Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model

doi: 10.1186/s13046-016-0326-y

Figure Lengend Snippet: In vivo effects of peptide R treatment of U87MG orthotopic mouse model. a Tumor volume measures obtained by MRI analyses performed on mice at day 10, 15 and 23 after U87MG cells implantation and drug administration. Curves represent mean value of tumor volumes measures of three independent experiments obtained from mice treated with vehicle (PBS) ● CTRL, ■ Plerixafor and ▲ peptide R ( n = 10 animals per group, error bars ± SD). b Representative brain sections of vehicle-treated (CTRL) or peptide R- or Plerixafor-treated mice stained by immunohistochemistry with the anti-vimentin antibody. Scale bars, 75 μM. c Representative CLSM images of Vimentin expression ( green ) of tumor-free contralateral hemispheres of vehicle-treated mice (CTRL), peptide R- and Plerixafor-treated mice. Arrows represent Vimentin + cells. Nuclei were stained with DAPI ( blue ). Scale bars 75 μM

Article Snippet: The following primary antibodies were used in overnight incubations: rat anti-mouse CD11b (1:100, Serotec), rabbit anti-human CD68 (1:200, able to detect mouse, rat and human CD68, Santa Cruz), mouse anti-mouse Arg-1 (1:200, BD Biosciences), rabbit anti-mouse anti-iNOS (1:2000, Millipore), goat anti-human vimentin (1:40, R&D Systems), rabbit anti-mouse/human glial fibrillary acidic protein, GFAP (1:200, Dako), rat anti-mouse CD31 (1:200, Serotec), mouse anti-human/mouse vascular endothelial growth factor, VEGF (1:50, R&D Systems).

Techniques: In Vivo, Staining, Immunohistochemistry, Expressing

Journal: Cancer Research

Article Title: Disruption of Prostaglandin E2 Signaling in Cancer-Associated Fibroblasts Limits Mammary Carcinoma Growth but Promotes Metastasis

doi: 10.1158/0008-5472.can-21-2116

Figure Lengend Snippet: Figure 1. mPGES-1 ablation limits tumorprogressionand increases CAFabundance. A,Tumorburden(percentageoftumor weightrelativetobodyweight)in20-week-oldWTand mPGES-1 KO PyMT mice is shown. Data are means SEM; n ¼ 6. B, Representative hematoxylin and eosin images of the center and invasive margin of WT and mPGES-1 KO PyMT tumors. Scale bars, 50 mm. C–J, Tumor sections were stained for CAF markers and analyzed using PhenOptics. C, Representative images show tissue segmentationintotumor(blue),stroma(green),andnotissue(red).Scalebars,100mm.D,Quantificationofstromalcontent.E,Representativeimages showfluorescence staining of CAF and proliferation markers in WT and mPGES-1 KO PyMT tumor sections, aSma (green), Vim (yellow), Col-1 (cyan), Fsp-1 (purple), Ki67 (red), and DAPI (white). Scale bars, 100 mm. F–I, Quantification of markers aSMA (F), Vim (G), Col-1 (H), and Ki67 (I) in stromal cells. J, Quantification of Ki67 I tumor cells. Data are means SEM; individual data points are high-power fields of at least six individual tumors per genotype. , P < 0.05; , P < 0.01; , P < 0.001; Mann–Whitney test.

Article Snippet: For immunofluorescence analysis of PyMT organoids, Organoids were washed and fixed overnight in 4% PFA at 4 C, then permeabilized/blocking using 0.3% Triton X-100 (Applichem) with 5% donkey serum (Sigma-Aldrich) in PBS for 12 hours at 4 C. Organoids were stained in PBS with 0.5% donkey serum and 0.1% Triton X-100, with the following antibodies: Anti–E-cad-AF647 (BioLegend), anti– vimentin-APC (R&D Systems), Ki67 (Abcam), phalloidin-AF488 (Thermo Fisher Scientific), and Hoechst 33342 (Sigma-Aldrich).

Techniques: Staining, MANN-WHITNEY

Journal: Cancer Research

Article Title: Disruption of Prostaglandin E2 Signaling in Cancer-Associated Fibroblasts Limits Mammary Carcinoma Growth but Promotes Metastasis

doi: 10.1158/0008-5472.can-21-2116

Figure Lengend Snippet: Figure 7. EP3/Tak1L in fibroblasts promotes tumor growth but limits EMT. A, Experimental setup. PyMT organoids were cultured in control medium alone, treated with 20 ng/mL TGFb, or cultured in supernatants of WT, EP3 KO, Map3k7cl KD MGFs for 96 hours; n ¼ 6 biological replicates each. Organoid phenotype was analyzed by immunofluorescence staining and confocal microscopy, or qPCR. B, Representative images of organoids after culturing for 96 hours. Scale bars, 30 mm. C, Quantification of Hoechst fluorescence, indicating organoid size. D, Quantification of Ki67þ cells by FACS. E, Ratio between vimentin (Vim) and E-cadherin (Ecad). F, Distance of Vim-positive cells from spheroid surface (mean). G andH, mRNA expression of Zeb2 and Lef1 normalized to organoids receiving supernatants of WT MGF. I–K, Lungs of 20-week-old WT or mPGES-1 KO (n ¼ 3) PyMT mice were harvested and analyzed for metastasis. I, PyMT mRNA expression quantified by qPCR (WT, n ¼ 4; mPGES-1 KO, n ¼ 3). J, Representative fluorescence images of metastasis in lung sections showing Ki67 (red), aSma (green), Pan-ck (yellow), and DAPI (white). Scale bars, 800 and 100 mm. K, Lung sections (10 sections for each animal) were analyzed using Inform software to determine the metastasis foci (n ¼ 6 each). All data are means SEM. , P < 0.05; , P < 0.01; , P < 0.001; n.s., nonsignificant; one-way ANOVA with Bonferroni’s correction (C–E), one-sample t test (F and G), or Mann–Whitney test (I and K).

