• 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. Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein belonging to the receptor tyrosine kinase family. It mainly participates in the processes of cell growth, proliferation, differentiation and survival. EGFR shows abnormal expression or mutation in various types of cancers, such as non-small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, etc., and thus becomes an important therapeutic target.
• Gene targeting strategy: The exogenous promoter and human PD-L1 CDS were inserted into the mouse Pd-l1 exon 3. The expression cassette containing an exogenous promoter, the extracellular region of human EGFR, and the transmembrane and intracellular regions of mouse EGFR was randomly inserted into the genome of B-hPD-L1 plus/Tg(hEGFR) MC38.
• Tumorigenicity: Confirmed in B-hPD-1 plus/hPD-L1/hEGFR mice.
• Application: The B-hPD-L1 plus/Tg(hEGFR) MC38 tumor models can be used for preclinical evaluation of bispecific antibody drugs targeting human PD-L1 and human EGFR.
• 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 and EGFR expression analysis in B-hPD-L1 plus/Tg(hEGFR) MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 plus/Tg(hEGFR) MC38 #2-H06 cultures were stained with anti-EGFR antibody (Biolegend, 352906) and anti-PD-L1 antibody (Biolegend, 329706; Biolegend, 124312). Human PD-L1 and human EGFR were detected on the surface of B-hPD-L1 plus/Tg(hEGFR) MC38 cells but not wild-type MC38 cells.

PD-L1 and EGFR expression evaluated on B-hPD-L1 plus/Tg(hEGFR) MC38 tumor cells by flow cytometry. B-hPD-L1 plus/Tg(hEGFR) MC38 cells were subcutaneously transplanted into B-hPD-1 plus/hPD-L1/hEGFR mice (n=6). Upon conclusion of the experiment, tumor cells were harvested and assessed for EGFR (Biolegend, 352904) and PD-L1 (Biolegend, 329706; Biolegend, 124312) expression by flow cytometry. As shown, human PD-L1 and human EGFR were highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 plus/Tg(hEGFR) MC38 cells can be used for in vivo efficacy studies evaluating novel EGFR therapeutics.

Subcutaneous tumor growth of B-hPD-L1 plus/Tg(hEGFR) MC38 cells. B-hPD-L1 plus/Tg(hEGFR) MC38 cells (2×10⁵) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into B-hPD-1 plus/hPD-L1/hEGFR 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.