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: bioRxiv

Article Title: Schlafen family member 5 (SLFN5) regulates LAT1-mediated mTOR activation in castration-resistant prostate cancer

doi: 10.1101/2020.09.17.301283

Figure Lengend Snippet: A , Immunofluorescence showing nuclear SLFN5 expression in LNCaP and LNCaP AI cells. Scale bar represents 10 μm. B , Venn diagrams highlighting genes commonly modulated (p-value < 0.05, FC = 2) in SLFN5 KO cells (top) and tumours (bottom) when compared to their respective controls. Up-regulated genes are on top; Down-regulated genes are into brackets. C , Schematic representation of the down-regulated genes (p-value < 0.05, FC = 2) in SLFN5 KO cells when compared to CTL cells. Pathway enrichment analysis was performed using the STRING database ( http://string-db.org ). D , RT-qPCR analysis of SLFN5 and top downregulated genes from B in SLFN5 KO and CTL cells. E , Volcano plots showing the proteins significantly modulated (p-value < 0.05, FC = 2) in the proteomic analysis of SLFN5 KO tumours. F , Pearson’s correlation analysis of NDNF , STRBP, UBAP2 and SLC7A5 with SLFN5 using the PRAD TCGA dataset. Results were obtained using the GEPIA website http://gepia.cancer-pku.cn/ . Panel D : Data are presented as mean values +/− SD. Panel D : *p-value < 0.05 using a 1-way ANOVA with a Dunnett’s multiple comparisons test. Panel F : statistical analysis was performed using a logrank test.

Article Snippet: 22Rv1 SLFN5 KO cells were further transfected with SLC7A5 (NM_003486) Human Tagged ORF Clone (RC207604, Origene, Rockville, MD, USA).

Techniques: Immunofluorescence, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Schlafen family member 5 (SLFN5) regulates LAT1-mediated mTOR activation in castration-resistant prostate cancer

doi: 10.1101/2020.09.17.301283

Figure Lengend Snippet: A , Western blot analysis of SLFN5, SLC7A5 and SLC3A2 expression in 22rv1 SLFN5 KO and CTL cells. B , Western blot analysis of SLFN5, SLC7A5 and SLC3A2 expression in 22rv1-derived SLFN5 KO and CTL tumour orthografts. C , Western blot analysis of SLFN5 and SLC7A5 expression in LNCaP AI SLFN5 KO and CTL cells. D , Western blot analysis of SLFN5 and SLC7A5 expression in LNCaP cells overexpressing SLFN5. E , Western blot analysis of SLFN5 and SLC7A5 expression in HN and matched CRPC cell lines. F , RT-qPCR analysis of SLFN5 , SLC7A5 and ATF4 expression in 22rv1 cells silenced for SLFN5, ATF4 or both. G , Gene set enrichment plots analysed from SLFN5-depleted tumours transcriptomics using ATF4-related gene set obtained from . H , Proximity ligation assay of SLFN5 and ATF4 performed on 22rv1 cells. Red dots represent co-localisation. Scale bar represents 11 μm. Panels A , B , C , D , E : HSC70 is used as a sample loading control. Panel F : Data are presented as mean values +/− SD. Panel F : *p-value < 0.05 using a 1-way ANOVA with a Tukey’s multiple comparisons test.

Article Snippet: 22Rv1 SLFN5 KO cells were further transfected with SLC7A5 (NM_003486) Human Tagged ORF Clone (RC207604, Origene, Rockville, MD, USA).

Techniques: Western Blot, Expressing, Derivative Assay, Quantitative RT-PCR, Proximity Ligation Assay, Control

Journal: bioRxiv

Article Title: Schlafen family member 5 (SLFN5) regulates LAT1-mediated mTOR activation in castration-resistant prostate cancer

doi: 10.1101/2020.09.17.301283

Figure Lengend Snippet: A , Steady-state levels of significantly regulated metabolites in SLFN5 KO cells when compared to CTL cells (FC > 1.2). Selected metabolites were significantly altered in at least one of the two KO cells (p < 0.05 using two-sided Student’s t-test). B , Western blot analysis of SLFN5, SLC7A5, SLC3A2, p-70S6K and p-S6 expression in 22rv1 SLFN5 KO and CTL cells. C , same analysis as B performed on 22rv1 SLFN5 KO and CTL cells overexpressing two different SLC7A5 constructs. D , Western blot analysis of p-S6 and p-4EBP1 expression in 22rv1-derived SLFN5 KO and CTL tumour orthografts. E , Volcano plot of the modulated proteins between high and low SLFN5 tumours using the TCGA PRAD dataset. Red and blue dots represent the proteins that are significantly up-regulated and down-regulated (FC = 1.2, p < 0.05) in high SLFN5 tumours respectively. F , Differential expression of p-S6 (Ser235-236 and 240-244) and p-4EBP1 (Thr37) in high and low SLFN5 tumours, according to the data generated in E . Centre line corresponds to median of data, top and bottom of box correspond to 90th and 10th percentile, respectively. Whiskers extend to adjacent values (minimum and maximum data points not considered outliers). Panels B , C , D : HSC70 is used as a sample loading control. Panel A : Data are presented as mean values +/− SD. Panel F : statistical analysis was performed using a two-tailed Mann-Whitney U test.

Article Snippet: 22Rv1 SLFN5 KO cells were further transfected with SLC7A5 (NM_003486) Human Tagged ORF Clone (RC207604, Origene, Rockville, MD, USA).

Techniques: Western Blot, Expressing, Construct, Derivative Assay, Quantitative Proteomics, Generated, Control, Two Tailed Test, MANN-WHITNEY