Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Endoglin is highly expressed on CAFs in human pancreatic tumors. ( A ) Representative images of human pancreatic cancer (representative from n = 25 PDAC patients) and normal pancreas stained for α-SMA, endoglin, cytokeratin, and vimentin. Endothelial cells (black arrow) and endoglin expressing CAFs (white arrow). ( B ) Immunofluorescent double staining for α-SMA and endoglin in human PDAC tumors. ( C ) Endoglin mRNA expression by human cells; ECRF endothelial cells, MIA PaCa-2, PANC-1 and BxPC-3 PDAC cells and 8 patient derived primary pancreatic CAFs. ( D ) Endoglin protein expression on human pancreatic fibroblasts. Basal and BMP9-induced downstream signaling (pSMAD1) was inhibited with TRC105 (full-length blot shown in Supplementary figure 4A – C ).

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Staining, Expressing, Double Staining, Derivative Assay

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Endoglin is highly expressed on CAFs in mouse pancreatic tumors. ( A ) Representative images of mouse pancreatic tumors (KPC) (representative from n = 5) and normal pancreas stained for α-SMA, endoglin, cytokeratin, and vimentin. Endothelial cells (black arrow) and endoglin expressing CAFs (white arrow). ( B ) Immunofluorescent double staining for α-SMA and endoglin in mouse KPC tumors. ( C ) Endoglin mRNA expression by mouse cells; 2H11 endothelial cells, MC38 colorectal cancer, and KPC-3 pancreatic cancer cells, CAFs isolated from colorectal and pancreatic tumors.

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Staining, Expressing, Double Staining, Isolation

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: TRC105 does not affect tumor growth in a murine KPC-3 model for pancreatic cancer. ( A ) Tumor volume in mm 3 and ( B ) tumor weight upon 13 days of therapy (28 days after tumor inoculation, n = 7 animals per group). ( C ) Percentage of intratumoral CD45+ cells (gated from live gate) by using flow cytometry. ( D ) Percentage of CD8+ T-cells (from CD45 gate, n = 6-7 mice per group. ( E ) Representative histological images and quantifications of endoglin ( F ), vimentin ( G ) and α-SMA ( H ) (n = 7 animals per group). ( I ) Intratumoral TRC105 levels in tumor lysates determined by ELISA (n = 5 control, n = 3 TRC105). All graphs represent mean ± SD. Student’s T -test was performed to calculate differences indicated in the graphs *p = <0.05 **p = <0.01.

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Early treatment with TRC105 does not affect tumor growth but changes the tumor microenvironment. ( A ) Tumor volume in mm 3 upon 27 days of therapy and 28 days after tumor inoculation (n = 5-8 mice per group). ( B ) Percentage of infiltrating immune cells (CD45+). ( C ) CD3+, ( D ) CD8+ and ( E ) CD4+ cells out of CD45 gate. ( F ) Intratumoral CD4+ CD25+ Treg-like cells out of CD4 gate (n = 5-8 mice per group). ( G ) Heatmap summarizing qPCR data normalized to the control group of different cytokines, growth factors and stromal markers (n = 5-8 mice per group). ( H ) Representative histological pictures of α-SMA, endoglin, cytokeratin and vimentin staining (n = 5-8 mice per group). All graphs represent mean ± SD. One-way ANOVA was used to calculate statistical differences. *p = <0.05.

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Control, Staining

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Col1a1 specific endoglin knock-out does not affect tumor growth but alters immune cell composition. ( A ) Tumor volume in mm 3 after 28 days of tumor inoculation (n = 7 mice per group). ( B ) Representative pictures of histological samples stained with α-SMA and endoglin (n = 6 mice per group). ( C ) CD45+ immune infiltrate and ( D ) CD3 + T-cells (from CD45+ gate). ( E ) CD8+ and ( F ) CD4+ cells from (from CD3+ gate). ( G ) Percentage CD8+ PD1+ cells (from CD8+ gate). ( H ) Percentage of CD8+ LAG-3+ cells (from CD8+ gate) (n = 6 mice per group). ( I ) Heatmap summarizing qPCR data normalized to the control group of different cytokines growth factors and stromal markers (n = 6 mice per group) ND in the graph indicates not-detectable. All graphs represent mean ± SD. Student’s T -test was performed to calculate significances indicated in the graphs **p = <0.01.

Article Snippet: Five μm sections wereimmunohistochemically stained using primary antibodies; goat anti-human endoglin (BAF 1097, R&D systems, Abington, UK) and goat anti-mouse endoglin (BAF 1320, R&D systems, Abington, UK), mouse anti-α-SMA (clone: 1A4/ASM-1, Progen, Heidelberg, Germany) mouse anti-pan-cytokeratin (clone: PKC-26, Sigma-Aldrich, Zwijndrecht, the Netherlands) and rabbit anti-vimentin (clone: D21H3, Cell Signaling Technologies, Leiden, the Netherlands).

