JONT Journal

Introduction

Circulating tumor cells (CTCs), play an important role during transition of epithelial to mesenchymal (EMT) cells and act as rich source of biomarker for early diagnosis in variety of tumors. CTCs shows genetic heterogeneous group of different subpopulation and may act as the promising tool for early monitoring of BC patients. BC, one of the most common cancer is associated with women health worldwide. Several studies have been shown in the literature that number of CTCs may be correlated with poor prognosis during progression of disease and metastasis in different cancer patients [1-7]. Liquid biopsy, is an integral part of CTCs, a minimal invasive technique is approved by FDA in cell-search system with limitations during monitoring of sub-population and their phenotypic expression using two different markers, CD45 and EpCAM [8-13]. Hence, CTCs, play a “central role” at all the stages of tumor including primary and metastatic event involving cytokeratin act as an active role to maintain tissue architecture and their plasticity [14-16].

5-Azacytidine (5AzaC), a DNA methylation cytostatic drug used by the clinicians for the management of acute leukemia and chronic myelogeneous leukemia belongs to pediatric agegroups [17- 19]. Besides this, it is also being used for solid tumors including adenocarcinoma during metastasis by interfering denovo pyrimidine synthesis. 5-AzaC, a DNA methyltransferase inhibitor that induces double strand breaks  (DSBs) including chromosome aberrations  (chromatid breaks) and segregation of chromosomes due to pronounced stickiness in proliferating cells in G2 and S phase of cell-cycle [20-22]. These neoplastic agents showing epigenetic modification of progenitor (stem) cells and makes the study more complex due to predisposition of tumor gene involving  several signaling pathways like p53 (tumor suppressor gene) and transforming growth factors (TGF) during tumor progression [23]. Sox4 gene is Sry-box transcription factor belongs to the high mobility group (HMG) region and their over-expression associated with cellular-proliferation in pre/post metastasis [24]. Epithelial cell adhesive molecule (EpCAM), a transmembrane conjugated glycoprotein that showed overexpression in variety of cancer including ovarian tumors [25]. Mechanism of transformation from epithelial to mesenchymal cells in CTCs has not been defined clearly in BC patients. However, the present study has been designed with the aims to evaluate the comparative sensitivity of Sox4 and EpCAM in CTCs population using monoclonal antibodies in flowcytometery. To validate of the above findings, the sensitivity was again evaluated using invitro system, where the peripheral blood lymphocytes (PBL) were exposed with 5-AzaC and compared with controls to evaluate genetic susceptibility towards drug sensitivity during therapeutics in breast cancer patients.

Materials and Methods

Peripheral blood lymphocytes (3.0 ml) samples were collected in EDTA sterile vials and stored at 4⁰C, till further study, after clinical diagnosis of breast cancer patients (n=47) from the OPD of All India Institute of Medical Sciences, Patna. Present study is dually approved by the IRC (Institute Research Committee) and Institute Ethical Committee (IEC).The samples were collected after informed consent from the patient or attendant.

Isolation of Circulating Tumour Cells using Ficoll’s Gradient Method

Short-term lymphocyte culture (n=47) were setup using RPMI1640 media, supplemented with 10% foetal bovine serum and phytohemagglutinin (PHA), and antibiotic. After harvesting, the cultured cells were used  for isolation of CTCs using Ficoll’s density gradient centrifugation from blood mononuclear cells (PBMC) ring at 400g for 30 min. Cells were washed twice with 1.0 ml PBS and again centrifuge at 1200rpm for 10 min at 4⁰C and stored at -20⁰C, till further characterization of CTCs using the standard protocol. The morphological characteristic of individual cells were also visualized under phase contrast microscope after staining with 1.0 % Giemsa to check the viability cells before flowcytometry.

CTCs staining for Sox4 and EpCAM markers

Sox4 (B-7) PE Lot # B2420, Catalog No sc-518016 monoclonal conjugated antibodies is used to detect intra cellular antigen (Santa Cruz Biotechnology USA). EpCAM PE (REF 12-579182, Lot 2384305) monoclonal antiMo-CD326 was purchased from Thermo fisher (USA) for extra cellular detection of antigen. Cell were fixed and permeabilization buffer is used according to the protocol of the kit (PerFix-nc, Beckman Coulter, France). Staining procedures were strictly followed by the manufacture’s protocol and 50 ul of CTCs were stained with 200 µg/ml for Sox4 PE and 100 µg for EpCAM PE monoclonal antibodies and incubated for 60 minutes. Cell were fixed for 10 minute in 5 µl of Per Fix-nc  buffer 1 followed by PerFix-nc  buffer 2 and then add antibodies , incubated for 30 minutes wash two times with 3.0 ml of phosphate buffer and centrifuge at 1600 rpm for 5 minutes to remove excess amount of residues. Now cell were allowed to keep in permeabilization buffer 3 (1:10 dilution) and centrifuge at 1600 rpm , finally resuspend pellet in 500 ul of PerFix-nc buffer 3 and cells are ready for acquisition on flowcytometry.

