human pdac cell lines mia paca 2  (ATCC)

Journal: Molecular Therapy Oncology

Article Title: Mechanistic and functional characterization of ABTB2 as a novel target for pancreatic cancer therapy

doi: 10.1016/j.omton.2025.201080

Figure Lengend Snippet: Gain- and loss-of-function experiments demonstrate the crucial role of ABTB2 in PDAC development in vitro (A) Establishment of stable Panc02 cells with ABTB2 knockout (KO) or overexpression (OE). A schematic illustrates the process of creating stable Panc02 cells with either ABTB2-OE or ABTB2-KO. Plasmids carrying ABTB2 or gRNA/Cas9 were co-transfected into HEK-293T cells with helper vectors psPAX2 and pMD2G to package lentivirus particles. Post overnight culture, the supernatant containing lentiviral particles was used to infect Panc02 cells. After 72 h, cells were subcultured at low density into 96 well-plates. Monoclonal cells that stably express ABTB2 or gRNA/Cas9 were selected using antibiotics for subsequent expansion. (B) Validation of ABTB2 KO and OE. The transduction efficiency of recombinant lentivirus in Panc02 cells was monitored by GFP expression under a microscope (upper left). ABTB2 KO or OE at the mRNA and protein levels was confirmed by RT-qPCR (right) and western blotting (lower left). (C) Impact of ABTB2 KO and OE on Panc02 cell colony formation. Panc02 cells with ABTB2-OE or ABTB2-KO were seeded into 6-well plates at a density of 200 cells per well and cultured for 7 or 10 days, then the cells were fixed with 6% v/v glutaraldehyde and stained with 0.5% w/v crystal violet. The colonies of Panc02 cells with ABTB2-OE (upper left) and Panc02-KO (lower left) were imaged and counted. (D) Impact of ABTB2 KO and OE on Panc02 cell migration: Panc02 cells with ABTB2-OE or ABTB2-KO were seeded into 24-well plate with a wound healing insert which was removed the second day. The wound closure was imaged under microscopy for assessing cell migration with ABTB2 OE (upper left) and ABTB2 KO (lower left); the accumulated gap diameters were counted (right). (E) Impact of ABTB2 KO and OE on Panc02 cell viability. Panc02 cells with ABTB2-OE or ABTB2-KO were seeded and cultured in 96-well plate. 16 h later, 10 μL of MTT labeling reagent was added to each well and incubated for 3 h, after which the absorbance of purple formazan crystals was measured. Cell viability with ABTB2-OE (left) and ABTB2-KO (right) was calculated as a percentage relative to the respective control. All cell culture experiments were conducted in at least three replicates ( n = 3). Statistical significance is denoted as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human PDAC cell lines Panc-1 (RRID:CVCL_0480) and Mia-paca-2 (RRID:CVCL_0428) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, Knock-Out, Over Expression, Transfection, Stable Transfection, Biomarker Discovery, Transduction, Recombinant, Expressing, Microscopy, Quantitative RT-PCR, Western Blot, Cell Culture, Staining, Migration, Labeling, Incubation, Control

Journal: Molecular Therapy Oncology

Article Title: Mechanistic and functional characterization of ABTB2 as a novel target for pancreatic cancer therapy

