The mouse Cd39 gene was replaced by human CD39 coding sequence in B-hCD39 MC38 cells. Human CD39 is highly expressed on the surface of B-hCD39 MC38 cells.

Gene targeting strategy for B-hCD39 MC38 cells. The promoter and human CD39 coding sequence were inserted into the targeting vector used in the lentivirus system.

CD39 and EGFP expression analysis in B-hCD39 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hCD39 MC38 cultures were stained with species-specific anti-CD39 antibody. Human CD39 was detected on the surface of B-hCD39 MC38 cells, but not on the surface of wild-type MC38 cells. EGFP was detected in B-hCD39 MC38 cells, but not in wild-type MC38 cells. The 1-F07 clone of B-hCD39 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hCD39 MC38 cells. B-hCD39 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hCD39 mice (female, 7-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 × long diameter × short diameter². As shown in panel A, B-hCD39 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hCD39 MC38 cells were subcutaneously transplanted into B-hCD39 mice (n=6). At the end of the experiment, tumor cells were harvested and assessed for human CD39 expression by flow cytometry. As shown, human CD39 was highly expressed on the surface of tumor cells. Therefore, B-hCD39 MC38 cells can be used for in vivo efficacy studies of novel CD39 therapeutics.

Subcutaneous homograft tumor growth of B-hCD39 MC38 cells. B-hCD39 MC38 cells (5x10⁵, 1x10⁶, 5x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hPD-1/hCD39 mice (female, 9-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-hCD39 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hCD39 MC38 cells were subcutaneously transplanted into B-hPD-1/hCD39 mice (n=5). At the end of the experiment, tumor cells were harvested and assessed for human CD39 expression by flow cytometry. As shown, human CD39 was highly expressed on the surface of tumor cells. Therefore, B-hCD39 MC38 cells can be used for in vivo efficacy studies of novel CD39 therapeutics.