U87-LUC xenograft model (Luciferase expressing U-87 cells; subcutaneous and metastatic)
Glioblastoma multiforme (GBM) is extremely aggressive brain malignancy, accounting for roughly 60 percent of all brain cancers, with a median survival after diagnosis from 12 to 15 months. U87-luc is a subline of the U-87 MG cell line, isolated in 1966 in Sweden from a 44-year-old Caucasian female patient with Stage 3 glioblastoma. The U87-luc cell line is luciferase modified U-87 MG that has been useful in a variety of bioluminescent imaging studies. A 2014 study by Marrero et al. published in Neoplasia, investigated the anti-cancer activity of serum albumin-binding doxorubicin (Doxo) and aldoxorubicin (Aldoxo) in vivo, using the U87-Luc xenograft model. These findings indicate that anti-tumor efficacy and low toxicity of Aldoxo demonstrate that it could be a potential innovative treatment for GBM patients. A 2007 study by Dinca et al. in the Journal of Neurosurgery used the U87-Luc cell line for bioluminescence imaging to monitor tumor cell growth and response to temozolomide (TMZ) chemotherapy in a rodent model. They concluded this method showed potential for predicting survival and assessing benefits from TMZ treatment which as a methylating agent is a highly toxic therapeutic regimen. A 2017 study used the U87-Luc xenograft tumors to demonstrate the efficacy of using iron oxide nanoparticles and a magnetic hyperthermia technique for a full disappearance of tumors in the mouse models.
The U87-luc cell line (human glioblastoma) is used in the creation of the CDX (Cell Line Derived Xenograft) U87-luc xenograft mouse model. The U87-luc xenograft has historical significance in the literature as a proven model for assessing angiogenesis and putative anti-angiogenic therapeutic agents.
Basic study design
- All flasks are maintained under aseptic conditions and at an exponential growth phase. The cells are then trypsinized for viability assessment via a trypan blue exclusion assay.
- One million cells (injection volume = 100 µL) are inoculated into each mouse (athymic BALB/c nude, 10 weeks old). Each injection contains a suspension of U87-luc cells plus Matrigel injected subcutaneously in the flank of a hind leg.
- As tumors become established, tumor size is continuously observed. As the average size of the tumors reach average size of 50-150 mm3, animals are sorted into treatment cohorts. Injections of the compound of interest follow the client supplied dosing schedule.
- Daily, tumors are calipered and body weights of the mice are documented. Animals are euthanized when tumor size approaches the study design tumor size limit.
- Final necropsies are performed as defined in the termination of experiment protocol. Immediately following resection, tumors are weighed and digitally imaged. All samples collected can be frozen, prepared in 10% NBF for histology or stabilized (in RNAlater)
- Animals are housed in an animal facility that is pathogen-free in accordance with an established Guide for Care and Use of Laboratory Animals, and also with the regulations of the Institutional Animal Care and Use Committee (IACUC).
CDX models are mouse xenografts used in pre-clinical therapeutic studies. However, as primary tumors proliferate they invade surrounding tissue, become circulatory, survive in circulation, implant in foreign parenchyma and proliferate in the distant tissue. This result leads to an extremely high percentage of death in cancer patients due to metastasis. Metastatic tumor mouse models are utilized to develop novel therapeutic agents that target metastasis (anti-metastatic therapeutics).
To create a metastatic model, the cell line of interest is transfected with vectors containing green fluorescent protein (GFP) or luciferase. Maintained under antibiotic selection, only cells containing the integrated vector will survive. The new cell line clones are capable of stably expressing the gene of interest and are used in metastatic mouse model studies. Although each new cell line clone may contain its own inherent difficulties, the new cell line contains the ability to track internal tumor progression via bioluminescence (luciferase fluorescence after injecting luciferin) or fluorescence (GFP). Internal orthotopic and metastatic tumor growth (not palpable) can now be measured throughout the study, enabling a researcher to gain more insight and additional data in contrast to relying on end of study tumor weight measurements.
Case Study: U87-luc Xenograft Model
An example of Altogen Labs research study utilizing a luciferase expressing cell line to monitor orthotopic tumor growth is exhibited below. The same ideology of tumor observation is incorporated in metastatic tumor models.
Luciferase expressing U87-luc cells were implanted and tumors allowed to grow. Tumor growth was monitored in a Night Owl (Berthold Technologies) imaging system 10 minutes after an intraperitoneal (IP) injection of the luciferin substrate. As seen in the example below, luciferase expression (measured as photons emitted) in the U87-luc model grants the researcher a visual image and quantifiable metric for orthotopic or metastatic tumor progression.
Figure 1. Luciferase expression in U87-luc orthotopic model. Control and implanted glioma mouse model luminescence was analyzed 10 minutes after intraperitoneal luciferin injection.
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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.
Xenograft animal models are used to assess the effectiveness of drugs against specific types of cancer. Novel 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.
Download Altogen Labs U87 Xenograft Model PowerPoint Presentation:
Following options are available for the U87-luc xenograft model:
- U87-luc Tumor Growth Delay (TGD; latency)
- U87-luc 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-luc 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