• Origin: The B16-F10 cell line is derived from C57BL/6 murine skin cells. The cell line is a commonly used murine model for melanoma.
• 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 C57BL/6 mice.
• Application: The B-hDLL3 low B16-F10 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 2E5.
         In the experiment, it is necessary to ensure that the number of animals inoculated subcutaneously is at least 1.4 times the actual grouping number.

DLL3 expression analysis in B-hDLL3 low B16-F10 cells by flow cytometry. Single cell suspensions from wild-type B16-F10 and B-hDLL3 low B16-F10 cultures were stained with anti-DLL3 antibody AMG-757 analog (in house). Human DLL3 was detected on the surface of B-hDLL3 low B16-F10 cells, but not on the surface of wild-type B16-F10 cells. The 2-A07 clone of B-hDLL3 low B16-F10 cells was used for in vivo tumor growth assays.

DLL3 expression evaluated on B-hDLL3 low B16-F10 tumor cells by flow cytometry. B-hDLL3 low B16-F10 cells were subcutaneously transplanted into C57BL/6 mice (n=6), and on 14 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 low B16-F10 cells can be used for in vivo efficacy studies of DLL3 therapeutics.

Subcutaneous tumor growth of B-hDLL3 low B16-F10. B-hDLL3 low B16-F10 (2×10⁵) and wild-type B16-F10 cells (2×10⁵) were subcutaneously implanted into C57BL/6 mice (female, 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 × long diameter × short diameter². Results indicate that B-hDLL3 low B16-F10 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

Subcutaneous tumor growth of B-hDLL3 low B16-F10. B-hDLL3 low B16-F10 (2×10⁵) and wild-type B16-F10 cells (2×10⁵) were subcutaneously implanted into C57BL/6 mice (female, 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 × long diameter × short diameter². Results indicate that B-hDLL3 low B16-F10 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.