bioRxiv2026
BackgroundStage III colorectal cancer poses a significant threat of metastasis development, as tumour resection and adjuvant chemotherapy do not guarantee prolonged disease-free survival. ObjectiveThe spatial, quantitative, and qualitative characteristics of various cell types within tumour tissues could be key to developing accurate prognostic AI models. DesignTissue microarrays created from primary tumour tissues collected during surgical resection from a cohort of 493 stage III colorectal cancer (CRC) patients were analysed for 61 protein markers at the single-cell level using multiplexed immunofluorescence imaging via the Cell DIVE platform. Subsequent cell-type classification enabled quantitative cell-type analyses, co-localisation neighbourhood assessments, and cell-type-specific protein signature discoveries that distinguish between early and late/non-recurring patient samples. ResultsThis study identifies a stem cell protein profile that drives tumour relapse. A deep neural network (DNN) model, based on a stem cell protein signature composed of BAX, MLKL, FLIP, GLUT1, and CDX2, provided accurate prognosis for stage III CRC patients in both discovery and validation cohorts and in an independent validation cohort. Nodal count-based metric further increased prognosis accuracy. Our study also revealed distinct spatial arrangements of immune, endothelial, and stem cells that were linked to early tumour recurrence. ConclusionOur findings propose a clinically promising prognostic tool based on a five-protein stem cell signature. These markers not only predict chemotherapy resistance in cancer stem cells but also suggest potential therapeutic strategies such as combinatorial treatments incorporating small molecule inhibitors targeting FLIP and GLUT1. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIMore than 20% of stage III colorectal cancer patients will experience early tumour recurrence within the first 3 years post treatment that includes surgery and adjuvant 5-FU based chemotherapy treatment. C_LIO_LISeveral studies pointed towards involvements of number of cell type specific spatial neighbourhoods in tumour progression where some immune tumour microenvironment promoting angiogenesis and intravasation events, some may provide immunosuppression. C_LIO_LICancer stem cells could be responsible for metastatic tumour spread, early recurrence and chemoresistance. C_LI What this study addsO_LISpatial single cell quantitative multiplex profiling of 45 cancer hallmark proteins and 15 cell identity markers in 493 stage III CRC patients tissue samples demonstrated significant differences in cellular proximity neighbourhoods, cell type specific abundance and expression between the early and late recurrence samples. C_LIO_LIWe discover that macrophages show spatial association with the blood vessels in early recurrence samples. Moreover, we observed conglomeration of B cells and macrophages with Tregulatory, Thelper and Tcytotoxic cells in association with early recurrences. C_LIO_LIWe showed that stromal abundance of Tregulatory, Thelper, Tcytotoxic cells and monocytes are significantly in late, and no recurrence samples compared to early recurrence samples. C_LIO_LIThe most differential expression profile that differentiates late and no recurrence samples from the early recurrence samples is related to the stem cell population. Particularly, we found overexpression of GLUT1, FLIP and downregulation of BAX, BAK, MLKL and CDX2 proteins in the cancer stem cell of early recurrence samples. C_LIO_LIWe built a neural network based on the cancer stem cell protein signature (BAX, MLKL, FLIP, GLUT1 and CDX2 proteins) that delivers a high-performance prognostic classifier. C_LI How this study might affect research, practice or policyO_LIOur results propose a clinically promising prognostic tool based on a five-protein stem cell signature that outperforms existing clinical and proposed transcriptomic based signatures for separation between risk groups. C_LIO_LIMoreover, our five-protein signature markers not only predict stem cell chemotherapy resistance and therefore tumour recurrence but also suggest potential therapeutic strategies. For instance, this approach could guide combinatorial treatments at high risk of chemoresistance, such as incorporating small molecule inhibitors targeting FLIP (currently in discovery phase) and GLUT1 (already under preclinical trial evaluation). C_LI