Western Blot analysis confirms nuclear localization of nodal ERα and PRB protein biomarkers in fractionated axillary node-positive breast cancer tissue: A Case Report

Abstract

Abstract We present herein a Western blot (WB) approach as an extension to our recently developed and published method termed "Fractionation of Nodal Cell Suspension" (FNCS). The method enables an efficient subcellular fractionation into nuclear (N) and cytosolic (C) compartments of extremely fibrous and problematic metastatic Axillary Lymph Node (mALN) tissue. For the purpose of present experiments, a case of an invasive lobular breast cancer (BC) patient, with pT2N3aMx clinico-pathological characteristics and defined primary tumor markers (ERα 8, PRB 8, and HER2 score 0), was selected. Initially, mALN tissue of this patient was analyzed by immunohistochemistry (IHC) and a positive correlation of nodal ERα, PRB and HER2 biomarkers to those of primary tumor was obtained. Subsequently, the mALN was FNCS-fractionated into N and C and WB analysis demonstrateted a single N band for nodal ERα, PRB biomarkers and nuclear loading control (HDAC1), but not the C band, revealing negligible compartmental cross-contamination. At the same time, HER2 bands were not observed in either of compartments, reflecting lack of HER2 expression consistent with IHC status in both primary tumor and mALN tissue. In conclusion, our results confirm the nuclear expression of ERα, and PRB biomarkers in metastatic loci. Finally, our results clearly demonstrate the purity of the FNCS-generated compartments - the protocol that offers reliable tool for further analysis of nuclear versus cytosolic content in downstream analysis of potential biomarkers in mALN of BC patients.