Journal: Advanced Science

Article Title: Spatial Transcriptomics Reveals Transcriptomic and Immune Microenvironment Reprogramming during Thyroid Carcinoma Dedifferentiation

doi: 10.1002/advs.202506925

Figure Lengend Snippet: SpRNAseq analysis of immune infiltration in ATC/PDTC‐DTC coexisting samples. A) Expression patterns of CD163, CD8, CD4, and CD20 across different histological subtypes of thyroid carcinoma identified by IHC. B) Single‐cell subcluster analysis of T cells and myeloid cells using public databases. C) Integrated analysis of spRNA‐seq and scRNA‐seq using the Cell2location algorithm revealed the spatial distribution of TYMP⁺ TAMs in ATC/PDTC‐DTC coexisting samples. D) MIF validation of TYMP⁺ TAMs in ATC‐DTC coexisting samples. E) Distribution of TYMP⁺ TAMs across different tumor niches in spRNAseq. F) Correlation between TYMP⁺ TAMs and pathway scores related to tumor and immune responses in spRNAseq analysis. G) Cell‐cell communication analysis of TYMP⁺ TAMs with T cells, endothelial cells, and fibroblast subpopulations based on scRNAseq data. ATC : Anaplastic Thyroid Carcinoma; DTC : Differentiated Thyroid Carcinoma; HGFCTC : High‐Grade Follicular Cell‐derived Thyroid Carcinoma; IHC : Immunohistochemistry; PDTC : Poorly Differentiated Thyroid Carcinoma; spRNA‐seq : Spatial RNA Sequencing; scRNA‐seq : Single‐Cell RNA Sequencing; TAM : Tumor‐Associated Macrophage; TYMP : Thymidine Phosphorylase; MIF : Multiplex Immunofluorescence. Significance in D was analyzed using the log‐rank test for survival analysis; Significance in A, E was determined using one‐way ANOVA and Bonferroni multiple comparison test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The antibodies used for IHC included: PDCD4 (Proteintech Cat#12587‐1‐AP), CD163 (OriGene Cat#TA506386), CD20 (Cell Signaling Technology Cat# 48750), CD4 (Proteintech Cat#67786‐1‐Ig), and CD8 (ZSGB‐Bio, Cat#ZA‐0508).

Techniques: Expressing, Biomarker Discovery, Derivative Assay, Immunohistochemistry, RNA Sequencing, Multiplex Assay, Immunofluorescence, Comparison

Journal: Biomarker Research

Article Title: RUNX1 promotes angiogenesis in colorectal cancer by regulating the crosstalk between tumor cells and tumor associated macrophages

doi: 10.1186/s40364-024-00573-1

Figure Lengend Snippet: RUNX1 regulates M2 polarization of TAMs. A Experimental scheme of different CMs preparation and in vitro model of cells co-culture. B THP-1 was stimulated with CMs derived from RUNX1 over-expressed (left; HCT116 vector/RUNX1 OE , RKO vector/RUNX1 OE ) or knockdown (right; SW480 shNC/shRUNX1, RKO shNC/shRUNX1) CRC cells, and the IL-10 in supernatant was analyzed by ELISA. C (left) Flow cytometry analysis of macrophage polarization phenotype stimulated by CMs derived from RUNX1 over-expressed HCT116 and RKO cells. (right) Quantification of percentage of CD68 + CD206 + or CD68 + CD200R + macrophages (of macrophages; n = 3). D RT-qPCR analysis of the expression of CD163 mRNA, CD206 mRNA, Arg1 mRNA and IL-10 mRNA in macrophages treated with CMs derived from RUNX1 over-expressed HCT116 (left) and RKO (right) cells. ( E ) (left) Flow cytometry analysis of macrophage polarization state stimulated by CMs derived from RUNX1 knockdown SW480 and RKO cells. (right) Quantification of percentage of CD68 + CD206 + or CD68 + CD200R + macrophages (of macrophages; n = 3). ( F ) RT-qPCR analysis of the expression of CD163 mRNA, CD206 mRNA, Arg1 mRNA and IL-10 mRNA in macrophages treated with CMs derived from RUNX1 knockdown SW480 (left) and RKO (right) cells. G CD206 and CD200R IF labeling of THP-1 cells treated with CRC cells-derived CMs. Data were recorded by a confocal laser scanning microscopy. Scale bars: 10 μm. H Quantification of mean fluorescence intensity of CD206 and CD200R (IntDen/Area; n = 3). I The expression of PTCH1, PTCH2, GLI1, SUFU and Shh in TAMs treated with CMs derived from RUNX1 over-expressed HCT116 cells was determined by western blotting. J (left) Flow cytometry analysis of macrophage phenotype induced by CMs derived from RUNX1 over-expressed HCT116 cells in the presence or absence of GDC-0449. (right) Quantification of percentage of CD68 + CD206 + or CD68 + CD200R + macrophages (of macrophages; n = 3)

Article Snippet: IHC staining was employed following the manufacturer’s instructions (PV-6001, ZSGB-BIO, Beijing, China) using RUNX1 (1:100, 25315-1-AP, Proteintech), CCL2 (1:300, 25542-1-AP, Proteintech), CD68 (ZM-0060, ZSGB-BIO), CD163 (ZM-0428, ZSGB-BIO), F4/80 (1:800, 29414-1-AP, Proteintech) and CD31 (1:500, 28083-1-AP, Proteintech).

