Additional file 1: of Crosstalk between cancer cells and tumor associated macrophages is required for mesenchymal circulating tumor cell-mediated colorectal cancer metastasis
Creators: name: Wei, Chen
name: Yang, Chaogang
name: Wang, Shuyi
name: Shi, Dongdong
name: Zhang, Chunxiao
name: Lin, Xiaobin
name: Liu, Qing
name: Dou, Rongzhang
name: Xiong, Bin
Publisher: name: figshare
Year: 2019
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer

description: Table S1. Correlation between the density of macrophages at non-invasive front and clinicopathologic parameters (n = 81). Table S2. Potential STAT3 binding site on miR-506-3p promoter. Table S3. The sequences of the primers for quantitative RT-PCR. Figure S1. CD68+ and CD163+ TAMs at non-invasive front are not associated with EMT, MCTC ratio, and poor prognosis in CRC patients. (A-B) Expression of E-cadherin and Vimentin in human CRC samples with low or high CD68 and CD163 expression at non-invasive front, respectively. (C-D) Correlation analysis between CD68 expression and CD163 at non-invasive front and MCTC ratio, respectively. (E-F) CD68 expression at non-invasive front and the patients’ recurrence-free survival and overall survival in CRC, respectively. (G-H) CD163 expression at non-invasive front and the patients’ recurrence-free survival and overall survival in CRC, respectively. Error bars, SEM. ns, not significant. Figure S2. Validation of in-vitro-generated TAMs. (A) Flow cytometry for analyzing the expression of HLA-DR, CD80, CD206, and CD163 in PMA-treated THP-1 macrophages incubated with conditioned media (CM) from CRC cell lines (HT-29 or HCT116) or normal cell line (NCM460) for 48 h. (B) ELISA for analyzing the secretion of IL-10 and IL-12 in PMA-treated THP-1 macrophages incubated with conditioned media (CM) from CRC cell lines (HT-29 or HCT116) or normal cell line (NCM460) for 48 h; Error bars, SD. (C) ELISA for analyzing the secretion of IL-1β, IFN-γ, and TNF-α in PMA-treated THP-1 macrophages incubated with conditioned media (CM) from CRC cell lines (HT-29 or HCT116) or normal cell line (NCM460) for 48 h; Error bars, SD. (D) The morphology of colorectal cancer cells (HCT116 and HT29) with or without TAMs-coculture. ns, not significant; *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001. Figure S3. STAT3 directly suppressed miR-506-3p expression in CRC cells. (A) A graphical illustration of five potential STAT3 transcriptional factor binding sites in the miR-506-3p promoter region. (B) Serially truncated and mutated miR-506-3p promoter constructs were cloned to pGL3-Basic luciferase reporters and transfected into HT29 cells. The relative luciferase activities were determined after IL-6 (50 ng/mL) treatment for 1 h; Error bars, SD. (C) Selective mutation analyses identified STAT3-responsive regions in the miR-506-3p promoter in HT29 cells; Error bars, SD. (D) ChIP assay demonstrated the direct binding of STAT3 to the miR-506-3p promoter, including nonspecific control (N.C), CHIP1, and CHIP2 in HT29 cells. Input, 5% of total lysate. (E) RT-PCR of the ChIP products confirmed the direct binding capacity of STAT3 to the miR-506-3p promoter in HT29 cells. Input, 5% of total lysate; Error bars, SD. ns, not significant; *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001 (DOCX 2752 kb)