Peripheral blood immune cell subsets as non-invasive biomarkers of colorectal cancer stage, laterality, metastasis and survival

Colorectal cancer (CRC) is a heterogeneous disease in which immune dynamics critically influence tumour progression and patient outcomes; however, the relationship between circulating immune cell subsets, immune checkpoints (ICs) expression, and clinical parameters remains poorly defined. In this study, we employed a high-dimensional spectral flow cytometry approach to analyse peripheral whole blood samples from 16 CRC patients, profiling 918 immune populations across both innate and adaptive compartments. Immune profiles were stratified by tumour stage (I vs. II–III), anatomical location (colon vs. rectum; right- vs. left-sided), and three-year metastasis and survival outcomes. Data analysis included unsupervised clustering, multiple t-tests, volcano plots, ROC curve analysis and logistic regression models. Preliminary patterns suggested associations between specific immune populations and clinical features: locally advanced-stage disease (stages II and III) tended to show higher frequencies of CD134+CD4+ conventional T cells and CCR6- CD161+CD57-CD8+natural killer T cells; rectal tumours were appeared enriched in regulatory T cells and basophils, and right- and left-sided colon cancers exhibited divergent CD4+CD8+ T cell distributions. Metastatic patients displayed an abundance of CD152+ CD14+ monocytes. Mortality correlated with the presence of CD141- CD1c+ myeloid dendritic cells and CD4-CD8- Conventional T cells. All immune signatures demonstrated perfect classification, with an area under the curve (AUC) value of 1.000. Despite the limited size of the cohort, these findings provide preliminary evidence for the potential of peripheral immune profiling as a non-invasive approach for prognostication and patient stratification in CRC.