• Origin: The B16-F10 cell line is derived from C57BL6 murine skin cells. The cell line is a commonly used murine model for melanoma.
• Background Information: VEGFA is the primary mediator of angiogenesis. It stimulates endothelial cell proliferation, migration, and survival, thereby promoting the growth of new blood vessels. Its overexpression is closely associated with tumor progression, as it enhances tumor vascularization, increases vascular permeability, and provides nutrients for cancer cell growth. PD-L1 is an immune checkpoint protein expressed on the surface of various cells, including tumor cells and immune cells. It binds to PD-1 on T cells, leading to T cell exhaustion, reduced cytokine production, and impaired anti-tumor immunity. Combining anti-VEGFA therapies with immune checkpoint inhibitors has emerged as a promising strategy to enhance therapeutic outcomes by simultaneously targeting tumor vasculature and immune evasion.
• Gene targeting strategy: The exogenous promoter, human PD-L1 and luciferase coding sequences were inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript. The human VEGFA coding sequence was inserted to replace part of murine exon 1 and all of exons 2-5. The insertion disrupts the endogenous murine Vegfa gene, resulting in a non-functional transcript.
• Tumorigenicity: Confirmed in C57BL/6 mice and B-hPD-1/hPD-L1/hVEGFA mice.
• Application: The B-hVEGFA/hPD-L1 B16-F10 tumor models can be used for preclinical evaluation of monoclonal antibody drugs and bispecific antibody drugs targeting human VEGFA and PD-1/PD-L1.
• Notes:

In the experiment, it is necessary to ensure that the number of animals inoculated subcutaneously is 1.6~2 times the actual grouping number.

Gene targeting strategy for B-hPD-L1 B16-F10 cells. The exogenous promoter and human PD-L1 coding sequence were inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript. The human VEGFA coding sequence was inserted to replace part of murine exon 1 and all of exons 2-5. The insertion disrupts the endogenous murine Vegfa gene, resulting in a non-functional transcript.

PD-L1 expression analysis in B-hVEGFA/hPD-L1 B16-F10 cells by flow cytometry. Single cell suspensions from wild-type B16-F10 and B-hVEGFA/hPD-L1 B16-F10 were stained with species-specific anti-human PD-L1 antibody (Biolgend, 329706) and anti-mouse PD-L1 antibody (Biolgend, 124312). Human PD-L1 was detected on the surface of B-hVEGFA/hPD-L1 B16-F10 cells but not wild-type B16-F10 cells. The 1-E02 clone of B-hVEGFA/hPD-L1 B16-F10 was used for in vivo experiments.

VEGFA expression analysis in B-hVEGFA/hPD-L1 B16-F10 by ELISA. Cell culture supernatant collected from B16-F10 and clone 1-E02 of B-hVEGFA/hPD-L1 B16-F10, and analyzed by ELISA with species-specific VEGFA kit (anti-mouse VEGFA antibody: R&D, MMV00; anti-human VEGFA antibody: R&D, DVE00). Mouse VEGFA was only detectable in B16-F10. Human VEGFA was detectable in B-hVEGFA/hPD-L1 B16-F10. Values are expressed as mean. ND: Not detected.

Subcutaneous tumor growth of B-hVEGFA/hPD-L1 B16-F10 cells. B-hVEGFA/hPD-L1 B16-F10 (1×10⁵) and wild-type B16-F10 cells (1×10⁵) were subcutaneously implanted into C57BL/6 mice (female, 6-week-old, n=8). 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-hVEGFA/hPD-L1 B16-F10 were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

Tumor cells were harvested at the end of ​​the​​ experiment and ​​assayed​​ for mouse and human VEGFA expression by ELISA. ​​As shown, human VEGFA was consistently highly expressed in the B-hVEGFA/hPD-L1 B16-F10 tumor homogenate, with an expression level of over 2000 pg per gram of total protein.​​ Data ​​are​​ presented as ​​mean ± SEM​​.

Subcutaneous tumor growth of B-hVEGFA/hPD-L1 B16-F10. B-hVEGFA/hPD-L1 B16-F10 (1×10⁵) and wild-type B16-F10 cells (1×10⁵) were subcutaneously implanted into B-hPD-1/hPD-L1/hVEGFA mice (male, 7-9 weeks old, n=8). 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-hVEGFA/hPD-L1 B16-F10 were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.