U-251MG Xenograft Model














U-251 MG xenograft model

An astrocytoma is a form of brain cancer that originates in astrocytes, star-shaped brain cells, and usually remains as a low-grade or benign growth inside the brain and spinal cord not harming other organs. Glioblastoma multiforme (GBM) the most aggressive tumor type, also known as grade IV astrocytoma) and is comprised of several different cell types. Various glioblastoma cancer cell lines have proven to be powerful tools to unravel molecular mechanisms related to brain tumors. The U-251 MG human cell line is derived from a 75-year-old brain cancer patient diagnosed with glioblastoma astrocytoma. Molecular Cancer Therapeutics published a 2017 study (Hilliard et al.) that demonstrated the efficacy of 15α-methoxypuupehenol, an anticancer drug that impacts cell viability of glioblastoma (using the U251 MG line) and breast cancer cells more than normal cells. Hashizume et al. (2010) demonstrated the benefits of using bioluminescence imaging for pre-clinical monitoring and testing of therapeutic regimens for patients with brainstem tumors; one of the xenograft brain tumor models used were U251 MG treated with temozolomide. In 2004, Ozawa et al. published results using U251 MG xenografts to demonstrate the efficacy of GRN163, a telomerase inhibitor, against growth and proliferation of malignant gliomas. It is important to note that the original U-251 cells were established in the 1960s and the modern subclone line (denoted as U-251 MG) have variations in genotype, phenotype and growth characteristics (Torsvik et al., 2014).  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

  1. 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 µL prior to injection.
  2. 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).
  3. Animals are randomized into treatment cohorts; administration of compounds of interest are dosed according to the treatment schedule.
  4. Whole body weights and tumor sizes are documented.  End of study is marked by tumors reaching the predetermined size limit.
  5. Necropsies and tissue collections are performed for termination of the study.  Tumors are excised & weighed; optional imaging is available.

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U-251 MG Xenograft Model

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).

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).

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

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U-251 MG Xenograft Model