Journal: Translational Cancer Research

Article Title: In vitro characterization of a novel anti-GD2/ROR1 bispecific antibody exhibiting tumor cell killing efficacy in neuroblastoma cross-cancer activity in ROR1-positive triple-negative breast cancer

doi: 10.21037/tcr-2026-0723

Figure Lengend Snippet: Molecular design schematic and purification gel image of bispecific antibody G/R-001. (A) Molecular design diagram of the light chain and heavy chain of bispecific antibody G/R-001. (B) Schematic diagram of GD2/ROR1 bispecific antibody G/R-001. (C) SDS-PAGE gel image of the bispecific antibody under reducing or non-reducing conditions. The left shows the SDS-PAGE gel image under reducing conditions, with two bands in the lane-the upper band representing the heavy chain and the lower band representing the light chain; the right shows the SDS-PAGE gel image under non-reducing conditions, with one band in the lane representing the intact antibody. CMV, cytomegalovirus; G/R-001, anti-GD2/ROR1 bispecific antibody; GD2, disialoganglioside 2; H, heavy chain; IgG, immunoglobulin G; L, light chain; ROR1, receptor tyrosine kinase-like orphan receptor 1; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; VH, variable domain of heavy chain; VK, variable domain of light chain.

Article Snippet: Isotype IgG (Cat#: HY-P99001, MedChemExpress, Princeton, NJ, USA), naxitamab (anti-GD2) (Cat#: HY-P99206, MedChemExpress), zilovertamab (anti-ROR1) (Cat#: HY-P99201, MedChemExpress), and GD2 × ROR1 BsAb were transiently expressed in HEK293 cells using the OPM-293 system (Cat#: 91070-010, OPM, Shanghai, China).

Techniques: Purification, SDS Page, Polyacrylamide Gel Electrophoresis

Journal: Translational Cancer Research

Article Title: In vitro characterization of a novel anti-GD2/ROR1 bispecific antibody exhibiting tumor cell killing efficacy in neuroblastoma cross-cancer activity in ROR1-positive triple-negative breast cancer

doi: 10.21037/tcr-2026-0723

Figure Lengend Snippet: ELISA detection of the binding activity of bispecific antibody G/R-001 to GD2 and ROR1. GD2 or ROR1 antigens were coated, and G/R-001, zilovertamab, naxitamab, and isotype IgG antibodies were added to the ELISA plate at concentrations of 0–150 nM with 2-fold serial dilutions. After secondary antibody incubation, absorbance values were detected at 450 nM wavelength by a microplate reader, curves were plotted, and EC 50 values were calculated. (A) Binding affinity of G/R-001 and naxitamab to GD2, with EC 50 values of 12.1 and 11.9 nM, respectively; (B) Binding affinity of G/R-001 and zilovertamab to ROR1, with EC 50 values of 3.5 and 3.1 nM, respectively. EC 50 , half maximal effective concentration; ELISA, enzyme-linked immunosorbent assay; G/R-001, anti-GD2/ROR1 bispecific antibody; GD2, disialoganglioside 2; IgG, immunoglobulin G; OD, optical density; ROR1, receptor tyrosine kinase-like orphan receptor 1.

Article Snippet: Isotype IgG (Cat#: HY-P99001, MedChemExpress, Princeton, NJ, USA), naxitamab (anti-GD2) (Cat#: HY-P99206, MedChemExpress), zilovertamab (anti-ROR1) (Cat#: HY-P99201, MedChemExpress), and GD2 × ROR1 BsAb were transiently expressed in HEK293 cells using the OPM-293 system (Cat#: 91070-010, OPM, Shanghai, China).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Incubation, Concentration Assay

