Distinct immune microenvironment of venous tumor thrombus in hepatocellular carcinoma at single-cell resolution

Abstract

Background and aims: Portal vein tumor thrombus (PVTT) worsens the prognosis of hepatocellular carcinoma by increasing intrahepatic dissemination and inducing portal vein hypertension. However, the immune characteristics of PVTT remain unclear. Therefore, this study aims to explore the immune microenvironment in PVTT.

Approach and results: Time-of-flight mass cytometry revealed that macrophages and monocytes were the dominant immune cell type in PVTT, with a higher proportion than in primary tumor and blood (54.1% vs. 26.3% and 9.1%, p<0.05). The differentially enriched clustering of inhibitory and regulatory immune cells in PVTT indicated an immune-suppressive environment. According to the single-cell RNA sequencing, TAM-C5AR1 was characterized by leukocyte chemotaxis and was the most common subpopulation in PVTT (36.7%). Multiplex fluorescent immunohistochemistry staining showed that the C5aR+ TAM/Mφ were enriched in PVTT compared to both the primary tumor and liver and positively correlated with C5a (r=0.559, p<0.001). Notably, THP-1 (monocyte cell line) was recruited by CSQT2 (PVTT cell line) and exhibited up-regulation of CD163, CD206, and PD-L1 upon stimulation. C5aR antagonist could reverse this. C5aR+ TAMs could also inhibit Granzyme B in CD8+ T cells. High infiltration of C5aR+ TAMs in PVTT correlated with poor differentiation (p<0.009) and was a risk factor for overall survival (p=0.003) and for reformation of PVTT after resection (p=0.007).

Conclusions: TAMs, especially C5aR+ TAMs, were enriched in PVTT. C5aR+ TAMs contribute to the development of PVTT and poor prognosis by reshaping the immunosuppressive environment.

Keywords: hepatocellular carcinoma; immune microenvironment; portal vein tumor thrombus/thrombosis; single-cell omics.

Graphical abstract

FIGURE 1

Distinct characteristics of infiltrating immune cells in PVTT. (A) Flow chart of the study. (B) A two-dimensional t-SNE illustration of the CyTOF data gated (with color coding) based on sample types (upper image) and major immune cell types (lower image) showing the heterogeneous distribution of immune cells. (C–E) The pairwise comparison of immune cell types (C), immune cell lineages (D), and immune response (E) (Wilcoxon signed-rank test, *p<0.05). (F) The trend of naïve T cells, memory T cells, and plasma B cells from Blood to PVTT to PT. (G) An ANOVA was performed for all 91 CyTOF clusters, and the detailed differentially enriched clusters among blood, PT, and PVTT are shown (Friedman test, p<0.05). Abbreviations: CyTOF, time-of-flight mass cytometry; DC, dendritic cell; FFPE, formalin-fixed and paraffin-embedded; GRAN, granulocytes; IHC, immunohistochemistry; MDSC, myeloid-derived suppressor cell; Mono/Macro, monocytes/macrophages; NK, natural killer; PT, primary tumor; PVTT, portal vein tumor thrombus; scRNA, single-cell RNA; TAM, tumor-associated macrophage.

FIGURE 2

Immunosuppressive role and heterogeneity of TAMs. (A) The correlation analysis of immune cell subtypes (Spearman test, blue-to-red colors represent the r scores from −1 to 1, *: p<0.05) (B) The composition of Mono/Macro clusters colored by sample showed the heterogeneity based on the sample origin. (C) The composition of each sample colored by cluster showed the heterogeneous contribution of TAM subtypes. (D) The correlation analysis of 91 CyTOF clusters in PVTT showed the heterogeneous correlation pattern of the TAM subtypes (Spearman test, p<0.05, each circle represents one cluster, the number of correlated clusters was measured by circle size, and the color of the lines between circles represents the r score, where the color of the circle describes the average $\mid$ r $\mid$ of correlated lines). Abbreviations: CTLA-4, cytotoxic T-lymphocyte associated protein 4; CyTOF, time-of-flight mass cytometry; DC, dendritic cell; HLA, human leukocyte antigen; MDSC, myeloid-derived suppressor cell; Mono/Macro, monocytes/macrophages; NK, natural killer; PT, primary tumor; PVTT, portal vein tumor thrombus; TAM, tumor-associated macrophage; Treg, regulatory T cell.

