U-87 MG xenograft model
Malignant glioblastoma is the most common type of primary brain tumors in adults that is incurable in most cases. According to the American Cancer Society, glioblastoma is one of the deadliest types of cancer that has a median survival time of fewer than 15 months. This form of cancer usually has a high recurrence rate with the 5-year survival rate of nearly 3% to 5% and is very challenging to eradicate. The U-87 MG epithelial cell line was isolated in 1966 at Uppsala University in Sweden from a 44-year-old Caucasian female patient with Stage 3 glioblastoma. It is an extensively studied cell line that has been analyzed in numerous publications for over four decades. In a 2010 study, published in PLOS Genetics, the U-87 MG cell line was evaluated and discovered to have a large number of chromosomal abnormalities. The modal chromosome number of 44 occurs in 48% of cells, only one copy of the normal X chromosome is present and N1, N6 and N9 are absent entirely. Additionally, a homozygous mutation in PTEN was identified. Kiaris et al. (2000) published a study in Clinical Cancer Research using the U-87 line to investigate potential inhibiters of somatostatin (SST) receptors in brain tumors. They identified the compound AN-238, an analogue derivative of doxorubicin, as a treatment that successfully inhibited growth of U-87 glioblastoma in nude mice. A 2008 study by Tseng et al. used a U-87 subcutaneous xenograft model in nude mice to test the preclinical efficacy of using microPET scanning to monitor 18F-FDG (a proliferation tracer) accumulation in tumors as a method of early therapy monitoring. They found that the c-Met inhibitor CE-355621 treated tumors exhibited lower levels of 18F-FDG, indicating a successful decrease in tumor growth and supporting the use of this method for human clinical trials.
In xenotransplantation, cancer cells are transplanted into immunodeficient mice to assess the response of cells to novel drugs and research the progress of the disease. The U-87 MG cell line is used to create the CDX (Cell Line Derived Xenograft) U87 xenograft mouse model. The U-87 MG xenograft model contains wild-type p53 status. The U87 xenograft has historical significance as a proven model when assessing glioblastoma angiogenesis and anti-angiogenic therapeutic agents.
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Basic study design
- Exponential growth of cells is maintained for U87 cells prior to injection. Trypsin-EDTA is used to collect cells, which is followed by trypan blue (cell viability) cell counting. The cell concentration is then diluted to 10,000 cells/µL.
- Twelve (12) weeks old athymic BALB/c nude mice receive an s.c. injection. All inoculations are made into the hind leg and contain Matrigel plus U-87 MG cells. Tumor measurements are made using calipers. Upon averages of 120-150 mm3, the animals are grouped into the number of treatment cohorts needed for the study. In-life dosing of all test compounds are performed following the client supplied schedule.
- Continual tumor measurements (e.g. daily) and whole body weights are logged. At the end of the study, tumors are weighed and then documented (optional digital imaging).
- In preparation for future downstream analysis, tumors/tissues are stored according to client instructions, including options such as samples being stabilized (RNAlater), snap frozen or prepared for histology (10% NBF).
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 (qPCR) and protein expression analysis using the WES system (ProteinSimple).
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).
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). 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 U87 xenograft model:
- U87 Tumor Growth Delay (TGD; latency)
- U87 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)
- U87 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