Journal: Journal of Extracellular Biology

Article Title: CD147 Mediates the Metabolic Reprogramming of Cancer Associated Fibroblasts Induced by EVs Released by Differentiating Cancer Stem Cells

doi: 10.1002/jex2.70039

Figure Lengend Snippet: (a) CAF activation after CR‐CSC derived sEV treatment for 48 h, assessed by increase of vimentin, α‐SMA and FAP levels. CTR = CAF untreated; sEVs‐1 CD147 low = CAF treated with sEVs with low CD147 expression isolated from CR‐CSC1; sEVs‐1 CD147 medium = CAF treated with sEVs with medium CD147 expression isolated from CR‐CSC1; sEVs‐1 CD147 high = CAF treated with sEVs with high CD147 expression isolated from CR‐CSC1; sEVs‐2 CD147 low = CAF treated with sEVs with low CD147 expression isolated from CR‐CSC2; sEVs‐2 CD147 medium = CAF treated with sEVs with medium CD147 expression isolated from CR‐CSC2. sEVs‐2 CD147 high = CAF treated with sEVs with high CD147 expression isolated from CR‐CSC2. (b) Western‐blot analysis of CAF activation markers after treatment with sEVs or with sEVs CD147 blocked with specific antibody. sEVs = CAF treated with sEVs‐CD147 high isolated from CR‐CSC1; sEVs + CD147BL = CAF treated with sEVs‐CD147 high blocked by CD147 antibody isolated from CR‐CSC1. (c, d) Values of extracellular lactate levels, ATP/ADP and NAD + /NADH intracellular ratio in CAF treated with sEVs isolated form CR‐CSC1 and CR‐CSC2 and expressing different CD147 levels. (e–f) Real time PCR of the gene encoding for glycolytic enzymes (HK2, PGM2, PKM2, ENO2) and genes encoding for glucose and lactate transporters (SLC2A1 and SLC16A3, respectively). (g) Western blot analysis of SLC16A3 (called also MCT4) lactate transporter in CAF conditioned with sEVs carrying different amount of CD147 release by CR‐CSC1 and CR‐CSC2 (sEVs–CD147 low, sEVs–CD147 medium, sEVs–CD147 high).

Article Snippet: MCT4 , A304‐398a‐M , Bethyl Laboratories , 1:200.

Techniques: Activation Assay, Derivative Assay, Expressing, Isolation, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Biology

Article Title: CD147 Mediates the Metabolic Reprogramming of Cancer Associated Fibroblasts Induced by EVs Released by Differentiating Cancer Stem Cells

doi: 10.1002/jex2.70039

Figure Lengend Snippet: (a–c) Western blot analysis of β‐catenin, pAkt, HIF1α, c‐Myc and NFKβ and gene expression of Myc, HIF1α and VEGF in CAF conditioned by sEVs with growing CD147 content. (d) Western blot analyses revealed that CAF treated with CD147 sEVs pre‐treated with AC73 underwent a reduction of some key proteins involved in CAF activation and in metabolism modulation (β‐catenin, NFKβ, pAkt, SLC16A3). (e) Analysis of pro‐inflammatory cytokines such as IL‐6 and IL‐8 and CD147 soluble form by Luminex assay in supernatant of CAF conditioned by sEVs with growing CD147 content at 24 and 48 h. (f) sEVs pre‐treated with anti‐CD147 antibodies or deleting its N‐glcosylation to analyse the CAF treatments to evaluate the secretion of analysed cytokines.

Article Snippet: MCT4 , A304‐398a‐M , Bethyl Laboratories , 1:200.

Techniques: Western Blot, Gene Expression, Activation Assay, Luminex

Journal: Journal of Extracellular Biology

Article Title: CD147 Mediates the Metabolic Reprogramming of Cancer Associated Fibroblasts Induced by EVs Released by Differentiating Cancer Stem Cells

doi: 10.1002/jex2.70039

Figure Lengend Snippet: Multiplex spatial imaging in the mouse model transplanted with CAF conditioned with sEVs carrying different amount of CD147 and CR‐CSC1. (a) Representative seven‐colour multispectral images of mouse sample stained with the mIF panel. Original magnification 20×. Immune markers and colour codes are indicated in the legend. (b) Significant differences in cell population (CD147+/PancK+; MCT1+/PancK+; α‐SMA/FAP+; α‐SMA/FAP+/CD147+; α‐SMA/MCT4+/CD147+) analysed according to mice groups described above. Data are presented as cell density (number of cells/mm 2 ). (c) Mean distance (µm) between: (1) Each PANCK‐MCT1 tumour cell and the nearest MCT4 CAF cells. (2) Each PANCK‐MCT1 tumour cell and the nearest MCT4 CAF cells. Representative image of cell–cell distance analysis. mIF, multiplex immunofluorescence; Untreated, mice treated with vehicle (PBS).

