Description

• Origin: The MC38 cell line is derived from C57BL6 murine colon adenocarcinoma cells. The cell line is a commonly used murine model for colorectal carcinoma.
• 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 B-hPD-1/hPD-L1 mice.
• Application: The B-hVEGFA/hPD-L1 plus MC38 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:

        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 5E5~1E6.

        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

PD-L1 expression analysis in B-hVEGFA/hPD-L1 plus MC38 by flow cytometry. Single cell suspensions from wild-type MC38 and B-hVEGFA/hPD-L1 plus MC38 #1-B07 cultures were stained with anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706) . Human PD-L1 was detected on the surface of B-hVEGFA/hPD-L1 plus MC38 but not wild-type MC38 cells.

VEGFA expression analysis in B-hVEGFA/hPD-L1 plus MC38 by ELISA. Cell culture supernatant collected from MC38 and clone 1-B07 of B-hVEGFA/hPD-L1 plus MC38, 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 MC38. Human VEGFA was detectable in B-hVEGFA/hPD-L1 plus MC38. Values are expressed as mean.

Tumor growth curve & body weight changes

Subcutaneous tumor growth of B-hVEGFA/hPD-L1 plus MC38 cells. B-hVEGFA/hPD-L1 plus MC38 (1×10⁶) were subcutaneously implanted into B-hVEGFA mice (male, 8-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 plus MC38 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-hVEGFA/hPD-L1 plus MC38 cells. B-hVEGFA/hPD-L1 plus MC38 (5×10⁵) were subcutaneously implanted into B-hPD-1/hPD-L1 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-hVEGFA/hPD-L1 plus MC38 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 tissue

PD-L1 expression was evaluated on B-hVEGFA/hPD-L1 MC38 cells by flow cytometry. These cells were subcutaneously transplanted into B-hPD-1/hPD-L1 mice (n = 6). At the end of the experiment, tumor cells were harvested and analyzed for both mouse and human PD-L1 expression by flow cytometry. The results demonstrated high surface expression of human PD-L1 on the tumor cells. Therefore, the B-hVEGFA/hPD-L1 MC38 model is suitable for evaluating the in vivo therapeutic efficacy of VEGFA- and PD-1-targeted drug combinations.

mVEGFA/hVEGFA expression analysis in tumor

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 plus MC38 tumor homogenate, with an expression level of approximately 1 pg per gram of total protein. Meanwhile, this level was 40-fold higher than that of mouse VEGFA in wild-type MC38 tumors. Data are presented as mean ± SEM.