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

GPNMB expression analysis in B-hGPNMB MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hGPNMB MC38 cultures were stained with species-specific anti-GPNMB antibody. Human GPNMB was detected on the surface of B-hGPNMB MC38 cells but not wild-type MC38 cells. The 3-B12 clone of B-hGPNMB MC38 cells was used for in vivo experiments.

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