Colorectal cancer liver metastasis involves coordinated remodelling of the tumour stroma and malignant epithelial compartments. The authors asked which specific fibroblast and malignant epithelial states are coupled in metastasis, and whether any identified coupling generalises beyond the dataset in which it was discovered.
The study analysed paired primary colorectal tumours and liver metastases in two sequential cohorts. The discovery cohort comprised six patients with single-cell RNA sequencing and spatial transcriptomics on three metastatic sections, together with protein staining from an independent tissue resource. Every analytic step from the discovery cohort was repeated in an independent paired cohort of five patients and two additional metastatic sections. In the replication cohort, cell identities were fixed by transfer from the discovery data rather than refitted.
Single-cell RNA sequencing was performed on paired primary and metastatic samples. Malignant cells were identified using copy-number-based approaches. Trajectory inference placed cell states along differentiation or progression axes; both the identified fibroblast state (F2) and the epithelial state (C2) occupied late positions in these inferred trajectories and were enriched in metastases.
Cell-cell communication analyses and ligand-target modelling nominated a directional programme from the RGS5-positive fibroblast/pericyte-like state (termed F2) toward a WNT-like malignant epithelial state (termed C2). Transforming growth factor beta (TGFB) emerged as the highest-ranked signalling pathway in this F2-to-C2 communication, and the TGFB receptor TGFBR2 was enriched in C2 cells. Hub genes implicated as downstream or programme components included TPT1, SLC1A5, SOX4 and TSC22D1.
Spatial transcriptomics on metastatic sections placed the F2 and C2 states in the same tissue neighbourhoods in some sections, supporting local paracrine signalling. The authors note, however, that spatial expression varied between sections, limiting how strictly the two states can be described as co-localised across all samples. For hub genes with available data, protein staining from an independent tissue resource was higher in tumour tissue than in normal colon.
When the analytic workflow was applied to a second, independent paired cohort with cell identities transferred from the discovery set, the same states reappeared. Specific replication observations reported by the authors include:
These findings support reproducibility of the directional F2→C2 TGFB programme across independently collected paired samples.
The communication programme between F2 and C2 highlighted TGFB signalling as the principal pathway. Hub genes identified within the programme were TPT1, SLC1A5, SOX4 and TSC22D1. For the subset of these genes with available protein staining data, tumour expression exceeded normal colon expression according to the independent tissue resource used.
The authors interpret the data as evidence for a directional TGFB axis from an RGS5-positive fibroblast state to a WNT-like epithelial state as a reproducible characteristic of colorectal liver metastasis rather than an idiosyncrasy of a single dataset. At the same time, spatial transcriptomics revealed heterogeneity between sections: while the two states co-occurred in the same neighbourhoods in some samples, their spatial relationship varied, which constrains claims about tight co-localisation across all metastases and sections.
The report is a preprint and has not been peer reviewed; the authors declared no competing interests. Details beyond those reported (for example, additional cohort clinical characteristics, exact numbers of cells analysed per cluster, or full methodological parameters) were not elaborated in the abstract-level source text and would require consulting the full manuscript or supplementary material.
Using paired single-cell and spatial analyses with independent replication, the study identifies a reproducible intercellular programme in colorectal liver metastasis: an RGS5-positive fibroblast (F2) state signals via TGFB to a WNT-like epithelial (C2) state. Hub genes of this programme and higher tumour protein expression for available targets support biological relevance. Spatial heterogeneity between sections means the two states are not uniformly tightly co-localised, but the directional TGFB axis itself replicated across independent paired cohorts, supporting its potential importance in metastatic tumour–stroma interactions.