Article Snippet: MCT4 , A304‐398a‐M , Bethyl Laboratories , 1:200.

Techniques: Multiplex Assay, Imaging, Staining, Immunofluorescence

Journal: Journal of Extracellular Biology

Article Title: CD147 Mediates the Metabolic Reprogramming of Cancer Associated Fibroblasts Induced by EVs Released by Differentiating Cancer Stem Cells

doi: 10.1002/jex2.70039

Figure Lengend Snippet:

Article Snippet: MCT4 , A304‐398a‐M , Bethyl Laboratories , 1:200.

Techniques: Incubation

Journal: Journal of Extracellular Biology

Article Title: CD147 Mediates the Metabolic Reprogramming of Cancer Associated Fibroblasts Induced by EVs Released by Differentiating Cancer Stem Cells

doi: 10.1002/jex2.70039

Figure Lengend Snippet:

Article Snippet: MCT4 , A304‐398a‐M , Bethyl Laboratories , 1:200.

Techniques:

Journal: Molecular Cancer Research

Article Title: Colorectal Cancer–Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis

doi: 10.1158/1541-7786.mcr-20-0827

Figure Lengend Snippet: Figure 3. Identification of CAT1 as a specific surface antigen on CEC-derived EVs. A, The result of differential analysis based on paired t test between proteome from tumor region–derived EVs (tumor Te-EVs) and that from normal mucosa-derived EVs (normal Te-EVs) is displayed as the volcano plot. Significantly upregulated 487 proteins (adjusted P< 0.05 and fold-change >5.0) or downregulated 88 proteins (adjusted P< 0.05 and fold-change <0.2) were indicated in red or blue dots. B, Principal component analysis was performed for top 100 upregulated proteins in (A). C, The line chart shows LC/MS-based relative protein abundances of CAT1 on EVs in 17 paired samples. D, A representative image of transmission electron microscopy (TEM) for tumor Te-EVs. The black dot on the surface of the EV indicates the signal of the gold particle-labeled anti-CAT1 antibody; bar, 200 nm. The expression of CAT1 was positive in 24 out of 74 (32.4%) of tumor Te-EVs whereas none (0/66) of normal Te-EVs were CAT1 positive. E, Expression of CAT1 and CD9 in normal (N) and tumor (T) Te-EVs was confirmed by western blotting. F, The CD9-normalized protein abundances of CAT1 in (E) were displayed with the box plot. G, Representative images of IHC staining of CAT1 in colorectal cancer tissues. The expression levels of CAT1 were classified into 3 groups as indicated; bars, 50 mmol/L. H, The result of IHC staining of CAT1 for 75 samples was summarized with the violin plot. N, normal colon mucosa tissue; I–IV, tissues of pathological stage I–IV colorectal cancer.

Article Snippet: The deparaffinization, rehydration, and IHC were automatically carried out on the Leica Bond III Automated IHC and ISH system (Leica Microsystems Ltd.) with polyclonal anti-CAT1 antibody (Proteintech) and anti-CD31 antibody.

Techniques: Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Transmission Assay, Electron Microscopy, Labeling, Expressing, Western Blot, Immunohistochemistry

Journal: Molecular Cancer Research

Article Title: Colorectal Cancer–Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis

doi: 10.1158/1541-7786.mcr-20-0827

Figure Lengend Snippet: Figure 4. The diagnostic potential of plasma EV-CAT1 for detection of colorectal cancer. A, The framework of anti-CAT1 and anti– CD81 EV-sandwich ELISA isshown. B, biotin; SA, streptavidin; HRP, horseradish peroxidase; TMB, 3,30,5,50-tetramethylben- zidine. B, The result of EV-CAT1 sandwich ELISA measuring 119 plasma samples is displayed as the box plot. N, normal donors; I–IV, plasma from pathological stage I–IV patients with colorectal cancer. The P values were calculated by Student t test. C, The diagnostic potential to distinguish patients with colorectal cancer (n ¼ 94) from normal donors (n ¼ 25) was assessed by ROC curve analysis. In addition to the single usage of EV-CAT1 or CEA, the combination bio- marker model, EV-CAT1 þ CEA, was also evaluated based on logistic regression. AUC, area under the curve.

Article Snippet: The deparaffinization, rehydration, and IHC were automatically carried out on the Leica Bond III Automated IHC and ISH system (Leica Microsystems Ltd.) with polyclonal anti-CAT1 antibody (Proteintech) and anti-CD31 antibody.

