• 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: Programmed Death-Ligand 1 (PD-L1) is a type I transmembrane protein, belonging to the B7 family of immunomodulatory molecules. In tumor cells, high expression of PD-L1 is associated with immune escape because of its ability to inhibit T cell activity and cytokine production, thereby helping tumor cells escape from the immune system attack. Therefore, PD-L1 has become an important target for tumor immunotherapy, and blocking the PD-1/PD-L1 signaling pathway can enhance the anti-tumor activity of T cells. GPC3 (Glypican-3) is highly overexpressed in various malignant tumors (such as hepatocellular carcinoma, malignant melanoma, ovarian cancer, etc.), while it is barely expressed in normal adult tissues (except in a few tissues like the placenta). This tumor-specific expression profile makes GPC3 an ideal therapeutic target.
• 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 GPC3 CDS was inserted into the mouse Gpc3 exon 1.
• Tumorigenicity: Confirmed in B-hGPC3 mice
• Application: B-hPD-L1 plus/hGPC3 MC38 tumor models can be used for preclinical evaluation of bispecific antibody drugs targeting human PD-L1 and human GPC3.
• 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.

GPC3 and PD-L1 expression analysis in B-hPD-L1 plus/hGPC3 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 plus/hGPC3 MC38 #1-C11 cultures were stained with anti-GPC3 antibody (in house, GC33-HuIgG1) and anti-PD-L1 antibody (Biolegend, 329706). Human GPC3 and human PD-L1 were detected on the surface of B-hPD-L1 plus/hGPC3 MC38 cells but not wild-type MC38 cells.

Subcutaneous tumor growth of B-hPD-L1 plus/hGPC3 MC38 cells. B-hPD-L1 plus/hGPC3 MC38 (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into homozygous B-hGPC3 mice (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 X long diameter X short diameter². Results indicate that B-hPD-L1 plus/hGPC3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

GPC3 and PD-L1 expression evaluated on B-hPD-L1 plus/hGPC3 MC38 cells by flow cytometry. B-hPD-L1 plus/hGPC3 MC38 cells were subcutaneously transplanted into homozygous B-hGPC3 mice (n=6). Upon conclusion of the experiment, tumor cells were harvested and analyzed with anti-GPC3 antibody (in house, GC33-HuIgG1) and anti-PD-L1 antibody (Biolegend, 329706) by flow cytometry. Therefore, B-hPD-L1 plus/hGPC3 MC38 cells can be used for in vivo efficacy studies evaluating novel GPC3 and PD-L1 therapeutics.