Breast cancer (BC) is a leading malignancy in women worldwide (1) with extremely heterogeneous cell morphology (2, 3) including more than 20 distinct subtypes that differ genetically and clinically(4). Distant metastasis is the main cause of death in BC patients. Axillary lymph nodes (ALN) are the main doorway for tumor cell escape from the primary site to other regions of the body (5). Consequently, metastatic ALN (mALN) are considered the most important prognostic factors and powerful source of biomolecules that may become reliable metastatic biomarkers. In spite of that, very few studies have been conducted to identify BC biomarkers associated with the ALN metastasis of BC. Therefore, inclusion of new mALN molecular biomarker profiles has been proposed to predict nodal status at the time of BC diagnosis (6).
Considerable research attention has been focused on a role of deregulation of Transforming Growth Factor β1 (TGFβ1) as tumor promoter step favoring BC invasion and metastasis(7). Moreover, accumulating evidence shows that FOS transcription factor binding motifs are critical for the regulation of TGFβ1 expression (8). Thus, cFOS elevation may have utility as a complementary candidate biomarker of BC invasiveness, co-expressed with TGFβ1. Consequently, we have, previously proposed that cFOS and TGFβ1 proteins may be considered as a pair of biomarkers of an early assessment of invasive BC(7, 8), providing adequate invasive BC specimens are available. In the past, primary tumor tissue had been fractionated into nuclear(9) or cytosolic(10, 11) extract to assess specific biomarkers of interest. Until recently, however, the protocol for fractionation of mALN nuclear and cytosolic extracts has not been available, presumably due to the specific tough, fibrous nature of mALN tissue.
To date, diameter of tumor deposits and proliferation index Ki67(6, 12, 13) are the most prominent clinically used features of mALN. Both parameters are detected by routine histology(14) involving the tissue paraffin blocks of 4-µm slices for each node(15, 16) and their staining with either Haematoxylin and Eosin (H&E) or Immunohistochemistry (IHC), respectively(17). Although indispensable, the histology methodology imposes some limitations(16). Namely, in a significant portion of cases, due to clustered spacial distribution of tumor cells within a lymph node, the histology sectioning generates sampling errors leading to false negative mALN sections(18). Consequently, the pool of biomarkers in various slices may be different. To overcome this problem, Edwards and co-workers(19) introduced mALN Cell Suspension as new specimens thereby covering the whole content of entire mALN. Based on this mALN specimen source, we developed the method, termed Fractionation of Nodal Cell Suspension (FNCS), which includes the mALN Cell Suspension approach and its fractionation into nuclear and cytosolic extracts to be suitable for determination of protein expression levels of respective native proteins. Using this experimental design, we have previously observed overexpression of TGFβ1 protein in cytosolic extracts of mALN(7). Likewise, we encountered a case of an advanced Triple Negative Breast Cancer (TNBC) patient with overexpressed both cytosolic TGFβ1 and nuclear cFOS proteins as a pair of mALN biomarkers for an early assessment of TNBC poor prognosis(8). However, in above mentioned studies(7, 8), apart from the outlined methods used, specific experimental protocols were not described. Having in mind that the FNCS design might help to generate an important predictive tool suitable for comparative analysis of individual patients in present era of genomics and personalized medicine(2), we undertook the present study. The main goal was to describe the full methodology of establishment and fractionation of mALN Cell Suspension thus providing FNCS specimens of nuclear and cytosolic extracts and determination of protein expression levels of respective cFOS and TGFβ1.
The workflow of the this study is presented in Figure 1. The following steps, included in the
experimental design, were: i ) mechanical disaggregation of mALN, chopped and filtered through 100 µm sieve devices, to obtain mALN cell suspension free from fat and connective tissue (mALN Cell Suspension); ii) model protocol of HeLa cell fractionation into nuclear and cytosolic extracts to be implemented on mALN Cell Suspension to obtain FNCS specimens and generate nuclear/cytosolic extracts; iii) determination of protein expression level of nuclear cFOS and cytosolic TGFβ1 by ELISA; iv) correlation of the respective cFOS and TGFβ1 biomarker levels with mALN diameter of tumor deposits for each BC patient.
Since mALN tissue sample is heterogeneous in terms of its cell content (including: various BC malignant clones, fibroblasts, macrophages, lymphocytes etc.) this protocol enables the use of autologous normal ALN (nALN) of each patient as an optimal choice of negative control. Taken together, this study provides tools to researchers termed FNCS, in which mALN and nALN tissue samples are used as initial ex vivo materials, to follow the protocol “from tough mALN/nALN tissue, through mALN/nALN Cell Suspension, to fractionationate the nuclear/cytosolic extracts” and enable ELISA determination of respective protein biomolecules. The method provides considerable advantages, when compared to current pathohistological BC diagnostics which is, during routine examination, rather limited to defined slices which cannot cover the complete volume of the nodal tissue.