Fig 1: TBX3 deficiency blocked tumor formation due to suppressed CXCR2-mediated MDSCs recruitment.a Flow cytometric analysis of MDSCs (CD45+CD11b+Gr-1+) in thyroid glands from mPTC (n = 14), mPTC/Tbx3+/- (n = 17) or mPTC/Tbx3-/- (n = 11) littermates at 5w. b, c Percentage of G-MDSC (CD45+CD11b+Ly6C-Ly6G+) and M-MDSC (CD45+CD11b+Ly6C+Ly6G-) of CD45+ tumor-infiltrating leukocytes (TILs) in mPTC tumors compared with mPTC/Tbx3-/- littermates at (b). Relative CD8+ T cells/MDSCs was plotted (c), n = 4. d IHC analysis of CD11b, MDSCs (Gr-1, S100A8), CD4+ T cells (CD4) and CD8+ T cells (CD8) in thyroid glands from mPTC/Tbx3-/- compared with mPTC littermates. Scale bars, 50µm. e, f Flow cytometric analysis of G-MDSC and M-MDSC in mPTC-TAM or mPTC-TAM/Tbx3-/- tumors induced with tamoxifen for 8 m. CD8+ T cells/MDSCs was plotted, n = 4. g Mice bearing mPTC and mPTC/Tbx3-/- tumors were treated with 200 µg anti-CD8 antibody per mouse twice weekly at age of 3w for 20d. Tumor weights were counted, n = 6. h Mice bearing tumors received daily oral doses of PLX4032 10 mg/kg body weight or SB265610 2 mg/kg body weight at age of 3w for 20d. Tumor weights were counted, n = 7. i, j Flow cytometric analysis of mPTC tumors underwent above treatments. Percentage of positive cells relative to total cells was plotted. MDSCs (i) and CD8+ T cells/MDSCs (j), n = 3. A representative of three independent experiments was shown (d). Data are shown as the mean ± s.d. (a–c, e–j). P values were calculated by unpaired two-tailed Student’s t test (a–c, e–j). Statistical source data are provided in Source Data.
Fig 2: Membrane protein composition of Neu-LPs. A SDS-PAGE analysis of neutrophils, NMVs, and Neu-LPs. B Western blotting analysis of characteristic protein bands (TNFaR, IL1ßR, IL6R, LFA-R, and CXCR2) in NMVs and Neu-LPs. NLWNM: Neutrophil lysate without neutrophil membrane protein
Fig 3: Single-cell transcriptomic analysis reveals the transcriptome of myeloid cells in the microenvironment of LUSC. a. tSNE plot of myeloid cells color-coded by their associated clusters. b. tSNE plot of myeloid cells color-coded by their associated sample origins. c. tSNE plot of myeloid cells color-coded by cell subtypes established by marker genes. d. The frequency and proportion of each cell subtype from tumor and normal tissues. e. The boxplot showed the proportion of neutrophils in tumor and normal tissues, respectively, in the TCGA LUSC cohort. (The difference between normal and tumor tissues was calculated by Wilcoxon-test). f. Module scores of genes related to inflammation and N2 signature of neutrophil cluster from tumor and normal tissues, respectively. g. Correlation between neutrophil and expression level of CXCR2(top)/CXCL8(bottom) in TCGA LUSC cohort, respectively (The correlation coefficient and difference were calculated by Pearson-test). h. Heatmap shows the difference in pathway activities scored by GSVA per cell between different monocyte/macrophage groups. i. The boxplot showed the proportion of SPP1+M, SEPP1+M and FABP4+M from tumor and normal tissues, respectively, in the TCGA LUSC cohort. (Significance of difference between tumor and normal tissues was calculated by Wilcoxon-test). j. Association between cell abundance and patient survival from TCGA LUSC cohort (P value was calculated with log-rank test).
Fig 4: CXCR2 ligands function downstream of TBX3 and promote tumor cell proliferation.a, b PTC cells were infected with specific shRNA against CXCL1/2 or CXCL8, and subjected to CCK8 (a) or colony formation assay (b). c, d TBX3 knock-down PTC cells with or without CXCLs over-expression were subjected to cell growth assays of CCK8 (c) or colony formation (d). e K1 cells with TBX3 knock-down and Vector or CXCLs over-expression were transplanted into athymic mice subcutaneously. Tumor weights as well as curves were generated and statistically compared, n = 6. f HE and IHC staining of indicated factors were performed on tumor sections in (e). Scale bars, 50µm. n = 3 biological independent samples (a–d). Data are shown as the mean ± s.d. (a–e). P values were calculated by unpaired two-tailed Student’s t test (a–e). Statistical source data are provided in Source Data.
Fig 5: CXCR2 is a functional target of miR-495-3p in glioma cells. A: miR-495-3p contains a potential binding site for CXCR2 as predicted by TargetScan (http://www.targetscan.org/vert_72/). B. RT-PCR was used to detect CXCR2 mRNA level in glioma and normal tissues. C and D. Pearson repression analysis was used to compare the relationships of circ_0000215 and CXCR2 (C), miR-495-3p and CXCR2 (D). E. Dual-luciferase reporter gene assay was used to verify the binding relationship between miR-495-3p and CXCR2. F. Enrichment of CXCR2 in immunoprecipitation of cell lysates were detected by qRT-PCR in RIP assay. G and H: The expression of CXCR2 was detected via qRT-PCR. I and J: The protein level of CXCR2, PI3 K, Akt were detected by western blot. NS, *,**,*** represents P > 0.05, P < 0.05, P < 0.01 and P < 0.001.
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