Journal: Translational Cancer Research

Article Title: In vitro characterization of a novel anti-GD2/ROR1 bispecific antibody exhibiting tumor cell killing efficacy in neuroblastoma cross-cancer activity in ROR1-positive triple-negative breast cancer

doi: 10.21037/tcr-2026-0723

Figure Lengend Snippet: Flow cytometry detection of the binding activity of bispecific antibody G/R-001 to GD2/ROR1 positive cells. GD2 − /ROR1 − HEK293 cells were used as a negative control. G/R-001, zilovertamab, naxitamab, and isotype IgG at 10 nM concentration showed significant binding activity to GD2 − /ROR1 + MDA-MB-468 cells, GD2 + /ROR1 − LAN-1 cells, and GD2 + /ROR1 + MHH-NB-11 cells. FITC, fluorescein isothiocyanate; G/R-001, anti-GD2/ROR1 bispecific antibody; GD2, disialoganglioside 2; IgG, immunoglobulin G; ROR1, receptor tyrosine kinase-like orphan receptor 1.

Article Snippet: Isotype IgG (Cat#: HY-P99001, MedChemExpress, Princeton, NJ, USA), naxitamab (anti-GD2) (Cat#: HY-P99206, MedChemExpress), zilovertamab (anti-ROR1) (Cat#: HY-P99201, MedChemExpress), and GD2 × ROR1 BsAb were transiently expressed in HEK293 cells using the OPM-293 system (Cat#: 91070-010, OPM, Shanghai, China).

Techniques: Flow Cytometry, Binding Assay, Activity Assay, Negative Control, Concentration Assay

Journal: Translational Cancer Research

Article Title: In vitro characterization of a novel anti-GD2/ROR1 bispecific antibody exhibiting tumor cell killing efficacy in neuroblastoma cross-cancer activity in ROR1-positive triple-negative breast cancer

doi: 10.21037/tcr-2026-0723

Figure Lengend Snippet: LDH assay detection of the in vitro killing activity of bispecific antibody G/R-001 against GD2/ROR1 positive neuroblastoma and other cells. Isotype IgG, naxitamab, zilovertamab, naxitamab + zilovertamab, and G/R-001 antibodies (50 µL) at different concentrations (0–100.0 nM) were added to the experimental wells and cultured for 48 h. Following the LDH kit procedure, absorbance was read at 490 nm using a microplate reader. (A) HEK293 cells (GD2 − /ROR1 − ), (B) LAN-1 cells (GD2 + /ROR1 − ), (C) MDA-MB-468 cells (GD2 − /ROR1 + ), and (D) MHH-NB-11 cells (GD2 + /ROR1 + ). With increasing concentrations, Naxitamab, Zilovertamab, Naxitamab+Zilovertamab, and G/R-001 antibodies showed significantly increased toxicity against target-positive cells, with G/R-001 demonstrating the most significant cytotoxicity. G/R-001, anti-GD2/ROR1 bispecific antibody; GD2, disialoganglioside 2; IgG, immunoglobulin G; LDH, lactate dehydrogenase; ROR1, receptor tyrosine kinase-like orphan receptor 1.

Article Snippet: Isotype IgG (Cat#: HY-P99001, MedChemExpress, Princeton, NJ, USA), naxitamab (anti-GD2) (Cat#: HY-P99206, MedChemExpress), zilovertamab (anti-ROR1) (Cat#: HY-P99201, MedChemExpress), and GD2 × ROR1 BsAb were transiently expressed in HEK293 cells using the OPM-293 system (Cat#: 91070-010, OPM, Shanghai, China).

