human crc cell lines (t84, lovo, sw480, hct116, hct8)  (Procell Inc)

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: mRNA and protein expression levels of selected candidates in human CRC cell lines and patients’ samples (A–F) mRNA expression levels of the six selected candidates for inclusion in the hybrid TSP. Quantitative PCR (qPCR) analysis was performed using specific primers for each candidate gene (vWA2, A33, FSCN1, NQO1, PDL1, and AQP5) in four human colorectal cancer cell lines (T84, HCT116, HT29, and LoVo) and in normal human colonic epithelial cells (CCD841). qPCR data are presented as relative quantification (RQ = 2 −ΔΔCt ). Statistical analysis was performed using one-way ANOVA. Data represent the mean of three independent experiments. (G) Immunohistochemical analysis of gpA33 and vWA2 expression was performed on paraffin-embedded primary and metastatic CRC tissue samples. (a and b) Representative images of primary tumor sections stained with an anti-gpA33 antibody and developed as described in the . (c and d) Representative images of primary tumor sections stained with an anti-vWA2 antibody under the same conditions. (e–g) Representative images of uterine, cutaneous, and liver metastases, respectively, stained with the anti-gpA33 antibody. (h–j) Corresponding metastatic tissue sections (uterus, skin, and liver, respectively) stained with an anti-vWA2 antibody. All images were acquired at an original magnification of 200×. Scale bar: 50 μm.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Immunohistochemical staining, Staining

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: The hybrid TSP vWA33 exhibits transcriptional activity across all human CRC cell lines tested (A and C) Identification of transcription factor (TF) binding motifs within the vWA2 (A) and A33 (C) promoter fragments. A comprehensive in silico analysis was performed on the six candidate promoter fragments (only vWA2 and A33 are shown). TFs and their positions were identified using a combination of PROMO, MATCH, and MEME Suite – FIMO and filtered for consensus hits across all three tools. ToppGene was used to rank the most statistically significant TFs, as described in the . (B) Transcriptional activity of the full-length vWA2 promoter (636 bp) vs. the shorter, transcriptionally silent 306 bp fragment lacking RNA polymerase binding motifs. Both fragments were cloned into a promoter-less luciferase vector and transfected into four CRC cell lines. Luciferase activity was used as a readout and is presented as a fold induction relative to the pGL3-Basic control. (D) Schematic representation of the vWA33 hybrid promoter, composed of the vWA2 fragment (−325 to −19 bp) followed by the A33 promoter fragment (−105 to +307 bp). Annotated TF binding sites are indicated. (E) Comparative transcriptional activity of the A33 promoter (Pr A33), vWA2 promoter (Pr vWA2), and vWA33 hybrid promoter (Pr vWA33) in four CRC cell lines, using luciferase as the reporter. The data shown represent one representative experiment out of at least three independent replicates. One-way ANOVA was used for statistical analysis.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Activity Assay, Binding Assay, In Silico, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Control

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: AR2015 lytic activity in vitro compared to single TSP-driven OAds (A–I) In vitro lytic activity of the OAds AR2015, AV22EL, and AV636, as well as Ad5WT (positive control), in CRC cell lines LoVo, T84, HT29, and HCT116, normal human colonic epithelial cells (CCD841), human fetal lung fibroblasts (WI-38), human microendothelial cells (HMEC-1), and human melanoma cells (A375 and SB2). Cells (1 × 10 4 ) were seeded in 24-well plates and infected 24 h later with increasing multiplicities of infection (MOIs: 0–100). After 6 days, cell viability was assessed using the MTS assay and expressed as mean ± SD ( n = 3), with the viability of uninfected control cells set to 100%. Two-way ANOVA was performed for statistical analysis, followed by Dunnett’s test (vs. Ad(F5)WT). (J) Comparative replication kinetics of AR2015 and AV22EL in CRC cell lines (LoVo, T84, HT29, and HCT116) and normal CCD841 cells. Cells were infected at an MOI of 100, and samples were collected at 5 h (baseline) and 72 h post-infection. Viral replication was quantified by qPCR targeting the adenoviral E4 gene. Data are presented as a fold increase in E4 levels (72 vs. 5 h). (K) Expression of adenoviral E1A protein in LoVo cells at 8 and 24 h post-infection with AV22EL, AR2015, or Ad5WT. Protein levels were assessed by western blot, with β-actin used as a loading control.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Activity Assay, In Vitro, Positive Control, Infection, MTS Assay, Control, Expressing, Western Blot

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: Systemically administered AR2015 exhibits superior efficacy against established liver metastases compared to single TSP-driven OAds (A) Schematic diagram of the experimental protocol. Nude mice were injected intrasplenically with 2 × 10 6 HCT116 CRC cells, and 15 days later, when liver metastases were established, mice were systemically treated with the indicated OAds (5 × 10 9 viral particles/mouse) with or without a suboptimal dose of 5-FU (30 mg/kg). (B) Kaplan-Meier survival plot of mice bearing HCT116 liver metastases treated with PBS ( n = 6), 5-FU ( n = 6), AV22EL ( n = 3), AR2015 ( n = 7), or AR2015 + 5-FU ( n = 6). Statistical significance was calculated using the log rank test. Mice were euthanized when the tumor burden reached 2 cm 3 , in accordance with ethical guidelines. (C and D) Presence (C) and quantification of area (D) of metastatic liver foci from the experimental groups in (B). (E and F) In an independent experiment, nude mice were injected intrasplenically with 2 × 10 6 HCT116 cells, and 15 days later, they were treated with PBS ( n = 6), AV636 ( n = 6), or AR2015 ( n = 6). Mice were euthanized at the endpoint and analyzed for the presence (E) and area (F) of hepatic metastatic lesions. One-way ANOVA was used for statistical analysis of metastatic burden.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Injection

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: Systemically delivered AR2015 demonstrates enhanced in vivo efficacy in combination with oxaliplatin and strong in vitro lytic activity in patient-derived xenograft CRC cells (A and B) Nude mice were injected intrasplenically with 2 × 10 6 LoVo CRC cells. 15 days post-injection, mice were treated with PBS ( n = 4), oxaliplatin (OXA) ( n = 3), AR2015 ( n = 5), or AR2015 + OXA ( n = 5). At the experimental endpoint, mice were euthanized and assessed for the presence of liver metastases (A) and metastatic lesion area (B). (C and F) qPCR analysis of vWA2, A33, CAR, and DSG-2 mRNA expression in RC-01 and RC-04 patient-derived xenograft (PDX)-derived CRC cells. Expression is presented as relative quantification (RQ = 2 −ΔΔCt ). (D and G) In vitro lytic activity of AR2015 and AR2015(F5/3) in RC-01 and RC-04 cells. Cells (1 × 10 4 per well) were seeded in 24-well plates and infected 24 h later with increasing MOIs (0–100). Cell viability was assessed after 6 days using the MTS assay. (E and H) Replication capacity of AR2015 and AR2015(F5/3) in RC-01 and RC-04 cells. Cells (1 × 10 4 ) were infected at an MOI of 100, and samples were collected at 5 (baseline) and 72 h. Viral replication was assessed by qPCR targeting the adenoviral E4 gene. Data are presented as a fold increase in E4 levels (72 vs. 5 h). One-way ANOVA was used for statistical analysis. p values are indicated where applicable.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: In Vivo, In Vitro, Activity Assay, Derivative Assay, Injection, Expressing, Quantitative Proteomics, Infection, MTS Assay