• 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. B7-H4 (also known as VTCN1, B7x, or B7S1) is a member of the B7 family of immune checkpoint molecules. In various cancers—including ovarian, breast, renal cell, and lung carcinomas—B7-H4 is highly expressed and contributes to tumor immune evasion, progression, and poor prognosis. Tumor-expressed B7-H4 can directly inhibit tumor-infiltrating T cells or indirectly promote immune suppression through tumor-associated macrophages. Consequently, B7-H4 has emerged as a promising immuno-oncology target.
• Gene targeting strategy: The exogenous promoter, human PD-L1 and luciferase coding sequences were inserted to replace part of murine exon 3. The exogenous promoter and human B7-H4 coding sequence were inserted to replace part of murine exon 3 and all of exon 4. The insertion disrupts the endogenous murine B7-h4 gene and Pd-l1 gene, resulting in a non-functional transcript.
• Tumorigenicity: Confirmed in B-hCD3E/hB7-H4 mice.
• Application: The B-hPD-L1 plus/hB7-H4 MC38 tumor models can be used for preclinical evaluation of bispecific antibody drugs targeting human PD-L1 and human B7-H4.
• Notes:

Inoculated cell lines can be suspended with DMEM stock solution.

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.

B7-H4 and PD-L1 expression analysis in B-hPD-L1 plus/hB7-H4 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 plus/hB7-H4 MC38 #1-B03 were stained with anti-B7-H4 antibody (Biolegend, 358108) and anti-PD-L1 antibody (human anti-PD-L1: Biolegend, 329706; mouse anti-PD-L1: Biolegend, 124312). Human B7-H4 and human PD-L1 were detected on the surface of B-hPD-L1 plus/hB7-H4 MC38 cells but not wild-type MC38 cells.

B7-H4 and PD-L1 expression evaluated on B-hPD-L1 plus/hB7-H4 MC38 tumor cells by flow cytometry. B-hPD-L1 plus/hB7-H4 MC38 cells were subcutaneously transplanted into B-hCD3E/hB7-H4 mice (n=6). Upon conclusion of the experiment, tumor cells were harvested and assessed for B7-H4 (eBioscience™, 17-5949-41) and PD-L1 (human anti-PD-L1: Biolegend, 329714; mouse anti-PD-L1: Biolegend, 124308) expression by flow cytometry. As shown, human B7-H4 and human PD-L1 were highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 plus/hB7-H4 MC38 cells can be used for in vivo efficacy studies evaluating novel B7-H4 therapeutics.

Subcutaneous tumor growth of B-hPD-L1 plus/hB7-H4 MC38 cells. B-hPD-L1 plus/hB7-H4 MC38 cells (5×10⁵) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into B-hCD3E/hB7-H4 mice (Female, 8-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/hB7-H4 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

Note: B-hCD3E/hB7-H4 mice were chosen for tumor inoculation to enable tumor establishment on a B7-H4 humanized background.