248 Word count, text (from the beginning of Introduction to the end of Discussion): 4,467 Reference count: 48 Table count: 3 Figure count: 3 Supplementary online material: 12 figures and 8 tables Disclosure of Potential Conflicts of Interest: M.C.L., J.B., S.O., and J.A.N. are listed as co-inventors on a provisional application for a patent titled "System for and Method of Discovering Spatially-Derived Signatures of Tumor-Immune Cell Interactions through Tumor-Immune Partitioning and Clustering" regarding novel methods for characterizing immune cell distributions in solid tumors that has been filed through Partners Healthcare. J.L.G. is a consultant for GlaxoSmithKline, Codagenix, Array BioPharma, and Verseau Therapeutics; and receives research support from GlaxoSmithKline, Eli Lilly and Array BioPharma for the study of the breast tumor microenvironment. A.T.

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Background Tumour budding and poorly differentiated clusters (PDC) represent forms of tumour invasion. We hypothesised that T-cell densities (reflecting adaptive anti-tumour immunity) might be inversely associated with tumour budding and PDC in colorectal carcinoma. Methods Utilising 915 colon and rectal carcinomas in two U.S.-wide prospective cohort studies, and multiplex immunofluorescence combined with machine learning algorithms, we assessed CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3 co-expression patterns in lymphocytes. Tumour budding and PDC at invasive fronts were quantified by digital pathology and image analysis using the International tumour Budding Consensus Conference criteria. Using covariate data of 4,420 incident colorectal cancer cases, inverse probability weighting (IPW) was integrated with multivariable logistic regression analysis that assessed the association of T-cell subset densities with tumour budding and PDC while adjusting for selection bias due to tissue availability and potential confounders, including microsatellite instability status. Findings Tumour budding counts were inversely associated with density of CD3 + CD8 + [lowest vs. highest: multivariable odds ratio (OR), 0.50; 95% confidence interval (CI), 0.35–0.70; P trend < 0.001] and CD3 + CD8 + CD45RO + cells (lowest vs. highest: multivariable OR, 0.44; 95% CI, 0.31–0.63; P trend < 0.001) in tumour epithelial region. Tumour budding levels were associated with higher colorectal cancer-specific mortality (multivariable hazard ratio, 2.13; 95% CI, 1.57–2.89; P trend < 0.001) in Cox regression analysis. There were no significant associations of PDC with T-cell subsets. Interpretation Tumour epithelial naïve and memory cytotoxic T cell densities are inversely associated with tumour budding at invasive fronts, suggesting that cytotoxic anti-tumour immunity suppresses tumour microinvasion.