SNB-19 xenograft model
The SNB-19 cell line (human brain; glioblastoma) is used to create the CDX (Cell Line Derived Xenograft) SNB-19 xenograft mouse model. The SNB-19 xenograft model is a high-grade glioma model that is a high expressor of uPAR and the cysteine protease cathepsin B, both of which are implicated in neovascularization. The SNB-19 model lends itself to studying anti-angionesis therapeutics (e.g. transcriptional supression via siRNAs against cathepsin-B or uPAR, anti-VEGF).
Basic study design
1. SNB-19 cells used for injection are maintained under conditions of exponential growth prior to injection
2. SNB-19 cells are prepared for injection by trypsinization and viable cell counts are determined using trypan blue exclusion (98% cell viability required). Cell suspension adjusted to appropriate density
3. Each mouse (athymic BALB/C or NOD/SCID, 10-12 w.o.) receive a subcutaneous injection in the flank of the hind leg of one million cells in a volume of 100 microliters of matrigel-SNB-19 cell suspension
4. The injection sites are palpated three times weekly until tumors are established. Tumors are then measured using digital calipers until they reach an average size of 50-150 mm3.
5. Animals are randomized into treatment cohorts and administration of compound of interest is performed according to the treatment schedule
6. Tumors are measured daily and mouse weights recorded 3 times weekly
7. Animals are euthanized when tumor size reaches 2,000 sq millimeters or predetermined size limit
8. Necropsy and tissue collection are performed as defined for termination of experiment
9. Tumors are excised, weighed and documented by digital imaging
10. Standard gross necropsies are performed and tissues collected for downstream analysis
11. Tumors and tissues can be snap frozen in LN2 and prepared for histology or gene expression analysis
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 SNB-19 xenograft model:
- SNB-19 Tumor Growth Delay (TGD; latency)
- SNB-19 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)
- SNB-19 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