Journal: Cancers

Article Title: Blu-Ray-Based Quantification of CD98+ Extracellular Vesicles for Early Detection of Hepatocellular Carcinoma

doi: 10.3390/cancers18071086

Figure Lengend Snippet: CD98 is highly expressed across all stages of HCC and is independent of survival and tumor size. ( A ) mRNA expression levels of CD98 in normal liver tissue ( n = 175) versus HCC ( n = 421) patient samples from GTEx and TCGA datasets are shown as box plots. ( B ) From the CLCA cohort obtained via the cBioPortal platform , CD98 mRNA expression in HCC tumors at early stages (BCLC 0 & A, n = 28) versus late stages (BCLC B, n = 81; BCLC C, n = 130) is shown as dot plots. Recurrence-free survival (RFS) of HCC patients with lower CD98 mRNA levels (T1 subgroup, n = 62) compared with those with higher CD98 levels (T3 subgroup, n = 62) was analyzed by log-rank test. ( C ) Scatter plot of CD98 mRNA z-scores showed no significant Pearson correlation with tumor size (cm). ( D ) Representative images of CD98 IHC in HCC TMA (LV1501a) across stages are shown. Scale bars = 100 µm and 50 µm, respectively. IHC staining intensity for CD98 was scored as 0 (negative), 1 (low), 2 (medium), or 3 (high) and plotted. ( E ) Dot plot of CD98 IHC scores. n.s. = not significant, ** = p < 0.01, **** = p < 0.0001 (Student’s t test).

Article Snippet: From the CLCA cohort [ ] via cBioPortal [ ], diagnostic sensitivity (%) of blood AFP (cutoffs: 200 ng/mL and 20 ng/mL) and PIVKA-II (cutoff: 40 mAU/mL) for early (BCLC 0 & A, n = 28) versus late HCC (BCLC B, n = 81; BCLC C, n = 130) were displayed as bar graphs; Figure S2: Comparison of three anti-CD98 antibodies for detecting extracellular CD98 on cells and EVs. (A) Surface CD98 on HepG2 cells quantified using three antibodies (Abcam ab307587, Novus NBP2-36491SS, NHRI#20D5) by flow cytometry.

Techniques: Expressing, Immunohistochemistry

Journal: Cancers

Article Title: Blu-Ray-Based Quantification of CD98+ Extracellular Vesicles for Early Detection of Hepatocellular Carcinoma

doi: 10.3390/cancers18071086

Figure Lengend Snippet: CD98 expression is independent of current etiological factors associated with HCC development. ( A , B ) From the CLCA cohort obtained via the cBioPortal platform , CD98 mRNA expression levels in HCC with diverse etiological factors, including fibrosis, cirrhosis, viral infection, alcohol consumption, and smoking history, were compared. Data are shown as dot plots. No statistical significance (n.s.) was observed between groups (Student’s t test).

Article Snippet: From the CLCA cohort [ ] via cBioPortal [ ], diagnostic sensitivity (%) of blood AFP (cutoffs: 200 ng/mL and 20 ng/mL) and PIVKA-II (cutoff: 40 mAU/mL) for early (BCLC 0 & A, n = 28) versus late HCC (BCLC B, n = 81; BCLC C, n = 130) were displayed as bar graphs; Figure S2: Comparison of three anti-CD98 antibodies for detecting extracellular CD98 on cells and EVs. (A) Surface CD98 on HepG2 cells quantified using three antibodies (Abcam ab307587, Novus NBP2-36491SS, NHRI#20D5) by flow cytometry.

