Fig 2: CD5L expression by TAMs is associated with poor prognosis in papillary lung adenocarcinoma. a) Clinical characteristics of patients diagnosed with Papillary Adenocarcinoma (PAC) who participated in this study. b) Representative immunohistochemistry images showing CD5L expression in early (I) and advanced (II–III) stages of papillary lung adenocarcinoma. Scale bar represents 10 µm. c) Graph shows the number of CD5L+ macrophages per field in early (I, n = 35) and advanced (II–III, n = 20) stages. Data are presented as the mean ± SEM, *p < 0.01 determined by the Mann–Whitney t-test. d) Kaplan–Meier analysis of recurrence-free survival in cases with lower and higher TAM CD5L expression. The mean number of CD5L+ macrophages from stage I was taken as the limit value, *p < 0.01 determined by the Log-rank (Mantel–Cox) test. e) Representative immunofluorescence image depicting CD68+ (red) (i) and CD5L+ (green) macrophages, and their co-expression (orange), (ii). Scale bar represents 25 µm.

Fig 3: Lung and liver cancer cell-conditioned media (CM) induce an IL10-like phenotype and CD5L expression in macrophages.a) Principal Component Analysis (PCA) scatterplot of PB monocytes treated for 72 h with medium alone (control), reference activation stimuli (IFN/LPS, IL4, and IL10), or cancer cell-CM. Projection based on the expression profile of surface markers characterized by multicolor flow cytometry. PC1 and PC2 represented in each axis depict the first and second principal components, respectively. b) Expression of CD80, CD163, CD206, VEGF, MERTK and c)CD5L mRNA was assessed by RT-qPCR in PB monocytes treated for 24 h with the indicated stimuli. mRNA levels normalized to GAPDH, and fold induction levels were calculated using the average expression of each gene in control macrophages as a reference. Data are represented as mean ± SEM (n = 5 to 9). d) CD5L immunofluorescence staining (green) in macrophages treated with the indicated stimuli for 72 h. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar represents 20 µm. CD5L mean fluorescence intensity (MFI) was calculated with ZenLite software and is represented as MFI ± SEM of 50 macrophages scored in random fields (right) (n = 3). Significance was calculated using the Mann–Whitney t-test (*p = 0.05, **p = 0.01, ***p = 0.001).

Fig 4: Blockade of CD5L slows tumor growth in vivo and reprograms TAMs towards an antitumor profile. a) Study design and timeline for mouse model. b) LLC tumor growth in mm3 in mice treated with control (PBS) or the anti-CD5L RImAb. Data are presented as the mean ± SEM (n = 8 per group). *p < 0.01 determined by the two-way repeated measures ANOVA test. c) Left: Immunofluorescence demonstrating CD5L expression (red) by F4/80+ TAMs (green) in control (PBS) and RImAb-treated mice. Nuclei were counterstained with Hoechst 33258 (blue). Scale bars represent 25 µm. Right: graph representing the number of F4/80+ TAMs and F4/80+ TAMs expressing CD5L per field in control and RImAb-treated animals. Data are presented as the mean ± SEM (n = 8 per group). *p < 0.01 determined by the Mann–Whitney t-test. d) Immunohistochemistry depicting expression of F4/80, iNOS, and Arg-1 in tumor samples from control (PBS) and RImAb-treated mice. Scale bar represents 10 µm. Average stained area obtained from five random areas was calculated using Image J (color deconvolution) software. Graphs illustrate the average stained area for F4/80, iNOS, Arg-1 and ratio of iNOS/Arg-1 in control (PBS) and RImAb-treated mice. Data are presented as the mean ± SEM (n = 8 per group). *p < 0.01 determined by the Mann–Whitney t-test.

Fig 5: Macrophages lacking HDAC3 upregulate CD5L and other efferocytosis-related genes in vitro.A Schematic of the in vitro efferocytosis experiment using bone marrow-derived macrophages (BMDMs) isolated from M-HDAC3−/− and HDAC3f/f mice. RNA-seq and Qiagen Ingenuity Pathway Analysis (IPA) were performed on BMDMs subjected to no treatment (no ttt) or incubation with apoptotic cells (ACs) or non-apoptotic cells (NACs) for 45 minutes, followed by washing of unengulfed cells & incubation for 5 h, n = 3 biological replicates per group. B Venn diagram showing 472 differentially expressed genes (DEG) in RNA-seq data from ACs-treated HDAC3−/− vs HDAC3f/f BMDMs. C Volcano plot showing CD5L as one of the up-regulated DEGs in AC-treated HDAC3−/− vs HDAC3f/f BMDMs using log2 fold change (logFC) and q value (adjusted p) cutoffs of 1.5 and 0.05, respectively. D Heatmap displaying the upregulated efferocytosis-related genes in HDAC3−/− BMDMs incubated with ACs. CD5L showed the highest logFC-value of 1.97 among the efferocytosis-related genes and a q value of 0.025. E–G RT-PCR validation of the efferocytosis-related genes CD5L, AXL, and LRP1 confirmed the RNA-Seq results showing upregulation of these genes in BMDMs derived from HDAC3−/− vs HDAC3f/f after incubation with ACs, n = 3–4, **P < 0.01 vs no ttt & NAC groups; #P < 0.05, ##P < 0.01 vs all other groups. H, I Western blot and quantification of CD5L protein abundance confirm the RNA-seq finding that HDAC3 deletion upregulates CD5L expression in macrophages incubated with ACs, n = 4, ##p < 0.01 vs HDAC3f/f groups. LRP1 protein levels did not differ between groups, whereas AXL was undetectable. J RT-PCR mRNA analysis of WT BMDMs treated with the HDAC3 inhibitor RGF966 shows upregulation of CD5L after incubation with ACs compared to DMSO-treated BMDMs, n = 3–4, *p < 0.05 vs DMSO. K ELISA confirms increased CD5L abundance in media from RGFP966-treated compared to DMSO-treated BMDMs after incubation with ACs, n = 8–9, *p < 0.05 vs DMSO.