• Origin: The CT26.WT cell line is derived from BALB/c murine colon adenocarcinoma cells. The cell line is a commonly used murine model for colorectal carcinoma.
• Background Information: The human epidermal growth factor receptor 2 (HER2), known as ERBB2, or proto-oncogene Neu, is a receptor tyrosine-protein kinase encoded by the ERBB2 (HER2) gene on chromosome 17q12. ErbB2 does not bind ligand, yet appears to be the major signaling partner for other ERBB receptors by forming heteromeric complexes with ErbB1, ErbB3, or ErbB4. Upon ligand-induced dimerization, the receptors autophosphorylate on specific tyrosine residues in their cytoplasmic tails. These residues provide docking sites for phosphotyrosine binding, cytoplasmic signaling molecules that activate numerous intracellular signaling pathways. HER2 overexpresses in a broad number of cancer types, such as bladder, breast, cervical, cholangio, colorectal, endometrial, esophageal, gastric, head and neck, liver, lung, ovarian, and salivary gland cancers. Especially, amplification and overexpression of HER2 occurs in 25% to 30% of the instances of human breast cancer and are associated with a poor prognosis. Several HER2-targeting therapies such as trastuzumab, pertuzumab, T-DM1, DS8201 and RC48 have been approved worldwide for patients with HER2-positive tumors.
• Gene targeting strategy: The mouse Her2 gene was replaced by human HER2 coding sequence in B-hHER2 CT26.WT cells. Human HER2 is highly expressed on the surface of B-hHER2 CT26.WT cells.
• Tumorigenicity: Confirmed in BALB/c mice.
• Application: The B-hHER2 CT26.WT tumor models can be used for preclinical evaluation of monoclonal antibody drugs and bispecific antibody drugs targeting human HER2.

Gene targeting strategy for B-hHER2 CT26.WT cells. The exogenous promoter and human HER2 coding sequence was inserted to replace part of murine exon 2 and all of exons 3-7. The insertion disrupts the endogenous murine Her2 gene, resulting in a non-functional transcript.

HER2 expression analysis in B-hHER2 CT26.WT cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hHER2 CT26.WT #1-A03 cultures were analyzed with anti-mouse HER2 antibody (RD, FAB6744P) and anti-human HER2 antibody (Biolegend, 324408) by flow cytometry. Human HER2 was detected on the surface of B-hHER2 CT26.WT cells but not wild-type MC38 cells.

Subcutaneous tumor growth of B-hHER2 CT26.WT cells. B-hHER2 CT26.WT cells (1x10⁵) and wild-type MC38 cells (1x10⁵) were subcutaneously implanted into BALB/c mice (female, 6-7-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 X long diameter X short diameter². Results indicate that B-hHER2 CT26.WT cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

HER2 expression evaluated on B-hHER2 CT26.WT tumor cells by flow cytometry. B-hHER2 CT26.WT cells were subcutaneously transplanted into BALB/c mice (n=6). Upon conclusion of the experiment, tumor cells were harvested and analyzed with anti-human HER2 antibody (Biolegend, 324406) by flow cytometry. As shown, human HER2 was highly expressed on the surface of tumor cells. Therefore, B-hHER2 CT26.WT cells can be used for in vivo efficacy studies evaluating novel HER2 therapeutics.

Antitumor activity of anti-human HER2 ADC (Trastuzumab analog-MMAE, in-house) in BALB/c mice. (A) Anti-human HER2 ADC inhibited B-hHER2 CT26.WT tumor growth in BALB/c mice. Murine colon cancer B-hHER2 CT26.WT cells were subcutaneously implanted into BALB/c mice (female, 9-week-old, n=6). Mice were grouped when tumor volume reached approximately 50 mm³, at which time they were intraperitoneally injected with anti-human HER2 ADC Trastuzumab analog-MMAE (in-house) indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human HER2 ADC was efficacious in controlling tumor growth in BALB/c mice, demonstrating that B-hHER2 CT26.WT provide a powerful preclinical model for in vivo evaluation of anti-human HER2 antibodies. Values are expressed as mean ± SEM.

Antitumor activity of anti-human HER2 ADC (Trastuzumab analog-MMAE, in-house) in BALB/c mice. Murine colon cancer B-hHER2 CT26.WT cells were subcutaneously implanted into BALB/c mice (female, 9-week-old, n=6). Mice were grouped when tumor volume reached approximately 50 mm³, at which time they were intraperitoneally injected with anti-human HER2 ADC Trastuzumab analog-MMAE (in-house) indicated in panel.

Anti tumor activity of anti-human HER2 antibody (DS8201, purchased from Daiichi Sankyo) in BALB/c mice. (A) Anti-human HER2 antibody inhibited B-hHER2 CT26.WT tumor growth in BALB/c mice. Murine colon cancer B-hHER2 CT26.WT cells were subcutaneously implanted into BALB/c mice (female, 8-week-old, n=6). Mice were grouped when tumor volume reached approximately 50 mm³, at which time they were intravenously injected with anti-human HER2 ADC DS-8201 (purchased from AstraZeneca/Daiichi Sankyo) indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human HER2 ADC DS-8201 (purchased from AstraZeneca/Daiichi Sankyo) was efficacious in controlling tumor growth in BALB/c mice in a dose-dependent manner, demonstrating that the BALB/C mice provide a powerful preclinical model for in vivo evaluation of anti-human HER2 antibodies. Values are expressed as mean ± SEM.

Antitumor activity of anti-human HER2 antibody (DS-8201, purchased from) in BALB/c mice. (A) Anti-human HER2 antibody inhibited B-hHER2 CT26.WT tumor growth in BALB/c mice. Murine colon cancer B-hHER2 CT26.WT cells were subcutaneously implanted into homozygous BALB/c mice (female, 8-week-old, n=6). Mice were grouped when tumor volume reached approximately 50 mm³, at which time they were intravenously injected with anti-human HER2 ADC DS-8201 (purchased from AstraZeneca/Daiichi Sankyo) indicated in panel.

Antitumor activity of anti-human HER2 ADC (ERBB2-trastuzumab-L-hIgG1-T1, in-house) in B-hHER2 mice(C). (A) Anti-human HER2 ADC inhibited B-hHER2 CT26.WT tumor growth in B-hHER2 mice(C). Murine colon cancer B-hHER2 CT26.WT cells were subcutaneously implanted into B-hHER2 mice(C) (female, 8-week-old, n=6). Mice were grouped when tumor volume reached approximately 80 mm³, at which time they were intraperitoneally injected with anti-human HER2 ADC ERBB2-trastuzumab-L-hIgG1-T1 (in-house) indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human HER2 ADC was efficacious in controlling tumor growth in B-hHER2 mice(C), demonstrating that B-hHER2 CT26.WT provide a powerful preclinical model for in vivo evaluation of anti-human HER2 antibodies. Values are expressed as mean ± SEM.

Passage stability analysis in B-hHER2 CT26.WT cells by flow cytometry. Single cell suspensions from wild-type CT26.WT and B-hHER2 CT26.WT cultures were stained with species-specific anti-HER2 antibody. Human HER2 was specifically detected on the surface of B-hHER2 CT26.WT cells and remained stable across multiple passages, with no significant change in expression levels observed. No expression was detected in wild-type CT26.WT cells.

The passage number for this cell line is calculated starting from the first subculture after revival from the master cell bank, which is designated as P1.