Journal: EBioMedicine

Article Title: Tumour-specific activation of a tumour-blood transport improves the diagnostic accuracy of blood tumour markers in mice.

doi: 10.1016/j.ebiom.2024.105178

Figure Lengend Snippet: Fig. 1: Intravenously injected iRGD increases blood AFP levels in HCC-bearing mice. (a) Experimental setup to study the effect of iRGD on blood AFP levels in Huh-7 xenografted nude mice. (b and c) Blood AFP levels before and after intravenous injection of iRGD (b, c, n = 12), RGD control peptide (c (n = 6)), and PBS (c (n = 6)) in mice with HepG2 xenografts. Human AFP was not detectable in the blood of mice without tumours. In (c) the fold increase of AFP with pre-injection level set to 1; lines and error bars represent geometric means and 95% CI. (d) Experimental setup to study the effect of iRGD on blood AFP levels in TGFα/c-myc HCC mice. (e and f) iRGD specifically increased the blood AFP levels in TGFα/c-myc HCC mice. TGFα/c-myc mice (20–24 weeks old) with HCC according to MRI or without HCC (f) were intravenously injected with iRGD (e, f), RGD control peptide (f) or PBS (f). Data are fold changes of blood AFP due to the treatments (n = 48: iRGD; RGD control peptide: n = 34; PBS: n = 15); lines and error bars indicate medians and 95% CI. Dashed line: upper 95% CI increase in blood AFP in the PBS-injected HCC mice. (g) AFP expression in HCCs of TGFα/c-myc mice. HCCs and liver tissues were excised from TGFα/c-myc mice. Pairs of the tissue lysates were analysed for AFP and β-actin content by immunoblotting. Band densities were measured densitometrically. The ratio of AFP/β-actin in the livers was set to 1. (h) iRGD-induced accumulation of Evans blue in HCCs in TGFα/c-myc mice with HCC. TGFα/c-myc mice with HCCs were co- injected with iRGD or PBS (n = 12 per group) and Evans blue (EB). The dye content of the tumours was related to that in the livers; lines and error bars indicate geometric means (b, c, and h) or medians (e and f) and 95% CI; dashed line: upper 95% CI of the measured EB content of a HCC from the PBS-injected animals. (b): paired t test for log-transformed data; (c) One-way ANOVA with multiple comparison post-hoc test for log-transformed data; (e): Wilcoxon signed-rank test for log-transformed data; (f) Kruskal–Wallis test with Dunn’s multiple comparison post- hoc test; (h): two-sample t test. The indicated fold increase in (b, e and h) is the ratio of the geometric means with 95% CI.

Article Snippet: Immunoblotting of lysates obtained from pairs of liver and HCC tissue from TGFα/c-myc mice was performed as described previously.38 Gel-resolved proteins were electrotransferred to nitrocellulose membranes and incubated with antibodies raised against mouse-AFP (#AF5369, R&D Systems, Minneapolis, MN, RRID:AB_2258018) and anti-β-actin (#A2066, Sigma– Aldrich/Merck, RRID:AB_476693).

Techniques: Injection, Control, Expressing, Western Blot, Transformation Assay, Comparison

Journal: EBioMedicine

Article Title: Tumour-specific activation of a tumour-blood transport improves the diagnostic accuracy of blood tumour markers in mice.

doi: 10.1016/j.ebiom.2024.105178

Figure Lengend Snippet: Fig. 3: iRGD-induced elevation of the blood AFP concentration depends on NRP-1 and the tumour blood concentration gradient of AFP. (a) Anti-NRP-1 prevented iRGD-induced increase in the blood AFP concentration. TGFα/c-myc tumour mice that displayed a robust iRGD-induced increase in blood AFP level one week earlier were injected with anti-NRP-1 and the effect of iRGD on blood AFP level was determined (n = 3 per group). Fold increase of AFP with pre-injection level at the first time point was set to 1. Lines and error bars represent medians and 95% CI. (b– e) iRGD-induced elevation of the blood AFP level correlated negatively with the pre-injection blood AFP level in TGFα/c-myc mice (b), DEN- CCl4-HCC mice (c), mice with Huh-7 (d) or HepG2 xenografts (e). Spearman correlation r (b and c) and Pearson correlation r (d and e) with 95% CI, two tailed p-values and the log–log regression lines. (f) iRGD increased the blood AFP levels in HepG2 xenografted nude mice and low basal AFP (<67 ng/ml, n = 36, left panel), but not in animals with high basal AFP (>67 ng/ml, n = 12, right panel). Lines and error bars indicate geometric means and 95% CI. [(g) iRGD increased the blood AFP levels in TGFα/c-myc mice with HCC and normal basal AFP (<67 ng/ml, n = 36, left panel), but not in mice with elevated basal AFP (>67 ng, n = 12, right panel). Lines and error bars represent medians (left) or geometric means (right) with 95% CI. Significance was calculated with one sample t test (a, left) and the unpaired t test (a, right), paired t test (f and g, right) and Wilcoxon matched-pairs signed-rank test (g, left). The indicated fold increase in (f) is the geometric mean ratio with 95% CI. The indicated fold increase in (g) is the median of the ratios with 95% CI.

Article Snippet: Immunoblotting of lysates obtained from pairs of liver and HCC tissue from TGFα/c-myc mice was performed as described previously.38 Gel-resolved proteins were electrotransferred to nitrocellulose membranes and incubated with antibodies raised against mouse-AFP (#AF5369, R&D Systems, Minneapolis, MN, RRID:AB_2258018) and anti-β-actin (#A2066, Sigma– Aldrich/Merck, RRID:AB_476693).

Techniques: Concentration Assay, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , General morphology of livers 2 days after PHx. b , H&E staining of liver sections 2 days after PHx. c , d , Ki67 + percentages in HNF4α + hepatocytes ( c ) and HNF4α − NPCs ( d ) 2 days after PHx. Means ± SEM are shown, n=3 per group, **P < 0.01 (two-tailed unpaired t test). n.s., not significant. e , EpCAM was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). Stellate cells (GFAP) were not altered around the colony. f , CK19 was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). CD133 was highly expressed in the colony. g , Sox9 and CD44 were highly expressed in biliary epithelial cells (arrowhead), but was not upregulated in the colony (dashed line) compared to surrounding hepatocytes. h , AFP showed no difference between the colony (dashed line) and the surrounding tissue. i , Liver/body weight ratios after PHx. Means ± SEM from 3 or more mice analyzed for each time point and group are shown. j , k , Immunofluorescent staining of SKO liver sections 3 weeks after PHx. Colonies were at various sizes and locations as shown by arrows in j . Macroscopic colonies were found as shown by arrows and dashed lines in k . Immunofluorescent image in k corresponds to the magenta arrow in the liver image. PV, portal vein; CV, central vein. l , Vasculature shown by PECAM did not distinguish the colony (arrow). m , HGF expressed by NPCs was not concentrated in the colony (dashed line). Scale bars, 1 cm ( a ) 100 μm ( b , e - g , i - l ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Staining, Two Tailed Test