Origin:

• The MC38 cell line is derived from C57BL/6 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.
• HER2, a receptor tyrosine kinase encoded by the ERBB2 gene, lacks ligand-binding ability but forms heterodimers with other ERBB receptors to activate intracellular signaling pathways via tyrosine phosphorylation. It is overexpressed in various cancers, including breast cancer, where amplification occurs in 25-30% of cases and correlates with poor prognosis. Several targeted therapies, such as trastuzumab, pertuzumab, and T-DM1, have been approved for HER2-positive tumors.

Gene targeting strategy:

• The exogenous promoter and human PD-L1 coding sequence was inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript.
• The exogenous promoter and human HER2 coding sequence was inserted to replace part of murine exon 2. The insertion disrupts the endogenous murine Her2 gene, resulting in a non-functional transcript.

Tumorigenicity: Confirmed in B-hPD-1/hPD-L1/hHER2 mice.

Application: B-hPD-L1 plus/hHER2 MC38 plus cells have the capability to establish tumors in vivo and can be used for efficacy studies.

Gene targeting strategy for B-hPD-L1 plus/hHER2 MC38 plus cells.

The exogenous promoter and human PD-L1 coding sequence was inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript.

The exogenous promoter and human HER2 coding sequence was inserted to replace part of murine exon 2. The insertion disrupts the endogenous murine Her2 gene, resulting in a non-functional transcript.

PD-L1 and HER2 expression analysis in B-hPD-L1 plus/hHER2 MC38 plus cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 plus/hHER2 MC38 plus cultures were stained with species-specific anti-PD-L1 and anti-HER2 antibody. Human PD-L1 and HER2 were detected on the surface of B-hPD-L1 plus/hHER2 MC38 plus cells but not wild-type MC38 cells. The 1-E07 clone of B-hPD-L1 plus/hHER2 MC38 plus cells was used for in vivo tumor growth assays.

Subcutaneous homograft tumor growth of B-hPD-L1 plus/hHER2 MC38 plus cells. B-hPD-L1 plus/hHER2 MC38 plus cells (1x10⁵) and wild-type MC38 cells (1x10⁵) were subcutaneously implanted into homozygous B-hPD-1/hPD-L1/hHER2 mice (female, 8-week-old, n=5). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B)  Body weight (Mean ± SEM). Volume was expressed in mm³ using the formula: V=0.5 X long diameter X short diameter². As shown in panel A, B-hPD-L1 plus/hHER2 MC38 cells plus were able to form tumors in vivo and can be used for efficacy studies.

HER2 expression evaluated on B-hPD-L1 plus/hHER2 MC38 plus tumor cells by flow cytometry. B-hPD-L1 plus/hHER2 MC38 plus cells were subcutaneously transplanted into homozygous B-hPD-1/hPD-L1/hHER2 mice (n=5). At the end of the experiment, tumor cells were harvested and assessed for human HER2 expression by flow cytometry. As shown, human HER2 were highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 plus/hHER2 MC38 plus cells can be used for in vivo efficacy studies of PD-L1 and HER2 therapeutics.

Note: The aim was to construct a humanized PD-L1 and HER2 MC38 cell line with high HER2 expression. After tumor formation, only HER2 expression was examined, while PD-L1 expression was not detected.