• 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. CLDN18.2 is a highly selective transmembrane protein, which belongs to the Claudin family. It is mainly expressed in well-differentiated gastric mucosal epithelial cells in normal tissues, and highly expressed in a variety of malignant tumors (such as gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, etc.), making it an ideal target for cancer treatment.
• 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 CLDN18.2 CDS was inserted in B-hPD-L1 plus/hCLDN18.2 MC38.
• Tumorigenicity: Confirmed in B-hPD-1 plus/hPD-L1 mice.
• Application: The B-hPD-L1 plus/hCLDN18.2 MC38 tumor models can be used for preclinical evaluation of bispecific antibody drugs targeting human PD-L1 and human CLDN18.2.
• 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.

CLDN18.2 and PD-L1 expression analysis in B-hPD-L1 plus/hCLDN18.2 high MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 plus/hCLDN18.2 high MC38 #1-E01 cultures were stained with anti-CLDN18.2 antibody (in house) and anti-PD-L1 antibody (Biolegend, 329706; Biolegend, 124312). Human CLDN18.2 and human PD-L1 were detected on the surface of B-hPD-L1 plus/hCLDN18.2 high MC38 cells but not wild-type MC38 cells.

CLDN18.2 and PD-L1 expression evaluated on B-hPD-L1 plus/hCLDN18.2 high MC38 tumor cells by flow cytometry. B-hPD-L1 plus/hCLDN18.2 high MC38 cells were subcutaneously transplanted into B-hPD-1 plus/hPD-L1 mice (n=6). Upon conclusion of the experiment, tumor cells were harvested and assessed for CLDN18.2 (in house) and PD-L1 (Biolegend, 329714; Biolegend, 124308) expression by flow cytometry. As shown, human CLDN18.2 and human PD-L1 were highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 plus/hCLDN18.2 high MC38 cells can be used for in vivo efficacy studies evaluating novel CLDN18.2 therapeutics.

Subcutaneous tumor growth of B-hPD-L1 plus/hCLDN18.2 high MC38 cells. B-hPD-L1 plus/hCLDN18.2 high MC38 cells (1×10⁵, 5×10⁵) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into B-hPD-1 plus/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-hPD-L1 plus/hCLDN18.2 high MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.