The mouse Ccr4 gene was replaced by human CCR4 coding sequence in B-hCCR4 MC38 cells. Human CCR4 is highly expressed on the surface of B-hCCR4 MC38 cells.

Gene targeting strategy for B-hCCR4 MC38 cells. The exogenous promoter and human CCR4 coding sequence were 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 on the surface of 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.