Figure 1.

C1q⁺ macrophage subset detected by scRNA-seq correlates with poor prognosis of MPE patients. (A–D) Seven MPE specimens of patients with untreated lung cancer were collected, and CD45⁺ immune cells were isolated using magnetic-activated cell sorting for scRNA-seq. A t-distributed stochastic neighbor embedding (t-SNE) plot based on scRNA-seq revealed six clusters of myeloid cells, comprising three subsets of macrophages and three subsets of dendritic cells (A). Heat map of gene signatures for macrophage subsets (B). Expression levels of C1QA, C1QB, and C1QC genes in t-SNE space (C). Expression levels of C1QA, C1QB, and C1QC genes in distinct myeloid subsets (D). (E) Immunofluorescence staining revealed co-localization of C1q and CD68⁺ macrophages in both human and mouse MPE. Red, CD68; green, C1q; and blue, DAPI for nucleus. Scale bar 10 µm. (F) The percentages of C1q⁺ TAMs (CD45⁺ CD68⁺ C1q⁺) in human MPE and C1q⁺ monocytes (CD45⁺ CD14⁺ C1q⁺) in corresponding peripheral blood were quantified by flow cytometry (n=6). (G) The percentages of C1q⁺ macrophages (CD45⁺ F4/80⁺ CD11b⁺ C1q⁺) in mouse MPE and corresponding spleen, as well as C1q⁺ monocytes (CD45⁺ Ly6C⁺ C1q⁺) in corresponding peripheral blood, were quantified by flow cytometry (n=5). (H) MPE patients were stratified into high tumor burden group (HTB, tumor cells >30%, n=12) and low tumor burden group (LTB, tumor cells <30%, n=6). The percentages of C1q⁺ TAMs in both groups were determined by flow cytometry analysis. (I) Correlation between C1q⁺ TAMs and survival in MPE was analyzed using Kaplan-Meier analysis (n=28–32). The median survival time of C1q⁺ TAMs^low was 19 months, while the median survival time of C1q⁺ TAMs^high was 10 months. (J and K) The MPE mouse model was established using LLC cells. Correlation analyses were conducted to investigate the relationship between the proportion of C1q⁺ TAMs and time (I, n=18), as well as the proportion of C1q⁺ TAMs and MPE volume (J, n=26). Data shown in (E–K) are representative of at least three independent experiments (mean±SD). Statistical analysis was performed using paired two-tailed Student’s t-test (F), unpaired two-tailed Student’s t-test (G, H), log-rank test (I), or Pearson correlation coefficient (J, K). *p<0.05, ***p<0.001, ****p<0.0001. C1q, component 1q; cDC, conventional dendritic cells; DAPI, 2- (4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; IL, interleukin; LLC, Lewis lung cancer cells; MPE, malignant pleural effusion; pDC, plasmacytoid dendritic cells; scRNA-seq, single-cell RNA sequencing; TAM, tumor-associated macrophage.