Techniques: Expressing, Infection

Journal: Cancers

Article Title: Blu-Ray-Based Quantification of CD98+ Extracellular Vesicles for Early Detection of Hepatocellular Carcinoma

doi: 10.3390/cancers18071086

Figure Lengend Snippet: CD98 is an abundant and detectable EV surface protein in HCC cell lines and patient plasma. ( A ) Protein levels of CD98, five EV markers (CD63, CD9, CD81, Flotillin1, TSG101), and two cellular proteins (Calnexin and β-actin) in HCC cell lines or their secreted EVs from HepG2, Hep3B, and PLC5 cells via ExoQuick-TC kit were analyzed by Western blot. ( B ) Protein levels of CD98, CD63, and ApoA1 with or without EV enrichment via Size Exclusion Chromatography (SEC) in HCC patient plasma were analyzed by Western Blot. ( C ) Illustration of EV morphology of plasma after EV enrichment via SEC from the healthy control and HCC patient using transmission electron microscopy (TEM). Scale bar is 100 nm. The uncropped blots are shown in .

Article Snippet: From the CLCA cohort [ ] via cBioPortal [ ], diagnostic sensitivity (%) of blood AFP (cutoffs: 200 ng/mL and 20 ng/mL) and PIVKA-II (cutoff: 40 mAU/mL) for early (BCLC 0 & A, n = 28) versus late HCC (BCLC B, n = 81; BCLC C, n = 130) were displayed as bar graphs; Figure S2: Comparison of three anti-CD98 antibodies for detecting extracellular CD98 on cells and EVs. (A) Surface CD98 on HepG2 cells quantified using three antibodies (Abcam ab307587, Novus NBP2-36491SS, NHRI#20D5) by flow cytometry.

Techniques: Clinical Proteomics, Western Blot, Size-exclusion Chromatography, Control, Transmission Assay, Electron Microscopy

Journal: Cancers

Article Title: Blu-Ray-Based Quantification of CD98+ Extracellular Vesicles for Early Detection of Hepatocellular Carcinoma

doi: 10.3390/cancers18071086

Figure Lengend Snippet: CD98 co-localizes with classical exosome markers CD63 and CD9 in HepG2-derived EVs. ( A ) Representative images of CD63, CD9, and CD98 surface staining, along with total EV staining by TFP ester–AF488, in HepG2-derived EVs using single-EV imaging at 20× ( upper panel) and 40× ( lower panel). Scale bars = 50 µm and 20 µm, respectively. ( B ) Illustration of the multiplex fluorescence imaging principle of the single-EV imaging technique. ( C ) Line scans (L1–L3) from ( A ) were analyzed by ImageJ 1.54p to display relative fluorescence intensity (RFU) of TFP ester, CD63, CD9, and CD98 per pixel. ( D ) Single-EV profiling of 20× images ( n = 3) quantifying the proportion of positive EVs for each marker (CD63, CD9, and CD98) or marker combination relay on TFP ester–AF488-positive signal (total EVs).

Article Snippet: From the CLCA cohort [ ] via cBioPortal [ ], diagnostic sensitivity (%) of blood AFP (cutoffs: 200 ng/mL and 20 ng/mL) and PIVKA-II (cutoff: 40 mAU/mL) for early (BCLC 0 & A, n = 28) versus late HCC (BCLC B, n = 81; BCLC C, n = 130) were displayed as bar graphs; Figure S2: Comparison of three anti-CD98 antibodies for detecting extracellular CD98 on cells and EVs. (A) Surface CD98 on HepG2 cells quantified using three antibodies (Abcam ab307587, Novus NBP2-36491SS, NHRI#20D5) by flow cytometry.

Techniques: Derivative Assay, Staining, Imaging, Multiplex Assay, Fluorescence, Marker

Journal: Cancers

Article Title: Blu-Ray-Based Quantification of CD98+ Extracellular Vesicles for Early Detection of Hepatocellular Carcinoma

doi: 10.3390/cancers18071086

Figure Lengend Snippet: Identification of plasma CD98+ EVs as a novel diagnostic biomarker for early-stage HCC. ( A ) Schematic illustrating plasma CD98+ EV quantification using ExoCounter technology. ( B ) Original CD98+ EV counts from patient plasma (left) and normalized CD98+ EV index (right) are shown as dot plots. ** = p < 0.01, *** = p < 0.001 (Student’s t test). ( C ) ROC curve showing diagnostic performance of the CD98+ EV index. ( D ) Sensitivity and specificity of the CD98+ EV index (cutoff = 6) were calculated from true/false positive and negative rates in early HCC. AFP (cutoff = 20 ng/mL) was compared as a reference, though AFP data were unavailable for healthy controls. TP = true positive; TN = true negative; FP = false positive; FN = false negative.

