Paolo Marzano

Supplementary Table 3. List of significant genes (FDR-adjusted p-value < 0.05, and |Log2-FoldChange| > 0.25, detected in not less than 10% of cells) identified from the comparison Liver vs Colon within the inflammatory or immunoregulatory TAMs. In green are indicated the genes up-regulated in the liver, while in orange are indicated the colon-associated ones. Table related to Fig. 5A.

Supplementary Figure 5. Differences between inflammatory and immunoregulatory TAMs in colon and liver

Supplementary Figure 2. Transcriptomic characterization of healthy liver and Kupffer cells

Supplementary Figure 4. Pseudotime trajectory analysis shows macrophage blood derivation

Supplementary Table S2: List of genes used to compute the gene module scores related to the antigen-presentation (n genes = 24), lipid-associated (n genes = 32), angiogenic (n genes = 31), and cytotoxic-inflammatory (n genes = 21) macrophage scores. Table related to Fig. 2E, 3E-G, supplementary figure 2F, and supplementary figure 3E.

Supplementary Figure 1. scRNA-sequencing analysis of early phases synchronous CRLM patients

Supplementary Table 4. List of genes used to compute the gene module scores related to the cell cycle proliferation score (n genes = 57). Table related to Fig. 5D.

Supplementary Table 1. List of significant genes (FDR-adjusted p-value < 0.05, and |Log2-FoldChange| > 0.25, detected in not less than 10% of cells) identified from in each tissue-specific cluster within pathologic colon, liver, and peripheral blood. Table related to Fig. 2D, 3D, and supplementary figure 3B.

Supplementary Figure 3. Transcriptomic characterization of pathologic and healthy monocytes in the peripheral blood

Early synchronous colorectal liver metastasis (CRLM) represents a clinical condition characterized by the simultaneous presence of primary colorectal cancer and metastatic liver lesions. In this study, we characterized the tissue-specific transcriptomes, phenotypes, and functional relevance of tumor-associated macrophages (TAM) within the tumor microenvironment (TME) of colorectal cancer and CRLM specimens from patients who underwent simultaneous surgical removal of these malignancies. The high-throughput single-cell transcriptional analysis revealed an inverse ratio of inflammatory and immunoregulatory TAMs in the colorectal cancer and CRLM TMEs, along with heterogeneity in both tumoral tissues. Furthermore, we found that inflammatory TAMs in colorectal cancer expressed inhibitory ligands that might support immune escape, thus favoring liver metastatic progression. In contrast, CRLM lesions possessed a highly immunosuppressive milieu characterized by large proliferative CTLA4+ immunoregulatory TAMs and the presence of IL7R+ cytotoxic TAMs. Higher frequencies of these specific TAM subsets in CRLM were associated with shorter disease-free survival and worse patient prognosis. The identification and characterization of immunoregulatory TAMs preferentially enriched in CRLM is key for the development of novel immunotherapeutic strategies aimed at boosting anticancer immune responses within the TME.