Fig 2: Circ-0034880-enriched TEVs facilitate the activation of SPP1highCD206+ pro-tumor macrophages by protecting SPP1 from miR-200a-3p and miR-141-3p-mediated degradation. A, B Volcano plots of log2 fold change (FC) and log10 adjusted p value of differentially expressed genes between MC38-EVs-treated BMDMs and control BMDMs or between MC38-EVs-treated BMDMs and MC38shRNA-EVs-treated BMDMs. MC38shRNA-EVs represents EVs derived from MC38 cells of circ-0034880 silencing. C Venn diagram assay of up-regulated genes in MC38-EVs-treated BMDMs and down-regulated genes in MC38shRNA-EVs-treated BMDMs. D Heatmap of top 30 upregulated and downregulated genes. E qPCR validation of the top 10 differentially expressed genes in BMDMs treated with or without circ-0034880 gene silencing. F qPCR detection of SPP1 gene in BMDMs that pretreated with IL-4, MC38-EVs, or MC38shRNA EVs, or none. G Immunoblotting and quantitative assays of SPP1 in BMDMs that pretreated with IL-4, MC38-EVs, or MC38shRNA EVs, or none. H Immunofluorescence and quantitative analysis of SPP1 in BMDMs that pretreated with IL-4, MC38-EVs, or MC38shRNA EVs, or none. Scale bar, 25 μm. I ELISA and quantitative assays of SPP1 levels in the supernatant of BMDMs that pretreated with IL-4, MC38-EVs, or MC38shRNA EVs, or none. J Immunofluorescence analysis of SPP1 and CD206 in the mice liver metastatic tissues from Fig. 2E. Antibody against SPP1 and CD206 were used in this part. Yellow arrow represents SPP1 in TAMs (CD206+), white arrow represents SPP1 in the whole mesenchyme of TME. Scale bar, 20 μm. K CircRNA interactome databases and RegRNA were used to predict the potential target miRNAs of circ-0034880 and SPP1, respectively. Venn diagram showed the mutual putative target miRNAs of circ-0034880 and SPP1. L MiR-200a-3p and miR-141-3p were selected as the potential bridge between circ-0034880 and SPP1. M Interaction between circ-0034880 and AGO2 protein was confirmed by RIP and qPCR assay in H293T cells. N Potential miRNAs (miR-200a-3p and miR-141-3p) that bind with circ-0034880 were detected by qPCR using the anti-AGO2 immunoprecipitates. O Luciferase reporter activity was measured in H293T cells after co-transfection with circ-0034880-WT or circ-0034880-MUT and miR-200a-3p/miR-141-3p mimics. P Luciferase reporter activity was measured in H293T cells after co-transfection with SPP1 3’UTR-WT or SPP1 3’UTR-MUT and miR-200a-3p/miR-141-3p mimics. All results were shown as mean ± SD. Student’s t-test was used to analyze the data. *, P < 0.05; **, P < 0.01

Fig 3: Distinct cell-cell interaction patterns in left-sided versus right-sided colon cancer microenvironments(A) Bar charts showing the intensity of communication between PShigh and PSlow malignant epithelium, respectively, with immune cells in LT. Circle plots showing the change in cell communication intensity between PShigh malignant epithelium and immune cell subpopulations in LT compared to PSlow malignant epithelium and immune cell subpopulations, where the red line represents an increase in signal intensity, while the blue line represents signal strength decreases. The thickness of the line is directly proportional to the strength of the interaction (discovery cohort).(B) Bar charts showing the intensity of communication between IShigh and ISlow malignant epithelium, respectively, with immune cells in RT. Circle plots showing the change in cell communication intensity between IShigh malignant epithelium and immune cell subpopulations in RT compared to ISlow malignant epithelium and immune cell subpopulations (discovery cohort).(C) Spatial feature plots of PS signature score, Mph-PLTP cells, CD8-LAYN cells, and Tregs in LCC tissue sections (top). Spatial feature plots of IS signature score, Mph-SPP1 cells, CD8-CD161 cells, and NK-XCL1 cells in RCC tissue sections (bottom).(D) Representative mIHC staining of ALDH1+ malignant epithelium and CD163+ macrophages in LT (validation cohort 5).(E) The upregulated signaling ligand-receptor pairs between PShigh malignant epithelium with Mph-PLTP cells in LT compared to PSlow malignant epithelium. Dot sizes represent p value (only p < 0.05 shown).(F) Top-ranked ligands inferred to regulate PShigh malignant epithelium by Mph-PLTP cells according to NicheNet. Dot plots show the expression percentage (dot size) and intensity (dot intensity) of top-ranked ligands in Mph-PLTP cells. Heatmap showing regulatory potential of top-ranked ligand and the downstream target genes in PShigh malignant epithelium.(G and H) Representative images of mIHC staining indicating CD74+ malignant epithelium, SPP1+ macrophages (G), and CD161+ CD8+ T cells (H) in the hypoxic area of RT samples (validation cohort 5).(I) The upregulated signaling ligand-receptor pairs between IShigh malignant epithelium with Mph-SPP1 cells and CD8-CD161 cells in RT compared to ISlow malignant epithelium. Dot sizes represent p value (only p < 0.05 shown). Arrows show the direction of regulatory relationships between cell populations.(J) Representative mIHC staining showing the crosstalk between PD-L1+ IShigh malignant epithelium and CD80+SPP1+ macrophages via the ligand-receptor of PD-L1-CD80 in the hypoxic area of RT.

