SJSA-1 xenograft model
Sarcoma is a cancer of bones and connective tissues that comprises approximately 20 percent of all childhood cancers, as indicated by the Sarcoma Foundation of America. Cell line derived xenografts (CDX) are based on direct implantation of cancer cell lines into different organs of nude mice and are valuable preclinical models for sarcoma research. The SJSA-1 cell line was isolated in 1982 from the primary tumor of a patient with sarcoma. The SJSA-1 cell line overexpresses MDM2 as a mechanism to restrict p53 function. Thus, inhibitors of p53-MDM2 binding that can reactivate p53 in cancer cells could be a useful treatment regimen for sarcoma patients. RG7112 is a potent MDM2 antagonist that binds MDM2, blocking its interactions with p53 in vitro. A 2013 study by Tovar et al. published in Cancer Research, investigated antitumor activity of RG7112, using the SJSA-1 xenograft model. The article indicates that RG7112 causes tumor inhibition and regression in the SJSA-1 xenograft model and is effective in the treatment of tumors expressing wild-type p53. The SJSA-1 cell line (human bone osteosarcoma) is used to create the CDX SJSA-1 xenograft mouse model. Two main benefits of the SJSA-1 xenograft model is 1) the overexpression of MDM2 that enables the study of small molecule MDM2 antagonists (e.g. nutlin-3), and 2) combination studies that significantly inhibit tumor growth such as ganitumab in combination with mTORC1 inhibitors for the treatment of sarcomas.
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Basic study design
- All flasks containing cells are maintained at a phase of exponential population growth.
- The SJSA-1 cells are collected for injection. Cell count and cell viability is determined by trypan blue. The concentration of the cell suspensions are adjusted to the required density and consists of matrigel + SJSA-1 cells.
- 1 x 10^6 cells (in 100 uL volume) of the cell suspension is injected into the hind leg of each mouse subcutaneously. NOD/SCID or athymic BALB/C mice are used that are 10-12 weeks old.
- Inoculation sites are observed for tumor establishment and digital calipers measure tumors until they reach 50-150 mm3. Animals are then sorted into treatment groups (randomization) and the in-life portion of the study begins when the compound of interest is administered to the mice. All injections follow the study treatment schedule.
- Daily measurements of the tumor and mouse whole-body weights are recorded (3 times weekly). The predetermined tumor size limit determines the end of the study (or 2,000 mm3). All animals are euthanized humanely for necropsies.
- All tumor tissue is removed and weighed. Digital imaging is available for tumors. Remaining tissues are collected and can be frozen (LN2), stabilized (RNAlater), submersed in 10% NBF 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 SJSA-1 xenograft model:
- SJSA-1 Tumor Growth Delay (TGD; latency)
- SJSA-1 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)
- SJSA-1 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