Cytometry by Time-of-Flight for Profiling Therapeutic Response in Colorectal Cancer: Strengths, Limitations, and Translational Challenges.

BACKGROUND/AIM: Response assessment in colorectal cancer (CRC) still relies largely on anatomic imaging, which does not capture dynamic immune or stromal adaptation. Techniques like cytometry by time-of-flight (CyTOF) and imaging mass cytometry (IMC) enable high-dimensional single-cell proteomic and spatial profiling, thereby enhancing our understanding of therapeutic responses in CRC. This narrative review summarizes current evidence from human, preclinical, and organoid studies describing how CyTOF and IMC have been applied to characterize therapeutic response in CRC. MATERIALS AND METHODS: Relevant publications applying suspension CyTOF or IMC to CRC tissue, blood, or model systems were identified through PubMed and major oncology journals. Studies linking single-cell features to treatment response or pharmacodynamics were prioritized and organized by biological theme and clinical applicability. RESULTS: natural killer (NK) cells on a STAT5-biased background. Organoid and phospho-signaling studies show that cellular differentiation states determine oncogenic ERK activation and therapy tolerance. Collectively, these datasets outline pharmacodynamic and predictive biomarkers relevant to immunotherapy and chemotherapy. CONCLUSION: monocytes, and NK-cell maturation profiles - that refine response assessment beyond imaging criteria. Harmonization of antibody panels, prospective validation, and integration into clinical workflows are required to apply these findings in clinical practice.