Techniques: In Vitro, Co-Culture Assay, Derivative Assay, Plasmid Preparation, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Quantitative RT-PCR, Expressing, Labeling, Confocal Laser Scanning Microscopy, Fluorescence, Western Blot

Journal: Biomarker Research

Article Title: RUNX1 promotes angiogenesis in colorectal cancer by regulating the crosstalk between tumor cells and tumor associated macrophages

doi: 10.1186/s40364-024-00573-1

Figure Lengend Snippet: RUNX1 promotes TAMs infiltration in CRC. A Circular plot of immune cells enriched for the RUNX1-associated genes in COAD. RUNX1 expression is well related to M2 macrophage in COAD. B Correlation analysis between RUNX1 and IL-10 (left), CD206 (middle) and CD163 (right) in the TCGA database. C RT-qPCR analysis of the RUNX1 mRNA expression in tumor specimens and matched normal tissues ( n = 66). D Correlation between the mRNA expression of RUNX1 and CD163, Arg1 in CRC tissues ( n = 30). E Representative IHC analysis of RUNX1, CD68, CD163 and CCL2 expression in 12 pairs of cancer and the adjacent normal tissue from CRC patients. Scale bars: 100 μm. F Correlation between the expression of RUNX1 and CD68, CD163 in the IHC analysis. G Multi-label immunofluorescence staining of tumor tissue in patients with CRC. H The expression of RUNX1 in CRC tumor tissues and matched normal tissues was determined by western blotting ( n = 24). I Multi-label immunofluorescence staining of tumor tissue from the nude mouse orthotopic CRC tumors

Article Snippet: IHC staining was employed following the manufacturer’s instructions (PV-6001, ZSGB-BIO, Beijing, China) using RUNX1 (1:100, 25315-1-AP, Proteintech), CCL2 (1:300, 25542-1-AP, Proteintech), CD68 (ZM-0060, ZSGB-BIO), CD163 (ZM-0428, ZSGB-BIO), F4/80 (1:800, 29414-1-AP, Proteintech) and CD31 (1:500, 28083-1-AP, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot

Journal: Journal of Virology

Article Title: Combinational Deletions of MGF360-9L and MGF505-7R Attenuated Highly Virulent African Swine Fever Virus and Conferred Protection against Homologous Challenge

doi: 10.1128/jvi.00329-22

Figure Lengend Snippet: ASFV-Δ9L/Δ7R induced T cell activation and macrophage infiltration in spleens. (A) Interferon gamma (IFN-γ) status in sections of spleens from one healthy animal (H), 5 vaccinated animals (V1, V2, V3, V5, and V6), and one contact animal (C3), as shown by IHC staining. Results for IFN-γ were negative in examined sections from healthy and contact pigs. (B) Integrated optical density (IOD) results from the experiment whose results are shown in panel A. (C, D) Immunohistochemical staining of CD4 + T cells (C) and CD163 + macrophages (D) in the spleens of two vaccinated animals (V2 and V3) and one contact animal (C3). Scale bars, 100 μm.

Article Snippet: Immunostaining for T-cell phenotypes was performed using the following reagents: interferon gamma polyclonal antibody (catalog no. 15365-1-AP; Proteintech Group), IHCeasyCD3 ready-to-use IHC kit (epsilon chain) (catalog no. KHC0013; Proteintech Group), anti-CD3 epsilon antibody (DF6594; Affinity Biosciences), anti-CD4 monoclonal antibody (catalog no. 67786-Ig; Proteintech Group), anti-CD4 mouse monoclonal antibody (clone UMAB64) (catalog no. UM800010CF; Origene), anti-CD8a antibody (RPA-T8) (catalog no. 65144-Ig; Proteintech Group), anti-CD163 antibody (EDHu-1) (NB110-40686; Novus), anti-CD68 antibody (Affinity Biosciences; DF7518), IHCeasyCD68 ready-to-use IHC kit (catalog no. KHC0006; Proteintech Group), anti-CD163 mouse monoclonal antibody (clone OTI3B4) (catalog no. TA506386; Origene), and anti-CD163 polyclonal antibody (catalog no. 16646-1-AP; Proteintech Group).

