CT26 xenograft model
Despite recent advances, colorectal cancer is often resistant to combination chemotherapy, highlighting the need for the development of potential novel treatment strategies. CT26 is an N-nitroso-N-methylurethane-(NNMU) induced undifferentiated colon carcinoma cell line established from BALB/c mice with aggressive colon carcinoma. Xenograft models that implant colon cancer cells into immunodeficient mice with eliminated T-cell functions have been extensively employed since the 1960s and remain the most widely used animal models for cancer drug development, as per a 2015 study published in Journal of Surgical Research. These models are essential for assessing effectiveness against human tumors in an animal setting. The CT26 cell line is used to create the CDX (Cell Line Derived Xenograft) CT26 xenograft mouse model. Along with the continual evaluation of small molecules (5-FU, oxaliplatin, 3-methyladenine), the CT26 tumor model also enables a preclinical model to investigate the potency of adenoviral vectors expressing immunostimulatory transgenes.
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Basic study design
- CT26 cells used for injection are continually maintained under exponential growth.
- The cells are prepared for injection via trypsinization. Viable cell counts are determined using a trypan blue exclusion (98% cell viability required). The cell suspension concentration is adjusted to the appropriate density.
- Each mouse (NCr-nu/nu mice; 10 weeks old) receives a single subcutaneous injection into the flank of one hind leg containing one million cells. The volume of the injection is 100 microliters of the matrigel + CT26 cell suspension.
- All injection sites are palpated up to three times a week until tumors are established. Tumors are measured using a digital caliper until an average size of 50-150 mm3 is reached.
- Animals are then randomized into treatment cohorts and the compound of interest is administered according to the treatment schedule.
- Daily, tumors are measured and mouse weights are recorded up to 3 times a week.
- Animals are euthanized as tumor size nears 2,000 cu millimeters, or predetermined study size limit.
- Tumor collections are performed as defined for termination of experiment.
- All tumors are excised and weighed, then documented with a digital image.
- Gross necropsies are performed and the tissues of interest are collected for downstream analysis.
- Tumors/tissues can be stablized in RNAlater, snap frozen and prepared for histology.
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 CT26 xenograft model:
- CT26 Tumor Growth Delay (TGD; latency)
- CT26 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)
- CT26 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