Article Snippet: From the CLCA cohort [ ] via cBioPortal [ ], diagnostic sensitivity (%) of blood AFP (cutoffs: 200 ng/mL and 20 ng/mL) and PIVKA-II (cutoff: 40 mAU/mL) for early (BCLC 0 & A, n = 28) versus late HCC (BCLC B, n = 81; BCLC C, n = 130) were displayed as bar graphs; Figure S2: Comparison of three anti-CD98 antibodies for detecting extracellular CD98 on cells and EVs. (A) Surface CD98 on HepG2 cells quantified using three antibodies (Abcam ab307587, Novus NBP2-36491SS, NHRI#20D5) by flow cytometry.

Techniques: Clinical Proteomics, Diagnostic Assay, Biomarker Discovery

Journal: Cancers

Article Title: Blu-Ray-Based Quantification of CD98+ Extracellular Vesicles for Early Detection of Hepatocellular Carcinoma

doi: 10.3390/cancers18071086

Figure Lengend Snippet: The CD98+ EV index is an independent and early detection biomarker. ( A ) In the non-viral early HCC cohort, CD98+ EV index values were compared across subgroups with different risk factors, including sex, smoking, alcohol consumption, and cirrhosis. Data are shown as dot plots. No significant differences (n.s.) were observed between groups (Student’s t test). ( B ) Bar graphs show diagnostic sensitivity (%) for blood AFP (200 ng/mL and 20 ng/mL), and CD98+ EV index in early HCC (stage I/II, n = 136) and small tumors (≤3 cm, n = 51). ** = p < 0.01, **** = p < 0.0001 (Z-score test for two proportions).

Article Snippet: From the CLCA cohort [ ] via cBioPortal [ ], diagnostic sensitivity (%) of blood AFP (cutoffs: 200 ng/mL and 20 ng/mL) and PIVKA-II (cutoff: 40 mAU/mL) for early (BCLC 0 & A, n = 28) versus late HCC (BCLC B, n = 81; BCLC C, n = 130) were displayed as bar graphs; Figure S2: Comparison of three anti-CD98 antibodies for detecting extracellular CD98 on cells and EVs. (A) Surface CD98 on HepG2 cells quantified using three antibodies (Abcam ab307587, Novus NBP2-36491SS, NHRI#20D5) by flow cytometry.

Techniques: Biomarker Discovery, Diagnostic Assay

Journal: Journal of the American Society of Nephrology

Article Title: B7–1 Is Not Induced in Podocytes of Human and Experimental Diabetic Nephropathy

doi: 10.1681/asn.2015030266

Figure Lengend Snippet: Figure 2. B7-1 is not expressed in glomeruli of BTBR WT and BTBR ob/ob mice euthanized at 20-22 weeks of age. (A and B) Representative images of glomerular B7–1 staining (red; with the polyclonal goat anti–mouse antibody) in kidney sections of BTBR WT and BTBR ob/ob mice. Nuclei were stained with 49,6-diamidino-2-phenylindole (DAPI; blue), and renal structures were stained with fluorescein wheat germ agglutinin (WGA; green). (A9 and B9) Each picture is also shown without WGA to point out B7–1 signal. (A and A9) Faint B7–1 glomerular expression was found in renal sections of BTBR WT mice comparable with the B7–1 expression observed in glomeruli of BTBR ob/ob mice (B and B9). (C and C9) Cos- taining for B7–1 (red) and PDX (green) in BTBR ob/ob glomeruli detected as just a few dots of colocalization (Inset) between the two markers. (D–F) Representative micrographs of B7–1 immunostaining (red) in BTBR murine renal samples using the monoclonal hamster anti–mouse B7–1 antibody. B7–1 glomerular staining was not detectable in glomeruli of (D) BTBR WT and (E) diabetic BTBR ob/ob mice. (E and F) Occasional B7–1-positive in- flammatory cells were found in diabetic renal samples (arrows). Scale bar, 20 mm. PDX, podocalyxin.