Techniques: Diagnostic Assay, Clinical Proteomics, Sandwich ELISA, Marker

Journal: Molecular Cancer Research

Article Title: Colorectal Cancer–Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis

doi: 10.1158/1541-7786.mcr-20-0827

Figure Lengend Snippet: Figure 5. CAT1-dependent promotion of cell growth and tubule formation of HUBEC via EVs. A, Representative images of IHC staining for CAT1 (A, C, E, G, I, K) or CD31 (B, D, F, H, J, L) are shown. CD31 (PECAM1) was stained as a marker for vascular endothelial cells. Serial sections were used for the comparison between the IHC staining of CAT1 and CD31. B, The rate of moderate to strong-CAT1 expression in vascular endothelial cells of or near colorectal cancer tissueswas significantly higher than that of adjacent normal tissues. C, The high expression of CAT1 in HCT116 cells transfected with pCAGGS-CAT1-FLAG (CAT1-overexpressing HCT116) compared with those transfected with pCAGGS-FLAG (Control HCT116) was observed. D, The expression level of CAT1 or CD9 was examined for EVs purified from CAT1-overexpressed HCT116 cells (CAT1þþ-EVs) or mock-transfected cells (mock-EV). E, A representative image of TEM for mock-EV or CAT1þþ-EV is shown. The black dot on the surface of the EV indicates the signal of the gold particle-labeled anti-CAT1 antibody; bar, 50 nm. F, Fluorescent microscopic images of HUVEC 4 hours after treatment with PBS or fluorescence-labeled CAT1þþ-EV; bar, 5 mm. G, Fluorescent microscopic images of HUVEC 72 hours after treatment with PBS or CAT1þþ-EV. The expression of CAT1 was detected by the Alexa 488-labeled secondary antibody; bar,5 mmol/L. H and I, The expression of CAT1 (H) or the growth activity (I) was measured in HUVEC 72 hours after treatment with PBS, mock-EV, or CAT1þþ-EV. Each error bars are presented as the mean SE (n ¼ 3). J, Representative images of tube formation assay. 72 hours after treatment with PBS, mock-EV, or CAT1þþ-EV, HUVEC was cultured in Matrigel plates for 24 hours. K, The relative total tube length in (J) was measured by ImageJ. Each error bar is presented as the mean SE (n ¼ 3).

Article Snippet: The deparaffinization, rehydration, and IHC were automatically carried out on the Leica Bond III Automated IHC and ISH system (Leica Microsystems Ltd.) with polyclonal anti-CAT1 antibody (Proteintech) and anti-CD31 antibody.

Techniques: Immunohistochemistry, Staining, Marker, Comparison, Expressing, Transfection, Control, Labeling, Activity Assay, Tube Formation Assay, Cell Culture

Journal: Molecular Cancer Research

Article Title: Colorectal Cancer–Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis

doi: 10.1158/1541-7786.mcr-20-0827

Figure Lengend Snippet: Figure 6. CAT1-dependent modulation of the NO metabolic pathway in vascular endothelial cells. A, Expression level of CAT1 was measured in mock-transfected HUVEC (HUVEC-mock) or CAT1-overexpressed HUVEC (HUVEC-CAT1). B, The results of quantitative metabolomic analysis for the arginine/NO/cGMP pathway were illustrated. Absolute quantification of the metaboliteswas performed for HUVEC-mock or HUVEC-CAT1 after stimulation with arginine for 15 minutes. Each error bar is presented as the mean SE (n ¼ 4). C, Cyclic GMP concentrations of HUVEC treated with CAT1þþ-EVs or mock-EVs were compared. Each error bars are presented as the mean SE (n ¼ 3). D, An illustration of CAT1 transfer from colorectal cancer cells to vascular endothelial cells via EVs in tumor microenvironment. Putative signaling pathway in a vascular endothelial cell from the arginine/NO/cGMP pathway to its downstream is shown. Arg, arginine; eNOS, endothelial nitric oxide synthase; NADPH, nicotinamide adenine dinucleotide phosphate; NADP, nicotinamide adenine dinucleotide phosphate; NHA, Nw-hydroxy-L-arginine; NO, nitric oxide; Cit, citrulline; sGC, soluble guanylatecyclase; GTP, guanosine triphosphate; cGMP, cyclic guanosine monophosphate; PRAK, p38-regulated/activated kinase; FAK, Focal adhesion kinase.

Article Snippet: The deparaffinization, rehydration, and IHC were automatically carried out on the Leica Bond III Automated IHC and ISH system (Leica Microsystems Ltd.) with polyclonal anti-CAT1 antibody (Proteintech) and anti-CD31 antibody.

Techniques: Expressing, Transfection