Gene targeting strategy for B-hENPP3 MC38 cells. The exogenous promoter and human ENPP3 coding sequence was inserted to replace part of murine exon 4 and all of exon 5. The insertion disrupts the endogenous murine ENPP3 gene, resulting in a non-functional transcript.

ENPP3 expression analysis in B-hENPP3 MC38 cells by flow cytometry. Single cell suspensions from B-hENPP3 MC38 cultures were stained with species-specific anti-ENPP3 antibody. Human ENPP3 was detected on the surface of B-hENPP3 MC38 cells. The 2-A12 clone of B-hENPP3 MC38 cells was used for in vivo experiments.

B-hENPP3 MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=6). At the end of the experiment, tumor cells were harvested and assessed for human ENPP3 expression by flow cytometry. As shown, human ENPP3 was highly expressed on the surface of tumor cells. Therefore, B-hENPP3 MC38 cells can be used for in vivo efficacy studies of novel ENPP3 therapeutics.

Subcutaneous homograft tumor growth of B-hENPP3 MC38 cells. B-hENPP3 MC38 cells (1x10⁶) were subcutaneously implanted into C57BL/6N mice (female, 10-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-hENPP3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hENPP3 MC38 tumor cells growth of individual mice. B-hENPP3 MC38 cells (1x10⁶) were subcutaneously implanted into C57BL/6 mice (female, 10-week-old, n=6). As shown in panel, B-hENPP3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.