Techniques: Lactate Dehydrogenase Assay, In Vitro, Activity Assay, Cell Culture

Journal: iScience

Article Title: Targeting ROR1 with humanized antibody drug conjugates and cytokine fusion proteins for cancer therapy

doi: 10.1016/j.isci.2026.115578

Figure Lengend Snippet: Generation of high-affinity antibodies against human ROR1 (A) The binding affinity of ROR1-targeted antibodies generated by the hybridoma technique was analyzed by ELISA against purified ROR1 extracellular domain proteins (ROR1-ECD) (replicate = 2). Amino acid sequences of variable regions were presented in . (B) Flow cytometry analysis of the binding between ROR1-targeted mouse antibodies and ROR1 presented on MDA-MB-231 cells (replicate = 2). (C) The antibodies’ isotypes and binding affinity measured by ELISA and FACS were summarized (data were represented as mean). (D) The binding affinity of chimeric antibodies was detected by ELISA against purified ROR1-ECD proteins (replicate = 2). (E) Flow cytometry analysis of the binding between chimeric antibodies and ROR1 presented on MDA-MB-231 cells (replicate = 2). The analysis of the binding on NCI-H226 cells was presented in . (F) The chimeric antibodies’ binding affinity measured by ELISA and FACS were summarized (data were represented as mean).

Article Snippet: Purified extracellular domain of human ROR1 protein (ROR1-ECD, Sino Biological) was used as immunogen.

Techniques: Binding Assay, Generated, Enzyme-linked Immunosorbent Assay, Purification, Flow Cytometry

Journal: iScience

Article Title: Targeting ROR1 with humanized antibody drug conjugates and cytokine fusion proteins for cancer therapy

doi: 10.1016/j.isci.2026.115578

Figure Lengend Snippet: Epitope analysis of ROR1 antibodies (A) The binding affinity of antibodies against truncated ROR1 variants expressed on CHO cells was detected by FACS (replicate = 2). (B) ELISA analysis of the antibodies’ binding activity to truncated ROR1 proteins coated on ELISA plates (replicate = 2). (C) The degradation of ROR1 protein after 5 μg/mL R-001c ∼ R-009c treatment of MDA-MB-231 cells for 18 h. (D) ROR1 internalization was monitored by immunofluorescence staining and confocal microscopy. Scale bars, 20 μm. (E) The GTP-Rac1 signaling pathway was detected by western blot after treatment with R-001c, R-002c, R-004c, R-005c, and UC-961 for 18 h, followed by Wnt5a simulation for 5 min.

Article Snippet: Purified extracellular domain of human ROR1 protein (ROR1-ECD, Sino Biological) was used as immunogen.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Immunofluorescence, Staining, Confocal Microscopy, Western Blot

Journal: iScience

Article Title: Targeting ROR1 with humanized antibody drug conjugates and cytokine fusion proteins for cancer therapy

doi: 10.1016/j.isci.2026.115578

Figure Lengend Snippet: Development and in vitro characterization of ROR1-humanized antibodies (A) The binding affinity of humanized antibodies was evaluated by ELISA against ROR1-ECD proteins (replicate = 2, data were represented as mean). Amino acid sequences of variable regions were presented in . (B) Flow cytometry analysis of the binding between ROR1-targeted humanized antibodies and ROR1 presented on MDA-MB-231 cells (replicate = 2, data were represented as mean). The analysis of the binding on NCI-H226 cells was presented in . (C) ROR1 internalization induced by Hu001-2 and Hu005-46 was detected by confocal microscopy. Scale bars, 50 μm. The ROR1 internalization induced by UC-961 was presented in A. (D) The internalization kinetics and binding affinity of humanized antibodies were monitored over 4 h by FACS. (E) The binding K D of Hu001-2 and Hu005-46 was evaluated by surface plasmon resonance (SPR) analysis. The binding K D of UC-961 was presented in B.

Article Snippet: Purified extracellular domain of human ROR1 protein (ROR1-ECD, Sino Biological) was used as immunogen.

