Journal: Biotechnology Journal

Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

doi: 10.1002/biot.70287

Figure Lengend Snippet: Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker distribution. CD133‐PE monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: CD133‐PE (Miltenyi Biotec, equal mixing of 130‐110‐962 and 130‐110‐962, 1:50–1:50 = 2:100) antibody was diluted 1:50 in CellO‐IF and incubation was done at 4°C for 20 min.

Techniques: Functional Assay, Cell Culture, Microscopy, Comparison, Confocal Microscopy, Marker, Inhibition, Sulforhodamine B Assay, Two Tailed Test

Journal: Biotechnology Journal

Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

doi: 10.1002/biot.70287

Figure Lengend Snippet: Comparative benchmarking of CSC and EMT‐associated traits in HGSOC cells cultured using ULA and photopatterned GelMA platforms. Representative western blot images and corresponding quantitative bar graphs showing expression of CSC‐associated proteins SOX‐2, ALDH1A1, NANOG, and OCT‐4 in OVCAR‐3 and OVSAHO cultures. Protein levels were normalized to Vinculin and Calnexin, with representative loading control bands shown. Different loading controls were selected depending on target abundance, membrane compatibility, and subcellular localization. Quantification was performed using ImageJ (a). Representative flow cytometry pseudo‐dot plots with gates defined using unstained controls, and bar graphs summarizing the percentage of CD133‐PE, CD44‐FITC, CD117‐PE, and CXCR4‐FITC positive populations. Representative gating strategy is provided in Figure (b). Representative western blot images and quantitative bar graphs of EMT‐associated proteins Slug, Snail, TWIST1, vimentin, and fibronectin in OVCAR‐3 cultures (normalized to calnexin). E‐cadherin and N‐cadherin were additionally analyzed (normalized to α‐tubulin), with quantitative N‐cadherin/E‐cadherin ratios shown (c). Representative western blot analysis of stemness‐associated proteins in OVCAR‐3 cells cultured as 2D monolayer, 2D monolayer in spheroid enrichment medium, UV‐treated monolayer, ULA spheroids, and photopatterned GelMA (GelMA‐Pm) spheroids. Due to limited cell yield obtained from GelMA‐Pm cultures, only SOX‐2 and ALDH1A1 expression could be evaluated in these samples. Protein expression was normalized to GAPDH. Corresponding densitometric quantification is shown on the right. Full‐length uncropped blots are provided in Figure . (d). RT‐qPCR analysis of the stemness‐associated genes SOX‐2, NANOG, ALDH1A1, and c‐MYC in OVCAR‐3 cells cultured under 2D monolayer, 2D + sphere medium, 2D + UV, photopatterned GelMA (GelMA‐Pm), and ULA spheroid conditions. Gene expression levels were normalized to β‐actin and are presented relative to 2D monolayer controls (set to 1). Values above 1 indicate increased expression relative to monolayer cultures. Data represent mean ± SD from three independent biological replicates ( n = 3). Statistical significance was determined using multiple t ‐tests; significance symbols are defined as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001. Western blot images were cropped for clarity; non‐adjacent lanes from the same membrane are indicated by spaces. Full‐length blots are provided in Figure .

Article Snippet: CD133‐PE (Miltenyi Biotec, equal mixing of 130‐110‐962 and 130‐110‐962, 1:50–1:50 = 2:100) antibody was diluted 1:50 in CellO‐IF and incubation was done at 4°C for 20 min.

Techniques: Cell Culture, Western Blot, Expressing, Control, Membrane, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Biotechnology Journal

Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

doi: 10.1002/biot.70287

Figure Lengend Snippet: Optimization of photopatterned GelMA hydrogels for spheroid formation and CSC enrichment in HGSOC cell lines. Representative brightfield microscopy images of photopatterned GelMA (GelMA‐Pm) cultures of OVCAR‐3 and OVSAHO cells (upper panels: 4× objective; lower panels: 10× objective), showing spheroid formation under optimized conditions (a). Schematic overview of the GelMA‐Pm fabrication workflow and representative image of square photomask‐generated polymerized GelMA structures visible to the naked eye (b). Quantitative comparison of spheroid number and spheroid diameter per mm 2 between ULA and GelMA‐Pm cultures (c). Representative brightfield microscopy images illustrating approximate spheroid diameter distributions within photopatterned GelMA cultures at Day 14, including representative OVCAR‐3 spheroids (∼100 µm) and smaller OVSAHO spheroids (∼50 µm) (d). Representative confocal microscopy images of GelMA hydrogels containing spheroids from OVCAR‐3 and OVSAHO cultures, demonstrating cell viability and spatial distribution (10× objective, with selected regions magnified to 20X as indicated by yellow boxes). Blue = DAPI (nuclei), green = α‐tubulin, red = CD133‐PE. Corresponding brightfield and fluorescence images of identical fields are shown (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: CD133‐PE (Miltenyi Biotec, equal mixing of 130‐110‐962 and 130‐110‐962, 1:50–1:50 = 2:100) antibody was diluted 1:50 in CellO‐IF and incubation was done at 4°C for 20 min.

Techniques: Microscopy, Generated, Comparison, Confocal Microscopy, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Cholesterol Laundry of Cell Membrane and Fatty Liver by Detergent Liposomes to Improve Anti‐Cancer Drug Responsiveness of Patient Liver Tissues

doi: 10.1002/advs.76144

Figure Lengend Snippet: Detergent effect of liposomes as a laundry key of membrane cholesterol to improve cellular drug uptake. (A) Detergent effect of liposome is strategized to launder cholesterol of plasma membrane so that cellular drug uptake is improved. As key mechanistic processes, liposomes penetrate the cholesterol‐rich plasma membrane which directly forms the endosomal membrane with the encapsulation of the liposomes. In this way, cholesterol is transferred from the plasma membrane to the endosomal membrane and finally to the liposomal membrane, which is defined as the soap‐like detergent mechanism. Because cholesterol aggregates with cholesterol through hydrophobic interactions, cholesterol is inserted into the liposomal membrane to form cholesterol (+)‐liposomes with the expectation to improve the laundry function. Moreover, as a real detergent component, Triton X‐100 is added to form Detergent (+)‐liposomes, and these two laundry‐specific liposomes are compared in addition to Cholesterol (−)‐liposomes or No treat for a series of applications (right box). (B) After culturing the hepatocellular carcinoma (HCC) cell line (Hep3B) for 24 h, treatment of either cholesterol (+)‐ or detergent (+)‐liposomes significantly reduces the cholesterol level of plasma membrane compared to cholesterol (−)‐liposomes (top left) and No treat (top right) by kit analysis. These results are validated by image analyses (middle and bottom), indicating the detergent‐like effect of cholesterol (+)‐liposomes. (C) An endosome model is produced by first increasing the cholesterol level of membrane through Hep3B culture in hypoxia, followed by serial filter extrusion to produce NVs. When the NVs are produced, green fluorescence (BODIPY)‐cholesterol is added to the self‐assembly process of membrane so that the detergent effect to transfer green cholesterol from the endosome NV to liposomes can be tracked. Moreover, CD133 of endosomal membrane is labeled with a red fluorescent to distinguish it from the test liposomes. When endosome NVs and test liposomes are incubated for 24 h, and nanoFACS analysis is carried out, green cholesterol is transferred from red endosome NVs to cholesterol (+)‐liposomes significantly more compared to No treat, which is equivalent to treatment of Detergent (+)‐liposomes. (D) After Hep3B cells are treated with test liposomes, subsequent 24‐h treatment of hydrophobic Sorafenib exerts non‐significant difference in the ant‐cancer effect between Cholesterol (+)‐ and Detergent (+)‐liposomes as shown by cell viability. In contrast, cholesterol (+)‐liposomes more effectively facilitates uptake of hydrophobic Coumarin‐6 with fluorescence label from subsequent 6‐h treatment compared to Detergent (+)‐liposomes by imaging analysis. The result indicates a superiority of cholesterol in addition to liposomal membrane to the detergent one. (E) This result is supported by the lower Hep3B cytotoxicity of cholesterol (+)‐liposome compared to Detergent ones when the liposomal concentrations are increased up to 0.8 mg/mL, thereby justifying the use of cholesterol (+)‐liposomes for the follow‐up experiments. Data = mean ± SEM. N = the number of dots as independent replicates in each graph. Statistical significance is denoted by * p < 0.033, ** p < 0.002, *** p < 0.001, and not significant (ns).

