Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Figure 8. Negative corre- lation between ecto- enzymes and CCND1 expression in CRC. (A) IHC staining of human normal colon (n ¼ 40) and colon adenocarcinoma (n ¼ 64) specimens in a tissue microarray for CCND1 and CD163 (a macrophage marker). (B) Relative intensities of CCND1 staining in the lamina propria of a normal and cancerous colon. **P < .01 (unpaired t test). (C) Correlation analysis of NT5E and CCND1 expres- sion in the TCGA colorectal adenocarcinoma dataset. ***P < .001 (Student t test).

Article Snippet: For the fluorescence-activated cell sorting analysis, cells were stained with APC,-anti human CD163 (clone: GHI/61), PD-anti human CTLA4 (clone: L3D10), Alexa 647-anti human IDO1 (clone: 2E2/IDO1), PE-anti human DR4 (clone: DJR1), FITC-anti human CD47 (clone: REA220, FITC-anti human MICA&B (clone: 6D4), PE-anti human PD-L1 (clone: MIH2), PD-anti human CD69 (clone: FNM50), FITCanti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421TM-anti mouse CD11b (clone: M1/70), Brilliant Violet 570TM-anti mouse CD45 (clone: 104), PE-anti-human CD45 (clone: HI30), monoclonal antibodies (Biolegend; San Diego, CA, USA), FITC-anti-human CD40 (clone: REA733), PE-anti human CD80 (clone: 2D10), PE/vio770-anti human CD206 (clone: DCN228), PE-anti human CD62E (clone: REA280), PE-anti human CD192 (clone: REA264), APC-anti human I-CAM (clone: REA266), APC-anti human HLA-DR, DP, DQ (clone: REA332), APC-anti human CCL2 (clone: REA485), APC-anti human CD14 (clone: HI30), Vioblue-anti human CD31 (clone: TUK4), FTIC-anti-human CD86 (clone: FM95), PE-anti human CD73 (clone: AD2), PE-anti mouse PD-L1 (clone: 60533), PE-anti mouse CD39, and PE-anti human CD39 (clone: REA739) monoclonal antibodies (Miltenyi Biotec; Bergisch Gladbach, Germany), and PE/CF594anti human CD3 (clone: UCHT1), PE-anti human CD44 (clone: G44-26), PE-anti human CD183 (clone: 150503), and PD-1 (clone: REA739) monoclonal antibodies (BD Biosciences; San Jose, CA, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Marker, Staining

Journal: Nature Communications

Article Title: Metabolic targeting of cancer associated fibroblasts overcomes T-cell exclusion and chemoresistance in soft-tissue sarcomas

doi: 10.1038/s41467-024-46504-4

Figure Lengend Snippet: a Absolute counts of CD90 + CAF by flow cytometry ( n = 5 mice). b Quantification of the distance from CD90 + CAF stroma to tumor invasive margin assessed by immunofluorescence staining. Dots represent individual distances from independent CD90+ cells to the tumor margin ( n = 16 Ccne1 + , n = 13 Vgll3 + ) taken from n = 3 independent mice. c Schematic of isolation of mouse CAF from Ccne1 + tumors for scRNA-seq ( n = 4 mice). d t-SNE depicting 4 major CAF clusters; inflammatory CAF (iCAF), matrix CAF (mCAF), antigen-presenting CAF (apCAF), and glycolytic CAF (glyCAF). e Dot plot of cell-typing markers used to confirm fibroblast identity in CAF clusters. f Expression of top marker genes differentially expressed by the CAF sub-clusters. Box denotes Nt5e (CD73) expression in CAF sub-clusters. g KEGG pathways enriched in the glyCAF cluster. h Flow cytometry gating strategy for glyCAF (CD90 + CD73 + ) and non-glyCAF (CD90 + CD73 − ) gated on dsRED − CD45 − CD31 − cells. i Median fluorescence intensity (MFI) of GLUT1 by flow cytometry ( n = 8 mice). j Proportions of glyCAF (CD73 + CD90 + ) by flow cytometry ( n = 9 mice). k Quantification of CD73 + CD90 + glyCAF in ROIs encompassing the tumor margin (Ccne1 + : n = 13 ROI, Vgll3 + : n = 8 ROI from n = 3 mice). l Immunofluorescence of glyCAF (top) and CD8 + T cells (right panel) at the tumor margin. Images are representative of two independent experiments. Unless otherwise indicated, results are presented as mean ± SEM and p -values are derived by a two-tailed unpaired Student’s t test. Source data are provided as a file.

