The mouse Pdl1 gene and Cd47 gene were replaced by human PD-L1 and CD47 coding sequence in B-hPD-L1 plus/hCD47 MC38 cells. Human PD-L1 and CD47 are highly expressed on the surface of B-hPD-L1 plus/hCD47 MC38 cells.

Gene targeting strategy for B-hPD-L1 plus/hCD47 MC38 cells. The exogenous promoter and human PD-L1 coding sequence were 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 CD47 coding sequence were inserted to replace part of murine exon 2 and all of exon 3. The insertion disrupts the endogenous murine Cd47 gene, resulting in a non-functional transcript.

PD-L1 and CD47 expression analysis in B-hPD-L1 plus/hCD47 MC38 cells by flow cytometry. Single cell suspensions from B-hPD-L1 plus/hCD47 MC38 cultures were stained with species-specific anti-PD-L1 and anti-CD47 antibodies. Human PD-L1 and CD47 were detected on the surface of B-hPD-L1 plus/hCD47 MC38 cells. The 3-D07 clone of B-hPD-L1 plus/hCD47 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hPD-L1 plus/hCD47 MC38 cells. B-hPD-L1 plus/hCD47 MC38 cells (5x10⁵) were subcutaneously implanted into B-hPD-L1/hSIRPA/hCD47 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/hCD47 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.