• 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. 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.
• Tumorigenicity: Confirmed in B-hPD-1 plus/hPD-L1/hTROP2 mice
• Application: B-hPD-L1 plus/hTROP2 low MC38 tumor models can be used for preclinical evaluation of bispecific antibody drugs targeting human PD-L1 and human TROP2.
• 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.

TROP2 and PD-L1 expression analysis in B-hPD-L1 plus/hTROP2 low MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 plus/hTROP2 low MC38 #5-B02 cultures were stained with anti-TROP2 antibody (R&D, FAB650P) and anti-PD-L1 antibody (Biolegend, 329706; Biolegend, 124312). Human TROP2 and human PD-L1 were detected on the surface of B-hPD-L1 plus/hTROP2 low MC38 cells but not wild-type MC38 cells.

TROP2 and PD-L1 expression evaluated on B-hPD-L1 plus/hTROP2 low MC38 cells by flow cytometry. B-hPD-L1 plus/hTROP2 low MC38 cells were subcutaneously transplanted into homozygous B-hPD-1 plus/hPD-L1/hTROP2 mice (n=6). Upon conclusion of the experiment, tumor cells were harvested and analyzed with anti-TROP2 antibody (R&D, FAB650P) and anti-PD-L1 antibody (Biolegend, 329706) by flow cytometry. Therefore, B-hPD-L1 plus/hTROP2 low MC38 cells can be used for in vivo efficacy studies evaluating novel TROP2 and PD-L1 therapeutics.

Subcutaneous tumor growth of B-hPD-L1 plus/hTROP2 low MC38 cells. B-hPD-L1 plus/hTROP2 low MC38 (2x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into homozygous B-hPD-1 plus/hPD-L1/hTROP2 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/hTROP2 low MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.