The chimeric CDS consist of human GUCY2C extracellular plus mouse Gucy2c intracellular region sequences was inserted to mouse Gucy2c gene exon 1 in B-hGUCY2C MC38 cells, so that the mouse GUCY2C was not expressed anymore, human GUCY2C is highly expressed on the surface of B-hGUCY2C MC38 cells.

Gene targeting strategy for B-hGUCY2C MC38 cells. The exogenous promoter and chimeric CDS consist of human GUCY2C extracellular plus mouse Gucy2c intracellular region sequences were inserted to mouse Gucy2c gene exon 1 in B-hGUCY2C MC38 cells.

B-hGUCY2C MC38 cells were subcutaneously transplanted into C57BL/6N mice (n=8). At the end of the experiment, tumor cells were harvested and assessed for human GUCY2C expression by flow cytometry. As shown, human GUCY2C was highly expressed on the surface of tumor cells. Therefore, B-hGUCY2C MC38 cells can be used for in vivo efficacy studies of novel GUCY2C therapeutics.

Subcutaneous homograft tumor growth of B-hGUCY2C MC38 cells. B-hGUCY2C MC38 cells (1x10⁶ ,5x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N 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 × long diameter × short diameter². As shown in panel A, B-hGUCY2C MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hGUCY2C MC38 tumor growth of individual mice. B-hGUCY2C MC38 cells (1x10⁶ ,5x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 7-week-old). As shown in panel, B-hGUCY2C MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hGUCY2C MC38 cells. B-hGUCY2C MC38 cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice mice (female, 9-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 × long diameter × short diameter². As shown in panel A, B-hGUCY2C MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hGUCY2C MC38 tumor growth of individual mice. B-hGUCY2C MC38 cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice mice (female, 9-week-old). As shown in panel, B-hGUCY2C MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.