An RGS5-positive fibroblast to WNT-like epithelial TGFB axis in colorectal liver metastasis, replicated in an independent paired cohort

Background: Colorectal cancer liver metastasis is accompanied by coordinated stromal remodelling, but which fibroblast and malignant epithelial states are coupled, and whether any such coupling holds outside the dataset it was found in, is rarely tested. Methods: Paired primary tumours and liver metastases from six patients were analysed by single-cell RNA sequencing with copy-number-based identification of malignant cells, trajectory inference, cell-cell communication and ligand-target modelling, and spatial transcriptomics of three metastatic sections. Protein staining was taken from an independent tissue resource. Every step was then repeated in a second, independent paired cohort of five patients and two further metastatic sections, with cell identities fixed by transfer from the discovery data rather than refitted. Results: An RGS5-positive fibroblast/pericyte-like state (F2) and a WNT-like malignant epithelial state (C2) were both enriched in metastases and occupied late trajectory positions. Communication analysis nominated a directional F2-to-C2 programme with transforming growth factor beta as the highest-ranked pathway and its receptor TGFBR2 enriched in C2. Spatial data placed the two states in the same neighbourhoods. Hub genes of the programme were TPT1, SLC1A5, SOX4 and TSC22D1, and protein staining was higher in tumour than normal colon for the three with available data. In the independent cohort the same states reappeared: F2 was enriched in metastases (Ro/E 1.22 versus 0.52; 32% versus 14% of fibroblasts), C2-like cells were the most metastasis-enriched epithelial state (1.10 versus 0.86), 92% of the cells that cohort's own authors called MCAM-positive fibroblasts mapped to F2, and F2 was again the strongest TGFB sender to C2-like epithelium at both sites. Conclusions: A directional TGFB axis from an RGS5-positive fibroblast state to a WNT-like epithelial state is a reproducible feature of colorectal liver metastasis rather than a property of one dataset. Its spatial expression varies between sections, which constrains how tightly the two states can be said to be co-localised.