Techniques: In Vitro, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Confocal Microscopy, SPR Assay

Journal: iScience

Article Title: Targeting ROR1 with humanized antibody drug conjugates and cytokine fusion proteins for cancer therapy

doi: 10.1016/j.isci.2026.115578

Figure Lengend Snippet: In vitro anti-tumor efficacy of humanized anti-ROR1 antibody-based ADCs (A) Schematic design of ADC drugs. The profiles of Hu001-2-vcMMAE were presented in . (B) Binding activity of ADC to ROR1 expressed on MDA-MB-231 and NCI-H226 cell membranes detected by FACS. (C) The correlation between ROR1 expression levels and anti-proliferation activity of Hu001-2-MMAE was measured in multiple cancer cell lines (replicate = 3, data were represented as mean). ROR1 mRNA expression levels were presented in . (D) Cell cycle analysis by FACS of MDA-MB-231 cells treated with Hu001-2-MMAE for 48 h. (E) Apoptosis induced by 48-h treatment of Hu001-2-MMAE was evaluated by Annexin V/PI-staining and detected by FACS. Data were presented as mean ± SD ( n = 3). Statistical analysis was performed using a two-tailed unpaired t test with Welch’s correction. ∗ p < 0.05 and ∗∗∗ p < 0.001.

Article Snippet: Purified extracellular domain of human ROR1 protein (ROR1-ECD, Sino Biological) was used as immunogen.

Techniques: In Vitro, Binding Assay, Activity Assay, Expressing, Cell Cycle Assay, Staining, Two Tailed Test

Journal: iScience

Article Title: Targeting ROR1 with humanized antibody drug conjugates and cytokine fusion proteins for cancer therapy

doi: 10.1016/j.isci.2026.115578

Figure Lengend Snippet: In vivo anti-tumor efficacy of ROR1-targeting ADC The tumor volume monitoring and final tumor growth inhibition of ROR1-ADC Hu001-2-MMAE in mouse models inoculated with (A and B) ROR1-high ( n = 8), (C and D) ROR1-medium ( n = 8), and (E and F) ROR1-low ( n = 10) expressing cancer cells, treated with 2.5 mg/kg or 5 mg/kg ADC (QW, intravenous injection). Representative tumor photographs and body weight measurements were presented in . Results were shown as means ± SD. Statistical analysis was performed using a two-tailed unpaired t test with Welch’s correction. ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, ns, not significant.

Article Snippet: Purified extracellular domain of human ROR1 protein (ROR1-ECD, Sino Biological) was used as immunogen.

Techniques: In Vivo, Inhibition, Expressing, Injection, Two Tailed Test

Journal: iScience

Article Title: Targeting ROR1 with humanized antibody drug conjugates and cytokine fusion proteins for cancer therapy

doi: 10.1016/j.isci.2026.115578

Figure Lengend Snippet: Development of immune-activating antibody-cytokine fusion proteins (A) Schematic diagram of the Hu001-2-IL15Rα-IL15 fusion protein. Purification and characterization of fusion proteins were presented in . (B) Binding affinity to ROR1-ECD and IL-15Rβ was detected by ELISA (replicate = 2, data were represented as mean). The binding affinity with ROR1 on MDA-MB-231 cells was presented in . (C) After 6-day culture with Hu001-2-IL15Rα-IL15, CD8 + T cell and CD56 + NK cell populations in PBMCs were analyzed by FACS. (D) IFN-γ and TNF-α secretion by PBMCs were measured by ELISA. (E) The cytotoxicity activity of fusion proteins was evaluated by co-culture of PBMCs with MDA-MB-231 cells at an effector-to-target ratio of 10:1 and 20:1 for 24 h. Results were expressed as means ± SD ( n = 3). Statistical analysis was performed using two-tailed unpaired t test with Welch’s correction, ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: Purified extracellular domain of human ROR1 protein (ROR1-ECD, Sino Biological) was used as immunogen.

