• Origin: The B16-F10 cell line is derived from C57BL/6 murine skin cells. The cell line is a commonly used murine model for melanoma.
• Background Information: GUCY2C (Guanylyl Cyclase C), also known as heat-stable enterotoxin receptor, is a type I transmembrane protein of the guanylate cyclase (gc) family that signal by producing cGMP. GUCY2C in epithelial cells plays an important role in cell dynamics and homeostatic balance of proliferation, metabolism, and differentiation that organizes the guanylyl cyclase C hormone axis. GUCY2C is also expressed in the brain and is implicated in attention deficiency and hyperactive behavior. CAR-T cell therapy utilizing GUCY2C to treat metastatic colorectal cancer is currently being explored.
• Gene targeting strategy: The exogenous promoter and chimeric GUCY2C coding sequence that include human extracellular region and mouse intracellular region were inserted to replace part of murine Gucy2c exon 1. The insertion disrupts the endogenous murine Gucy2c gene, resulting in a non-functional transcript.
• Tumorigenicity: Confirmed in C57BL/6 mice.
• Application: The B-hGUCY2C B16-F10 tumor models can be used for preclinical evaluation of ADC and monoclonal antibody drugs targeting human GUCY2C.
• Notes: 

Inoculated cell lines can be suspended with DMEM stock solution. 

Before implementing the project, it is recommended to perform tumor growth experiments. The recommended cell inoculation amount is between 2E5. 

In the experiment, it is necessary to ensure that the number of animals inoculated subcutaneously is at least 1.6 times the actual grouping number.

GUCY2C expression analysis in B-hGUCY2C B16-F10 #4-D10 by flow cytometry. Single cell suspensions from wild-type B16-F10 and B-hGUCY2C B16-F10 #4-D10 cultures were stained with human GUCY2C Antibody (R&D, FAB650P). Human GUCY2C was detected on the surface of B-hGUCY2C B16-F10 #4-D10 cells, but not on the surface of wild-type B16-F10 cells. The clone of B-hGUCY2C B16-F10 #4-D10 cell were used for in vivo tumor growth assays.

GUCY2C expression evaluated on B-hGUCY2C B16-F10 tumor cells by flow cytometry. B-hGUCY2C B16-F10 cells were subcutaneously transplanted into C57BL/6 mice (n=6). Upon conclusion of the experiment, tumor cells were harvested and assessed for human GUCY2C expression by flow cytometry (Human GUCY2C Antibody, R&D, FAB650P). As shown, human GUCY2C was highly expressed on the surface of tumor cells. Therefore, B-hGUCY2C B16-F10 cells can be used for in vivo efficacy studies evaluating novel GUCY2C therapeutics.

Subcutaneous tumor growth of B-hGUCY2C B16-F10. B-hGUCY2C B16-F10 (2×10⁵) and wild-type B16-F10 cells (2×10⁵) were subcutaneously implanted into C57BL/6 mice (female, 7-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that B-hGUCY2C B16-F10 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.