Article Snippet: The surface marker CD133 and membrane cholesterol of the nanovesicles were labeled with a red fluorescence conjugate (ab226355, Abcam, Cambridge, UK) and BODIPY‐cholesterol (HY‐125746, MedChemExpress, Monmouth Junction, NJ, USA), respectively, following the suppliers’ protocols.

Techniques: Liposomes, Membrane, Clinical Proteomics, Encapsulation, Produced, Fluorescence, Labeling, Incubation, Imaging

Journal: JHEP Reports

Article Title: AI-guided additive scoring model for differential diagnosis of primary liver cancer

doi: 10.1016/j.jhepr.2026.101826

Figure Lengend Snippet: Serological parameters and CD133/2 + sEVs in HCC and iCCA. (A) Overview of routinely assessed standard serum parameters in biliary and hepatic malignancies, including albumin, ALP, CA19-9, and CRP.(B) Platelet (thrombocyte) counts. (C) Total numbers of CD133/2 + sEVs and their CD9 + , CD63 + , and CD81 + subpopulations immobilized on Human ExoView® Tetraspanin Chips precoated with anti-CD9, anti-CD63, and anti-CD81 EV capture antibodies, including MIgG isotype controls. Bars represent median values with 95% CIs. Associated AUROC values, sensitivity, specificity, and cut-off values were calculated using GraphPad PRISM and subsequently applied in the analogous additive scoring system. Statistical analyses were performed using GraphPad PRISM (version 10.4.1). Group comparisons were conducted using two-sided Mann-Whitney U tests because of non-normal data distribution. Exact p values are shown and were as follows: albumin, p = 0.06; ALP, p <0.001; CA19-9, p = 0.03; and CRP, p <0.001 (A); thrombocytes, p = 0.002 (B); CD9 + CD133/2 + , p = 0.0014; CD63 + CD133/2 + , p = 0.0009; and CD81 + CD133/2 + , p = 0.0001 (C). Post-hoc effect size (Cohen’s d) and power analyses were derived from the Mann-Whitney U test results to quantify the discriminatory sensitivity of each biomarker comparison . ALP, alkaline phosphatase; CRP, C-reactive protein; EV, extracellular vesicle; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; MIgG, mouse IgG; sEV, small extracellular vesicle.

Article Snippet: Anti-CD133/2 antibodies (130-112-195, Miltenyi Biotec) and anti-CD44v6 antibodies (130-111-238, Miltenyi Biotec) were added and titrated against their matching isotype (REA293 phycoerythrin, 130-107-771 and REA293 allophycocyanin, 130-113-446, Miltenyi Biotec) and used in concentrations according to the respective antibodies; 7-AAD (BD Pharmingen, NJ, USA, 559925) was used for dead cell exclusion.

Techniques: MANN-WHITNEY, Derivative Assay, Biomarker Discovery, Comparison

Journal: JHEP Reports

Article Title: AI-guided additive scoring model for differential diagnosis of primary liver cancer

doi: 10.1016/j.jhepr.2026.101826

Figure Lengend Snippet: Selection of the final marker set using LASSO and PCA. (A) The LASSO path plot illustrates the selection process of candidate markers using LASSO regression first. Early entrants, such as ALP, CRP, and CD9 + CD133/2 + small EVs, achieved high coefficient values, indicating consistent relevance within the fitted model. By contrast, thrombocytes, AFP, and albumin contributed with lower coefficients, whereas late entrants, including CA19-9 and CD63 + CD133/2 + small EVs, had the weakest contributions. (B) PCA biplot: PCA was applied to the full feature set to visualize marker distribution along the first two PCs (PC1 and PC2). The biplot depicts how the investigated markers cluster in distinct regions, with CRP, AFP, and thrombocytes forming separate groups from CA19-9 and EV subpopulations. AFP, alpha-fetoprotein; ALP, alkaline phosphatase; CRP, C-reactive protein; EV, extracellular vesicle; PC, principal component.

