Gene targeting strategy for B-hJAM3 MC38 cells. The exogenous promoter and human JAM3 coding sequence was inserted to replace part of murine exon 5 and all of exon 6. The insertion disrupts the endogenous murine JAM3 gene, resulting in a non-functional transcript.

JAM3 expression analysis in B-hJAM3 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hJAM3 MC38 cultures were stained with species-specific anti-JAM3 antibody. Human JAM3 was detected on the surface of B-hJAM3 MC38 cells but not wild-type MC38 cells. The 2-H12 clone of B-hJAM3 MC38 cells was used for in vivo tumor growth assays.

Subcutaneous homograft tumor growth of B-hJAM3 MC38 cells. B-hJAM3 MC38 cells (1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 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-hJAM3 MC38 cells were able to form tumors in vivo and can be used for efficacy studies.

B-hJAM3 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 JAM3 expression by flow cytometry. As shown, human JAM3 was highly expressed on the surface of tumor cells. Therefore, B-hJAM3 MC38 cells can be used for in vivo efficacy studies of JAM3 therapeutics.