The mouse Cd73 gene was replaced by human CD73 coding sequence in B-hCD73 MC38 cells. Human CD73 is highly expressed on the surface of B-hCD73 MC38 cells.

Gene targeting strategy for B-hCD73 MC38 cells. The exogenous promoter and human CD73 coding sequence were inserted to replace part of murine exon 2 and all of exon 3. The insertion disrupts the endogenous murine Cd73 gene, resulting in a non-functional transcript.

CD73 expression analysis in B-hCD73 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hCD73 MC38 cultures were stained with species-specific anti-CD73 antibody. Human CD73 was detected on the surface of B-hCD73 MC38 cells but not wild-type MC38 cells. The 1-D02 clone of B-hCD73 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hCD73 MC38 cells. B-hCD73 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 6-8-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 × long diameter × short diameter². As shown in panel A, B-hCD73 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hCD73 MC38 tumor growth of individual mice. B-hCD73 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 6-8-week-old, n=5). As shown in panel, B-hCD73 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.