Fig 2: 25-HC induced Kupffer cells polarization into M2 type to promote liver metastasis.a RAW264.7 cells treated with 25-HC and vehicle were subjected to RNA-seq and GSEA, showing enrichment of genes set related to macrophage pathway (Permutation test, n = 3 independent batches per group). b Quantification of purified Kupffer cells with CD11b+ and F4/80+ expression by flow cytometry in indicated groups. c IHC of F4/80 in liver and lung sections in indicated groups. Scale bar, 50 μm. d Representative bioluminescence images of liver and lung metastasis sites in indicated groups. e IHC of Ki67 in liver and lung sections in indicated groups. Scale bar, 50 μm. f Purified Kupffer cells were selected with TIM4+, I-A/I-Eint expression and stained and further analyzed for CD163 and CD11c expression. Representative flow cytometry results were shown. g Quantification of the M2/M1 ratio in flow cytometry results (one-way ANOVA, n = 3 independent batches). h RNA level in liver tissue of mice from indicated groups by qRT-PCR (unpaired two-tailed t test, n = 5 mice per group). i Heatmap showing the relative levels of selected genes related to tolerant macrophage pathway based on the RNA-seq data. j Relative mRNA level of Mafk in liver from indicated mice (unpaired two-tailed t test, n = 6 mice per group) (top); immunoblot analysis of liver in indicated mice (bottom). k The qRT-PCR of Mafk in the RAW264.7 cells after 25-HC treatment with a dose dependent for 48 h (unpaired two-tailed t test, n = 3 independent batches) (top); immunoblot analysis of Raw264.7 cells after 25-HC treatment with a dose dependent for 48 h (bottom). l Different expression and quantification of various chemokines in media derived from 25-HC treated or control-RAW264.7 cells was detected by a mouse chemokine array kit (n = 2 independent batches). Red boxes designate the chemokines whose expression was altered by 25-HC treatment. m TGF-β and IL-10 secreted from BMDM cells treated with vehicle and 25-HC were detected by ELISA kit (unpaired two-tailed t test, n = 6 independent batches). Data are presented as mean ± s.d. Source data and exact P value are provided as a Source data file.

Fig 3: PCS enhanced the mitophagy of Tim4+ macrophages by binding Cdh1. (A) Tumour‐conditioned RAW264.7 cells were treated with PCS, and these cells were stained with MitoTracker Green and MitoTracker Deep Red to assess the damaged mitochondria by flow cytometry. The histograms and statistical results were depicted (n = 6). (B) Relative protein expression of mitophagy‐associated molecules in tumour‐conditioned RAW264.7 cells after PCS stimulation measured by Western blot (n = 3). (C) Annexin V‐PI staining detected by flow cytometry showed the effects of PCS on apoptosis in Tim4+ cells in tumour‐conditioned RAW264.7 cells (n = 6). (D) KEGG analysis of DEPs between two groups of Tim4+ TAMs (mentioned in Figure 5) arranged by rich factor. (E) Venn diagram depicted the concordant proteins in DEPs from Hippo signalling pathway from promoted/inhibited OC and down‐/up‐regulated proteins in OC. (F) Relative mRNA expression of 7 genes for further validation in primary Tim4+ TAMs from the murine model of OC after PCS stimulation in vitro measured by qPCR (n = 3). (G) Relative protein expression of Cdh1 for further validation in Tim4+ macrophages in tumour‐conditioned RAW264.7 cells after PCS stimulation measured by Western blot (n = 3). (H) Relative protein expression of mitophagy‐associated molecules in tumour‐conditioned RAW264.7 cells after different treatments: Transfecting pcDNA3.1/pcDNA3.1‐Cdh1, with/without PCS stimulation (n = 3). (I) Representative TEM pictures and statistical results of autophagosomes containing mitochondria in tumour‐conditioned RAW264.7 cells. Black arrows indicate mitophagosomes (n = 3). (J) The two‐dimensional binding model of PCS and Cdh1. (K) The three‐dimensional binding model of PCS on the molecular surface of Cdh1, yellow colour represented hydrogen bonds. Data represent the mean scores ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: The mitigative effect of sh‐CD81 on OC was reversed by PCS. (A) The flow chart of establishing model, and the body weight changes in mice of four groups from the beginning to the end of constructing peritoneal metastasis model, and the final volume of ascites in the peritoneal metastasis murine tumour model of these four groups. (B) Representative fluorescent images of mice using the in vivo imaging system in four groups, and the statistical results of radiance were showed. (C) Representative images of the abdomen and diaphragm after sacrifice, and the number of tumour nodules was quantified. (D) The omentum weight in mice of the four groups. (E) HE staining of the omentum in peritoneal metastasis murine tumour model of the four groups. (F) Representative images of Tim4+ TAMs in four groups, and the percentage of Tim4+ TAMs was quantified. (G) Tim4+ TAMs from the four groups were stained with MitoSOX Green, as detected by flow cytometry to measure the production of superoxide by mitochondria. The histograms and statistical results were depicted. (H) Tim4+ TAMs from the four groups were stained with MitoTracker Green to detect the number of mitochondria. The histograms and statistical results were depicted. (I) Tim4+ TAMs from the four groups were stained with MitoTracker Green and MitoTracker Deep Red, and the representative images and statistical results were depicted. (J) Representative TEM pictures of autophagosomes containing mitochondria in Tim4+ TAMs from four groups. Black arrows indicate mitophagosomes. (K) Relative protein expression of mitophagy‐associated molecules in Tim4+ TAMs from four groups. (L) Annexin V‐PI staining detected by flow cytometry showed the effects of PCS on cell apoptosis in Tim4+ TAMs from four groups. n = 10 per group. (M) A schematic diagram showing how CD81 regulated tumour progression and immune environment. Data represent the mean scores ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: PCS exhibited potential in regulating Tim4+ TAMs survival. (A) Representative images of Tim4+ TAMs in peritoneal metastasis murine tumour model of NC and sh‐CD81 groups, and the percentage of Tim4+ TAMs was quantified (n = 6). (B) The number of Tim4+ TAMs in the peritoneal metastasis murine tumour model of NC and sh‐CD81 groups was quantified (n = 6). (C) Relative protein expression of Tim4 in RAW264.7 cells after co‐culture with TCM from NC and sh‐CD81 ID8 cells measured by Western blot (n = 3). (D) Representative images of Tim4+ macrophages in tumour‐conditioned RAW264.7 cells after PCS stimulation; the percentage of Tim4+ macrophages and MFI of Tim4 were quantified (n = 4). (E) The working flow of integrated analysis between RNA sequencing and database analysis. (F) Volcano plot for DEGs in the two groups of Tim4+ TAMs: Purified from peritoneal metastasis murine tumour model of NC and sh‐CD81 groups (n = 3). (G) GO enrichment analysis of DEPs, including biological process, arranged by the number oFf genes. (H) Correlation analysis of PCS and L‐Tyrosine with apoptotic process related molecules. (I) Venn diagram depicted the concordant proteins in DEPs from unfavourable/favourable and down‐/up‐regulated in OC. (J) Survival curves of three typical cell death‐associated molecules (Bcl2, Fasl and Bnip3) in OC. (K) Relative mRNA expression of 12 vital molecules for further validation in the ascites isolated from NC and sh‐CD81 ID8 cells measured by qPCR (n = 8). Data represent the mean scores ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.