Fig 4: Detection of odontoblast-like cells in the regenerated dental pulp-like tissue. Representative images of hematoxylin and eosin (H&E)-stained sections and immunohistochemistry (IHC) sections stained with nestin, osteopontin (OPN), and green fluorescent protein (GFP) antibodies, as well as osteocalcin. Nestin (odontoblast marker) is not detected in regenerative endodontic procedure (REP) groups, whereas in the REP + dental pulp cell (DPC) groups, the regenerated tissue contains nestin-stained cells that co-express GFP (arrows). OPN and osteocalcin were detected in both groups, with the former being more abundant in the cellular matrix of the REP + DPC group (n = 6 per group). Scale bars = 50 µm (arrows indicate nestin and GFP co-expressing cells). 4′,6-diamidino-2-phenylindole: DAPI, Osteopontin: OPN.

Fig 5: Rb1 administration inhibits CRLM via impeding circ-0034880-enriched TEVs mediated activation of SPP1highCD206+ pro-tumor TAMs. A Flow chart of Rb1 intervention in mice model of experimental liver metastasis. B, C Luciferase-based bioluminescence imaging on experimental liver metastasis of the indicated mice treated without EVs or with EVs derived from circ-0034880-enriched MC38 cells, or circ-0034880-silenced MC38 cells, or Rb1. n = 6 for each group. Representative bioluminescent images of mice in indicated groups were shown in Fig. 8B, and the quantification data (fluorescence intensity) was presented in Fig. 8C. D, E, F, G Photograph and HE staining to observe the effect of Rb1 on liver metastasis of mice colon cancer. Scale bar, 2 mm (up), 500 μm (down). n = 6 for each group. Representative liver tissues images of mice in indicated groups were shown in Fig. 8D, and the quantification data (liver/body weight, %) was presented in Fig. 8F. Representative HE staining pictures of liver tissue sections from indicated mice were shown in Fig. 8E (up and middle). In addition, the representative immunohistochemical results using anti-CK20 and anti-glypican-3 (GPC-3) antibody were also shown in Fig. 8E to indicate that the metastatic origin of tumor in liver tissues are from colon tissues (down). The quantification data (tumor burden, %) for HE staining results was presented in Fig. 8G. Scale bar, 2 mm (up), 500 μm (middle), 40 μm (down). H Immunofluorescence analysis of TAMs in the liver metastatic tissues from above Rb1 intervene model. Scale bar, 50 μm. I FISH assay of circ-0034880 in the liver metastatic tissues from above Rb1 intervene model. Scale bar, 25 μm. J Immunofluorescence analysis of SPP1 in the liver metastatic tissues from above Rb1 intervene model. Scale bar, 20 μm. K, L, M The quantitative assay for Fig. 8H, I, J. N A schematic mechanism underlying interactions between primary CRC cells and macrophages in distant liver organs. All results were shown as mean ± SD. Student’s t-test was used to analyze the data. *, P < 0.05; **, P < 0.01