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.

Passage stability analysis in B-hGUCY2C MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hGUCY2C MC38 cultures were stained with species-specific anti-GUCY2C antibody. Human GUCY2C was specifically detected on the surface of B-hGUCY2C MC38 cells and remained stable across multiple passages, with no significant change in expression levels observed. No expression was detected in wild-type MC38 cells.

The passage number for this cell line is calculated starting from the first subculture after revival from the master cell bank, which is designated as P1.

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.

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.

Endocytosis and binding experiment results analysis in B-hGUCY2C MC38 cells by flow cytometry. Single cell suspensions from B-hGUCY2C MC38 #2-B03 cultures were analyzed with anti-human GUCY2C antibody GCC-TAK164 analog (in house) and Fab-human pH-sensor Red Antibody Labeling Reagent for FACS (in house) by flow cytometry. Results indicate that GCC-TAK164 exhibits normal internalization function in B-hGUCY2C MC38 cells (A, B). Human GUCY2C was detected on the surface of B-hGUCY2C MC38 cells (C).