doi: 10.1016/j.omton.2025.201080

Figure Lengend Snippet: Impact of ABTB2 ectopic expression or depletion on PDAC growth in vivo (A) Establishment of orthotopic PDAC-bearing mice: A schematic illustrates the preparation of orthotopic PDAC and endpoint euthanasia. Wild-type C57BL/6 mice received intra-pancreatic injections of Panc02 cells with ABTB2-OE (5 × 10 4 per mouse) or ABTB2-KO (2.5 × 10 4 per mouse), with corresponding vehicle as controls. Post 35 days, mice were euthanized, and tumors were harvested for further analysis. (B) Tumor size and weight measurement: Macroscopic photographs of tumors from each mouse are shown on the left, with cumulative tumor weights displayed on the right. The date indicated an inverse relationship between ABTB2 expression and tumor growth. (C) Immunohistochemistry (IHC) for detection of ABTB2 expression: IHC was performed to validate ABTB2 expression levels in the tumors. Tumors induced by Panc02 cells with ABTB2-OE or ABTB2-KO, along with their corresponding vehicles, were processed, sectioned, and stained with an ABTB2 antibody to assess ABTB2 expression levels. (D) Kaplan-Meier survival analysis of tumor-bearing mice: viable mice with tumors induced by ABTB2-OE or ABTB2-KO cells were documented daily. Survival rates in each group were calculated over time to construct the Kaplan-Meier Curve. (E) IHC for detection of cell growth and apoptosis markers: Tumors induced with ABTB2-OE or ABTB2-KO cells were processed, sectioned, and stained with antibodies against Bcl2, ki67, and cleaved capase-3. The area of positive staining was quantified using ImageJ software. ( n = 10). Statistical significance is indicated by ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human PDAC cell lines Panc-1 (RRID:CVCL_0480) and Mia-paca-2 (RRID:CVCL_0428) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, In Vivo, Immunohistochemistry, Staining, Construct, Software

Journal: Molecular Therapy Oncology

Article Title: Mechanistic and functional characterization of ABTB2 as a novel target for pancreatic cancer therapy

doi: 10.1016/j.omton.2025.201080

Figure Lengend Snippet: ABTB2-recombinant AAV2 therapeutically suppresses orthotopic PDAC tumor growth in mice (A) Schematic diagrams depicting the preparation of ABTB2-recombinant AAV2 (AAV2-ABTB2) and outlining the administration protocol. Recombinant AAV2-ABTB2 was prepared through cDNA subcloning, amplification, CsCl ultracentrifugation, and qPCR titration, achieving a final concentration of 2 × 10 13 vg/ml. Orthotopic PDAC-bearing mice were established in wild-type C57BL/6 mice by intra-pancreatic injection of the indicated mouse PDAC cells at 5 × 10 4 cells/mouse. Subsequently, the mice were randomly divided into three groups and received injections of recombinant AAV2-ABTB2 at 10 11 vg/mouse. AAV2 vector and PBS were used as controls. All mice were euthanized on day 35 post-cell inoculation. (B to D) Recombinant AAV2-ABTB2 injection inhibits the growth of PDAC tumors induced by mouse UN-KPC-961 cells (B), human Panc-1 cells (C), and mouse Panc02 cells (D). On day 35 post-cell inoculation, all mice were euthanized. Macroscopic tumors along with their corresponding weights for each mouse were meticulously recorded. (E) Recombinant AAV2-ABTB2 treatment leads to enhanced ABTB2 expression. Western blotting was used to assess ABTB2 expression levels in Panc02-induced tumors with or without AAV2-ABTB2 treatment. ( n = 6). Statistical significance is indicated by ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human PDAC cell lines Panc-1 (RRID:CVCL_0480) and Mia-paca-2 (RRID:CVCL_0428) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Recombinant, Subcloning, Amplification, Titration, Concentration Assay, Injection, Plasmid Preparation, Expressing, Western Blot

Journal: Molecular Therapy Oncology

Article Title: Mechanistic and functional characterization of ABTB2 as a novel target for pancreatic cancer therapy

