Abstract：
We present herein an extension to our recently developed and published method termed \"Fractionation of Nodal Cell Suspension\" (FNCS). The method enables efficient subcellular fractionation into nuclear (N) and cytosolic (C) compartments of extremely fibrous and problematic metastatic axillary lymph node (mALN) tissue, using the entire nodule. For the purpose of the present study, a case of invasive lobular breast cancer (BC) patient with pT2N3aMx status and defined primary tumor markers (ERα 8, PR-B 8, and HER2 score 0) was available. Initially, the mALN tissue of this patient was analyzed by immunohistochemistry (IHC), and a positive correlation of nodal ERα, PR-B and HER2 biomarkers to those of the primary tumor was obtained. Subsequently, the mALN was FNCS fractionated into N and C, and Western blot (WB) analysis demonstrated a single band for ERα, PR-B and nuclear loading control (HDAC1) in nuclear, but not in the cytosolic compartments, confirming the efficiency of our fractionation protocol. At the same time, HER2 bands were not observed in either compartment, in accordance with HER2 negativity determined by IHC in both primary tumor and mALN tissue. In conclusion, by confirming the nuclear expression of ERα and PR-B biomarkers in metastatic loci, we demonstrate the purity of the FNCS-generated compartments - the protocol that offers a reliable tool for further analysis of nuclear versus cytosolic content in downstream analysis of novel biomarkers in the whole mALN of BC patients.
摘要：
我们在此介绍了我们最近开发和公开的方法的扩展，称为“结节细胞悬浮液的分级分离”（FNCS）。该方法能够有效地将亚细胞分级分离成非常纤维和有问题的转移性腋窝淋巴结（mALN）组织的核（N）和胞质（C）隔室，使用整个结节。就本研究而言，1例浸润性小叶乳腺癌(BC)患者具有pT2N3aMx状态和确定的原发肿瘤标志物(ERα8,PR-B8和HER2评分0).最初，通过免疫组织化学（IHC）分析该患者的mALN组织，和淋巴结ERα的正相关，获得了原发肿瘤的PR-B和HER2生物标志物。随后，MALN被FNCS分为N和C，和蛋白质印迹（WB）分析显示ERα的单个条带，PR-B和核负荷控制（HDAC1），但不是在胞质区室，确认我们的分馏方案的效率。同时,在任一区室均未观察到HER2条带，根据IHC在原发性肿瘤和mALN组织中确定的HER2阴性。总之,通过证实ERα和PR-B生物标志物在转移位点的核表达，我们证明了FNCS产生的区室的纯度-该方案为在BC患者整个mALN的新型生物标志物的下游分析中进一步分析细胞核与细胞溶质含量提供了可靠的工具.
