences γδ T-cell functionality.
• Engineered γδ T cells showed enhanced survival and proliferation in vivo, and B7-H3 knockout experiments confirmed the antigen-specific nature of their ADCC mechanism.

Research Significance and Prospects
This study reveals the multimodal cytotoxic mechanisms of γδ T cells in colorectal cancer therapy and demonstrates their ability to overcome cancer cell immunomodulation after engineering. Future research should focus on further optimizing engineered γδ T-cell designs, exploring their application in other solid tumors, and integrating 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 sustain cytotoxicity autonomously and overcome cancer cell immunosuppression through B7-H3-targeted ADCC. Additionally, the dual-mode (AIC + ADCC) killing strategy effectively eliminates chemotherapy-resistant cancer stem cells, offering a novel immunotherapeutic approach for colorectal cancer, especially the MSS subtype. These findings not only enhance 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.