• Origin: The MC38 cell line is derived from C57BL/6 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. TROP2 is a tumor-associated calcium ion signal transduction protein and a cell surface glycoprotein, which is highly expressed in tumor tissues. It can promote the growth, proliferation and metastasis of tumor cells by regulating the calcium signaling pathway, the expression of cyclin and reducing the adhesion of fibronectin.
• Gene targeting strategy: The exogenous promoter and human PD-L1 CDS was inserted into the mouse Pd-l1 exon 3. The exogenous promoter and human TROP2 CDS was inserted into the mouse Trop2 exon 1. The mouse Vegfa gene was replaced by human VEGFA coding sequence in B-hVEGFA/hPD-L1/hTROP2 MC38.
• Tumorigenicity: Confirmed in B-hPD-1 plus/PD-L1 mice.
• Application: B-hVEGFA/hPD-L1/hTROP2 MC38 cells have the capability to establish tumors in vivo and can be used for multiple antibodies drugs of efficacy studies.
• 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 1E5-5E5. 

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.

PD-L1,TROP2 and VEGFA expression analysis in B-hVEGFA/hPD-L1/hTROP2 MC38 cells by flow cytometry and ELISA. Single cell suspensions from wild-type MC38 and B-hVEGFA/hPD-L1/hTROP2 MC38 #2-B11 cultures were stained with anti-human PD-L1 antibody (Biolegend, 329706), anti-TROP2 antibody (Biolegend, 363804). Cell culture supernatant collected from MC38 and B-hVEGFA/hPD-L1/hTROP2 MC38, analyzed by ELISA with species-specific VEGFA kit (anti-mouse VEGFA antibody: R&D, MMV00; anti-human VEGFA antibody: R&D, DVE00). Human PD-L1 and TROP2 were detected on the surface of B-hVEGFA/hPD-L1/hTROP2 MC38 cells but not wild-type MC38 cells. Human VEGFA was detectable in B-hVEGFA/hPD-L1/hTROP2 MC38. Mouse VEGFA was only detectable in MC38. Values are expressed as mean. Values are expressed as mean.

Subcutaneous tumor growth of B-hVEGFA/hPD-L1/hTROP2 MC38 cells. B-hVEGFA/hPD-L1/hTROP2 MC38 (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hPD-1 plus/PD-L1 mice (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 X long diameter X short diameter². Results indicated that B-hVEGFA/hPD-L1/hTROP2 MC38 cells were able to establish tumors in vivo and could be used for efficacy studies. Values are expressed as mean ± SEM.

PD-L1 and TROP2 expression analysis in B-hVEGFA/hPD-L1/hTROP2 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hVEGFA/hPD-L1/hTROP2 MC38 #2-B11 cultures were stained with anti-mouse and human PD-L1 antibody (Biolegend, 124312; Biolegend, 329706) and anti-TROP2 antibody (Biolegend, 363804). Human PD-L1 and TROP2 were detected on the surface of B-hVEGFA/hPD-L1/hTROP2 MC38 cells but not wild-type MC38 cells.

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/hTROP2 MC38 tumor homogenate, with an expression level of over 2000 pg per milligram of total protein.​​ Data ​​are​​ presented as ​​mean ± SEM​​. ND: Not detected.