Tertiary lymphoid structures in colorectal cancer: Clinical significance and their interaction with CAFs and the immune microenvironment

Colorectal cancer (CRC) is a major malignancy with high global mortality. The tumor immune microenvironment, particularly tertiary lymphoid structures (TLSs), has gained increasing attention for its role in antitumor immunity. However, their specific functions in CRC and the prognostic relevance of TLS-associated cells remain unclear. We analyzed 166 CRC patients who underwent curative resection, evaluating TLSs by hematoxylin and eosin (H&E) staining and immunohistochemistry for CD20, alpha-SMA, CD8, CD4 and CD103. We additionally performed Opal-based multiplex immunofluorescence (mIF) with a two-plex panel (CD20 and alpha-SMA) to validate the presence of cancer-associated fibroblasts (CAFs) within TLSs. Patients were categorized by TLS density and immune-cell infiltration levels. TLSs were predominantly located at invasive margins, and higher TLS density correlated with better prognosis (P < 0.05). In contrast, high CAF density within TLSs was associated with poorer outcomes (P < 0.01), whereas a high abundance of CD8⁺ T cells predicted improved survival. Furthermore, using Xenium Explorer spatial transcriptomics, we confirmed the spatial coexistence of CAFs and immune cells within TLSs in colorectal cancer. In particular, CAF accumulation within TLSs may reshape the immune microenvironment and potentially impair antitumor T-cell activity.