Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

Abstract

Background Metastatic Axillary Lymph Node (mALN) status is currently the most important prognostic factor in the management of primary breast cancer (BC). Thus, development of specimens which enable identification of new mALN markers, involved in the progression of the disease, are of considerable interest. The specific aim of this work was to describe the method of establishment of Metastatic Axillary Nodal Cell Suspension and its fractionation, termed Fractionated Nodal Cell Suspension (FNCS), into nuclear and cytosolic extracts to enable determination of protein expression levels of nuclear cFOS and cytosolic Transforming Growth Factor β1 (TGFβ1) in BC patients. Results To standardize the procedure, HeLa cells were successfully fractionated into nuclear/cytosolic extracts with confirmed presence of nuclear cFOS and cytosolic TGFβ1 proteins. Subsequently, the ALN Cell Suspension specimens were obtained and further fractionated from a pilot sample of six ALN tissue pairs, mALN versus autologous normal ALN (nALN), dissected from invasive BC patients. The mALN/nALN results revealed overexpression of both nuclear cFOS and cytosolic TGFβ1 protein levels. However, only the TGFβ1 data exhibited statistically significant overexpression, which was proportional to the respective values of mALN diameter of tumor deposits. Conclusions Detailed protocol for establishment and fractionation of mALN cell suspension specimens, termed FNCS, into nuclear and cytosolic extracts is here described for the first time. This approach might be a convenient ex vivo model for simultaneous analysis of protein, RNA and DNA biomarkers from nuclear/cytosolic extracts of the same mALN tissue sample. It might have potential to enable, in the age of genomics and personalized medicine, an identification of novel mALN biomarkers and thus improve the screening, diagnosis and prognosis of invasive BC.

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

“…Consequently, the pool of biomarkers in various slices may be different. In contrast, in developed FNCS method (17) we used mALN cell suspension preparation as a specimen source, and its fractionation into nuclear and cytosolic extracts suitable for further analysis of native protein expression. Using this experimental design, we have previously observed overexpression of Transforming Growth Factor Beta 1 (TGFβ1) in cytosolic extracts of mALN (17).…”

“…In contrast, in developed FNCS method (17) we used mALN cell suspension preparation as a specimen source, and its fractionation into nuclear and cytosolic extracts suitable for further analysis of native protein expression. Using this experimental design, we have previously observed overexpression of Transforming Growth Factor Beta 1 (TGFβ1) in cytosolic extracts of mALN (17). Likewise, we encountered a case of an advanced Triple Negative Breast Cancer (TNBC) patient with concomitant overexpression of both, cytosolic TGFβ1 and nuclear cFOS proteins as a pair of potential mALN biomarkers for an early assessment of TNBC poor prognosis (17).…”

“…The fractionation was performed as we previously described in details (17). Initial mALN tissue total weight was 497 mg with total viable cell number of 7.8x10 6 , and nuclear and cytosolic total protein yield of 1.58 mg and 2.13 mg, respectively.…”

“…Protein concentration of N and C compartments was analyzed in microplates by a micro Lowry assay((20) as we previously described (17). (Western blotting was performed essentially as described by Ivkovic et al (21).…”

“…The main objective of the present study was to con rm the e cacy of our recently developed and published method (17), termed Fractionation of Nodal Cell Suspension (FNCS), for subcellular compartmentalization of mALN. The method includes the protocols "from tough mALN tissue, through mALN cell suspension, to fractionationated nuclear (N) and cytosolic (C) compartments".…”