Fig 2: CAF heterogeneity. A. Analysis of the individual transcriptomes of 4,397 CAFs identified two main populations (ECM- and Ctr-CAFs) and four more specialized populations. B. Expression of the 30-gene signatures for each CAF population and subpopulation. C. Biological processes showing the different activity levels in the CAF populations. D. LTBP2 and ACTA2 expression in CAFs. E. Protein expression of Pan-CK (cancer cells), α-SMA (all CAFs) and LTBP2 (ECM-CAFs) in CRC-LM. Representative image of nine CRC-LM samples; scale bars,100 μm. Nuclei were counterstained with DAPI.

Fig 3: A. Expression profiles of ECM- and Ctr-CAF signature genes in different liver tumor types. B. ML-inferred CAF composition in CRC-LM, hepatocellular carcinoma (HCC), and intrahepatic cholangiocarcinoma (iCCA). P-values by multinomial testing (#P < 7E-11). C. Survival curves in function of LTBP2 and FAP expression. D. CAF origin model. CAFs mainly derive from three major sources: hepatic stellate cells (HSC), portal fibroblasts (PF), and vascular smooth muscle cells (VSMC). CRC-LM initiate by the arrival of CRC cells from the colon through the portal vein into the portal space of liver. There, they extravasate, activate PFs, and orient them to a CAF phenotype. The lesions grow and invade the liver parenchyma where they subsequently convert HSCs into CAFs. In HCC, the opposite occurs. Tumors originate in the liver parenchyma where they first activate HSCs.

Fig 4: Expression of Pan-CK (cancer cells), α-SMA (CAFs) and LTBP2 (ECM-CAFs) in CRC-LM samples at the invasion front of replacement, and in the pushing and desmoplastic HGPs. LM, liver metastasis; D, desmoplasia; and NL, normal liver. Representative images of nine CRC-LM samples; scale bars, 100 μm. Nuclei were counterstained with DAPI. Histograms indicate the mean values from nine samples and two different locations per sample; error bars are the standard deviations; *P ≤ 0.05, **P ≤ 0.01 (Mann-Whitney-U-Test).

Fig 5: CAF origin. A. Machine learning (ML) prediction of CAF origin using mesenchymal cell transcriptome data from healthy and cirrhotic human livers (the table on the right shows the rate of closest mesenchymal phenotype assignment to each CAF population.) B. RNA velocity analysis indicating a seamless flow between ECM-CAF subtypes, in agreement with a common SAMes origin. Conversely, transitions between ECM- and Ctr-CAFs, and Ctr-CAF-II and Ctr-CAF-II seem more random, in agreement with their distinct origins. C. Portal fibroblast genes not expressed by HSCs strongly associate with ECM-CAFs. D. Multiplexed immunofluorescence staining of Pan-CK (hepatocytes), α-SMA (HSCs), and LTBP2. The presence of LTBP2+ CAFs in CRC-LM samples is limited to portal regions of the normal adjacent liver tissue. LM, liver metastasis; D, desmoplasia; and NL, normal liver. Representative images of nine CRC-LM samples; scale bars, 100μm. Nuclei were counterstained with DAPI. E-F. RT-qPCR analysis of C3, POSTN, and LTBP2 expression in LX2 and CCD18Co cells, and CRC-LM-CAFs incubated with CM from CRC cell lines (HT29, SW1222, and LoVo). Basal levels (E) and gene fold-change relative to control (F). G. Western blot analysis of LTBP2 abundance in conditioned medium samples. Panels E and F: *P < 0.05, **P < 0.01, ***P < 0.001.