Expression cassettes containing the exogenous promoter and human TROP2 CDS were randomly inserted into the genome of B-hTROP2 CT26.WT cells. Human TROP2 was detected to be highly expressed on the surface of B-hTROP2 CT26.WT cells.

Gene targeting strategy for B-hTROP2 CT26.WT cells. Expression cassettes containing the exogenous promoter and human TROP2 CDS were randomly inserted into the genome of CT26.WT cells that do not express the mouse TROP2.

TROP2 expression analysis in B-hTROP2 CT26.WT cells by flow cytometry. Single cell suspensions from wild-type CT26.WT and B-hTROP2 CT26.WT cultures were stained with species-specific anti-TROP2 antibody. mTROP2 was not detected on the surface of wild-type CT26.WT cells. Human TROP2 was detected on the surface of B-hTROP2 CT26.WT cells but not wild-type CT26.WT cells. The 2-B04 clone of B-hTROP2 CT26.WT cells was used for in vivo experiments.

B-hTROP2 CT26.WT cells were subcutaneously transplanted into BALB/c mice (n=6), and on 24 days post inoculation, tumor cells were harvested and assessed for human TROP2 expression by flow cytometry. As shown, human TROP2 was highly expressed on the surface of tumor cells. Therefore, B-hTROP2 CT26.WT cells can be used for in vivo efficacy studies of novel TROP2 therapeutics.

Subcutaneous homograft tumor growth of B-hTROP2 CT26.WT cells. B-hTROP2 CT26.WT cells (1x10⁵) and wild-type CT26.WT cells (1x10⁵) were subcutaneously implanted into BALB/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-hTROP2 CT26.WT cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hTROP2 CT26.WT tumor growth of individual mice. B-hTROP2 CT26.WT cells (1x10⁵) and wild-type CT26.WT cells (1x10⁵) were subcutaneously implanted into BALB/c mice (female, 8-week-old, n=6). As shown in panel, B-hTROP2 CT26.WT cells were able to establish tumors in vivo and can be used for efficacy studies.