A20 Xenograft Model













A20 xenograft model (subcutaneous and metastatic)

Chronic lymphocytic leukemia (CLL) is caused by a defect in apoptosis that involves the overexpression of antiapoptotic B-cell lymphoma-2 (BCL-2) family members BCL-2. Preclinical xenograft models are instrumental for investigating novel treatment regimens. The A20 cell line was isolated from a spontaneously developed tumor in a BALB/c mouse in 1979. A 2010 study by Samuel et al. published in Molecular Therapy examined the combination treatment of vesicular stomatitis virus (VSV) with obatoclax (GX15-070)-a small-molecule BCL-2 inhibitor using the A20 lymphoma xenograft model. The study indicated that the combination treatment of obatoclax and VSV significantly reduces tumor growth in the A20 xenograft model and could be a promising strategy to overcome apoptosis resistance in CLL. A 2012 review by Donnou et al. in Advances in Hematology highlighted the A20 model and summarized the different effects of cell implantation into varies organs/microenvironments and resulting antigen/T-cell infiltration. The authors explored the advantages and limitations of various conditions with this model for optimal use as research models. A 2016 study (Bascuas et al.) published in the Journal of Translational Medicine used the A20 cell line to establish an appropriate pre-clinical non-Hodgkin lymphoma mouse model to study minimal disease resistance (MRD). This is an important issue to address as many pre-clinical mouse models are under high tumor burden whereas patients are likely under remission when treated with immunotherapies as this is a treatment following chemotherapy. A 2016 study in Clinical Cancer Research (Ren et al.) used the A20 xenograft model to investigate the limits of anti-CD20 therapy for B-cell lymphoma. They looked at immune response-related resistance in this type of treatment and how T-cells are involved in mediating CD-20 therapy. Results demonstrated that anti-CD20 treatment is mediated by CD8+ T-cells and that resistance can be overcome by CTLA-4 blockage. The A20 cell line (mouse lymphoma) is used for the CDX (Cell Line Derived Xenograft) A20 xenograft mouse model. The A20 xenograft model can be utilized as a preclinical model to study the immunotherapy combinatorial effects of small molecules (e.g. ibrutinib) and PD-L1 inhibitors.

Download Altogen Labs A20 Xenograft Model PowerPoint Presentation: PPT2

Basic study design

  1. A20 cells used for injection cultured under conditions of exponential growth prior to injection.
  2. A20 cells are prepared for injection and then viable cell counts are determined using the trypan blue exclusion assay (98% cell viability required). The cell suspension is then adjusted to the appropriate density.
  3. The mice (athymic BALB/C or NOD/SCID, 10-12 weeks old) receive subcutaneous injections in the flank of one hind leg containing one million viable A20 cells suspended in a volume of 100 µL of Matrigel.
  4. The injection sites are repeatedly palpated multiple times weekly until tumors are established. Tumors are then measured with digital calipers an average size of 50-150 mm3is reached.
  5. Animals are randomized into the specified treatment cohorts and administration of the test compound is performed according to the customer’s treatment schedule.
  6. Daily measurements and mouse weights are recorded 3 times weekly.
  7. When tumor size reaches 2,000 mm3 or the predetermined size limit, the animals are euthanized.
  8. Necropsy and tissue collection is performed as defined in experimental design.
  9. Tumors are excised, weighed and also documented by digital imaging.
  10. Standard gross necropsies are then performed and tissues are collected for downstream analysis.
  11. Tumors and tissues can be stabilized in RNAlater, snap frozen in LN2 or prepared for histology or gene expression analysis.

Metastatic Model

CDX models are mouse xenografts used in pre-clinical therapeutic studies.  However, as primary tumors proliferate they invade surrounding tissue, become circulatory, survive in circulation, implant in foreign parenchyma and proliferate in the distant tissue.  This result leads to an extremely high percentage of death in cancer patients due to metastasis.  Metastatic tumor mouse models are utilized to develop novel therapeutic agents that target metastasis (anti-metastatic therapeutics).

