Gene targeting strategy for B-Tg(hMICA) MC38. The coding sequence of human MICA CDS was randomly inserted into the genome of B-Tg(hMICA) MC38. Human MICA is highly expressed on the surface of B-Tg(hMICA) MC38.

Human MICA expression analysis in B-Tg(hMICA) MC38 by flow cytometry. Single cell suspensions from wild-type MC38 and B-Tg(hMICA) MC38 cultures were stained with species-specific anti-MICA antibody. Human MICA was detected on the surface of B-Tg(hMICA) MC38 cells but not wild-type MC38 cells. The 2-G12 clone of B-Tg(hMICA) MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-Tg(hMICA) MC38 cells. B-Tg(hMICA) MC38  cells (5x10⁵) were subcutaneously implanted into B-hNKG2D mice (male, 7-week-old, n=6). Tumor volume and body weight were measured three times 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-Tg(hMICA) MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-Tg(hMICA) MC38 cells were subcutaneously transplanted into B-hNKG2D mice (n=6). At the end of the experiment, tumor cells were harvested and assessed for human MICA expression by flow cytometry. As shown, human MICA was highly expressed on the surface of tumor cells. Therefore, B-Tg(hMICA) MC38 cells can be used for in vivo efficacy studies of novel MICA therapeutics.

Shedding MICA expression in the cell supernatant and tumor cells. Shedding MICA was detected in the B-Tg(hMICA) MC38 derived from the cell supernatant but not the K562 and MC38 cells(A). Shedding MICA was detected in the tumor tissues derived from the 2-G12 clone but not the MC38 cells(B).