The mouse Cd24 gene was replaced by human CD24 coding sequence in B-hCD24 MC38 cells. Human CD24 is highly expressed on the surface of B-hCD24 MC38 cells.

Gene targeting strategy for B-hCD24 MC38 cells. The exogenous promoter and human CD24 coding sequence was inserted to replace part of murine exon 1 and all of exon 2. The insertion disrupts the endogenous murine Cd24 gene, resulting in a non-functional transcript.

CD24 expression analysis in B-hCD24 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hCD24 MC38 cultures were stained with species-specific anti-CD24 antibody. Human CD24 was detected on the surface of B-hCD24 MC38 cells but not wild-type MC38 cells. The 1-F02 clone of B-hCD24 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hCD24 MC38 cells. B-hCD24 MC38 cells (1x10⁶) and wild-type MC38 cells (1x10⁶) were subcutaneously implanted into C57BL/6 mice (female, 6-7-week-old, n=5). 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-hCD24 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hCD24 MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=5). At the end of the experiment, tumor cells were harvested and assessed for human CD24 expression by flow cytometry. As shown, human CD24 was highly expressed on the surface of tumor cells. Therefore, B-hCD24 MC38 cells can be used for in vivo efficacy studies of novel CD24 therapeutics.