Trogocytosis, a process involving the transfer of membrane fragments between interacting cells, has recently emerged as an important mechanism in tumor–immune cell communication. Through this process, tumor cells can acquire immune regulatory molecules from immune cells, thereby contributing to immune evasion and resistance to immunotherapy. However, whether specific cancer cell subtypes preferentially engage in trogocytosis and the molecular mechanisms underlying this process remain poorly understood.

Cancer stem cells (CSCs) are known to play critical roles in tumor initiation, recurrence, and therapeutic resistance due to their self-renewal and differentiation capacities. Given their distinct cellular properties, we hypothesized that CSCs may exhibit enhanced trogocytosis compared to non-CSC tumor cells and that this phenomenon may be associated with alterations in membrane biophysical properties.

To investigate this hypothesis, we established a CSC-enriched spheroid model using the colorectal cancer cell line Caco-2. Three-dimensional spheroid culture was employed to increase the CSC population and to better mimic the tumor microenvironment. Trogocytosis between cancer cells and T cells was quantified using a co-culture system followed by flow cytometric analysis. Furthermore, differences in membrane fluidity between CSC and non-CSC populations were assessed using the polarity-sensitive fluorescent probe di-4-ANEPPDHQ, allowing quantification of membrane lipid order through generalized polarization (GP) analysis. Candidate genes associated with membrane dynamics and trogocytosis were further evaluated by quantitative PCR and functional perturbation approaches.

Finally, publicly available colorectal cancer single-cell RNA-sequencing datasets were analyzed to determine whether the identified candidate genes are enriched in CSC clusters in patient tumors.

This study aims to identify cancer cell subtypes with enhanced trogocytosis and to uncover membrane fluidity-dependent molecular mechanisms regulating this process. Our findings may provide new insights into CSC-mediated immune evasion and suggest potential therapeutic targets for improving cancer immunotherapy.