The mouse Fn14 gene was replaced by human FN14 coding sequence in B-hFN14 MC38 cells. Human FN14 is highly expressed on the surface of B-hFN14 MC38 cells.

Gene targeting strategy for B-hFN14 MC38 cells. The exogenous promoter and human FN14 coding sequence were inserted to replace part of murine exon 1 and all of exons 2-4. The insertion disrupts the endogenous murine Fn14 gene, resulting in a non-functional transcript.

FN14 expression analysis in B-hFN14 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hFN14 MC38 cultures were stained with species-specific anti-FN14 antibody. Human FN14 was detected on the surface of B-hFN14 MC38 cells, but not on the surface of wild-type MC38 cells. The 3-G11 clone of B-hFN14 MC38 cells was used for in vivo experiments.

B-hFN14 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 FN14 expression by flow cytometry. As shown, human FN14 was highly expressed on the surface of tumor cells. Therefore, B-hFN14 MC38 cells can be used for in vivo efficacy studies of novel FN14 therapeutics.

Subcutaneous homograft tumor growth of B-hFN14 MC38 cells. B-hFN14 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 mice (female, 6-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-hFN14 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hFN14 MC38 cells. B-hFN14 MC38 cells (5x10⁵, 1x10⁶, 5x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hCD3E mice (female, 7-week-old). 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-hFN14 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hFN14 MC38 cells. B-hFN14 MC38 cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice (female, 9-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-hFN14 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

B-hFN14 MC38 tumor growth of individual mice. B-hFN14 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 6-week-old, n=5). As shown in panel, B-hFN14 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hFN14 MC38 tumor growth of individual mice. B-hFN14 MC38 cells (5x10⁵, 1x10⁶, 5x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hCD3E mice (female, 7-week-old). As shown in panel, B-hFN14 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hFN14 MC38 tumor growth of individual mice. B-hFN14 MC38 cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice (female, 9-week-old, n=6). As shown in panel, B-hFN14 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.