Article Snippet: Anti-CD133/2 antibodies (130-112-195, Miltenyi Biotec) and anti-CD44v6 antibodies (130-111-238, Miltenyi Biotec) were added and titrated against their matching isotype (REA293 phycoerythrin, 130-107-771 and REA293 allophycocyanin, 130-113-446, Miltenyi Biotec) and used in concentrations according to the respective antibodies; 7-AAD (BD Pharmingen, NJ, USA, 559925) was used for dead cell exclusion.

Techniques: Selection, Marker

Journal: JHEP Reports

Article Title: AI-guided additive scoring model for differential diagnosis of primary liver cancer

doi: 10.1016/j.jhepr.2026.101826

Figure Lengend Snippet: Kaplan–Meier OS analysis in LR-M patients stratified by CD133/2 small EV subpopulations. Kaplan–Meier curves depict OS (days since diagnosis) stratified by above-median versus ≤ median marker levels within disease-specific subgroups (HCC and iCCA; each n = 25). ‘Event’ indicates death. All survival analyses are exploratory and tests were two sided. (A) CD9 + CD133/2 + small EVs. Patients with iCCA (n = 25; deaths = 23; censored = 2) were stratified into above-median (n = 13) versus ≤ median (n = 12). Patients with above-median CD9 + CD133/2 + EV levels showed shorter median OS (91.0 vs . 389.5 days; median ratio: 4.28, 95% CI: 1.877–9.761). Survival differed significantly by log-rank (Mantel–Cox) test (χ 2 = 7.829, p = 0.005) and by Gehan–Breslow–Wilcoxon test (χ 2 = 5.324, p = 0.020). The log-rank HR was 2.802 (95% CI:1.164–6.744) for above-median versus ≤ median groups. Patients with HCC (n = 25; deaths = 20; censored = 5) were stratified into above-median (n = 10) versus ≤ median (n = 15). Median OS was 760.0 vs . 907.0 days. No significant survival differences were observed (log-rank χ 2 = 0.119, p = 0.730; Gehan–Breslow χ 2 = 0.0077, p = 0.930). The log-rank HR was 1.162 (95% CI: 0.482–2.798). (B) CD81 + CD133/2 + small EVs. Patients with iCCA patients (n = 25; deaths = 23; censored = 2) were stratified into above-median (n = 12) versus ≤ median (n = 13). Median OS was 114.5 vs . 173.0 days. Differences did not reach statistical significance (log-rank χ 2 = 2.840, p = 0.090; Gehan–Breslow χ 2 = 1.205, p = 0.270). The log-rank HR was 1.914 (95% CI: 0.818–4.476). Patients with HCC (n = 25; deaths = 20; censored = 5) were stratified into above-median (n = 12) versus ≤ median (n = 13). Median OS was 730.0 vs . 1460.0 days. Survival differed by log-rank test (χ 2 = 4.488, p = 0.034), whereas the Gehan–Breslow–Wilcoxon test did not reach significance (χ 2 = 3.048, p = 0.0808). The log-rank HR was 2.457 (95% CI: 0.996–6.064). Vertical dotted lines indicate the estimated time points at which survival reached 50% (median OS). EV, extracellular vesicle; HCC, hepatocellular carcinoma; HR, hazard ratio; iCCA, intrahepatic cholangiocarcinoma; OS, overall survival.

Article Snippet: Anti-CD133/2 antibodies (130-112-195, Miltenyi Biotec) and anti-CD44v6 antibodies (130-111-238, Miltenyi Biotec) were added and titrated against their matching isotype (REA293 phycoerythrin, 130-107-771 and REA293 allophycocyanin, 130-113-446, Miltenyi Biotec) and used in concentrations according to the respective antibodies; 7-AAD (BD Pharmingen, NJ, USA, 559925) was used for dead cell exclusion.

Techniques: Biomarker Discovery, Marker