Samples Acquisition and Analysis of the Data analysis

Beckman Coulter Life Science DxFLEX software (Serial No BG10007 Model No DxFLEX) equipment is used for detection of CTCs with blue argon laser at 488 nm. FlowClean Cleaning Agent (100 ul) from Beckman Coulter is used for daily clean of instrument and QC/Standardization is passed using DxFLEX Daily QC Fluorospheres (REF C39283, LOT 15AQHF Beckman Coulter) before performing experiments. FSC and SSC data were analysed using CytExpert version 2.2 for DxFLEX software. Gating strategy were selected the Sox4 and EpCAM positive circulating tumor cells, exclude lymphocytes and erythrocytes. More explicitly on a CD326/SSC dot plot a gate was drawn to select only CD326 population.Sox4/SSC dot plot a gate was drawn to select Sox4 population. In another set of experiment, where, the cells were exposed with 5-AzaC for detection of Sox4 and EpCAM positive population of CTCs and compare with controls.

Statistical Analysis

Significance difference (p values) for odd ratio and confidence interval (C.I) at 95% was calculated between cases and controls using SPSS 27.0 version software (USA). X²- test (two tailed) was also used to observe the p values between experiment and controls.

Results

Table-1 showing the details statistical analysis data of two EMT markers Sox4 & EpCAM  in CTCs of  BC patients.

Sox4, a pluripotent stem cell marker  and compare to EpCAM (intracytoplasmic) EMT markers were assessed by flowcytometer using two different specific monoclonal antibodies for CTCs population that varying significantly findings between cases  and controls. Forward cell scattering (FCS) showing cell-size in mix population including CTCs, while pure population after gating is more than 98.27 % cells were score for EpCAM with (+) or without (-) antibodies labeled cells as shown in figure-1 A,B,C&D . The calculated values of odd ratio and C.I at 95% for EpCAM showing highly significant (p<0.001) difference with respect to controls. The sensitivity of these two markers Sox4 and EpCAM were again evaluated by in - vitro exposure with 5AzaC (24 hour) exposure in CTCs. EpCAM cell number (mean values) decrease more than two times i.e. +ve5AzaC 45540 to -ve5AzaC 17357 (controls). Graphically and histogram represents total cell population +veEpCAM and –ve EpCAM that showing the frequency of CTCs decrease significantly (p<0.05) in + EpCAM (98.27%) and - EpCAM (89%) as depicted in figure 2 A,B,C &

2D due to selective specificity towards antigen. The sensitivity of EpCAM was further evaluated using 5AzaC (1.0 µg/ml) in -vitro PBL exposed in CTCs of BC patients and compare with controls (unexposed), cell population again showing decrease (p<0.05) significantly. Interestingly, Sox4, cells number increases in treated (exposed) group 5+ ve AzaC 251443, as compare with controls (unexposed cells) 5AzaC -ve 12882.66 (fig.3 A,B,C&D). Age wise distribution of CTCs showing  that highest frequency (40%) of EpCAM in CTCs were observed between 51-60 & 61-70 year of age groups, but frequency of Sox4 decreases (30%) in 61-70 age groups in breast cancer patients (fig 4 A&B).

Figure-1A,B,C&D: Flowcytometric analysis for the detection of EpCAM in CTCs population from breast cancer patients. Forward cell scattering (FCS) showing cell-size in mix CTC population (fig.1A) and pure CTC population after gating (fig.1B). Graphically represents total cell population +ve EpCAM increase (blue) and –ve EpCAM (red)   as shown in fig.1C. Histogram showing frequency of cell population is 98.27 (%) in + EpCAM and - EpCAM (89%) as depicted in fig.1D.

Figure-2A,B,C & D: In-vitro 5-Azacytidine showing mix and pure CTCs population of EpCAM after gating (fig. 2A& B). Graphically represents total cell population +ve EpCAM (blue) and –ve EpCAM (red) as shown in fig. 2C. Histogram showing frequency of + EpCAM (98.27 %) cell population (fig.2D) with decrease trend significantly, when compare with controls due to cytotoxic nature of the drug.

Figure 3A,B,C &D: Sox4 showing after gating pure CTCs population from breast cancer patients (fig3A) visualize by two different colors, red –ve mAbSox4 and +vemAbSox4 (blue) as shown in fig.3B. In-vitro detection of CTCs population after exposure with 5-AzaC showing cell population after gating (fig.3C) and graphically represents total cell population significantly decrease with +vemAbSox4 (blue) and –ve mAbSox4 (red) as shown in fig.3D.

Figure-4A &B: Age-wise distribution and their percentage (%) frequency of transcription factor and epithelial-mesenchymal transition marker-Sox4 and EpCAM, respectively in circulating tumor cells isolated from breast cancer patients.

Table-1: Statistical analysis showing mean and their (%) frequency between two different markers Sox4 & EpCAM in circulating tumor cells population of breast cancer patients.