Abstract
Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed PANC-1 cells. Among the established clones, C(44)Mab-18 (IgM, kappa) reacted with CHO/CD44v3-10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that C(44)Mab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that C(44)Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, C(44)Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant (K(D)) of C(44)Mab-18 for CHO/CD44v3-10 and HSC-3 was 1.6 × 10(-7) M and 1.7 × 10(-7) M, respectively. Furthermore, C(44)Mab-18 detected CD44v3-10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that C(44)Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.