To create a metastatic model, the cell line of interest is transfected with vectors containing green fluorescent protein (GFP) or luciferase.  Maintained under antibiotic selection, only cells containing the integrated vector will survive.  The new cell line clones are capable of stably expressing the gene of interest and are used in metastatic mouse model studies.  Although each new cell line clone may contain its own inherent difficulties, the new cell line contains the ability to track internal tumor progression via bioluminescence (luciferase fluorescence after injecting luciferin) or fluorescence (GFP).  Internal orthotopic and metastatic tumor growth (not palpable) can now be measured throughout the study, enabling a researcher to gain more insight and additional data in contrast to relying on end of study tumor weight measurements.

Case Study: U87-luc Xenograft Model

An example of Altogen Labs utilizing a luciferase expressing cell line to monitor orthotopic tumor growth is exhibited below.  The same ideology of tumor observation is incorporated in metastatic tumor models.

Luciferase expressing U87-luc cells were implanted and tumors allowed to grow.  Tumor growth was monitored in a Night Owl (Berthold Technologies) imaging system 10 minutes after an intraperitoneal (IP) injection of the luciferin substrate.  As seen in the example below, luciferase expression (measured as photons emitted) in the U87-luc model grants the researcher a visual image and quantifiable metric for orthotopic or metastatic tumor progression.

Figure 1. Luciferase expression in U87-luc orthotopic model.  Control and implanted glioma mouse model fluorescence was analyzed 10 minutes after intraperitoneal luciferin injection.

View full details of the case study by clicking here.

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A20 Xenograft Model

Xenograft animal models are used to assess the effectiveness of drugs against specific types of cancer. New medicines are tested on staged tumor growths that have been engrafted via subcutaneous or orthotopic inoculation in an immunocompromised mouse or rat model. All clinically approved anti-cancer agents have been evaluated with conventional preclinical in vivo models. Xenograft studies can be highly complex, starting with the selection of the appropriate animal model, choice of tumorigenic cell line, administration method, dosing, analysis of tumor growth rates and tumor analysis (histology, mRNA and protein expression levels).

The dosing of the experimental compound of interest is initiated, for a staged study, when the mean tumor size reaches a specified volume (typically 50-100 mm3). In an unstaged study, the dosing of the compound of interest is initiated immediately after xenografting. Mice are dosed once or twice a day for 28 days (or other desired study duration) via the chosen route of administration. Tumor volume (mm3) is calculated via the “(W x W x L) / 2” formula, where W is tumor width and L is tumor length.

Animal handling and maintenance at the Altogen Labs facility is IACUC-regulated and GLP-compliant. Following acclimation to the vivarium environment, mice are sorted according to body mass. The animals are examined daily for tumor appearance and clinical signs. We provide detailed experimental procedures, health reports and data (all-inclusive report is provided to the client that includes methods, results, discussion and raw data along with statistical analysis). Additional services available include collection of tissue, histology, isolation of total protein or RNA and analysis of gene expression. Our animal facilities have the flexibility to use specialized food or water systems for inducible gene expression systems.

Following options are available for the A20 xenograft model:

  • A20 Tumor Growth Delay (TGD; latency)
  • A20 Tumor Growth Inhibition (TGI)
  • Dosing frequency and duration of dose administration
  • Dosing route (intravenous, intratracheal, continuous infusion, intraperitoneal, intratumoral, oral gavage, topical, intramuscular, subcutaneous, intranasal, using cutting-edge micro-injection techniques and pump-controlled IV injection)
  • A20 tumor immunohistochemistry
  • Alternative cell engraftment sites (orthotopic transplantation, tail vein injection and left ventricular injection for metastasis studies, injection into the mammary fat pad, intraperitoneal injection)
  • Blood chemistry analysis
  • Toxicity and survival (optional: performing a broad health observation program)
  • Gross necropsies and histopathology
  • Positive control group employing cyclophosphamide, at a dosage of 50 mg/kg administered by intramuscular injection to the control group daily for the study duration
  • Lipid distribution and metabolic assays
  • Imaging studies: Fluorescence-based whole body imaging, MRI

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A20 Xenograft Model