**Background:** Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, with the adenoma-carcinoma sequence being the accepted model of sporadic tumourigenesis. Inflammation is a hallmark of cancer, and immune cell composition of the tumour microenvironment influences prognosis. High Mobility Group Box 1 (HMGB1) is a ubiquitous nuclear protein that stabilises DNA, regulates transcription, and enhances DNA repair. Under cellular stress, HMGB1 undergoes post-translational modification, shuttles to the cytoplasm, and is released extracellularly where it acts as a damage-associated molecular pattern cytokine. While HMGB1 has been implicated in many epithelial malignancies, its dynamic subcellular expression and biological significance across colorectal neoplastic progression had not been defined in large cohorts.
**Methods:** A total of 846 human colonic tissue samples yielding 6242 immunostained sections were analysed, including a CRC tissue microarray (TMA, n = 650 with 50 matched normal pairs), 75 normal colonic biopsies, 52 adenomatous polyps, and 69 cancer polyps (CaP; 28 initial plus 41 validation). Epithelial HMGB1 expression was assessed by immunohistochemistry using a semi-quantitative scoring system (intensity: absent, weak, moderate, strong) by two independent observers. Stromal immune cell phenotyping (CD3, CD4, CD8, CD20, FOXP3, ICOS, IDO-1, PDL1) was performed on the CRC TMA using QuPath digital image analysis, with immune cells categorised as 'immune cold' versus immune NOS based on prior published criteria. Cancer polyps additionally underwent phenotyping for CD68, CD20, CD4, CD8, and FOXP3 at the invasive cancer margin. Statistical analysis used Pearson's chi-square, Fisher's exact, Mann–Whitney U, Kruskal–Wallis, and log-rank/Kaplan–Meier tests with alpha = 0.05.
**Key Results:** Normal colonic epithelium showed strong nuclear and absent cytoplasmic HMGB1. In CRC, nuclear HMGB1 was reduced (p < 0.001), with the switch occurring between normal and T1 stage, and cytoplasmic HMGB1 emerged (p < 0.001). Cytoplasmic HMGB1 was associated with lymph node positivity (p < 0.001) and male sex (p = 0.009). Absent (p = 0.001) or weak (p = 0.010) nuclear HMGB1 was associated with mismatch repair protein expression. In cancer polyps, moderate/strong nuclear and cytoplasmic HMGB1 at the invasive cancer margin was seen in 77% of the initial cohort and 82.1% of the validation cohort. No relationship was demonstrated with p53, RUNX3, KRAS, BRAF, or MSI status. In the CRC TMA, stronger nuclear (p = 0.011) and cytoplasmic (p = 0.002) HMGB1 was associated with increased CD4+ T-cell density. Strong nuclear HMGB1 was also associated with greater FOXP3+ (p < 0.001) and ICOS+ (p = 0.018) lymphocyte density, and with reduced CD8+ T-cell density (p = 0.022). HMGB1 expression was not directly associated with overall survival (nuclear p = 0.213; cytoplasmic p = 0.498). However, patients with 'immune cold' tumours had poorer survival (p < 0.001), and strong cytoplasmic HMGB1 was associated with the 'immune cold' phenotype (24% vs 15%, p = 0.029), as well as reduced IDO-1 (p = 0.010), ICOS (p = 0.002), and stromal PDL1 (p = 0.027) expression.
**Clinical Implications:** The findings reveal a pathogenic HMGB1 expression profile switch in early CRC, with reduced nuclear HMGB1 potentially leaving the genome vulnerable to DNA damage and linking to mismatch repair protein expression. The association with mismatch repair status is clinically relevant because microsatellite unstable tumours have high neo-epitope loads and may respond favourably to immunotherapy. The pro-tumour immune profile (increased CD4+, FOXP3+, ICOS+; reduced CD8+) suggests HMGB1 orchestrates an immunosuppressive microenvironment, while the link to 'immune cold' phenotype—conferring poor survival—positions HMGB1 as a candidate therapeutic target, potentially in combination with immunotherapy or chemotherapy to remodel the tumour microenvironment. The association of cytoplasmic HMGB1 with male sex parallels the established male disadvantage in CRC outcomes and warrants further investigation. The lack of direct survival association in this cohort, in contrast to two smaller Chinese studies (n = 369 and n = 72), highlights potential geographic and molecular heterogeneity in CRC. Overall, HMGB1 emerges as a novel biomarker and therapeutic candidate requiring further translational investigation.