Graphical abstract graphic file with name fx1.jpg [31]Open in a new tab Highlights * • The integrated scRNA-seq data portrait the mononuclear phagocytes in HCC * • XGBoost model identified 445 infiltration-associated genes in tumors * • MIF is one of the upstream regulators of SPP1 to promote macrophage migration * • MIF and SPP1 promote tumor metastasis and invasion __________________________________________________________________ Microenvironment; Cancer; Transcriptomics Introduction Hepatocellular carcinoma (HCC) is an aggressive malignancy accounting for >80% of primary liver cancers. HCC is the fourth most common cause of cancer-related death worldwide, posing a serious threat to people’s health and lives.[32]^1 In addition to tumor cells, fibroblasts, endothelial cells, and immune cells in the tumor comprise the tumor microenvironment (TME). Tumor-associated macrophages are considered a major component of the TME and are related to patient prognosis and tumor resistance to therapy. Macrophages in the liver are categorized into tissue-resident macrophages (Kupffer cells) and bone-marrow-derived macrophages according to their origin.[33]^2 The TME can induce these macrophages to undergo a series of phenotypic and functional alterations, yielding tumor-associated macrophages (TAMs). Monocytes extravasate into the tissue and interact with the TME during infiltration and differentiation, becoming TAMs that can promote tumor progression. Mononuclear phagocytes (MNPs) include macrophages and monocytes, and their precursors are dispersed across various organs and tissues throughout the body. It has previously been demonstrated that macrophages can be divided into two groups, M1 and M2.[34]^3 M1 macrophages are considered “classically activated” macrophages that can be activated with lipopolysaccharide and IFN-γ. M1 macrophages express TNF-α, iNOS, IL-1β, and CXCL9/10, recruit T cells, and promote inflammation. M2 macrophages are “alternatively activated” by IL-4 and IL-13. M2 macrophages express CD206, CD163, TREM2, and other anti-inflammatory markers and do not play a pro-inflammatory role.[35]^3^,[36]^4 However, these two distinct types do not comprehensively represent the highly heterogeneous nature of macrophages[37]^5 such as TAMs, a heterogeneous group of cells expressing both M1 and M2 markers. Many TAMs with immunosuppressive effects exhibit an M2-like phenotype and do not inhibit tumors.[38]^6 These TAMs secrete IL-10, TGF-β, VEGFA, and other tumor-promoting cytokines, hampering T cell cytotoxicity and promoting tumor metastasis.[39]^7 Cancer patient single-cell RNA-seq (scRNA-seq) data have revealed previously unrecognized dynamics and heterogeneity among TAMs.[40]^8^,[41]^9^,[42]^10 Due to cellular plasticity, numerous MNPs exhibit distinct phenotypes, implying a diverse role in disease. Many studies have now demonstrated that macrophages in the TME promote tumor development[43]^11^,[44]^12 and targeting TAMs has emerged as a promising direction for drug development and therapeutic strategies.[45]^13 There are two main TAM-targeting approaches: changing the function of the TAMs or inhibiting TAMs infiltration.[46]^14 Several cytokines, including IL-6, IL-1β, CSF-1, and VEGFA, and chemokines CCL2, CCL3, CCL4, and CXCL12, have been demonstrated to promote monocyte recruitment in animal models of lung, breast, and pancreatic cancer.[47]^15 This finding has led to the development of many drugs targeting MNP infiltration, predominately targeting the CCR2–CCL2 and CXCR4–CXCL12 pathways.[48]^16 Strategies targeting these pathways have shown efficacy in HCC mouse models.[49]^13 However, due to the complexity of the TME, additional signaling pathways that mediate intercellular interactions within the TME need to be illustrated. Limited targets and unclear mechanisms impede the development of clinical treatment strategies. To further understand the role of MNPs in HCC and identify potential drug targets influencing MNP infiltration, we integrated single-cell transcriptomic data from patients with HCC and healthy individuals. We used a gradient-boosted decision tree (GBDT) approach to distinguish different spatial locations of MNPs. A multi-classifier was trained to identify genes affecting the spatial distribution of cells, and the effects of these genes were further confirmed with microarray and RNA-seq data. Through data mining and experimental validation, we demonstrated that the migration inhibitory factor (MIF) gene could influence TAM infiltration, providing a potential target for treating HCC. Results MNPs show phenotype and function heterogeneity in HCC To better understand the heterogeneity of the immune system in liver cancer, we evaluated data from 75,696 immune cells from two