Article Snippet: For immunofluorescence analysis of PyMT organoids, Organoids were washed and fixed overnight in 4% PFA at 4 C, then permeabilized/blocking using 0.3% Triton X-100 (Applichem) with 5% donkey serum (Sigma-Aldrich) in PBS for 12 hours at 4 C. Organoids were stained in PBS with 0.5% donkey serum and 0.1% Triton X-100, with the following antibodies: Anti–E-cad-AF647 (BioLegend), anti– vimentin-APC (R&D Systems), Ki67 (Abcam), phalloidin-AF488 (Thermo Fisher Scientific), and Hoechst 33342 (Sigma-Aldrich).

Techniques: Cell Culture, Control, Staining, Confocal Microscopy, Expressing, Software, MANN-WHITNEY

Journal: OncoTargets and therapy

Article Title: An isolated orbital mass with proptosis and diplopia: an uncommon metastasis from intracranial olfactory neuroblastoma in a chinese patient

doi: 10.2147/OTT.SI09929

Figure Lengend Snippet: Immunohistochemical staining of the mass. Notes: The tumor cells were positive for ( A ) SYN; original magnification: ×400; a reaction in the cytoplasm of the neoplastic cells; ( B ) CGA; original magnification: ×400; a reaction in the cytoplasm of the neoplastic cells; (C) CD56 (cluster of differentiation 56); original magnification: ×400; a strong and heavy membrane-type staining; ( D ) CK; original magnification: ×400; a reaction in the cytoplasm of the neoplastic cells; ( E ) VIM; original magnification: x400; a reaction in the cytoplasm of the neoplastic cells; ( F ) S-100 protein; original magnification: ×400; a sustentacular reaction. The tumor cells were negative for EMA and CD99 (data not shown). Abbreviations: CD, cluster of differentiation; CGA, chromogranin; CK, cytokeratin; EMA, epithelial membrane antigen; SYN, synaptophysin; VIM, vimentin.

Article Snippet: Immunohistochemical analyses for synaptophysin (SYN) (rabbit monoclonal antibody, OriGene, Rockville, MD, USA), chromogranin A (CGA; mouse monoclonal antibody; OriGene), cluster of differentiation (CD56; mouse monoclonal antibody; OriGene), cytokeratin (CK; mouse monoclonal antibody; OriGene), vimentin (VIM; mouse monoclonal antibody; OriGene), S-100 protein (mouse monoclonal antibody, OriGene), epithelial membrane antigen (EMA; mouse monoclonal antibody; OriGene), and cluster of differentiation 99 (CD99; mouse monoclonal antibody; OriGene) were performed on the paraffin sections.

Techniques: Immunohistochemical staining, Staining, Membrane

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: LAMC2 marks a tumor-initiating cell population with an aggressive signature in pancreatic cancer

doi: 10.1186/s13046-022-02516-w

Figure Lengend Snippet: Generation of LAMC2 -EGFP knock-in human pancreatic cancer cells. a Design of LAMC2‐EGFP donor and CRISPR/Cas9 sgRNA vectors. Blue circle represents the CRISPR/Cas9 protein complex and the yellow box underneath illustrates the guide RNA. b qPCR analysis for LAMC2 gene expression in EGFP + and EGFP − cells. Data are normalized to GAPDH and are presented as fold change in gene expression relative to the EGFP − counterpart. c Sphere formation capacity for EGFP + versus EGFP − cells. d Representative images of gelatin degradation for EGFP + versus EGFP − cells. Nuclei were stained with Hoechst 33342 (blue), green represents actin (Alexa Fluor™ 488 Phalloidin) and red illustrates gelatin (Rodhamine). The white dashed line circles indicates the areas of degradation. e Invasive potential of sh empty versus LAMC2 knockdown cells. f qPCR analysis for EMT and MMP2 and MMP10 gene expression for EGFP + versus EGFP − cells. Data are normalized to GAPDH and are presented as fold change in gene expression relative to the EGFP − cells. g Western blot analysis of VIM and MMP2 in EGFP + and EGFP − cells. Parallel Tubulin immunoblotting was performed. * p < 0.05, ** p < 0.005, *** p < 0.0005. n \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge 3.$$\end{document} ≥ 3 . Statistical significance was assessed by Student's t-test

Article Snippet: PVDF membranes were hybridized with antibodies against LAMC2 (AMAB 91,098, Sigma-Aldrich), SMAD2 (cell signaling – 5339), pSMAD2 (cell signaling – 3108S), GAPDH (cell signaling – 2118), β-actin (E-AB-20058, Elabscience), MMP-2 (4022S, Cell Signaling), Vimentin (3932, Cell Signaling) and α-tubulin (2144, Cell Signaling) and subsequently hybridized with peroxidase-conjugated goat anti-mouse or anti-rabbit Ig secondary antibodies (DPVR-HRP, Immunologic), and then visualized by enhanced chemiluminescence (ECL Nova 2.0 XLS071, 2050 Cyanagen). n \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge 3.$$\end{document} ≥ 3 .

Techniques: Knock-In, CRISPR, Expressing, Staining, Western Blot