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 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 in the A498 xenograft model. Also, the inhibition of both VEGF and FGF signaling pathways by the combination proves its anti-angiogenic activity in the A498 RCC xenograft model. 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
- A498 cells are cultured in asceptic 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 uL injection contains 1 x 10^6 cells (matrigel + A498 cells).
- 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 mm3 the animals are sorted into study groups. Injections of the test compound follows the dose schedule.
- Daily tumor measurements and tri-weekly body weights are recorded. When tumor size approaches reaches 2,000 mm2 or the predetermined size limit the mice are euthanized.
- 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.
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).
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).
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 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