published HCC single-cell datasets, Zhang Q et al. and Sharma A et al. The cells were obtained from distinct spatial regions: adjacent normal (AN) tissue, the tumor periphery (TP), and the tumor core (TC) ([50]Figures S1A–S1C). We divided the cells into 12 groups based on the expression of classical immune cell marker genes, such as CD8A, IL-7R, TCLA4, IFG, and CD79A ([51]Figures S1D, S1E, and [52]Table S1). We further focused on myeloid cells, comprising dendritic cells (DCs), monocytes, and macrophages, and classified the cells into 13 subtypes, including seven MNP subtypes, five conventional dendritic cells subtypes, and one plasmacytoid dendritic cells subtype after re-clustering ([53]Figure 1A and 1B). To exclude the effect of ribosomal proteins (RPs), we compared cell clusters before and after ribosome removal and found that DC-CD1C-RPs belonged to the DC-CD1C subtype and Macr-RPs represented a separate subtype ([54]Figures S1F and S1G). Figure 1. [55]Figure 1 [56]Open in a new tab Identification of the MNPs in HCC (A) UMAP plot showing the clusters of the 10,381 HCC myeloid cells. (B) Dim plot showing the marker genes expression of the HCC myeloid cells. (C) Heatmap showing ssGSEA results of the 6,939 HCC MNPs in the BP gene set in the GO term enrichment. Heatmap colors show the normalized gene enrichment scores (NES), and label “∗” represents the adjusted p value <0.05. The different color blocks of row names distinguish the pathway types, from top to bottom, the pathway types are “inflammation & immune”, “antigen presentation”, “phagocytosis”, “angiogenesis”, “remodeling cell adhesion and matrix”, “cell migration”, and “oxidative stress”. “▲” represents “Antigen Processing and Presentation”, “●” represents “Sprouting Angiogenesis”. (D) Bar plot showing the average expression of CCR2 and CX3CR1 in each type of MNPs. (E) Heatmaps showing the expression levels of cell polarization, chemotaxis, and immune-related genes in each MNP subtypes. (F) Dim plot showing the expression of CXCLs in each type of MNP. Gene Ontology (GO) enrichment analysis[57]^17 was performed for the 7 MNP subtypes ([58]Figure 1C). The Mono-VCAN subtype has high motility and is enriched in the cell extravasation pathway; thus, these cells might migrate during the inflammatory response. In contrast, the Mono-CD16 subtype with low motility exhibited a more mature phenotype ([59]Figure 1C). The Macr-STAT1 and Macr-APOE subtypes have a strong antigen-presenting functions and are significantly enriched in myeloid-mediated immunity and immune effects ([60]Figure 1C). The Macr-APOE subtype expressed M2 markers, such as CD163, MRC1, and IL-10, and classical complement C1q (C1QA/B/C) ([61]Figure S1H). It has been reported that tumors with a high number of macrophages and a high level of C1q expression establish an immunosuppressive environment.[62]^18 Another type of immunosuppressive macrophage was the Macr-secreted phosphoprotein 1 (SPP1) subtype. These cells strongly expressed SPP1 and TREM2 ([63]Figures 1B and [64]S1H). The Macr-SPP1 subtype demonstrated overexpression of pathways involved in cell adhesion, extracellular matrix remodeling, and angiogenesis ([65]Figure 1C). To identify direct factors affecting MNP infiltration, we evaluated the expression of chemokines and their ligands in each cell subtype. The inflammatory Mono-VCAN subtype showed the highest level of CCR2 expression, indicating the initial event of CX3CR1^LoCCR2^Hi monocytes recruitment from the vasculature to the tumor; infiltrating monocytes are then further transformed into CX3CR1^HiCCR2^Lo monocytes (the Mono-CD16 subtype) ([66]Figure 1D). The CCR2^+ Macr-STAT1 subtype expressed many chemokine receptors and M1 marker genes, suggesting that the Macr-STAT1 subtype is present in the early stages of monocyte differentiation into macrophages ([67]Figures 1D and 1E). The Macr-SPP1 subtype was also highly enriched in cell migration-related pathways and had low expression of chemokine receptors but higher chemokine levels. These cells primarily secrete CXCL1/2/3/8 ([68]Figure 1F), which are CXCR1/2 ligands. The aforementioned chemokines primarily recruit myeloid cells, such as granulocytes, promote the development of immunosuppressive cells, and inhibit T and NK cell cytotoxicity.[69]^19^,[70]^20 In addition, secretion of IL-5 by Macr-SPP1 cells may also inhibit NK cell function by recruiting eosinophils.[71]^21 Due to the low enrichment scores in the immune-related pathways ([72]Figure 1C), Macr-SPP1 cells may not secrete inflammatory factors such as IL-6 and TNF-α. Overall, we distinguished immunosuppressive and activated MNP subtypes in the tumor microenvironment using their distinct gene expression characteristics. MNPs show spatial distribution preferences