Article Snippet: Primary antibodies anti-CD8 (ebioscience, 4SM15), anti-CD4 (R&D, GK1.5), anti-CD90 (Sino Biological, Cat #50461-T44), anti-CD73 (Sino Biological, Cat # 50231-T56), anti-GranzymeB (ebioscience, 16G6), anti-CD90.1 (BioLegend, OX-7), anti-CD45.1 (Invitrogen, A20) diluted to 1:200 were incubated overnight at 4°C.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Isolation, Expressing, Marker, Fluorescence, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Metabolic targeting of cancer associated fibroblasts overcomes T-cell exclusion and chemoresistance in soft-tissue sarcomas

doi: 10.1038/s41467-024-46504-4

Figure Lengend Snippet: a Glucose uptake of glyCAF quantified by flow cytometry of 2-NBDG (FITC) MFI relative to non-glyCAF ( n = 5 mice). b Multiplex immunofluorescence staining of CAF marker CD90 (red) glyCAF marker CD73 (green), and CD3 (white) at the tumor margin. Images are representative of two independent experiments with n = 3 mice. c Quantifications of the number of CD73 + CD90 + cells (glyCAF) in ~1 mm 2 ROIs encompassing the tumor margin (Ctrl: n = 17 ROI, GLUT1i: n = 14 ROI from n = 3 mice). d Proportion of CD73 + glyCAF (dsRED − , CD45 − , CD31 − , CD90 + , CD73 + ) by flow cytometry ( n = 10 mice). e Proportion of CD90 + CAF (dsRED − , CD45 − , CD31 − , CD90 + ) by flow cytometry ( n = 10 mice). f Averaged expression of mCAF, iCAF, glyCAF, and apCAF signature genes (top 10 DEG per cluster) in CAFs from mouse tumors treated with GLUT1i or control (NT) ( n = 4 mice). g Immunofluorescence staining of CD8 + cells (green) in Ctrl and GLUT1i treated Ccne1 + tumors (Scale bars 500 μm (top) and 100 μm (bottom)). Images are representative of two independent experiments with n = 3 mice. h Quantifications of CD8 + cells/field in ROIs encompassing the tumor margin or the tumor parenchyma in Ctrl and GLUT1i treated Ccne1 + tumors ( n = 3 mice) and i relative proportions of tumor infiltrating CD8 + T cells (CD45 + CD8 + ) cells determined by flow cytometry ( n = 10 mice). j Quantifications of CD8 + cells/field in ROIs encompassing the tumor margin or the tumor parenchyma in WT and Glut1-KD tumors (Ctrl, Parenchyma: n = 6 ROI, Margin: n = 7 ROI; GLUT1i, Parenchyma: n = 8, Margin n = 8 ROIs from n = 3 mice) and k relative proportions of tumor infiltrating CD8 + T cells (CD45 + CD8 + ) cells determined by flow cytometry ( n = 8 mice). l Multiplex immunofluorescence staining of CAF marker CD90 (red), glyCAF marker CD73 (green), and CD8 (white) at the tumor margin. White arrows illustrate association between glyCAF and CD8 + T cells. Scale bars 100 μm. Images representative of three independent experiments with n = 3 mice. m Quantifications of the distance to the nearest CD8 + T cell using multiplex immunohistochemistry images. Dots represent individual cell-cell interactions ( n = 24 CD73 + CD90 + , n = 17 CD73 − CD90 + , n = 24 CSF1R + ) acquired from n = 3 mice each group. P -values determined by one-way ANOVA with Tukey’s multiple comparisons test. Unless otherwise indicated, results are presented as mean ± SEM and p -values are derived by a two-tailed unpaired Student’s t test. Source data are provided as a file.

Article Snippet: Primary antibodies anti-CD8 (ebioscience, 4SM15), anti-CD4 (R&D, GK1.5), anti-CD90 (Sino Biological, Cat #50461-T44), anti-CD73 (Sino Biological, Cat # 50231-T56), anti-GranzymeB (ebioscience, 16G6), anti-CD90.1 (BioLegend, OX-7), anti-CD45.1 (Invitrogen, A20) diluted to 1:200 were incubated overnight at 4°C.

Techniques: Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining, Marker, Expressing, Immunohistochemistry, Derivative Assay, Two Tailed Test

Journal: Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics

Article Title: RNA Interference of Biot2 Induces G₁ Phase Arrest and Apoptosis in Mouse Colorectal Cancer Cell Line

doi: 10.3727/096504014x14146137738583

Figure Lengend Snippet: Figure 2. Biot2 genes downregulated arrest of CT26 cell cycle. The effect of Biot2-shRNA on inducing G1 cell cycle arrest in CT26 cells. After treatment with Biot2-shRNA for 48 h, (A) the cell cycle distributions were determined by flow cytometry. (B) The protein expressions of cycle regulatory proteins and cyclin-dependent kinase cyclin D1, CDK2 and (C) the expression of several CKIs, p16, p21, and p27, were measured by Western blotting.

Article Snippet: Rabbit anti-CDK2 (1:1,000), rabbit anti-p16 (1:1,000), rabbit anti-p27 (1:1,000), and mouse anti-PCNA (1:1,000) were purchased from BOSTER (China).

Techniques: shRNA, Flow Cytometry, Expressing, Western Blot