Techniques: Knock-Out, Staining, Control

Journal: Cancer cell

Article Title: Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity.

doi: 10.1016/j.ccell.2021.06.017

Figure Lengend Snippet: Figure 4. Phenotypic plasticity of mesenchymal marker expression (A) Flow cytometry analysis of PDPN and CD105 in purified and in-vitro-cultured CD105pos and CD105neg pancreatic fibroblasts (PaFs) after 1 and 7 weeks. Plots are representative of n = 4 experiments. Relative frequencies shown in relevant quadrants. (B) Normalized Eng mRNA expression in purified CD105pos (n = 4) and CD105neg PaFs (n = 4) treated with control (top) or KPC PDA conditioned medium (bottom). Data displayed as mean ± SD. (C) Representative flow cytometry analysis (n = 4) of CD105 on GFPposCD105pos and GFPposCD105neg PaFs in mono- or co-culture with RFPpos KPC PDA tu- mor cells. (D) Representative flow cytometry analysis (n = 3) of CD105 in isolated CD105pos and CD105neg human PaFs after >3 weeks of in vitro culture. (E and F) MC analysis of primary PaFs treated with the indicated ligands for 3 days. Representative plots displaying relative frequencies of CD105pos and CD105neg PaFs. (G and H) Heatmap of median marker intensity (MMI) displayed as column Z scores for each phenotypic marker on CD105pos (G) and CD105neg (H) PaFs after 3 days of treatment as indicated. Boxplots show MMI with upper and lower boundary of the interquartile range and whiskers denoting maximum and minimum values minus outliers, across all conditions. (I and J) Representative flow cytometry analysis (n = 3) of CD105pos (I) and CD105neg (J) PaFs with IFN-g, IFN-g + KPC PDA conditioned medium, or IFN-g + TGF- b1 treatment. Samples are compared using unpaired t tests (B) (top and bottom). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5 and Table S4.

Article Snippet: The first staining round used mouse anti-human CD105 antibody (CST clone 3A9) at 1/200 and TSA570 (FP1488001KT).

Techniques: Marker, Expressing, Flow Cytometry, In Vitro, Cell Culture, Control, Cytometry, Co-Culture Assay, Isolation

Journal: Clinical Cancer Research

Article Title: Mesenchymal Stem Cell Targeting of Microscopic Tumors and Tumor Stroma Development Monitored by Noninvasive In vivo Positron Emission Tomography Imaging

doi: 10.1158/1078-0432.ccr-05-0876

Figure Lengend Snippet: Fig. 5. In situ characterization of EGFP+ cells in HT-29 (s.c.) + TG-hMSC (i.v.) tumors. A, agarose gel electrophoresis of PCR-amplified EGFP sequence (717 bp) done using1 Ag of cellular DNA prepared from loosely associated cell fraction (loose), stromal matrix associated fraction (stroma), and the residue (residue) of HT-29 (s.c.)/TG-hMSC (i.v.) tumor and HT-29 (s.c.) tumor,TG-hMSC, and HT-29 cells. B, cytofluorometric histograms comparing the stromal matrix cell fractions of HT-29 (s.c.) + TG-hMSC (i.v.) tumors (red) and HT-29 (s.c.) tumors (black), examined with anti-GFP specific antibody. EGFP+ cells in the gated area represented 11.5% of total cells. C, histograms showing the presence of human endothelial cell markers but absence of hMSC markers in EGFP+ cells obtained from the HT-29 (s.c.) + TG-hMSC (i.v.) tumor after 30 days of growth in vivo. Stromal matrix ^ associated cell fractions were double stained with FITC-labeled anti-GFP mouse monoclonal antibody, and with a PE-labeled anti-vWF, or anti-CD31, or anti-CD90, or anti-CD105 to assess the expression of these cell surface markers on EGFP+ cells (red). PE-labeled mouse isotype immunoglobulins were included in parallel to serve as the negative control (black).

Article Snippet: © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from anti-mouse IgG antibody or PE-labeled anti-hvWF; and PE-labeled mouse monoclonal antibodies against human CD14 (PharMingen, San Diego, CA), CD31, CD90, CD105 (Serotec, Raleigh, NC), and smooth muscle actin (Chemicon, Temecula, CA).

Techniques: In Situ, Agarose Gel Electrophoresis, Amplification, Sequencing, Residue, In Vivo, Staining, Labeling, Expressing, Negative Control

Journal: Bone marrow transplantation

Article Title: Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads.

doi: 10.1038/sj.bmt.1705358

Figure Lengend Snippet: Figure 2 Immunophenotype of mesenchymal stem cells isolated by fibrin microbeads (FMB). (a) FACS analysis of the immunophenotype profile for hematopoietic marker CD45 and for mesenchymal antigens CD90 and CD105 of the initial peripheral blood progenitor cell sample before isolation. Cells isolated by FMB technique were harvested 18–19 days following harvesting and cultured for further passages. Shaded histograms represent the fluorescence from control cells stained with only second antibody; non-filled histograms represent positive staining of cells with the indicated antibody. (b) Cells from passages 2–3 were harvested and labeled with antibodies against CD45, CD34, CD90, CD105, fibronectin (FN) and vimentin (Vim) and analyzed by FACS. Shaded histograms represent the fluorescence from control cells stained with only second antibody; non-filled histograms represent positive staining of cells with the indicated antibody. (c) Cells from passage 5 were harvested and immunostained with antibodies to CD45 (Cy3 labeled), CD34 (Cy3 labeled), CD90 (Cy3 labeled), CD105 (Cy3 labeled), FN (Cy3 labeled) and Vim (Cy2 labeled). The fluorescence images were merged with dim-light images of the same field to show cell contours.

Article Snippet: The cell suspension was then incubated with the following primary antibodies to the different markers : mouse anti-human CD45 (1:10), mouse anti-human CD34 (1:10), mouse anti-human CD90 (1:10), mouse anti-human CD105 (1:10), all from Serotec, Oxford, UK, mouse anti-human fibronectin (1:500) and rabbit antihuman vimentin (1:50) both from Sigma-Aldrich, Rehovot, Israel.

Techniques: Isolation, Marker, Cell Culture, Control, Staining, Labeling