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

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

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

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

Subcutaneous homograft tumor growth of B-hTF MC38 cells. B-hTF 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-hTF MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hTF MC38 tumor growth of individual mice. B-hTF MC38 cells (2x105, 5x105, 1x106) and wild-type MC38 cells (5x105) were subcutaneously implanted into C57BL/6 mice (female, 7-week-old, n=5). As shown in panel, B-hTF MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hTF MC38 cells. B-hTF 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-hTF MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hTF MC38 tumor growth of individual mice. B-hTF MC38 cells (5x105, 1x106, 5x106) and wild-type MC38 cells (5x105) were subcutaneously implanted into B-hCD3E mice (female, 7-week-old, n=6). As shown in panel, B-hTF MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hTF MC38 cells. B-hTF 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-hTF MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.