Tumor budding and poorly differentiated clusters as a biological continuum in colorectal cancer invasion and prognosis.

Tumor budding (TB) and poorly differentiated clusters (PDCs) are features of infiltrative growth patterns and powerful independent prognostic factors in colorectal cancer (CRC), yet the underlying biological mechanisms behind their role in CRC invasion is less understood. The aim of this study was to investigate the molecular background and prognostic role of tumor cluster size at the invasive margin (IM) of CRC, and determine whether a biological continuum between TB and PDCs exists. Using a combination of spatial transcriptomic and immunohistochemical (IHC) techniques, we demonstrated a biological continuum from larger to smaller tumor clusters, with TB possessing greater invasive potential than PDCs. We deployed artificial intelligence on a cohort of 1134 Stage I-III CRC resections to automatically detect nearly 400,000 isolated tumor cells/clusters of any particular size across the IM. We determined that 2-celled clusters were the most abundant feature at the IM, and the simultaneous assessment of TB and PDCs yielded a prognostic performance stronger than either independently. Our study provides a deeper understanding of the mechanisms behind CRC invasion while improving risk stratification for Stage I-III CRC.