Techniques: Protein Purification, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Co-Culture Assay, Two Tailed Test

Journal: iScience

Article Title: Targeting ROR1 with humanized antibody drug conjugates and cytokine fusion proteins for cancer therapy

doi: 10.1016/j.isci.2026.115578

Figure Lengend Snippet: In vivo anti-tumor efficacy of the ROR1 antibody-IL15Rα-IL15 fusion (A) Body weight changes of mice during the treatment ( n = 5). (B) Tumor volume (mm 3 ) monitored over time. (C) Representative images of excised tumors and the final tumor growth inhibition. (D) IFN-γ and TNF-α expression in tumor tissues were measured by real-time RT-PCR from three independent tumors ( n = 3), with each sample measured in triplicate. Data were presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s or Dunnett’s multiple comparison test (C) or two-tailed unpaired t test (D), ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: Purified extracellular domain of human ROR1 protein (ROR1-ECD, Sino Biological) was used as immunogen.

Techniques: In Vivo, Inhibition, Expressing, Quantitative RT-PCR, Comparison, Two Tailed Test

Journal: Cell Death & Disease

Article Title: ROR1-PI3K/AKT signaling drives adaptive resistance to cell cycle blockade in TP53 mutated ovarian cancer

doi: 10.1038/s41419-026-08501-x

Figure Lengend Snippet: a UMAP plots showing the distribution of drug-naïve and long-term resistant cells for each HGSOC cell line. Colors denote cell line and treatment condition. b Bar charts showing the proportion of cells (%) in each cell cycle phase across cell lines and treatment conditions. c Heatmap illustrating the scaled signaling pathway activity scores of PROGENy pathways in PTX lt-res and ADA lt-res cell lines and parental cells. Red color indicates higher pathway activity, and blue color indicates lower pathway activity. d Heatmaps of gene modules for each cell line. Letters A-D indicate the gene modules identified within each cell line. Columns represent signatures (gene sets derived from cell clusters), and rows represent the genes within each module. Color indicates z-scored expression of module genes across signatures (capped at ± 3). The stacked bars below each heatmap show the percentage of cells from each condition present in each signature. e Dot plot showing shared significantly enriched pathways among the gene modules identified in panel d (JHOS2 ADA module A, Kuramochi ADA modules A, B, C, Ovsaho ADA module A, JHOS2 PTX modules B, C and COV318 PTX modules C, D). Dot size represents the gene ratio, and dot color corresponds to the adjusted p -value. f Immunoblot analysis for signaling pathways of interest in drug-naïve and lt-res models. β-actin was used as loading control. Below, a schematic representation of the two PI3K/AKT-regulated drug resistance mechanisms, with background colors matching the lt-res models where the mechanisms were active. Left (PI3K/AKT OFF): Nuclear FOXO3 represses MYC, activates DNA Damage Response (DDR, ATR/CHK1), and together with active WEE1 stalls cells in G2, leading to replication stress responses. Right (PI3K/AKT ON): AKT phosphorylates FOXO3 (pFOXO3) causing its cytoplasmic retention, induction of FOXM1/MYC, reduced WEE1 function, and RTK upregulation (e.g., ROR1), enabling mitotic escape and survival.

Article Snippet: Protein in the lysates was quantified using Pierce 660 nm Protein Assay reagent and Nanodrop (Thermo Fisher Scientific), then mixed with 4X Laemmli sample buffer (#1610747, Bio-Rad Laboratories, California, USA) and β-mercaptoethanol, separated in SDS-PAGE with 4-20% gradient gels (Bio-Rad Laboratories), and transferred to 0.45 μm nitrocellulose membranes, followed by incubation with the following primary antibodies (all used 1:1,000 dilution, if not stated otherwise): β-tubulin (#2146, Cell Signaling Technology (CST), Danvers, MA, USA), β-actin (#4967, CST), caveolin 1 (#3267, CST), FoxO3a (#99199, CST), c-MYC (#13987, CST), AKT (#2920, CST), AKT-pSer473 (#4060, CST), E-cadherin (#14472S, CST), E-cadherin (#3195, CST), ERK1/2 (#4696, CST), ERK1/2-pThr202/Tyr204 (#9101, CST), GAPDH (#G8795, Sigma-Aldrich, Burlington, MA, USA or #AHP1628, Bio-Rad Laboratories), NF-kB p65 (#6956, CST), NF-kB p65-pSer536 (#3033, CST), STAT3 (#9139, CST), STAT3-pTyr705 (#9145, CST), STAT1 (#65917, CST), STAT1-pTyr701 (#88845, CST), PTK7 (#25618, CST), ROR1 (#16540, CST, 1:500), EGFR (#4267, CST), WEE1 (#13084, CST).

