HT29 xenograft model (subcutaneous and metastatic)
Colon cancer represents the third most prevalent malignancy among both males and females and the second primary cause of cancer-related deaths in the United States. HT29 is a human colorectal adenocarcinoma cell line that was derived from a tumor of a 44-year-old Caucasian woman in 1964 and is helpful in gaining insight into the etiology of human colon cancers. The HT29 colorectal cell line is vital for examining biochemical pathways that aid in the development of colon carcinoma. Many colorectal rodent models have been developed to facilitate studies of human malignancies, allowing a better understanding of mechanisms contributing to tumor growth. The HT29 cell line is used to create the CDX (Cell Line Derived Xenograft) HT29 xenograft mouse model. The HT29 xenograft model enables analysis of the anti-tumor efficacy of EGFR inhibitors (e.g. cetuximab) and to elucidate the mechanism of acquired resistance to tyrosine kinase inhibitors (TKIs), such as using a sunitinib-resistant HT29 cell line.
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Basic study design
- Prior to cellecting the cells, exponential growth is maintained.
- All cells are collected by trypsinizing the cells in flasks and combining all cell suspensions. Cell count and viability is properly determined using trypan blue (min 98% viability). Cell suspensions are adjusted to an appropriate density for inoculation.
- One million cells of the matrigel plus HT29 cell suspension (vol=100 uL) is injected s.c. into the flank of one hind leg of each mouse (10 to 12 weeks; NOD/SCID or athymic BALB/C).
- All injection sites are monitored until tumors (palpated) are established. Calipers (digital) are used to measure the tumors pending average sizes of 50-150 mm3.
- After sorting (randomization), the test materials are administered according to the client supplied treatment schedule.
- Daily measurements of the tumor are logged and mouse weights are recorded (up to 3 times weekly).
- The animals are euthanized humanely as the study’s maximum tumor size is reached (or 2,000 mm2).
- A necropsy is performed to remove the tumors, record their weight and then document with digital imaging.
- Tissues are collected as discussed and are stabilized in RNAlater, snap frozen, nucleic acids isolated or tissues are prepared for histological analysis.
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 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 fluorescence was analyzed 10 minutes after intraperitoneal luciferin injection.
View full details of the case study by clicking here.
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 HT29 xenograft model:
- HT29 Tumor Growth Delay (TGD; latency)
- HT29 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)
- HT29 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