rituximab  (MedChemExpress)

Journal: bioRxiv

Article Title: Cancer cell-selective ecotopic expression of CD20 as an antigen enables rituximab repurposing for solid tumor immunotherapy

doi: 10.1101/2025.10.16.682764

Figure Lengend Snippet: a Schematic of the TRAP system. The CD20 coding sequence was placed under the control of an NF-κB–responsive decoy minimal promoter (DMP) containing multiple NF-κB binding sites, with WPRE downstream to enhance expression. In tumor cells with constitutive NF-κB activity, DMP drives CD20 surface expression, enabling recognition by rituximab (RTX) and Fcγ receptor III (CD16)–mediated ADCC, whereas normal cells with low NF-κB activity remain unrecognized. b qPCR analysis of RELA (p65) mRNA in tumor (HCT116, MDA-MB-231, PANC-1, MC38) and normal (MCF-12A, HL7702, GES-1, NIH-3T3) cell lines. Mean ± SD, n = 3 independent experiments. One-way ANOVA (two-sided) with Tukey’s test. c Flow-cytometry analysis of murine CD20 (mCD20) expression in MC38 and NIH-3T3 cells transfected with pAAV-mCD20 or pAAV-MCS. Representative histograms (left) and mean fluorescence intensity (MFI; right) are shown. Mean ±SD, n = 3. Unpaired two-tailed Student’s t-test. d Flow-cytometry analysis of human CD20 (hCD20) expression in tumor (HCT116, MDA-MB-231, PANC-1) and normal (MCF-12A, HL7702, GES-1) cell lines transfected with pAAV-hCD20 or pAAV-MCS. Representative histograms (top) and MFI quantification (bottom). Mean ± SD, n = 3. Unpaired two-tailed Student’s t-test.

Article Snippet: Following viral transduction, spheroids were treated with 10 μg/mL rituximab (MCE, USA) at 37 °C for 30 minutes, and then co-cultured with 5 × 104 PBMCs (pre-activated overnight with 50 U/mL IL-2; R&D Systems) per well for 24 hours.

Techniques: Sequencing, Control, Binding Assay, Expressing, Activity Assay, Flow Cytometry, Transfection, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Cancer cell-selective ecotopic expression of CD20 as an antigen enables rituximab repurposing for solid tumor immunotherapy

doi: 10.1101/2025.10.16.682764

Figure Lengend Snippet: a Representative flow-cytometry histograms of CD107a expression in NK cells (CD3 − CD56 + ; gating in Supplementary Fig. S1) after co-culture with normal (MCF-12A, HL7702, GES-1) and tumor (MDA-MB-231, PANC-1, HCT116) cells transfected with pAAV-MCS or pAAV-hCD20, with or without rituximab (RTX). Percentages of CD107a + NK cells are indicated. b Quantification of CD107a + NK cells in ( a ). Mean ±SD, n = 3 independent experiments. One-way ANOVA (two-sided) with Tukey’s test within each cell line. c Representative histograms of intracellular IFN-γ expression in NK cells under the same conditions as in ( a ). d Quantification of IFN-γ + NK cells in ( c ). Mean ± SD, n = 3. Statistical analysis as in ( b ). e ADCC assessed by LDH release after co-culture of IL-2–activated PBMCs with CD20-expressing or control cells in the presence or absence of rituximab (RTX). Mean ±SD, n = 3. Statistical analysis as in ( b ). PBMCs were pre-activated overnight with IL-2 and co-cultured with targets at an effector-to-target (E:T) ratio of 30:1 for 4 h after RTX pre-incubation.

Article Snippet: Following viral transduction, spheroids were treated with 10 μg/mL rituximab (MCE, USA) at 37 °C for 30 minutes, and then co-cultured with 5 × 104 PBMCs (pre-activated overnight with 50 U/mL IL-2; R&D Systems) per well for 24 hours.

