• 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: FOLR1 is a folate receptor that binds folic acid and its derivatives, transporting 5-methyltetrahydrofolate into cells. It exists either anchored to cell membranes via GPI linkage or in a soluble form, and mutations in its gene cause cerebral folate transport deficiency, leading to neurodegeneration. PD-L1 is an immune checkpoint protein expressed on the surface of variouscells, including tumor cells and immune cells. It binds to PD-1 on T cells, leading to T cell exhaustion, reduced cytokine production, and impaired anti-tumor immunity. Combining anti-FOLR1 therapies with immune checkpoint inhibitors has emerged as a promising strategy to enhance therapeuticoutcomes by simultaneously targeting tumor and immune evasion.
• Gene targeting strategy: The exogenous promoter, human PD-L1 and luciferase coding sequences were inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript. The human FOLR1 coding sequence was inserted into the exon 4 of mouse Folr1. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript.
• Application: The B-hPD-L1 plus/hFOLR1 MC38 tumor models can be used for preclinical evaluation of monoclonal antibody drugs and bispecificantibody drugs targeting human FOLR1 and PD-1/PD-L1.

PD-L1 and FOLR1 expression analysis in B-hPD-L1 plus/hFOLR1 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 plus/hFOLR1 MC38 cultures were stained with species-specific anti-mouse PD-L1(Biolegend, 124312), anti-human PD-L1 (Biolegend, 329706) and anti-hFOLR1(Biolegend, 908304) antibodies. Human PD-L1 and human FOLR1 was detected on the surface of B-hPD-L1 plus/hFOLR1 MC38 cells but not wild-type MC38 cells. The 1-F02 clone of B-hPD-L1 plus/hFOLR1 MC38 cells was used for in vivo experiments.

B-hPD-L1 plus/hFOLR1 MC38 cells were subcutaneously transplanted into B-hFOLR1/hFOLR2 mice (female, 9-week-old, n=6), and on 28 days post inoculation, tumor cells were harvested and assessed for human PD-L1 and human FOLR1 expression with anti-mouse PD-L1(Biolegend, 124312), anti-human PD-L1 (Biolegend, 329706) and anti-hFOLR1(Biolegend, 908304) antibodies by flow cytometry. As shown, human PD-L1 and human FOLR1 was highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 plus/hFOLR1 MC38 cells can be used for in vivo efficacy studies of novel PD-L1 and FOLR1 therapeutics.

Subcutaneous homograft tumor growth of B-hPD-L1 plus/hFOLR1 MC38 cells. B-hPD-L1 plus/hFOLR1 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hFOLR1/hFOLR2 mice (female, 9-week-old, n=6). 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/hFOLR1 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.