Single-Cell RNA Sequencing Reveals the Heterogeneity of Tumor-Associated Macrophage in Non-Small Cell Lung Cancer and Differences Between Sexes

Abstract

Non-Small Cell Lung Cancer (NSCLC) is a disease with high morbidity and mortality, which has sex-related differences in prognosis and immunotherapy efficacy. However, the difference in the mechanisms remains unclear. Macrophages, characterized by high plasticity and heterogeneity, act as one of the key cells that exert anti-tumor effects in the tumor microenvironment (TME) and play a complicated role in the process of tumor progression. To elucidate the subtype composition and functional heterogeneity of tumor-associated macrophages (TAMs) in NSCLC and further compare the sex-mediated differences, we conducted a single-cell level analysis in early-stage smoking NSCLC patients, combined with ssGSEA analysis, pseudotime ordering, and SCENIC analysis. We found two universally presented immune-suppressive TAMs with different functional and metabolic characteristics in the TME of NSCLC. Specifically, CCL18+ macrophages exerted immune-suppressive effects by inhibiting the production of inflammatory factors and manifested high levels of fatty acid oxidative phosphorylation metabolism. Conversely, the main metabolism pathway for SPP1+ macrophage was glycolysis which contributed to tumor metastasis by promoting angiogenesis and matrix remodeling. In terms of the differentially expressed genes, the complement gene C1QC and the matrix remodeling relevant genes FN1 and SPP1 were differentially expressed in the TAMs between sexes, of which the male upregulated SPP1 showed the potential as an ideal target for adjuvant immunotherapy and improving the efficacy of immunotherapy. According to the early-stage TCGA-NSCLC cohort, high expression of the above three genes in immune cells were associated with poor prognosis and acted as independent prognostic factors. Moreover, through verification at the transcription factor, transcriptome, and protein levels, we found that TAMs from women showed stronger immunogenicity with higher interferon-producing and antigen-presenting ability, while men-derived TAMs upregulated the PPARs and matrix remodeling related pathways, thus were more inclined to be immunosuppressive. Deconstruction of the TAMs at the single-cell level deepens our understanding of the mechanism for tumor occurrence and progress, which could be helpful to achieve the precise sex-specific tumor treatment sooner.

Macrophage diversity in cancer revisited in the era of single-cell omics

“…IFN-TAMs have high expression of IFN-regulated genes, such as CXCL10 [25,27,28], PDL1 [12,29], and ISG15 [25], and M1-like markers, such as CD86 and MHCII [12]. These TAMs were identified across many tumor types, including breast cancer (BRCA), colorectal cancer (CRC), hepatocellular carcinoma (HCC), head and neck cancer (HNC), lymphoma, melanoma, nasopharyngeal carcinoma (NPC), nonsmall cell lung cancer (NSCLC), ovarian cancer (OVC), pancreatic ductal adenocarcinoma (PDAC), thyroid carcinoma (THCA), uterine corpus endometrial carcinoma (UCEC) [12,25,30,31], multiple myeloma, osteosarcoma, glioblastoma, and spinal ependymomas [32][33][34].…”

“…These TAMs have been identified in BRCA, CRC, NSCLC, NPC, OVC, PDAC, THCA, and UCEC [25,30,31]. Through SCENIC and single-sample gene-set enrichment analysis (ssGSEA) analysis of scRNA-seq data from these cancer types, transcription factors CEBPB, FOSL2, and HIFA were predicted to regulate the expression of this gene signature [25,27,28,30,31,49,50]. In some cancer types, instead of expressing SPP1, Angio-TAMs express other marker genes, including VCAN in BRCA and melanoma, INHBA in gastric cancer and ESCA, and FN1 in kidney cancer [25].…”

“…Notably, TAM clusters feature various metabolic patterns, as inferred from scRNA-seq data through pathway analysis. For example, studies on human NSCLC, PDAC, and OVC indicated that Angio-TAMs highly express hypoxia-related metabolic genes, such as HIFA and SLC2A1 [28,49,67]. In addition, IFN-TAMs were reported to express genes of tryptophan catabolism in a human pan-cancer study [12].…”

Fatty Acid Metabolic Signaling Pathway Alternation Predict Prognosis of Immune Checkpoint Inhibitors in Glioblastoma

“…These new tumor antigens can not only enhance the immune response of T cells, but also increase the content of new antigen-specific T cells, thus improving the immune system's ability to identify tumors (32). Studies have shown that the mutation of the DDR pathway will result in the production of higher TMB and neoantigen loads, thus significantly improving the effectiveness of ICI treatment (33)(34)(35).…”

The Role of Metabolic Plasticity of Tumor-Associated Macrophages in Shaping the Tumor Microenvironment Immunity

“…Myeloid cells are a heterogeneous [ 3 , 21 ] population of innate immune cells that constitute more than 70% of all immune cell populations in the TME [ 22 ]. Common myeloid progenitor cells give rise to macrophages, myeloid-derived suppressor cells (MDSC), granulocytes, dendritic cells (DC), and neutrophils [ 23 , 24 ].…”