CT26 Orthotopic Mouse Model Service for Colon Cancer

The CT26 orthotopic mouse model offers a robust, immunocompetent preclinical platform for colon cancer research, enabling evaluation of immunotherapies, chemotherapeutic agents, and combination regimens within a syngeneic tumor microenvironment that preserves intact host immune surveillance and stromal crosstalk. Alfa Cytology constructs and manages this orthotopic allograft system with authenticated CT26 cell stocks, standardized surgical cecal wall implantation protocols, and integrated immune profiling capabilities, delivering mechanistically rich datasets that capture tumor-immune dynamics, metastatic progression, and therapeutic response in an in vivo context unmatched by subcutaneous or immunodeficient alternatives.

Overview of CT26 Orthotopic Mouse Model for Colon Cancer

The CT26 cell line is a murine colon adenocarcinoma originally established in 1975 by Corbett and colleagues following intrarectal administration of N-nitroso-N-methylurethane (NMU) to a female BALB/c mouse. Genetically, CT26 harbors a homozygous Kras activating mutation (p.G12D) and a homozygous deletion of the Cdkn2a tumor suppressor locus, alongside elevated expression of proliferation and stem-cell markers including Top2a, Birc5, and Mki67. The line exhibits an aneuploid karyotype and secretes high levels of proinflammatory and proangiogenic mediators such as IL-6, VEGF, and matrix metalloproteinases, reflecting the aggressive, undifferentiated phenotype characteristic of refractory human colorectal carcinoma. As a fully murine cell line, CT26 can be implanted into syngeneic, immunocompetent BALB/c hosts, preserving endogenous T-cell, B-cell, NK-cell, and myeloid compartments—an attribute that renders it indispensable for immunotherapy development, tumor microenvironment dissection, and chemo-immune combination studies.

Fig 1. Comprehensive workflow for establishing a CT26.WT-luc orthotopic colorectal cancer model via rectal submucosal injection in BALB/c mice, integrating surgical technique, bioluminescence imaging (BLI) longitudinal monitoring, and MRI validation to ensure reproducible tumor engraftment and growth kinetics for preclinical drug efficacy studies. (Chen, Wen, et al., 2025)

Orthotopic implantation of CT26 cells into the cecal wall of BALB/c mice reconstructs the anatomical positioning, peritoneal environment, and lymphatic drainage patterns of clinical colorectal malignancy. The surgical approach typically involves a midline laparotomy, exteriorization of the cecum, and submucosal injection of a Matrigel-suspended cell inoculum (5 × 10⁵ cells in 10 µL) between the serosa and mucosa, followed by two-layer closure. Tumors establish consistently within 10–14 days and progress to endpoint volumes over 3–5 weeks, with documented potential for spontaneous metastasis to regional mesenteric lymph nodes, liver, and lungs. This configuration is particularly powerful for assessing anti-angiogenic agents, immune checkpoint inhibitors, antibody-cytokine fusions, and autophagy modulators, as the intact immune system enables meaningful interrogation of both direct tumor cytotoxicity and host-mediated antitumor immunity.

Cell Line Information: CT26

CT26 (Colon Tumor #26) is among the most widely employed murine colorectal cancer cell lines in preclinical oncology, distinguished by its syngeneic compatibility with immunocompetent BALB/c mice and its well-characterized genetic driver landscape. The table below consolidates its essential biological, genetic, and culture parameters:

Our Services

Alfa Cytology delivers a comprehensive CT26 orthotopic model service that spans authenticated cell line banking, surgical cecal wall implantation, longitudinal tumor monitoring, therapeutic intervention, immune profiling, and metastasis assessment. Our platform is optimized for immunotherapy and combination therapy studies, leveraging the intact BALB/c immune system to generate translationally relevant efficacy, pharmacodynamic, and biomarker datasets that inform your colon cancer drug development strategy.

Workflow of CT26 Orthotopic Mouse Model Construction

Establishment of the CT26 orthotopic model requires precise surgical technique and strict aseptic conditions to achieve consistent engraftment, minimize perioperative complications, and enable meaningful metastasis readouts. The protocol proceeds through the following stages:

1. Cell Expansion and Authentication: CT26 cells are propagated in RPMI 1640 with 10% FBS, harvested during logarithmic growth, and authenticated via STR profiling against the BALB/c reference. Mycoplasma negativity is confirmed by PCR. Cells are washed, counted, and resuspended in ice-cold PBS at a concentration of 5 × 10⁷ cells/mL for in vitro preparation.
2. Matrigel Suspension Preparation: The cell suspension is mixed 1:1 by volume with ice-cold Matrigel Basement Membrane Matrix immediately prior to injection. Matrigel provides an extracellular scaffold that prevents cell leakage into the peritoneal cavity, minimizes carcinomatosis, and supports initial tumor colonization within the cecal wall.
3. Surgical Cecal Wall Implantation: Female BALB/c mice (6–8 weeks old) are anesthetized, and the abdominal wall is shaved and sterilized. A midline laparotomy (~10 mm) is performed, and the cecum is exteriorized onto sterile gauze moistened with PBS. A small cecotomy is made at the cecal tip, and 10 µL of the Matrigel-cell suspension (5 × 10⁴ cells) is injected submucosally into the cecal wall from the luminal side using a 28-gauge insulin syringe. The cecotomy is closed with interrupted 5-0 silk sutures in Lembert fashion, the cecum is returned to the abdominal cavity, and the abdominal wall is closed in two layers.
4. Postoperative Care and Monitoring: Mice receive sustained-release buprenorphine for analgesia and are monitored closely during the 48-hour perioperative period for signs of pain, dehydration, or intestinal obstruction. Body weights are recorded daily for the first week and twice weekly thereafter to detect early cachexia or treatment-related toxicity.
5. Tumor Monitoring and Cohort Randomization: Tumor establishment is assessed by palpation of the abdominal cavity and, where applicable, by ultrasound imaging beginning on day 7–10 post-surgery. Once tumors reach 50–100 mm³, mice are randomized into treatment cohorts based on tumor volume to ensure balanced baseline distributions.
6. Therapeutic Intervention and Longitudinal Assessment: Test compounds are administered according to the predefined regimen. Tumor dimensions are measured twice weekly by caliper or high-frequency ultrasound. Body weights and clinical signs are recorded concurrently. For immunotherapy studies, peripheral blood may be collected at intervals for circulating immune cell phenotyping and cytokine analysis.
7. Endpoint Necropsy and Multi-Organ Collection: Upon reaching study endpoints (tumor volume ~1,500 mm³, humane moribund criteria, or scheduled termination), mice are euthanized. The primary cecal tumor is excised, weighed, and photographed. The liver, lungs, mesenteric lymph nodes, and spleen are harvested for metastasis assessment, immune profiling, and histopathological analysis.
8. Histopathological, Immune, and Molecular Analysis: Primary tumors and metastatic lesions are fixed for H&E and immunohistochemistry (CD31, Ki-67, cleaved caspase-3), or processed for flow cytometry to characterize tumor-infiltrating lymphocytes (CD8⁺ T cells, CD4⁺ T cells, Tregs, MDSCs, dendritic cells), NK cells, and myeloid populations. Snap-frozen tissues are reserved for Western blot, qPCR, and RNA-seq. Serum cytokines (IL-6, TNF-α, IFN-γ) are quantified by multiplex assay.

Fig 2. CT26 Orthotopic Mouse Model construction workflow.

Case Study-CT26 Orthotopic Mouse Model Development

In a preclinical program investigating chemo-immunotherapy combinations for microsatellite-stable colorectal cancer, the CT26 orthotopic model was employed to evaluate the efficacy of a novel anti-VISTA monoclonal antibody administered as monotherapy and in combination with 5-fluorouracil (5-FU). Following surgical cecal wall implantation and postoperative recovery, tumor-bearing mice were randomized into treatment arms receiving escalating doses of the test antibody, 5-FU alone, the combination regimen, or vehicle control. Longitudinal tumor monitoring revealed significant growth suppression in the combination cohort compared to either monotherapy arm, with the highest dose combination group exhibiting the most pronounced tumor volume reduction. Endpoint flow cytometric analysis of tumor-infiltrating leukocytes demonstrated increased CD8⁺ cytotoxic T-cell infiltration and diminished polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) accumulation in the combination-treated tumors, alongside elevated serum IFN-γ levels. Histopathological examination confirmed reduced Ki-67 proliferation indices and increased apoptotic signaling in the treated groups. These findings illustrate the model's capacity to capture both direct antitumor effects and immune microenvironment remodeling, providing actionable insights for optimizing chemo-immunotherapy strategies in the preclinical setting.

Why Choose Alfa Cytology?

Selecting Alfa Cytology for your CT26 orthotopic colon cancer study ensures access to a specialized CRO platform with deep expertise in syngeneic model construction and immune-oncology endpoint analysis. Our core differentiators include:

• Authenticated CT26 cell banking with documented STR profiling, mycoplasma clearance, and low-passage expansion to preserve genetic stability and immunogenicity across study cohorts.
• Skilled surgical team experienced in cecal wall orthotopic implantation with high take rates and low perioperative mortality, ensuring robust statistical power and reproducible tumor growth kinetics.
• In-house flow cytometry infrastructure for comprehensive tumor immune profiling, including T-cell subsets, NK cells, dendritic cells, MDSCs, and tumor-associated macrophages with validated antibody panels.
• Customizable combination study designs supporting immune checkpoint inhibitors, chemotherapeutics, targeted agents, and novel biologics with flexible dosing schedules and route optimization.
• Integrated metastasis assessment capabilities encompassing systematic organ harvest, macroscopic nodule counting, histopathological confirmation, and molecular validation of disseminated disease.
• Dedicated project scientists providing weekly tumor monitoring updates, interim immune profiling reports, and collaborative data interpretation to align preclinical findings with your clinical development roadmap.

Contact Us

If your preclinical pipeline demands an immunocompetent colon cancer model that preserves intact host immunity and enables robust evaluation of immunotherapies and combination strategies, we invite you to reach out to us to discuss how Alfa Cytology can design and execute a tailored CT26 orthotopic study aligned with your therapeutic hypotheses. From surgical implantation and immune profiling through comprehensive metastasis assessment, our team is committed to delivering mechanistically rich, translationally relevant data that advance your colon cancer drug development program. Contact us today to initiate your project and leverage our syngeneic model expertise for your next-generation therapeutic candidate.

For research use only. Not intended for any clinical use.