Gene targeting strategy for B-hCCR4 MC38 cells. The exogenous promoter and human CCR4 coding sequence was inserted to replace part of murine exon 2. The insertion disrupts the endogenous murine Ccr4 gene, resulting in a non-functional transcript.

CCR4 expression analysis in B-hCCR4 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hCCR4 MC38 cultures were stained with species-specific anti-CCR4 antibody. Human CCR4 was detected on the surface of B-hCCR4 MC38 cells but not wild-type MC38 cells. The 2-F05 clone of B-hCCR4 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hCCR4 MC38 cells. B-hCCR4 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 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 X long diameter X short diameter². As shown in panel A, B-hCCR4 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. (A) Average tumor volume ± SEM. (B)  Body weight (Mean± SEM). 

B-hCCR4 MC38 cells were subcutaneously transplanted into C57BL/6N mice (n=5). At the end of the experiment, tumor cells were harvested and assessed for human CCR4 expression by flow cytometry. As shown, human CCR4 was highly expressed on the surface of tumor cells. Therefore, B-hCCR4 MC38 cells can be used for in vivo efficacy studies of novel CCR4 therapeutics.