• 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. Tissue Factor (TF), also known as CD142, TFA, F3, is a member of the tissue factor family and a single-pass transmembrane protein. In cancer biology, TF plays a role in tumor signaling and angiogenesis. It is overexpressed in most patients with cervical cancer and many other solid tumors including ovarian, lung, pancreatic, colorectal, and head & neck cancers. Given its high expression levels on many solid tumors coupled with rapid internalization while being absent from vasculature makes it an ideal target for ADC drug development.
• 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 F3 coding sequence were inserted to replace part of murine exon 3 and all of exons 4-5. The insertion disrupts the endogenous murine F3 gene and Pd-l1 gene, resulting in a non-functional transcript.
• Tumorigenicity: Confirmed in B-hPD-1 plus/hPD-L1 mice.
• Application: The B-hPD-L1 plus/hF3 MC38 tumor models can be used for preclinical evaluation of bispecific antibody drugs targeting human PD-L1 and human F3.
• 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.

F3 and PD-L1 expression analysis in B-hPD-L1 plus/hF3 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 plus/hF3 MC38 #1-A06 were stained with anti-F3 antibody (R&D, FAB23391A) and anti-PD-L1 antibody (human anti-PD-L1: Biolegend, 329706; mouse anti-PD-L1: Biolegend, 124312). Human F3 and human PD-L1 were detected on the surface of B-hPD-L1 plus/hF3 MC38 cells but not wild-type MC38 cells.

Subcutaneous tumor growth of B-hPD-L1 plus/hF3 MC38 cells. B-hPD-L1 plus/hF3 MC38 cells (5×10⁵, 1×10⁶) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into B-hPD-1 plus/hPD-L1 mice (Male, 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/hF3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

F3 and PD-L1 expression evaluated on B-hPD-L1 plus/hF3 MC38 tumor cells by flow cytometry. B-hPD-L1 plus/hF3 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 F3 (Biolegend, 365206) and PD-L1 (human anti-PD-L1: Biolegend, 329706; mouse anti-PD-L1: Biolegend, 124312) expression by flow cytometry. As shown, human F3 and human PD-L1 were highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 plus/hF3 MC38 cells can be used for in vivo efficacy studies evaluating novel F3 therapeutics.