Hepa 1-6 xenograft model
Hepatocellular carcinoma (HCC) is a significant cause of cancer mortality worldwide. Liver cancer is often associated with a poor prognosis, and the exact mechanisms leading to its development are still unclear. The Hepa 1-6 tumorigenic epithelial cell line is derived from liver cells of the BW7756 mouse (Mus musculus) with hepatoma. Hepa 1-6 has been negatively tested for mousepox and is widely used in mammalian liver tissue functions research. A 2013 study in PLoS demonstrates that Hydroxysteroid sulfotransferase 2B1b SULT2B1 is overexpressed in the human hepatocarcinoma tumorous tissues compared to their adjacent tissues, which promotes the growth of the mouse hepatocarcinoma cell line Hepa1-6. Also, knock-down of SULT2B1b inhibits cell growth and cyclinB1 levels in human hepatocarcinoma cells and suppresses xenograft growth in vivo. The Hepa 1-6 cell line (mouse liver) is used to create the CDX (Cell Line Derived Xenograft) Hepa 1-6 xenograft mouse model. The Hepa 1-6 xenograft model enables the study of tumor growth inhibition of therapeutic agents such as expression of hTERTC27 by a recombinant adenovirus or RNA replacement therapy through miRNA regulation (i.e. miR-122 or miR-34).
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Basic study design
1. Exponential growth of cells is maintained prior to collection.
2. The cells are collected for inoculation by trypsinizing the cells in the flasks. Cell count and viability is then determined following a trypan blue exclusion test (98% minimum viability). Suspensions are then adjusted to the density required for inoculation.
3. One million cells, in a volume of 100 uL of the matrigel+Hepa 1-6 suspension, is inoculated subcutaneously (s.c) into the hind leg of each mouse (NOD/SCID or athymic BALB/C, 10-12 weeks).
4. The injection sites are observed until palpated tumors are established. Calipers are used for measuring tumors until average sizes of 50-150 mm3 are reached.
5. After sorting (randomization) into appropriate treatment cohorts, the test compounds are administered according to an agreed upon treatment schedule.
6. Daily tumor measurements are recorded and mouse weights are documented 3 times weekly.
7. As the study’s tumor size limit is reached (or 2,000 mm3), the animals are euthanized humanely.
8. As predetermined for the end of the study, a necropsy is performed.
9. The tumors are removed, their weight recorded, and then the tumor is documented using digital imaging.
10. Predetermined tissues are collected (snap frozen, submersed in RNAlater, isolation of nucleic acids or prepared for histological analysis) by performing standard gross necropsies.
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 Hepa 1-6 xenograft model:
- Hepa 1-6 Tumor Growth Delay (TGD; latency)
- Hepa 1-6 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)
- Hepa 1-6 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