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.

Keywords: Non-small cell lung cancer; immune microenvironment; sex; single-cell RNA sequencing; tumor-associated macrophage.

Figure 1

Overview of the Cell Types in the Microenvironment of Non-small-cell Lung Cancer. (A) Workflow of the study design. (B) Bar plot showing the different response situations to immune checkpoint inhibitors of females and males, split by smoking status. Fisher’s exact test was used to compare the significance of the association between sexes and immune response. NR: no response; OR: objective response. (C) t-SNE plot showing the cell composition in the microenvironment of NSCLC, colored according to cell types. (D) Bubble heatmap showing expression of cell-type markers across cell clusters. Dot size indicates the percentage of expressed cells, colored by their relative expression levels. (E) Bar plot showing the fraction of each cell type according to the origin of samples. (F) t-SNE plot showing the cell distribution originated from tumor and normal lung samples. (G) Bar plot showing the overall cell composition of normal and tumor samples, colored by cell types.

Figure 2

Re-clustering of Immune Cells and Preliminary Exploration of Myeloid Cells. (A) t-SNE plot of the tumor-infiltrating immune cells. (B) Violin plot showing the relative expression of immune cell-type-specific markers in each immune cluster. (C) Lollipop plot showing the fraction of immune cells in male and female, colored by sex. Unpaired Wilcoxon test showed no statistical differences between sexes. (D) A t-SNE plot of the tumor-infiltrating myeloid cells. (E) The composition of myeloid clusters in patients from the discovery scRNA-seq data. (F) Composition of the TAMs in patients from the validation scRNA-seq data 1. (G) Heatmap showing the relative expression of selective gene sets across all TAM subtypes in the discovery data. (H) Boxplot showing the comparison of Myeloid cell fraction between sexes in the discovery data. P-values were calculated by the unpaired two-sided Wilcoxon test. No significantly differently expressed myeloid cell types were found.

Figure 3

Heterogeneity of Tumor-associated Macrophages in the Microenvironment of NSCLC. (A) Heatmap shows the top 5 differentially expressed genes of the nine myeloid clusters. (B) Heatmap showing the relative regulon activity for different TAM clusters. (C) Pseudotime trajectory of TAMs subtypes with high variable genes, colored by cell types, root cells of the trajectory were marked by the black circle. Each point represents a single cell, analyzed by Monocle2. (D) Differentially expressed pathways between CCL18+ and SPP1+ macrophage in the discovery data. Pathway activity scores were calculated by ssGSEA and compared using the limma package. T values fitted the linear models, and p-values were adjusted by the Benjamini-Hochberg method. In CCL18+Macrophage, red indicates fatty acid metabolism pathways; In SPP1+Macrophage, green indicates the glycolysis associated pathways.

Figure 4

Differentially Expressed Genes (DEGs) in TAMs by Sex. (A) Venn diagram showing the overlapped genes between the discovery and validation scRNA-seq data. (B) Boxplot showing the 5 DEGs with the same trends in each sex. p <0.05 was considered to be statistically significant, ****p < 0.001. Unpaired two-sided Wilcoxon test. (C, D) t-SNE plot showing the normalized expression of C1QC, FN1, and SPP1 in the tumor microenvironment (TME) and tumor immune microenvironment (TIME) of NSCLC. (E) Violin plot showing the relative expression of the 5 DEGs with the same trends between sexes, colored by immune cell type. (F) Boxplot comparing the TPMs of C1QC, CD45 normalized expression of FN1, and SPP1 in the early stage TCGA-NSCLC cohort (370 women vs. 541 men). Asterisk indicated different levels of p-values, *p < 0.05, ***p < 0.005, ****p < 0.001. (G) Boxplot comparing the expression variance of C1QC, FN1, and SPP1 in macrophages from normal and tumor samples. ****p < 0.001. (H) Kaplan–Meier curves based on the expression levels of C1QC, FN1, and SPP1 for TCGA NSCLC patients. P-value was calculated with a log-rank test.

Figure 5

Functional Heterogeneity of TAMs by Sex. (A) Boxplot comparing the M1 and M2 gene-sets expression of TAMs in the discovery scRNA-seq data, grouped by sex. (B) t-SNE plot showing the overall distribution of immune cells in 8 NSCLC patients, color-coded by corresponding cell type. (C) Split-violin plot showing the overall M1 and M2 associated proteins expressed by TAMs from female (left violins) and male (right violins) in the CyTOF data. (D) Boxplot showing the specific M1 and M2 associated proteins expressed by TAMs in the CyTOF data, grouped by sex. (E) Heatmap showing the relative regulon activity of TAMs from different sexes in the discovery scRNA-seq data. (F) Differential pathways enriched in TAMs originated from different sexes. ssGSEA was used to calculate gene-set scores of every single cell, while the limma package executed differential analysis. (G) Heatmap showing the relative expression variance of Toll-like receptor and IRFs-associated genes in TAMs between sexes. (H) Boxplot showing the MHC I molecules of TAMs from different sexes in the discovery scRNA-seq data. (I) Boxplot showing the male up-regulated gene-sets of TAMs in the validation scRNA-seq data 1, colored by sex. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 6

Overview of the Key Transcriptome and Functional Changes in TAMs between Sexes. Female-derived TAMs presented higher immunogenicity, stronger anti-tumor ability with increased expression of TLR-mediated inflammatory factors, and enhanced antigen presentation capacity; Male-derived TAMs were more immunosuppressed, to which upregulated PPAR-associated pathways contributed considerably. A higher level of complement-related C1QC was found in female-derived TAMs, while male-derived TAMs elevated the expression of matrix remodeling-relevant genes—SPP1 and FN1. Chromosome and sex hormone differences may be the main reasons for the above heterogeneity; TAMs: Tumor-Associated Macrophages; TLR: Toll-like Receptors; PPAR: Peroxisome Proliferator-Activated Receptors Gamma. This picture was created with BioRender.com (Agreement number: QS22TH8BY8).