H460 xenograft model
Although lung cancer treatment has dramatically improved within the last two decades, it is the primary cause of cancer death in both males and females worldwide. It results in over 158,000 fatalities in the U. S. yearly, as per the American Cancer Society. The H460 epithelial cell line was isolated in 1982 from a male patient with large cell lung carcinoma. It is tumorigenic in nude mice and expresses p53 mRNA. The H460 cell line is negative for neurofilament triplet protein, but stains positively for vimentin and keratin. H460 does not show significant structural DNA abnormalities. A 2016 study published in Molecular Cancer Therapeutics, investigated the efficacy of peloruside, a microtubule-stabilizing agent isolated from a New Zealand marine sponge, compared with standard anticancer agents such as paclitaxel, docetaxel, and doxorubicin in the H460 xenograft model. According to the article, peloruside shows an impressive dose-dependent single-agent antitumor activity in the H460 xenograft model. Peloruside is effective in preventing the growth of lung tumors in vivo and could be a clinical candidate for the treatment of lung cancer. The H460 cell line (human lung) is used to create the CDX (Cell Line Derived Xenograft) H460 xenograft mouse model. The H460 xenograft model enables tumor growth inhibition studies of small molecules such as gemcitabine, paclitaxel or doxorubicin.
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Basic study design
- All flasks are maintained at exponential growth prior to collection.
- The H460 cells are collected by trypsinization. Cell count and viability are then determined by trypan blue exclusion (req 98% viability). Cell suspensions are then adjusted to the density required for inoculation.
- One million cells (volume of 100 uL) of the H460 cell suspension (+ matrigel) is subcutaneously injected into the flank of the hind leg per mouse (NOD/SCID or athymic BALB/C, 10-12 weeks old).
- Injection sites are monitored for tumor establishment. Digital calipers are utilized for tumor measurement until tumors reach average sizes of 50-150 mm3.
- After sorting into treatment cohorts (randomization), the test compound is administered following the treatment schedule.
- Daily tumor measurements are recorded, along with mouse weights 3 times weekly.
- Upon reaching the predetermined tumor size limit (or 2,000 mm3), the mice are euthanized.
- As defined for termination of the study, a necropsy is performed.
- Tumors are removed and weighed, and then documented by digital imaging.
- Specified tissues are removed by performing standard gross necropsies.
- Tumors and tissues are snap frozen, stabilized in RNAlater, prepared for histology, or a nucleic acid isolation performed for genetic 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 acclimatization 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 H460 xenograft model:
- H460 Tumor Growth Delay (TGD; latency)
- H460 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)
- H460 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