S100A10 promotes cancer metastasis via recruitment of MDSCs within the lungs

Figures & data

Figure 1. S100A10 deficiency prevents lung metastasis.

a) Representative images of B16/F10 lung metastasis in S100A10-deficient or WT mice via luciferase bioluminescence in vivo. b) Representative images of B16/F10 and E0771 lung metastasis nodules in S100A10-deficient or WT mice. c) Quantitation of B16/F10 and E0771 tumor cell focia within the lung tissue (n = 6 individual mice in each group, **p < 0.01). d) Quantitation of B16/F10 and E0771 metastasis lung weight (n = 6 individual mice in each group, **p < 0.01, *p < 0.05). e) Representative images of B16/F10 lung metastasis tissue in WT or KO mice via luciferase bioluminescence and GFP-labeled fluorescence. f) Immunofluorescent analysis S100A10-deficient or WT mice lung sections after GFP-labeled B16/F10 and E0771 inoculation (blue-DAPI, green-GFP, Scale bar, 100 μm). g) The percentage of GFP-positive B16/F10 and E0771 cells in the lung tissue of WT or KO mice detected by flow cytometry (n = 5 individual mice in each group, ***p < 0.001, **p < 0.01). h) Schematic diagram for the lung metastasis of the in situ tumor. i) Representative luciferase bioluminescence images of B16/F10 lung metastasis from in situ S100A10-deficient or WT mice. j) Representative images of B16/F10 lung metastasis tissue from in situ S100A10-deficient or WT mice via luciferase bioluminescence. k) H&E-stained lung sections of S100A10-deficient or WT mice after tail-vein inoculation of B16/F10 cell. l) H&E-stained lung sections of KO or WT mice after subcutaneous injection of B16/F10 cell.

Figure 2. S100A10 deficiency prevents lung pre-metastatic microenvironment formation.

a) Schematic diagram for the tumor-derived exosome induced formation of the pre-metastatic microenvironment. 10 μg of B16/F10 and E0771-derived exosomes in 50 μl of PBS were intravenously injected into the tail vein of S100A10-deficient or WT mice every 2 d. Pre-metastatic microenvironment formation in the lungs of mice after 28 d. b and c) Pro-metastatic gene expression in the lungs of S100A10-deficient or WT mice after B16/F10-derived exosome b) and E0771-derived exosomes c) induction was quantified by qPCR. d and e) Immunofluorescent analysis of lung tissue sections d) and IHC stained lung tissue sections e) of the fibronectin expression of S100A10-deficient mice or WT mice. f) Top 10 biological processes with enrichment of different expression gene sets in S100A10-deficient or WT mice lungs by GO analysis (n = 3 individual mice in each group). g) Top 10 signaling pathways with enrichment of different expression gene sets in S100A10-deficient or WT mice lungs by KEGG analysis (n = 3 individual mice in each group).

Figure 3. S100A10 deficiency inhibits MDSCs recruitment to the lung pre-metastatic microenvironment.

a) The proportion of MDSCs in the lungs of KO or WT mice after B16/F10 or E0771-derived exosome inoculation was measured via flow cytometry (n = 6 individual mice in each group, *p < 0.05, **p < 0.01). b) The proportion of PMN-MDSCs and M-MDSCs in the lungs of KO or WT mice after B16/F10-derived exosome inoculation was measured via flow cytometry (n = 6 individual mice in each group, *p < 0.05, **p < 0.01). c) The proportion of CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells in the lungs of KO or WT mice after B16/F10-derived exosome inoculation was measured by flow cytometry (n = 6 individual mice in each group, *p < 0.05, **p < 0.01). d) Immunofluorescent analysis of Gr-1⁺ cells in WT or KO mice lung sections after B16/F10-derived exosome inoculation (blue-DAPI, green-Gr-1, Scale bar, 25 μm). e) Model diagram of the co-culture system of the T cell, S100A10^−/− MDSCs, and S100A10^+/+ MDSCs. f) Representative images of CFSE-base proliferation assay of T cells co-cultured with S100A10^−/− MDSCs and S100A10^+/+ MDSCs by flow cytometry. g) Quantitative T cell proliferation analysis after co-cultured with S100A10^−/− MDSCs and S100A10^+/+ MDSCs. h) The ARG1, iNOS, and IDO expression of S100A10^−/− and S100A10^+/+ MDSCs in the lung pre-metastatic microenvironment were quantified by qPCR. i) Anti-Gr-1 Abs or isotype control was injected into the tail-vein of WT and KO mice at 200 µg/mouse the day before the B16/F10^LUC were implanted. Subsequently, Gr-1 Abs or isotype control was injected every 3 d into tail veins of WT and KO mice. B16/F10 lung metastasis was monitored via luciferase bioluminescence in vivo.

