Gene targeting strategy for B-hGPC3 CT26.WT cells. The exogenous promoter and human GPC3 coding sequence was inserted into the exon 1 of mouse Gpc3 in B-hGPC3 CT26.WT cells. The insertion disrupts the endogenous murine Gpc3 gene, resulting in a non-functional transcript.

GPC3 expression analysis in B-hGPC3 CT26.WT cells by flow cytometry. Single cell suspensions from wild-type CT26.WT and B-hGPC3 CT26.WT cultures were stained with species-specific anti-GPC3 antibody. Human GPC3 was detected on the surface of B-hGPC3 CT26.WT cells but not wild-type CT26.WT cells. The 2-F01 clone of B-hGPC3 CT26.WT cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hGPC3 CT26.WT cells. B-hGPC3 CT26.WT cells (1x10⁵) and wild-type CT26.WT cells (1x10⁵) were subcutaneously implanted into BABL/c 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 X long diameter X short diameter². As shown in panel A, B-hGPC3 CT26.WT cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hGPC3 CT26.WT tumor cells growth of individual mice. B-hGPC3 CT26.WT cells (1x10⁵) and wild-type CT26.WT cells (1x10⁵) were subcutaneously implanted into BABL/c mice (female, 8-week-old, n=6). As shown in panel, B-hGPC3 CT26.WT cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hGPC3 CT26.WT cells were subcutaneously transplanted into BABL/c mice (n=6), and on 21 days post inoculation, tumor cells were harvested and assessed for human GPC3 expression by flow cytometry. As shown, human GPC3 was highly expressed on the surface of tumor cells. Therefore, B-hGPC3 CT26.WT cells can be used for in vivo efficacy studies of novel GPC3 therapeutics.