组织学生长模式决定肝转移灶中免疫细胞的空间分布

Background: Hepatic metastases from colorectal cancer represent one of the leading causes of mortality associated with this disease, and their management remains a clinical challenge that requires increasingly precise diagnostic and therapeutic strategies. We have previously reported colorectal liver metastases (CRCLM) with desmoplastic histological growth pattern (dHGP) to have more cytotoxic immune environment, enriched by CD8⁺ lymphocytes over tumor nests. FoxP3⁺ cells followed the CD8⁺, but CD4⁺FoxP3⁺ (Tregs) did not demonstrate difference. Here we focused on rare lymphocyte subset: CD8⁺FoxP3⁺ cells. Methods: We applied multiplex IHC panel with immune cell markers for tissue samples from 100 patients with CRCLM, characterized by HGP. Cell classes included Cytokeratin (CK)-positive cells, CD8, CD4, CD4⁺FoxP3⁺, CD8⁺FoxP3⁺, CD20⁺ and HSA (Hepatocyte specific antigen). Cell density defined as number of cells per mm 2 . To resolve spatial patterns, we computed neighborhood enrichment (ENR) and the pair correlation function (PCF). ENR, evaluated distance to k-nearest neighbors (k=5-100), and quantified attraction or avoidance between cell classes while controlling for cell density. PCF, computed over radial distances (10-50 µm). Results: The initial cell density analysis demonstrated that CD8⁺FoxP3⁺ cells were significantly more abundant in dHGP (p=0.003), following similar arrangement of larger CD8+ cell set (p=0.007). Spatial analysis, adjusted to cell density, demonstrated more complex patterns. First , CD8⁺FoxP3⁺ showed strong spatial attraction to CD8 cells in non-dHGP, while in dHGP, where both cell types were significantly more abundant, they tended to disperse (confirmed by agreement across five spatial metrics, with k=5-50, r=50, p=0.01-0.004). Second, In dHGP, the CD8⁺FoxP3⁺ cells had strong tendency to neighbor to CD20⁺ (B-cells) (confirmed by agreement across five spatial metrics, with k=5-25, p=0.05-0.005). Third, CD8⁺FoxP3⁺ cells demonstrated attraction to cancer cells, in dHGP, detected by ENR at k=5-25 (p=0.01), indicating nearest vicinity or direct contact. Conclusions: The neighboring tendencies observed between CD8⁺FoxP3⁺ and CD8⁺ cells in non-dHGP, suggest either a strong biological interaction between these two T-cell subsets or dynamic conversion between states of CD8⁺ linage. In dHGP, CD8⁺FoxP3⁺ cells were instead redirected toward CD20⁺ B-cell rich areas and showed nearest-neighbor attraction to CK-positive tumor cells, consistent with positioning in or near tertiary lymphoid structure (TLS)-like niches and at the tumor-stroma interface. Altogether, this provides the first spatially resolved description of CD8⁺FoxP3⁺ cells in CRCLM and highlights their abundance and neighborhood context as potential contributors to the distinct immune architectures and clinical behavior of dHGP versus non-dHGP liver metastases.