Hep3B Xenograft Model













Hep3B xenograft model

Liver cancer is the second leading cause of cancer deaths worldwide that accounts for over 800,000 new cases annually, making it a global public health challenge. Hepatocellular carcinoma (HCC) is the most common type of liver cancer in adults, accounting for more than 70 percent of all liver malignancies. The Hep3B epithelial cell line is isolated from the liver tissue of an 8-year-old Black male patient with hepatocellular carcinoma. Hep-3B cells are tumorigenic in nude mice and contain Hepatitis B. Also, the Hep-3B cell line has the Hep B virus integrated into its genome. A 2013 study published in International Journal of Hepatology investigated the antitumor efficacy of allosteric mTOR inhibitor everolimus, a rapamycin analog, both alone or in combination with the microtubule-destabilizing agent, patupilone. In vivo studies show that everolimus treatment for two weeks inhibits the growth of Hep3B xenografts. The study demonstrates the antitumor activity of everolimus in both in vitro and in vivo models of HCC as a potential therapeutic strategy for liver cancer. Also, the combination of everolimus with patupilone could be a highly effective regimen for the treatment of HCC. The Hep3B cell line (human liver) is used to create the CDX (Cell Line Derived Xenograft) Hep3B xenograft mouse model. The Hep3B xenograft model of human hepatocellular carcinoma (HCC) enables studies consisting of tumor growth inhibition of a therapeutic agent such as sorafenib, along with studies monitoring acquired sorafenib resistance.

*NOTE: All IP rights to the Hep3B cell line belongs to Wistar Institute. Clients will need to obtain licensing from Wistar Institute prior to the initiation of a xenograft study using the Hep3B cell line.

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Basic study design

1. Exponential growth of cells is maintained prior to collection.
2. The Hep3B cells are collected by trypsinization, viability determined by a trypan blue exclusion test (req 98% viability) and cell count determined.  At this piont, cell suspension concentrations are adjusted to the required density.
3. One million cells of the matrigel/Hep3B cell suspension (vol = 100 uL) is injected subcutaneously into the flank of a hind leg per each mouse (10-12 weeks old, NOD/SCID or athymic BALB/C).
4. Injection sites are monitored for tumor growth.  Calipers are used for measuring tumors with an expected start size of 50-150 mm3.
5. After sorting into treatment cohorts, the compounds of interest are injected according to the treatment design schedule.
6. Daily tumor measurements are recorded and mouse weights are logged 3 times weekly.
7. Upon reaching the tumor size limit (or 2,000 mm3), the mice are humanely euthanized.
8. As defined in the experimental design, necropsy and tissue collections are performed.
9. Tumors are removed, weighed, documented, and imaged digitally.
10. Tissues are then collected for downstream analysis; standard gross necropsies are performed.
11. All collected tumors/tissues have the option to be snap frozen, submerged in RNAlater, nucleic acids isolated for genetic analysis, or fixed for histological analysis.

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Hep3B Xenograft Model

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 Hep3B xenograft model:

  • Hep3B Tumor Growth Delay (TGD; latency)
  • Hep3B 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)
  • Hep3B 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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Hep3B Xenograft Model