Description

• Origin: The CT26.WT cell line is derived from BALB/c murine colon cells. The cell line is a commonly used murine model for colon carcinoma.
• Background Information: DLL3 (Delta-like ligand 3), a non-canonical ligand of the Notch signaling pathway, belongs to the Delta/Serrate/LAG-2 (DSL) protein family. In neuroendocrine tumors (e.g., small cell lung cancer, neuroblastoma), DLL3 overexpression inhibits the Notch pathway, leading to the maintenance of stem cell-like properties, promoting cell proliferation, suppressing differentiation, and driving malignant tumor progression. Within the tumor immune microenvironment, DLL3 may facilitate immune evasion by modulating tumor-associated macrophages (TAMs) or suppressing T cell activity. Additionally, DLL3 promoting tumor growth and metastasis. DLL3-targeted therapies, such as antibody-drug conjugates (ADCs) and chimeric antigen receptor T-cell (CAR-T) therapies, have demonstrated clinical potential, offering new directions for improving treatment outcomes for refractory tumors.
• Gene targeting strategy: The exogenous promoter and chimeric DLL3 coding sequence that include human extracellular region and mouse intracellular region were inserted to replace part of murine Dll3 exon 2 and all of exons 3-5. The insertion disrupts the endogenous murine Dll3 gene, resulting in a non-functional transcript.
• Tumorigenicity: Confirmed in BALB/c mice.
• Application: The B-hDLL3 CT26.WT tumor models can be used for preclinical evaluation of monoclonal antibody drugs targeting human DLL3.
• Notes:

        Inoculated cell lines can be suspended with DMEM stock solution.

        Before implementing the project, it is recommended to perform tumor growth experiments. The recommended cell inoculation amount is between 1E5.

        In the experiment, it is necessary to ensure that the number of animals inoculated subcutaneously is at least 1.6 times the actual grouping number.

Protein expression analysis

DLL3 expression analysis in B-hDLL3 CT26.WT cells by flow cytometry. Single cell suspensions from wild-type CT26.WT and B-hDLL3 CT26.WT cultures were stained with anti-DLL3 antibody AMG-757 analog (in house). Human DLL3 was detected on the surface of B-hDLL3 CT26.WT cells, but not on the surface of wild-type CT26.WT cells. The 1-E05 clone of B-hDLL3 CT26.WT cells was used for in vivo tumor growth assays.

Tumor growth curve & body weight changes

Subcutaneous tumor growth of B-hDLL3 CT26.WT. B-hDLL3 CT26.WT (1×10⁵) and wild-type CT26.WT cells (1×10⁵) were subcutaneously implanted into BALB/c mice (female, 8-10-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 × long diameter × short diameter². Results indicate that B-hDLL3 CT26.WT cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

Protein expression analysis of tumor cells

DLL3 expression evaluated on B-hDLL3 CT26.WT tumor cells by flow cytometry. B-hDLL3 CT26.WT cells were subcutaneously transplanted into BALB/c mice (n=6), and on 23 days post inoculation, tumor cells were harvested and assessed for human DLL3 expression by flow cytometry using anti-human DLL3 antibody AMG-757 analog (in house). As shown, human DLL3 was expressed on the surface of tumor cells. Therefore, B-hDLL3 CT26.WT cells can be used for in vivo efficacy studies of DLL3 therapeutics.