Techniques: Activity Assay, Derivative Assay, Expressing, Western Blot, Protein-Protein interactions, Control

Journal: Cell Death & Disease

Article Title: ROR1-PI3K/AKT signaling drives adaptive resistance to cell cycle blockade in TP53 mutated ovarian cancer

doi: 10.1038/s41419-026-08501-x

Figure Lengend Snippet: a Box plots illustrating WEE1 and ROR1 expression (variance-stabilizing transformed, VST) in DECIDER tissue samples across treatment phases: diagnostic ( n = 158), post-NACT ( n = 69 ) , and relapse ( n = 31). Statistical comparisons between treatment phases were performed using a two-tailed Wilcoxon test, with asterisks indicating significance levels ( ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 ). b Immunoblot analysis of ROR1 in HGSOC cell lines, where β-actin was used as a loading control. c Immunoblot analysis of ROR1 and WEE1 in parental, control plasmid-transfected, and HA-tagged ROR1-plasmid transfected (ROR1 high ) JHOS2 and OVCAR8 cells. GAPDH served as a loading control. ROR1-HA indicates the HA-tag present in the plasmid. d ROR1 and WEE1 protein levels in doxycycline-induced shROR1 knockdown (KD) and parental cells. Protein expression was normalized to GAPDH and to untreated controls (set to 1). e Proliferation assay comparing parental, ROR1 high , and the ADA or PTX-res cells. The parental, ROR1 high and their ADA- or PTX-res counterparts were treated with increasing concentrations of ADA or PTX for 3 days. Significance was calculated using two-tailed Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001), n = 3 or 4 replicates. f Proliferation assay comparing ROR1 knockdown (shROR1) and parental cells, and their respective ADA-res models. The parental, shROR1 and their ADA-res counterparts were treated with increasing concentrations of ADA for 3 days. Significance was calculated using two-tailed Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001), n = 3 or 4 replicates. g Proliferation assay of JHOS2, Kuramochi, and Ovsaho parental and ADA lt-res cells treated with zilovertamab vedotin (Zilo-VT; 25 µg/mL), ADA (250 nM for JHOS2 and Ovsaho, 500 nM for Kuramochi), or their combination. Cell viability was measured after 3 days using the CTG assay. Data represent mean ± SD ( n = 3 or 4 replicates). Statistical significance was determined using two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001 ).

Article Snippet: Protein in the lysates was quantified using Pierce 660 nm Protein Assay reagent and Nanodrop (Thermo Fisher Scientific), then mixed with 4X Laemmli sample buffer (#1610747, Bio-Rad Laboratories, California, USA) and β-mercaptoethanol, separated in SDS-PAGE with 4-20% gradient gels (Bio-Rad Laboratories), and transferred to 0.45 μm nitrocellulose membranes, followed by incubation with the following primary antibodies (all used 1:1,000 dilution, if not stated otherwise): β-tubulin (#2146, Cell Signaling Technology (CST), Danvers, MA, USA), β-actin (#4967, CST), caveolin 1 (#3267, CST), FoxO3a (#99199, CST), c-MYC (#13987, CST), AKT (#2920, CST), AKT-pSer473 (#4060, CST), E-cadherin (#14472S, CST), E-cadherin (#3195, CST), ERK1/2 (#4696, CST), ERK1/2-pThr202/Tyr204 (#9101, CST), GAPDH (#G8795, Sigma-Aldrich, Burlington, MA, USA or #AHP1628, Bio-Rad Laboratories), NF-kB p65 (#6956, CST), NF-kB p65-pSer536 (#3033, CST), STAT3 (#9139, CST), STAT3-pTyr705 (#9145, CST), STAT1 (#65917, CST), STAT1-pTyr701 (#88845, CST), PTK7 (#25618, CST), ROR1 (#16540, CST, 1:500), EGFR (#4267, CST), WEE1 (#13084, CST).