doi: 10.1016/j.omton.2025.201080

Figure Lengend Snippet: ABTB2-recombinant AAV2 inhibits human PDAC tumor growth in a patient-derived xenograft model (A) Schematic diagram illustrates ABTB2-recombinant AAV2 preparation (what difference with Figure 3 or overlap), PDX model establishment, and AAV2-ABTB2 treatment. The ABTB2-recombinant virus was engineered using AAV2, as depicted in A. For the creation of PDX mice, each NSG mouse received a subcutaneous injection of human PDAC tumor cells from two patients (MPC02 or MPC25) at 1 ×10 6 cells per mouse. The resulting tumors were serially transplanted into subsequent NSG mice. In this study, fourth-generation PDX mice harboring human patient MPC02 tumors (4PDX-MPCO2) and fifth-generation PDX mice with human tumors derived from patient MPC25 (5PDX-MPC25) were treated with AAV2-ABTB2 on day 8. PBS was used as the control. 4PDX-MPC02 mice were euthanized on day 41 and 5PDX-MPC25 mice on day 33. (B and C) AAV2-ABTB2 treatment led to a reduction in tumor volume. The sizes of human MPC02 (B) and MPC25 tumors (C) throughout the duration of the experiment were measured at the indicated intervals using a Vernier Caliper. The results were graphically represented to depict changes in tumor volume. (D and E) Recombinant AAV2-ABTB2 treatment led to the reduction in tumor weight. On day 33, mice in each group were euthanized to harvest tumors. Tumor weights from both 4PDX-MPCO2 (D) and 5PDX-MPC25 (E) mice were decreased following treatments. (F) AAV2-ABTB2 treatment increased mRNA expression of ABTB2 in PDX tumors. RNA was isolated from tumors of treated and untreated mice, and ABTB2 mRNA expression was determined by RT-qPCR. (G) Recombinant AAV2-ABTB2 treatment inhibited tumor growth, induced apoptosis, and suppressed vascularization. A portion of tumors was sectioned for IHC. Results indicated that AAV2-ABTB2 reduced Ki67 and CD31 expression and increased cleaved caspase-3 production. n = 6. Significance levels are denoted as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human PDAC cell lines Panc-1 (RRID:CVCL_0480) and Mia-paca-2 (RRID:CVCL_0428) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Recombinant, Derivative Assay, Virus, Injection, Control, Expressing, Isolation, Quantitative RT-PCR

Journal: Molecular Therapy Oncology

Article Title: Mechanistic and functional characterization of ABTB2 as a novel target for pancreatic cancer therapy

doi: 10.1016/j.omton.2025.201080

Figure Lengend Snippet: LNP-mRNA-ABTB2 in combination with 5-FU induced synergistic therapeutic suppression of PDAC tumors (A) Construction of LNP-encapsulated ABTB2-mRNA. Schematics show ABTB2-mRNAs encapsulated in LNPs, named LNP-mRNA-ABTB2. (B) Transduction efficacy of LNP-mRNA-ABTB2 in KRAS G12D cell. KRAS G12D cells was seeded into 6-well plates at a dose of 5 × 10 5 /well and incubated at 37°C with 5% CO 2 and saturated humidity. After overnight culture, LNP-mRNA-ABTB2 was added to each well at varying doses from 100 ng to 2 μg. after 48 h, cells were harvested to isolate total RNA, which was reversed transcribed into cDNA to evaluate ABTB2 mRNA expression levels by RT-qPCR. Stable KRAS G12D cells with ABTB2-OE were used as a positive control. (C) LNP-mRNA-ABTB2 transduction led to ABTB2 production. As described in (B), KRAS G12D cells transduced with LNP-mRNA-ABTB2 for 48 h showed increased ABTB2 production, which was detected by western blotting. (D) LNP-mRNA-ABTB2 treatment design. KRAS G12D cells suspended in 15% Matrigel was subcutaneously injected into the left flank of 8-week-old C57BL/6 mice. From day 5, mice received injections of 5 μg LNP-mRNA-ABTB2 and 25 mg/Kg 5-FU every 3 days for three treatments. Mice receiving monotherapy with LNP, 5FU, or PBS were used as controls. (E) LNP-mRNA-ABTB2 and 5-FU alone led to a reduction in tumor volumes with a great effect seen in their combination. From the first treatment, the longest (L) and shortest (W) diameters of the subcutaneous implants were regularly measured. Tumor volume was calculated using the formula: L/2 × (W 2 ). (F and G). LNP-mRNA-ABTB2 and 5-FU alone led to a reduction in tumor weights, with an increased effect seen in their combination. (G) Twenty-two days after cell inoculation, tumors were isolated from mice receiving the indicated treatments, and tumor weights were measured. All cell culture experiments were conducted in at least four replicates ( n = 4), and animal experiment in three replicates ( n = 3). Statistical significance is denoted as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human PDAC cell lines Panc-1 (RRID:CVCL_0480) and Mia-paca-2 (RRID:CVCL_0428) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transduction, Incubation, Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Injection, Isolation, Cell Culture