It has been shown that overexpression of activated leukocyte cell adhesion molecule (ALCAM) is involved in development of resistance to tamoxifen therapy and promotion of cell invasion, migration and metastasis in ER+ breast cancer cells. Thus, we hypothesized that blockade of ALCAM interconnections with antibodies could be an effective approach for reversing mentioned negative events associated with ALCAM overexpression in breast cancer cells. Here, an anti‐ALCAM scFv was recombinantly expressed and used throughout study for examination of the putative anticancer effects of ALCAM blockade. The anti‐ALCAM scFv coding sequence was obtained from GenBank database and after addition of a 6× His‐tag moiety, signal peptide and flanking sequences, the whole construct was expressed in Escherichia coli . Tamoxifen resistant MCF7 cells were then pretreat for 24 hours with purified recombinant anti‐ALCAM scFv prior to administration of tamoxifen. In parallel, the cytotoxicity profile of anti‐ALCAM scFv and tamoxifen co‐treatments against tamoxifen resistant and sensitive MCF7 cell lines was also evaluated using CompuSyn software. The invasion/migration inhibitory effects of anti‐ALCAM scFv on MDA‐MB‐231 cells were also evaluated. Pretreatment with anti‐ALCAM scFv could successfully enhance anti‐proliferative effects of tamoxifen against resistant MCF‐7 cell lines. Furthermore, the combination of 19.2:1 of tamoxifen to anti‐ALCAM scFv demonstrated synergistic cell inhibitory effect against tamoxifen resistant MCF7 cell lines. Also, incubating MDA‐MB‐231 cell lines with anti‐ALCAM scFv resulted in a 30% and 25% reduction in number of invaded and migrated cells respectively. Overall, application of anti‐ALCAM scFv could significantly suppress cancer cells metastasis in vitro and modulate tamoxifen resistant ER+ MCF7 cell line's sensitivity to tamoxifen.

中文翻译：

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重组抗ALCAM scFv对乳腺癌细胞的双重体外侵袭/迁移抑制和他莫昔芬应答调节作用

已经显示，活化的白细胞粘附分子（ALCAM）的过表达参与了对他莫昔芬疗法的抗性的发展以及促进ER +乳腺癌细胞的细胞侵袭，迁移和转移。因此，我们假设用抗体阻断ALCAM互连可能是逆转上述与乳腺癌细胞中ALCAM过表达相关的阴性事件的有效方法。在这里，抗ALCAM scFv重组表达并在整个研究中用于检查ALCAM阻滞的假定抗癌作用。抗ALCAM scFv编码序列从GenBank数据库获得，并添加6x His-tag部分，信号肽和侧翼序列后，整个构建体在大肠杆菌中表达。然后在使用他莫昔芬之前，用纯化的重组抗ALCAM scFv对他莫昔芬耐药的MCF7细胞进行预处理24小时。同时，还使用CompuSyn软件评估了抗ALCAM scFv和他莫昔芬共同治疗对他莫昔芬耐药和敏感的MCF7细胞系的细胞毒性谱。还评估了抗ALCAM scFv对MDA-MB-231细胞的侵袭/迁移抑制作用。抗ALCAM scFv预处理可以成功增强他莫昔芬对耐药MCF-7细胞系的抗增殖作用。此外，他莫昔芬19.2：1与抗ALCAM scFv的组合显示出对他莫昔芬耐药MCF7细胞系的协同细胞抑制作用。也，将MDA-MB-231细胞系与抗ALCAM scFv一起孵育，分别可使入侵和迁移的细胞数量减少30％和25％。总体而言，抗ALCAM scFv的应用可以显着抑制体外癌细胞的转移，并调节抗他莫昔芬的ER + MCF7细胞系对他莫昔芬的敏感性。