CT26 Syngeneic Mouse Model

CT26 Xenograft Model
Validated CT26 Syngeneic Xenograft Model | Altogen Labs

CT26 syngeneic murine 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. They have been used in many studies such as one by Lechner et al. (2013) which used CT26 to study the immune profile of colon cancer in order to identify novel therapy targets. They looked at T-cell co-stimulatory molecules, immunosuppression markers, tumor infiltrating cytotoxic T-lymphocytes and mature dendritic cells treated with various immunotherapies and concluded that it is possible to utilize immune profiles to identify patients that may best respond to immunotherapy treatment. A 2017 Oncotarget article (Zhao et al.) used CT26 as a colorectal cancer (CRC) model to improve immunotherapy strategies; they developed a new orthotopic murine model of tumor injection resulting in higher levels of tumor-infiltrating T cells, NK cells and B cells as well as increased levels of immune-stimulating cytokines (interleukin 2 and 6, interferon-gamma and granzyme B) with lower levels of immunosuppressive myeloid cells. Lastly, a 2005 report by Cho et al. used CT26 to study a novel technique for improving the clinical relevance of endostatin, an angiogenesis inhibitor with a limiting half-life. Results suggested that when endostatin is fused to an antibody specific to HER2, together called anti-Her2 IgG3-endostatin, the resulting fusion molecule is effective at inhibiting angiogenesis and tumor growth. This has clinical relevance of increasing the viability of endostatin as a therapy, as well as increasing the versatility of anti-cancer therapy drugs and reducing their shortcomings.

In a syngeneic CT26 model, cancer cells from a particular mouse strain are implanted back into mice of the same strain, allowing researchers to study the interaction between the tumor and the immune system in a genetically matched system. The CT26 cell line is used to create the CT26 syngeneic murine 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

  1. CT26 cells used for injection are continually maintained under exponential growth.
  2. 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.
  3. 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 µL of the Matrigel + CT26 cell suspension.
  4. 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.
  5. Animals are then randomized into treatment cohorts and the compound of interest is administered according to the treatment schedule.
  6. Daily, tumors are measured, and mouse weights are recorded up to 3 times a week.
  7. Animals are euthanized as tumor size nears 2,000 cu millimeters, or predetermined study size limit.
  8. Tumor collections are performed as defined for termination of experiment.
  9. All tumors are excised and weighed, then documented with a digital image.
  10. Gross necropsies are performed and the tissues of interest are collected for downstream analysis.
  11. Tumors/tissues can be stabilized in RNA-Later, snap frozen and prepared for histology.

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CT26 Xenograft Model

Altogen Labs provides an array of laboratory services using over 90 standard Cell Line Derived Xenograft (CDX) models and over 30 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 (qRT-PCR) and protein expression analysis using the WES system (ProteinSimple).

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. Additional services available include collection of tissue, histology, isolation of total protein or RNA and analysis of gene expression.

The following options are available for the CT26 syngeneic mouse 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)
  • Imaging studies: Fluorescence-based whole body imaging, MRI

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CT26 Xenograft Model