FIGURE 3

Identification of cell types in PVTT and paired HCC by scRNA-seq. (A) The clustering of scRNA-seq data identified 26 clusters. (B) The single-cell populations were projected by UMAP and nominated into 15 cell types. (C) The distribution of immune cells, HCC cells, stromal cells, and erythrocytes was similar between PVTT and PT. (D) The distribution of immune cell types showed a tendency for a higher percentage of macrophage/monocyte cells in PVTT. (E) The myeloid cell population was further grouped into 5 TAM clusters, 2 monocyte clusters, 3 DC clusters, and 2 cycling MP clusters. (F) Biological functions of TAM clusters analyzed by GO enrichment analysis. Abbreviations: DC, dendritic cell; GO, Gene Ontology; HLA, human leukocyte antigen; Mono/Macro, monocytes/macrophages; MHC, major histocompatibility complex; NK, natural killer; PT, primary tumor; PVTT, portal vein tumor thrombus; scRNA, single-cell RNA; TAM, tumor-associated macrophage.

FIGURE 4

Monocytes and TAMs were recruited by PVTT through the C5a-C5aR interaction. (A) Proportions of five TAM clusters and their changes in PVTT. (B) The mIHC staining assays (left) and the comparison of C5aR⁺ TAMs proportion (middle) and density (right) among PVTT, PT, and liver tissue. (C) The screening strategy of potential ligand-receptor pairs between HCC cells and TAM-c2-C5AR1 cells. (D) The correlation analysis between C5a⁺ HCC cells and C5aR⁺ TAMs according to IHC/mIHC staining assays (Spearman test, r=0.559, p<0.001). (E) The comparison of C5a level in the conditioned medium from a PVTT cell line (CSQT2), 3 metastatic HCC cell lines (LM3, 97H, and HLF), and negative control (t test, 9 replicates). (F) The chemotaxis assays of a monocyte cell line (THP-1) in conditioned medium from CSQT2 cells with or without a C5aR antagonist (PMX-53) (t test, 7 replicates). (G) The morphological assessment of THP-1 monocytes cultured with conditioned medium from CSQT2, LM3, 97H, and HLF cells (t test, 7 replicates). (H) The comparison of CD163 and CD206 (markers of M2-like macrophages) on THP-1 monocytes when cultured with different conditioned medium (t test, 6 replicates). (I) The effect of C5a concentration on the expression of CD163 and CD206. (J) The comparison of M2-like phenotype on THP-1 co-cultured in CSQT2 with or without C5aR antagonist (t test, 6 replicates). (*p<0.05, **p<0.01). Abbreviations: CM, conditioned media; IHC, immunohistochemistry; mIHC, multiplex fluorescent immunohistochemistry; MFI, mean fluorescence intensity; PT, primary tumor; PVTT, portal vein tumor thrombus.

FIGURE 5

Function and clinical significance of C5aR⁺ TAMs. (A) The flow cytometry gating and sorting scheme for primary C5aR⁺ TAMs. (B) CD8⁺ T cells co-cultured with C5aR⁺ TAMs expressed a higher level of granzyme B than those co-cultured with C5aR^- TAMs. (C) The TCGA HCC data showed that the gene signature of exhausted T cells was positively correlated with the gene signature of TAM-c2-C5AR1. (D) The interaction pair analyses of suppressive immune checkpoint between TAM-c2-C5AR1 and T-CD8-c1-GZMK showed 3 significant ligand-receptor pairs. (E) PD-L1 was expressed more often by C5aR⁺ TAMs than C5aR^- TAMs. (F) Recombinant C5a increased the level of PD-L1 in a dose-dependent manner. (G) The increase in PD-L1 expression could be reversed by treating cells with a C5aR antagonist. (H, I) The log-rank survival analysis showed that patients with high enrichment of C5aR⁺ TAMs had a poorer overall survival (H) and shorter time to PVTT relapse (I) than those with low enrichment of C5aR⁺ TAMs. (Wilcoxon signed-rank test, *p<0.05, **p<0.01). Abbreviations: MFI, mean fluorescence intensity; PD-L1, programmed cell death 1 ligand 1; PMX-53, AcPhe(ornithine-Pro-cyclohexylamine-Trp-Arg); PVTT, portal vein tumor thrombus; TAM, tumor-associated macrophage; TCGA, The Cancer Genome Atlas Program.

FIGURE 6

Characteristics of malignant cells in PVTT. (A) The differential characteristics of PVTT and PT malignant cells according to the Hallmark enrichment analyses of scRNA-seq data. (B) A comparison of gene signatures of malignant cells in PVTT and PT. (C) The expression of HABP2, a hypoxia-related gene, was upregulated in malignant cells from PVTT. (D) A schematic diagram showing how PVTT tumor cells educated C5aR⁺ TAMs and contributed to the immunosuppressive microenvironment. Abbreviations: FASP, factor VII-activating protease; PD-L1, programmed cell death 1 ligand 1; PT, primary tumor; PVTT, portal vein tumor thrombus; scRNA, single-cell RNA.