Techniques: Expressing, Transformation Assay, Diagnostic Assay, Two Tailed Test, Western Blot, Control, Plasmid Preparation, Transfection, Knockdown, Proliferation Assay, CTG Assay

Journal: Cell Death & Disease

Article Title: ROR1-PI3K/AKT signaling drives adaptive resistance to cell cycle blockade in TP53 mutated ovarian cancer

doi: 10.1038/s41419-026-08501-x

Figure Lengend Snippet: a Immunoblot analysis of ROR1, WEE1 and pWEE1 expression in three primary tumor-derived HGSOC cultures (PDC#1-3). β-actin was used as a loading control. b Representative brightfield images of drug-naïve (parental) and intermediate-resistant ADA (ADA i-res) or PTX (PTX i-res) PDCs. Scale bar: 100 µm. c Proliferation assay of parental and ADA or PTX i-res PDCs treated with ADA or PTX, respectively. Data represent mean ± SD ( n = 3 or 4 replicates). Significance was determined with one-tailed Student’s t-test ( *P < 0.05, **P < 0.01, ***P < 0.001) . d Representative images of tetramethylrhodamine ethyl ester (TMRE)-stained parental, ADA res or PTX i-res PDCs organoids embedded in VitroGel® 3D matrix and cultured for 14 days with zilovertamab vedotin (25 µg/mL), ADA (100 nM/PDC1, 2000nM/PDC2 and 500 nM/PDC3) PTX (10 nM/PDC1, 50 nM/PDC2 and 10 nM/PDC3), or their combination as indicated. Grid square width: 100 µm. e Bar charts illustrating the mean volume in pixels (px) across n = 3-4 replicates, where error bars correspond to standard deviation. Significance was determined with one-tailed Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Protein in the lysates was quantified using Pierce 660 nm Protein Assay reagent and Nanodrop (Thermo Fisher Scientific), then mixed with 4X Laemmli sample buffer (#1610747, Bio-Rad Laboratories, California, USA) and β-mercaptoethanol, separated in SDS-PAGE with 4-20% gradient gels (Bio-Rad Laboratories), and transferred to 0.45 μm nitrocellulose membranes, followed by incubation with the following primary antibodies (all used 1:1,000 dilution, if not stated otherwise): β-tubulin (#2146, Cell Signaling Technology (CST), Danvers, MA, USA), β-actin (#4967, CST), caveolin 1 (#3267, CST), FoxO3a (#99199, CST), c-MYC (#13987, CST), AKT (#2920, CST), AKT-pSer473 (#4060, CST), E-cadherin (#14472S, CST), E-cadherin (#3195, CST), ERK1/2 (#4696, CST), ERK1/2-pThr202/Tyr204 (#9101, CST), GAPDH (#G8795, Sigma-Aldrich, Burlington, MA, USA or #AHP1628, Bio-Rad Laboratories), NF-kB p65 (#6956, CST), NF-kB p65-pSer536 (#3033, CST), STAT3 (#9139, CST), STAT3-pTyr705 (#9145, CST), STAT1 (#65917, CST), STAT1-pTyr701 (#88845, CST), PTK7 (#25618, CST), ROR1 (#16540, CST, 1:500), EGFR (#4267, CST), WEE1 (#13084, CST).

Techniques: Western Blot, Expressing, Derivative Assay, Control, Proliferation Assay, One-tailed Test, Staining, Cell Culture, Standard Deviation