The mouse Cdh17 gene was replaced by human CDH17 coding sequence in B-hCDH17 MC38 cells. Human CDH17 is highly expressed on the surface of B-hCDH17 MC38 cells.

Gene targeting strategy for B-hCDH17 MC38 cells. The exogenous promoter and human CDH17 coding sequence was inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Cdh17 gene, resulting in a non-functional transcript.

CDH17 expression analysis in B-hCDH17 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hCDH17 MC38 cultures were stained with anti-CDH17 antibody. Human CDH17 was detected on the surface of B-hCDH17 MC38 cells but not wild-type MC38 cells. Mouse CDH17 was not detected on the surface of wild-type MC38 cells (data not shown).

B-hCDH17 MC38 cells (5x106) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 8-week-old, n=6). At the end of the experiment, tumor cells were harvested and assessed for human CDH17 expression by flow cytometry. As shown, human CDH17 was highly expressed on the surface of tumor cells. Therefore, B-hCDH17 MC38 cells can be used for in vivo efficacy studies of novel CDH17 therapeutics.

Binding of an anti-human CDH17 antibody to B-hCDH17 MC38 cells. B-hCDH17 MC38#1-B09, B-hCDH17 MC38#1-H03 cells and the positive control SNU-5 cell line were collected and analyzed by flow cytometry using anti-hCDH17 antibody (CDH17H2, in-house). The antibody bound to both B-hCDH17 MC38 and SNU-5 cells.

Endocytosis experiment results analysis in B-hCDH17 MC38 cells by flow cytometry. B-hCDH17-MC38#1-B09 cells were incubated for a set time with a pre-formed complex of anti-hCDH17 antibody (CDH17H2, in-house ) and a fluorophore-conjugated secondary antibody. Following incubation, cellular uptake of the antibody complex was measured by flow cytometry to assess internalization capability. The results demonstrate efficient uptake in B-hCDH17-MC38 cells (A, B).

Endocytosis experiment results analysis in B-hCDH17 MC38 cells by flow cytometry. B-hCDH17-MC38#1-H03 cells were incubated for a set time with a pre-formed complex of anti-hCDH17 antibody (CDH17H2, in-house ) and a fluorophore-conjugated secondary antibody. Following incubation, cellular uptake of the antibody complex was measured by flow cytometry to assess internalization capability. The results demonstrate efficient uptake in B-hCDH17-MC38 cells (A, B).

Endocytosis experiment results analysis in SNU-5 cells by flow cytometry. SNU-5 cells were incubated for a set time with a pre-formed complex of anti-hCDH17 antibody (CDH17H2, in-house ) and a fluorophore-conjugated secondary antibody. Following incubation, cellular uptake of the antibody complex was measured by flow cytometry to assess internalization capability. The results demonstrate efficient uptake in SNU-5 cells (A, B).

Subcutaneous homograft tumor growth of B-hCDH17 MC38 cells. B-hCDH17 MC38 cells (5x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 8-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 × long diameter × short diameter². As shown in panel A, B-hCDH17 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hCDH17 MC38 cells. B-hCDH17 MC38 cells (3x10⁶) were subcutaneously implanted into B-hCDH17 mice (female, 6-7-week-old, n=7). 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-hCDH17 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.