Suzuki Hiroyuki, Ozawa Kazuki, Tanaka Tomohiro, Kaneko Mika K, Kato Yukinari
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan.
Biomedicines. 2023 Apr 5;11(4):1099. doi: 10.3390/biomedicines11041099.
Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3-10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (CMab-34; IgG, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that CMab-34 is a specific mAb for CD44v7/8. Moreover, CMab-34 reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent of CMab-34 for CHO/CD44v3-10 and HSC-3 was 1.4 × 10 and 3.2 × 10 M, respectively. CMab-34 could detect CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that CMab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.
分化簇44（CD44）已被作为一种癌症干细胞（CSC）标志物进行研究，因为它在肿瘤恶性进展中起关键作用。剪接变体在许多癌症中过度表达，尤其是在鳞状细胞癌中，并且在促进肿瘤转移、获得CSC特性以及抵抗治疗方面发挥关键作用。因此，为了建立新的肿瘤诊断和治疗方法，应阐明每种CD44变体（CD44v）在癌症中的功能和分布。在本研究中，我们用CD44变体（CD44v3 - 10）胞外域免疫小鼠，并建立了多种抗CD44单克隆抗体（mAb）。其中一个建立的克隆（CMab - 34；IgG，κ）识别一个覆盖变体7和变体8编码区域的肽段，表明CMab - 34是CD44v7/8的特异性单克隆抗体。此外，通过流式细胞术，CMab - 34与过表达CD44v3 - 10的中国仓鼠卵巢-K1（CHO）细胞或口腔鳞状细胞癌（OSCC）细胞系（HSC - 3）发生反应。CMab - 34对CHO/CD44v3 - 10和HSC - 3的表观解离常数分别为1.4×10和3.2×10 M。CMab - 34在蛋白质印迹中可检测到CD44v3 - 10，并且在免疫组织化学中可对福尔马林固定石蜡包埋的OSCC进行染色。这些结果表明，CMab - 34在各种应用中可用于检测CD44v7/8，有望在OSCC诊断和治疗应用中发挥作用。