Figure 4. The S100A10 of lung fibroblasts activates CXCL1/CXCL8 expression and increases the migration of myeloid lineage via B16/F10-derived exosome induction.

a) Immunofluorescent analysis of the location of S100A4⁺ fibroblast and PKH26 marked B16/F10-derived exosome in the lungs of WT mice. b) S100A10 expression in NIH3T3 fibroblast cells stimulated with B16/F10-derived exosomes was detected via immunoblot. c) Chemokine gene expression in NIH3T3 fibroblast cells after B16-derived exosome induction was quantified by qPCR (n = 6 individual cells in each group, *p < 0.05, **p < 0.01). d) Numbers of MDSCs migrating in response to B16/F10 exosome-stimulated fibroblasts conditioned medium (FCM) (n = 6 individual cells in each group, **p < 0.01). e) S100A10 gene knockdown efficiency in NIH3T3 fibroblast cells was detected via immunoblot. f) Chemokine gene expression in S100A10 knockdown NIH3T3 fibroblast cells after B16-derived exosome induction was quantified via qPCR (n = 6 individual cells in each group, *p < 0.05, **p < 0.01). g) Numbers of MDSCs migrating in response to B16/F10 exosome-stimulated S100A10 knockdown NIH3T3 fibroblasts conditioned medium (n = 6 individual cells in each group, **p < 0.01). h) Chemokine gene expression in NIH3T3 fibroblast cells after S100A10 recombinant protein induction was quantified via qPCR (n = 6 individual cells in each group, *p < 0.05, **p < 0.01). i) Numbers of MDSCs migrating in response to the recombinant protein S100A10 stimulated NIH3T3 fibroblasts conditioned medium (n = 6 individual cells in each group, ***p < 0.001).

Figure 5. Upregulation of S100A10 expression in lung fibroblasts promotes lung metastasis of tumor in KO mice.

a) The schematic diagram for AAV6-NC or AAV6-S100A10 infection in the lungs of WT and KO mice. Six-week-old WT and KO mice were intratracheally injected with AAV6-NC and AAV6-S100A10. Four weeks after AAV administration, the B16/F10^LUC cells were injected into the tail-vein of WT and KO mice. Two weeks after the B16/F10^LUC cells were injected, tumor metastasis was assessed via luciferase bioluminescence in vivo. b) qRCR analysis of S100A10 mRNA expression in the lungs of mice from the AAV6-NC and AAV6-S100A10 groups (n = 6 individual cells in each group, ***p < 0.001). c) Western blot analysis of S100A10 expression in the lungs of mice from the AAV6-NC and AAV6-S100A10 groups (n = 3 per group). d) Immunofluorescence staining with S100A4⁺ fibroblasts and Flag-marked fibroblast-specific AAV6-S100A10 in the lungs of WT mice. e) Statistical analysis of the percent of AAV6-S100A10 transduction in S100A4⁺ fibroblasts (n = 3 mice, five fields assessed per mouse). f) Representative luciferase bioluminescence images of B16/F10 lung metastasis in WT or KO mice via AAV6-NC and AAV6-S100A10 infection. g and h) The proportion of MDSCs in the lungs of KO or WT mice after AAV6-NC and AAV6-S100A10 infection was measured via flow cytometry (n = 6 individual mice in each group, *p < 0.05, **p < 0.01).

Figure 6. S100A10 inhibitor prevents lung metastasis.

a) Chemical structures of the S100A10 inhibitor 1-substituted 4-aroyl-3-hydroxy-5-phenyl-1 H-pyrrol-2(5 H)-ones. b) Representative images of B16/F10 lung metastasis nodules of C57 mice in treated with vehicle or S100A10 inhibitor 30 mg/kg/d via BFI. c) In vivo quantitation of B16/F10 metastasis lung weight of C57 mice treated with vehicle or S100A10 inhibitor 30 mg/kg/d (n = 5 individual mice in each group, *p < 0.05). d) H&E-stained lung sections of C57 mice treated with vehicle or S100A10 inhibitor 30 mg/kg/d. e) Schematic activation model of S100A10 in host lung fibroblast cells via tumor exosome initiated MDSCs recruitment promotes lung pre-metastatic microenvironment formation and cancer metastasis.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.