Integrating bulk and single‐cell RNA sequencing reveals cellular heterogeneity and immune infiltration in hepatocellular carcinoma

Abstract

Efficacy of immunotherapy in hepatocellular carcinoma (HCC) is blocked by its high degree of inter‐ and intra‐tumor heterogeneity and immunosuppressive tumor microenvironment. However, the correlation between tumor heterogeneity and immunosuppressive microenvironment in HCC has not been well addressed. Here, we endeavored to dissect inter‐ and intra‐tumor heterogeneity in HCC and uncover how they contribute to the immunosuppressive microenvironment. We performed consensus molecular subtyping with non‐negative matrix factorization (NMF) clustering to stratify the inter‐heterogeneity profile of HCC tumors. We grouped HCC tumors from the Cancer Genome Atlas (TCGA) patients into three subtypes (S1, S2 and S3), where S1 was characterized as a ‘hot tumor’ profile with high expression level of T cell genes and rate of immune scores. S2 was characterized as a ‘cold tumor’ profile with the highest tumor purity score, and S3 as an ‘immunosuppressed tumor’ profile with the poorest prognosis and a high expression level of immunosuppressive genes such as cytotoxic T‐lymphocyte‐associated protein‐4, TIGIT, and PDCD1 . Moreover, we combined weighted gene co‐expression network analysis and single‐cell regulatory network inference and clustering (SCENIC) in the single‐cell dataset of the S3‐like subtype (CS3) and identified a transcription factor, BATF , which could upregulate immunosuppressive genes. Finally, we identified a cell interaction network in which a myeloid‐derived suppressor cell‐like macrophage subtype could promote the formation of immunosuppressive T‐cells.

Identification of a CD4+ conventional T cells-related lncRNAs signature associated with hepatocellular carcinoma prognosis, therapy, and tumor microenvironment

“…Negative regulation of immune checkpoint molecules was one of the major factors leading to the exhaustion or dysfunction of effector memory CD8+ T cell, and late exhausted or dysfunctional T cells, representing impaired proliferation ability and reduced production of cytokines, further led to a weakened anti-tumor response ( 61 ). High expression levels of TIGIT and PDCD1 were recognized as immunosuppressed tumors with the poorest prognosis ( 62 ). Meanwhile, we further predicted the response to immunotherapy in high- and low-risk groups of HCC patients using IPS, with patients in the low-risk group associated with increased immunogenicity.…”

T cells in the heterogeneous tumour immune microenvironment of hepatocellular carcinoma: Implications for immune checkpoint inhibitor therapy

“…The molecular subtypes may be associated with different aetiologies, survival outcomes and immune responses 99,100,102–104 . Immune classifications have largely been based on inference of immune cell subsets from transcriptome data in HCC TME 31,105–108 . Thorsson et al identified six general cancer immune subtypes spanning several cancer entities in The Cancer Genome Atlas (TCGA) study.…”

“…The presence of T cells as well as cytotoxic T cells, the active immune response and CD45‐high but FoxP3‐low tumours were individually related to prolonged OS 30,31,107 . In contrast, the expression of immunosuppressive or exhausted features of infiltrating immune cells dominates the immune‐exhausted subclass that was associated with poorer prognosis among all immune subtypes 31,105,107 . It is characterized by signatures of immunosuppressive or exhausted cell populations such as Tregs, Th17 cells, regulatory B cells (Bregs), tumour‐associated macrophages (TAMs) and TEX, higher expression of immunoregulatory molecules (e.g., VEGFA, CTLA4, TIM‐3 and TIGIT) and is associated with intratumour steatosis (Figure 2).…”

“…Another distinct subclass related to rather low OS has been termed immune‐excluded due to the paucity of infiltrating lymphocytes and chemokine and cytokine expression (Figure 2). 31,106,107 It is frequently related to CTNNB1 mutations and overlaps with the non‐proliferation‐Wnt/β‐catenin molecular subclass 31,105,108,115 . Canonical Wnt‐activation may lead to immune exclusion by limiting dendritic cell and T‐cell recruitment in vivo suggesting a direct impact of specific oncogenic alterations on the immune response 116 .…”

“…Tumours are classified into immune‐active, immune‐exhausted and immune‐depleted subtypes depending on their immune characteristics. Predominate T‐cell infiltrate and molecular signalling pathways related to the immune classes according to 31 ,105,106,108,110 are shown. CCL, chemokine (C‐C motif) ligand; CTLA‐4, cytotoxic T lymphocytic antigen 4; CXCL, chemokine (C‐X‐C motif) ligand; DC, dendritic cell; iNOS, inducible nitric oxide synthase; LAG‐3, Lymphocyte‐activation gene 3; MDSC, myeloid‐derived suppressor cell; NK, natural killer cell; PD‐1, programmed cell death protein 1; PD‐L1, programmed cell death ligand 1; TAM, tumour‐associated macrophages; TEX, exhausted CD8+ T cells; TGF‐β, transforming growth factor β; TIM‐3, T‐cell immunoglobulin and mucin‐domain containing‐3; TNF‐α, tumour necrosis factor α; Treg, regulatory T cell; TRM, tissue‐resident memory T cell; VEGF, vascular endothelial growth factor.…”