Article Snippet: After blocking nonspecific sites with 1% BSA, acetone-fixedcryosectionswere incubated with polyclonal goat anti–human B7–1 (di- luted 1:200; catalog no. AF140; R&D Sys- tems, Minneapolis, MN), polyclonal goat anti–mouse B7–1 (1:200; catalog no. AF740; R&D Systems), or monoclonal ham- ster anti–mouse B7–1 (1:20; clone 16–10A1; catalog no. 553766; BD Pharmingen, San Diego, CA) and mouse anti–human podo- calyxin (1:150; gift from Robert Atkins, Monash Medical Centre, Clayton, Victoria, Australia) or rabbit anti–mouse podocalyxin (1:100; Novus Bio, Littleton, CO) followed by the specific human IgG–adsorbed Cy3– and FITC–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) previously exposed to ei- ther BSA or strain–specific murine serum.

Techniques: Staining, Expressing, Immunostaining

Journal: Journal of the American Society of Nephrology

Article Title: B7–1 Is Not Induced in Podocytes of Human and Experimental Diabetic Nephropathy

doi: 10.1681/asn.2015030266

Figure Lengend Snippet: Figure 3. B7-1 is not expressed in glomeruli of C57BL/6 mice with STZ-induced diabetes euthanized at 16-20 weeks after disease induction. (A and B) Representative micrographs of B7–1 immunostaining (red; with the polyclonal goat anti–mouse antibody) in renal samples. Nuclei were stained with 49,6-diamidino-2-phenylindole (DAPI; blue), and renal structures were stained with fluorescein wheat germ agglutinin (WGA; green). (A9 and B9) Each picture is also shown without WGA to highlight the B7–1 signal. (A and A9) Spots of B7–1 staining were observed in glomeruli of control C57BL/6 mice, and almost the same glomerular B7–1 expression was detected in C57BL/6 mice after STZ injection (B and B9). (C and C9) Double staining for B7–1 (red) and PDX (green) in glomeruli of STZ–injected C57BL/6 mice showed very few spots of colocalization (Inset) between the two markers. (D–F) Representative micrographs of B7–1 immunostaining (red) in C57BL/6 murine renal samples using the monoclonal hamster anti–mouse B7–1 antibody. No glomerular B7–1 staining was observed in (D) control or (E) diabetic C57BL/6 mice, whereas (F) B7– 1-positive inflammatory cells were detected in the renal interstitium (arrow). Scale bar, 20 mm. PDX, podocalyxin.

Article Snippet: After blocking nonspecific sites with 1% BSA, acetone-fixedcryosectionswere incubated with polyclonal goat anti–human B7–1 (di- luted 1:200; catalog no. AF140; R&D Sys- tems, Minneapolis, MN), polyclonal goat anti–mouse B7–1 (1:200; catalog no. AF740; R&D Systems), or monoclonal ham- ster anti–mouse B7–1 (1:20; clone 16–10A1; catalog no. 553766; BD Pharmingen, San Diego, CA) and mouse anti–human podo- calyxin (1:150; gift from Robert Atkins, Monash Medical Centre, Clayton, Victoria, Australia) or rabbit anti–mouse podocalyxin (1:100; Novus Bio, Littleton, CO) followed by the specific human IgG–adsorbed Cy3– and FITC–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) previously exposed to ei- ther BSA or strain–specific murine serum.

Techniques: Immunostaining, Staining, Control, Expressing, Injection, Double Staining