The mouse Tnfr2 gene was replaced by human TNFR2 coding sequence in B-hTNFR2 MC38 cells. Human TNFR2 is highly expressed on the surface of B-hTNFR2 MC38 cells.

Gene targeting strategy for B-hTNFR2 MC38 cells. The exogenous promoter and human TNFR2 coding sequence were inserted to replace the extracellular region of the mouse Tnfr2 gene. The insertion disrupts the endogenous murine Tnfr2 gene, resulting in a non-functional transcript.

TNFR2 expression analysis in B-hTNFR2 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hTNFR2 MC38 cultures were stained with species-specific anti-TNFR2 antibody. Mouse TNFR2 was detected on the surface of wild-type MC38 cells. Human TNFR2 was detected on the surface of B-hTNFR2 MC38 cells, but not on the surface of wild-type MC38 cells. The 6-D05 clone of B-hTNFR2 MC38 cells was used for in vivo experiments.

B-hTNFR2 MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=5), and on 27 days post inoculation, tumor cells were harvested and assessed for human TNFR2 expression by flow cytometry. As shown, human TNFR2 was expressed on the surface of tumor cells. Therefore, B-hTNFR2 MC38 cells can be used for in vivo efficacy studies of novel TNFR2 therapeutics.

Subcutaneous homograft homograft tumor growth of B-hTNFR2 MC38 cells. B-hTNFR2 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 6-9-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 X long diameter X short diameter². As shown in panel A, B-hTNFR2 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hTNFR2 MC38 tumor growth of individual mice. B-hTNFR2 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 6-9-week-old, n=5). As shown in panel, B-hTNFR2 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.