The mouse F3 gene was replaced by human F3 coding sequence in B-hF3 MC38 cells. Human F3 is highly expressed on the surface of B-hF3 MC38 cells.

Gene targeting strategy for B-hF3 MC38 cells. The exogenous promoter and human F3 coding sequence were inserted to replace part of murine exon 3 and all of exons 4~5. The insertion disrupts the endogenous murine F3 gene, resulting in a non-functional transcript.

F3 expression analysis in B-hF3 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hF3 MC38 cultures were stained with species-specific anti-F3 antibody. Human F3 was detected on the surface of B-hF3 MC38 cells, but not on the surface of wild-type MC38 cells. The 2-D03 clone of B-hF3 MC38 cells was used for in vivo experiments.

B-hF3 MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=5). At the end of the experiment, tumor cells were harvested and assessed for human F3 expression by flow cytometry. As shown, human F3 was highly expressed on the surface of tumor cells. Therefore, B-hF3 MC38 cells can be used for in vivo efficacy studies of novel F3 therapeutics.

Subcutaneous homograft tumor growth of B-hF3 MC38 cells. B-hF3 MC38 cells (2x10⁵, 5x10⁵, 1x10⁶) and wild-type MC38 cells (5x105) were subcutaneously implanted into C57BL/6 mice (female, 7-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-hF3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hF3 MC38 cells. B-hF3 MC38 cells (5x10⁵, 1x10⁶, 5x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hCD3E mice (female, 7-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-hF3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hF3 MC38 cells. B-hF3 MC38 cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice (female, 7-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume. (B)  Body weight. Volume was expressed in mm³ using the formula: V=0.5 X long diameter X short diameter². As shown in panel A, B-hF3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.