A498 Xenograft Model

 

 

 

 

 

 

 

 

 

 

 

 

 

A498 xenograft model

Renal cancer is responsible for nearly 63,000 new cases diagnosed annually with an average age at diagnosis of 64 years, as per the National Cancer Institute. The exact cause of renal cancer is unknown, and scientists often rely on preclinical research in the development of innovative kidney therapeutics. Despite the improvements in outcomes for patients with advanced RCC, the 5-year relative survival rate remains low, signaling that improved therapeutic regimens are still required. Xenograft studies have a significant impact on the development of cancer treatment. The A498 epithelial cell line was isolated from the kidney carcinoma of a 52-year-old male by D.J. Giard in 1973. The A498 cell line is tumorigenic in nude mice. A 2017 study in Cancer Science demonstrates with the A498 model line that simultaneous targeting of tumor cell growth and angiogenesis by a combination of lenvatinib and everolimus resulted in enhanced antitumor activity and led to tumor regression and that the inhibition of both VEGF and FGF signaling pathways by the combination proves its anti-angiogenic activity in the A498 RCC xenograft model. The 2008 study by Wu et al. used the A498 cell model to characterize the drug YC-1 ({3-(5′-hydroxy methyl-2′-furyl)-1-benzylindazole}) which was first discovered for its antiplatelet activity and was later shown to target HIF-1 pathways including VEGF and erythropoietin. Results from the study showed increased phosphorylation of JNK, cytotoxicity induced by YC-1 and an increase in apoptosis via mitochondria and caspase mediated pathways. In 2013 Fang et al. released a study using the A498 cells to test 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors to improve renal cell carcinoma prognosis. Data demonstrated the ability of simvastatin to reduce proliferation, motility and in vivo tumor growth; results also suggest this mechanism is via inhibition of IL-6 induced phosphorylation of STAT3 AND JAK2 (AKT/mTOR and ERK were also downregulated) that results in reduction metastasis and promotion of apoptosis. A 2011 study (Zhang et al.) used A498 cells to demonstrate that the cytokine interleukin-22 (IL-22) suppresses proliferation of renal cell carcinoma via a STAT1 pathway and cell cycle arrest. It was previously thought that the mechanism was through apoptosis induction and upregulation of inflammatory cytokines; understanding drug mechanisms is important for the development of appropriate therapies. The A498 cell line (human kidney) is used to create the CDX (Cell Line Derived Xenograft) A498 xenograft mouse model. The A498 xenograft model is currently utilized in preclinical studies involving apoptosis thru activation of JNK pathway (e.g. YC-1), inhibitors targeting hypoxia-inducible factor (HIF; e.g. ELR510444) and anti-tumor efficacy (e.g. IL-22).

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Basic study design

  1. A498 cells are cultured in aseptic conditions growing at exponential phase.  The cells are trypsinized and then viable cell counts are determined using trypan blue exclusion.  The cell suspension is adjusted such that a 100 µL injection contains 1 x 106 cells (Matrigel + A498 cells).
  2. The mice, athymic BALB/C or NOD/SCID (10 to 12 weeks) receive single, subcutaneous injection in the hind leg.  Manual palpation of injection sites aids in tumor establishment observation.  At 50-150 mm3the animals are sorted into study groups.  Injections of the test compound follows the dose schedule.
  3. Daily tumor measurements and tri-weekly body weights are recorded.  When tumor size approaches 2,000 mmor the predetermined size limit (per IACUC protocol) the mice are euthanized.
  4. Necropsies enable the collection of all tissues required for downstream analysis.  Excised tumors are weighed and documented via digital imaging.  All collected tissues are stored according to customer defined parameters: frozen, prepared for histology or nucleic acids isolated.

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A498 Xenograft Model

Altogen Labs provides an array of laboratory services using over 30 standard Cell Line Derived Xenograft (CDX) models and over 20 PDX models. Researchers investigating the role of specific proteins or gene products in regulating tumor growth can benefit from development of protein overexpression (genetically engineered to ectopically express proteins, tumor suppressors, or oncogenes) and RNAi cell lines with long term gene silencing. Altogen Labs provides quantitative gene expression analysis of mRNA expression (RT-PCR) and protein expression analysis using the WES system (ProteinSimple).

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 A498 xenograft model:

  • A498 Tumor Growth Delay (TGD; latency)
  • A498 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)
  • A498 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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A498 Xenograft Model