Techniques: Flow Cytometry, Expressing, Co-Culture Assay, Transfection, Control, Cell Culture, Incubation

Journal: bioRxiv

Article Title: Cancer cell-selective ecotopic expression of CD20 as an antigen enables rituximab repurposing for solid tumor immunotherapy

doi: 10.1101/2025.10.16.682764

Figure Lengend Snippet: a Representative brightfield and fluorescence images of HCT116 spheroids treated with rAAV-MCS, rAAV-hCD20, rAAV-MCS + rituximab (RTX), or rAAV-hCD20 + RTX. Spheroids were generated under serum-free suspension conditions, transduced with rAAV vectors, and co-cultured with IL-2–activated PBMCs for 24 h. Live and dead cells were stained with Calcein AM (green) and propidium iodide (PI) (red), respectively. Scale bar, 50 μm. b Quantification of spheroid area under the indicated conditions. Bars represent mean ± SD (n = 3 independent experiments). Statistical significance was determined using one-way ANOVA (two-sided) with Tukey’s multiple-comparison test. c Quantification of Calcein (live) and PI (dead) fluorescence intensities in spheroids across treatment groups. Data represent mean ± SD (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA (two-sided) with Tukey’s test. d Representative brightfield and fluorescence images of patient-derived colorectal cancer organoids treated with rAAV-MCS, rAAV-hCD20, rAAV-MCS + RTX, or rAAV-hCD20 + RTX. Organoids were transduced with rAAV, pre-incubated with RTX, and co-cultured with PBMCs for 24 h. Organoids were pre-labeled with CellTracker™ Red CMTPX (red), and apoptotic cells were visualized using Caspase-3/7 Green (green). Scale bar, 20 μm. e Quantification of Caspase-3/7 fluorescence intensity within organoid regions of interest (ROIs) defined by CellTracker Red. Data represent mean ± SD (n = 3 independent experiments). Statistical significance was assessed using one-way ANOVA (two-sided) with Tukey’s multiple-comparison test.

Article Snippet: Following viral transduction, spheroids were treated with 10 μg/mL rituximab (MCE, USA) at 37 °C for 30 minutes, and then co-cultured with 5 × 104 PBMCs (pre-activated overnight with 50 U/mL IL-2; R&D Systems) per well for 24 hours.

Techniques: Fluorescence, Generated, Suspension, Transduction, Cell Culture, Staining, Comparison, Derivative Assay, Incubation, Labeling

Journal: bioRxiv

Article Title: Cancer cell-selective ecotopic expression of CD20 as an antigen enables rituximab repurposing for solid tumor immunotherapy

doi: 10.1101/2025.10.16.682764

Figure Lengend Snippet: a Schematic of experimental design. NCG mice were subcutaneously (s.c.) implanted with HCT116 cells and human PBMCs, followed by intravenous (i.v.) injection of rAAV-hCD20 and rituximab (RTX). b Mouse body weight monitored during treatment (n = 6). c Tumor growth curves of each treatment group (n = 6). d Images of excised tumors collected from each group at endpoint. e Quantification of tumor size (left) and tumor weight (right) (n = 6). f TUNEL staining of tumor sections and quantification of apoptotic cells. Scale bar, 100 μm. g Schematic of tumor-infiltrating lymphocyte (TIL) collection on day 4 after RTX administration. h Flow cytometry plots showing intratumoral NK cells, defined as CD3 − CD56 + cells within the viable CD45 + population, and quantification of NK-cell frequency (n = 3). Data are shown as mean ± SD. Statistical significance was analyzed by one-way ANOVA with Tukey’s test in c, e and f , and by the unpaired two-tailed Student’s t-test in h.

Article Snippet: Following viral transduction, spheroids were treated with 10 μg/mL rituximab (MCE, USA) at 37 °C for 30 minutes, and then co-cultured with 5 × 104 PBMCs (pre-activated overnight with 50 U/mL IL-2; R&D Systems) per well for 24 hours.

Techniques: Injection, TUNEL Assay, Staining, Flow Cytometry, Two Tailed Test