Single-Cell RNA Sequencing Reveals The Heterogeneity Of Tumor-Associated Macrophage In Non-Small Cell Lung Cancer And Differences Between Sexes

BackgroundNon-Small Cell Lung Cancer(NSCLC)is a disease with high morbidity and mortality,which has sex-related differences in prognosis and immunotherapy efficacy.Whereas the exact diverging trends and differences in the mechanisms remain 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.Elucidating the subtype composition and functional heterogeneity of tumor-associated macrophages(TAMs)in NSCLC and further comparing the sex-mediated differences would not only deepen our understanding of the occurrence and progression of NSCLC from the perspective of TAMs,but also provide a theoretical basis for explaining the sexual-associated clinical differences.Methods and ResultsWe conducted a single-cell level analysis,combined with ssGSEA analysis,pseudotime ordering,and SCENIC analysis in an early-stage smoking NSCLC cohort to explore the subtype composition and functional heterogeneity of TAMs and further compare the sex-mediated differences.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 efficiency 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.Conclusionwe expanded upon the different subtypes of TAMs in early smoking NSCLC patients at the single-cell level and revealed two immunosuppressive TAMs with disparate functions and metabolic characteristics,which would provide a theoretical basis for targeting TAMs for more precise tumor therapy.Furthermore,C1QC,SPP1,and FN1 were differentially and stably expressed between male and female-derived TAMs.Of these genes,SPP1 showed potential as a adjuvant immunotherapy target in NSCLC.Meanwhile,we proved that female-derived TAMs were more likely to exhibit the pro-inflammatory phenotypes,while male-derived TAMs tended to be immunosuppressive.Elucidating the sex-based immune efficacy distinctions in TAMs suggested that sexual dimorphism should be considered in NSCLC-relevant research in the future.