U-251 MG xenograft model
Glioblastoma (GBM) represents a tumor that comprises several different cell types. Various glioblastoma cancer cell lines have proven to be powerful tools to unravel molecular mechanisms related to brain tumors. An astrocytoma is a form of brain cancer that originates in astrocytes, star-shaped brain cells, and usually remains inside the brain and spinal cord not harming other organs. The U-251 MG human glioblastoma astrocytoma cell line is derived from a 75-year-old brain cancer patient. The U-251 MG cell line is used to create the CDX (Cell Line Derived Xenograft) U-251 MG xenograft mouse model. The U-251 MG xenograft model is a highly invasive pre-clinical model that contains mu-p53 and mu-PTEN. The model yields itself to therapies targeting angiogenesis (e.g. anti-VEGF, vadimezan) and telomerase inhibitors (e.g. GRN163).
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Basic study design
- U-251 MG cells for inoculation are continually grown under conditions of exponential growth. All cells are prepared for injection using trypsin-EDTA, with viability and cell concentration determined by flow cytometry Guava-PCA cell viability assay. Total cell concentrations are adjusted to 10,000 cells per uL prior to injection.
- T-cell deficient athymic nude mice strain (Foxn1nu/Foxn1+) about 9-12 week old receives a subcutaneous injection (s.c.) in the hind leg of a total of one million cells (50% matrigel plus U251 MG cell suspension). Injection sites are palpated (tri-weekly) until tumor establishment is determined (100-150 mm3).
- Animals are randomized into treatment cohorts; administration of compounds of interest are dosed according to the treatment schedule.
- Whole body weights and tumor sizes are documented. End of study is marked by tumors reaching the predetermined size limit.
- Necropsies and tissue collections are performed for termination of the study. Tumors are excised & weighed; optional imaging is available.
- Tissues are collected and 1) snap frozen in LN2, 2) prepared for histology in 10% NBF, or 3) nucleic acids isolated.
Xenograft animal models are used to assess the effectiveness of experimental compounds against specific types of cancer. All new anti-cancer medicines are tested on staged tumor growths that have been engrafted via subcutaneous or orthotopic inoculation in an immunocompromised rodent 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 (pathology, 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 facilities are 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 U-251 MG xenograft model:
- U-251 MG Tumor Growth Delay (TGD; latency)
- U-251 MG 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)
- U-251 MG 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