Techniques: Activation Assay, Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Biomarker Research

Article Title: RUNX1 promotes angiogenesis in colorectal cancer by regulating the crosstalk between tumor cells and tumor associated macrophages

doi: 10.1186/s40364-024-00573-1

Figure Lengend Snippet: RUNX1 regulates M2 polarization of TAMs. A Experimental scheme of different CMs preparation and in vitro model of cells co-culture. B THP-1 was stimulated with CMs derived from RUNX1 over-expressed (left; HCT116 vector/RUNX1 OE , RKO vector/RUNX1 OE ) or knockdown (right; SW480 shNC/shRUNX1, RKO shNC/shRUNX1) CRC cells, and the IL-10 in supernatant was analyzed by ELISA. C (left) Flow cytometry analysis of macrophage polarization phenotype stimulated by CMs derived from RUNX1 over-expressed HCT116 and RKO cells. (right) Quantification of percentage of CD68 + CD206 + or CD68 + CD200R + macrophages (of macrophages; n = 3). D RT-qPCR analysis of the expression of CD163 mRNA, CD206 mRNA, Arg1 mRNA and IL-10 mRNA in macrophages treated with CMs derived from RUNX1 over-expressed HCT116 (left) and RKO (right) cells. ( E ) (left) Flow cytometry analysis of macrophage polarization state stimulated by CMs derived from RUNX1 knockdown SW480 and RKO cells. (right) Quantification of percentage of CD68 + CD206 + or CD68 + CD200R + macrophages (of macrophages; n = 3). ( F ) RT-qPCR analysis of the expression of CD163 mRNA, CD206 mRNA, Arg1 mRNA and IL-10 mRNA in macrophages treated with CMs derived from RUNX1 knockdown SW480 (left) and RKO (right) cells. G CD206 and CD200R IF labeling of THP-1 cells treated with CRC cells-derived CMs. Data were recorded by a confocal laser scanning microscopy. Scale bars: 10 μm. H Quantification of mean fluorescence intensity of CD206 and CD200R (IntDen/Area; n = 3). I The expression of PTCH1, PTCH2, GLI1, SUFU and Shh in TAMs treated with CMs derived from RUNX1 over-expressed HCT116 cells was determined by western blotting. J (left) Flow cytometry analysis of macrophage phenotype induced by CMs derived from RUNX1 over-expressed HCT116 cells in the presence or absence of GDC-0449. (right) Quantification of percentage of CD68 + CD206 + or CD68 + CD200R + macrophages (of macrophages; n = 3)

Article Snippet: IHC staining was employed following the manufacturer’s instructions (PV-6001, ZSGB-BIO, Beijing, China) using RUNX1 (1:100, 25315-1-AP, Proteintech), CCL2 (1:300, 25542-1-AP, Proteintech), CD68 (ZM-0060, ZSGB-BIO), CD163 (ZM-0428, ZSGB-BIO), F4/80 (1:800, 29414-1-AP, Proteintech) and CD31 (1:500, 28083-1-AP, Proteintech).

Techniques: In Vitro, Co-Culture Assay, Derivative Assay, Plasmid Preparation, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Quantitative RT-PCR, Expressing, Labeling, Confocal Laser Scanning Microscopy, Fluorescence, Western Blot

Journal: Biomarker Research

Article Title: RUNX1 promotes angiogenesis in colorectal cancer by regulating the crosstalk between tumor cells and tumor associated macrophages

doi: 10.1186/s40364-024-00573-1

Figure Lengend Snippet: RUNX1 promotes TAMs infiltration in CRC. A Circular plot of immune cells enriched for the RUNX1-associated genes in COAD. RUNX1 expression is well related to M2 macrophage in COAD. B Correlation analysis between RUNX1 and IL-10 (left), CD206 (middle) and CD163 (right) in the TCGA database. C RT-qPCR analysis of the RUNX1 mRNA expression in tumor specimens and matched normal tissues ( n = 66). D Correlation between the mRNA expression of RUNX1 and CD163, Arg1 in CRC tissues ( n = 30). E Representative IHC analysis of RUNX1, CD68, CD163 and CCL2 expression in 12 pairs of cancer and the adjacent normal tissue from CRC patients. Scale bars: 100 μm. F Correlation between the expression of RUNX1 and CD68, CD163 in the IHC analysis. G Multi-label immunofluorescence staining of tumor tissue in patients with CRC. H The expression of RUNX1 in CRC tumor tissues and matched normal tissues was determined by western blotting ( n = 24). I Multi-label immunofluorescence staining of tumor tissue from the nude mouse orthotopic CRC tumors

Article Snippet: IHC staining was employed following the manufacturer’s instructions (PV-6001, ZSGB-BIO, Beijing, China) using RUNX1 (1:100, 25315-1-AP, Proteintech), CCL2 (1:300, 25542-1-AP, Proteintech), CD68 (ZM-0060, ZSGB-BIO), CD163 (ZM-0428, ZSGB-BIO), F4/80 (1:800, 29414-1-AP, Proteintech) and CD31 (1:500, 28083-1-AP, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot