Suzuki Hiroyuki, Tanaka Tomohiro, Goto Nohara, Kaneko Mika K, Kato Yukinari
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-Machi, Aoba-Ku, Sendai 980-8575, Japan.
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-Machi, Aoba-Ku, Sendai 980-8575, Japan.
Curr Issues Mol Biol. 2023 Feb 25;45(3):1875-1888. doi: 10.3390/cimb45030121.
CD44 has been known as a marker of tumor-initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in the malignant progression of cancers by promoting stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and the establishment of the therapy. However, the function of the variant 4-encoded region has not been elucidated. Therefore, specific monoclonal antibodies (mAbs) against variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) mAbs by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterize them. One of the established clones (CMab-108; IgG, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The of CMab-108 for CHO/CD44 v3-10 was 3.4 × 10 M. In western blot analysis, CMab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, CMab-108 stained formalin-fixed paraffin-embedded (FFPE) oral squamous carcinoma tissues in immunohistochemistry. These results indicated that CMab-108 is useful to detect CD44v4 in immunohistochemistry using FFPE tissues.
CD44一直被认为是肿瘤起始细胞的标志物，在许多癌症中发挥促肿瘤发生的功能。剪接变体通过促进干性、癌细胞侵袭或转移以及对化疗和放疗的抗性，在癌症的恶性进展中发挥关键作用。了解每个CD44变体（CD44v）的功能对于了解癌症的特性和治疗方法的建立至关重要。然而，变体4编码区域的功能尚未阐明。因此，针对变体4的特异性单克隆抗体（mAb）对于基础研究、肿瘤诊断和治疗不可或缺。在本研究中，我们用含有变体4编码区域的肽免疫小鼠，建立了抗CD44变体4（CD44v4）单克隆抗体。接下来，我们进行了流式细胞术、蛋白质印迹和免疫组织化学来对它们进行表征。其中一个建立的克隆（CMab-108；IgG，κ）与CD44v3-10过表达的中国仓鼠卵巢-K1细胞（CHO/CD44v3-10）反应。CMab-108对CHO/CD44 v3-10的亲和力为3.4×10 M。在蛋白质印迹分析中，CMab-108在CHO/CD44v3-10细胞的裂解物中检测到CD44v3-10。此外，在免疫组织化学中，CMab-108对福尔马林固定石蜡包埋（FFPE）的口腔鳞状癌组织进行了染色。这些结果表明，CMab-108可用于在使用FFPE组织的免疫组织化学中检测CD44v4。
