Wound fluids collected postoperatively from patients with breast cancer induce epithelial to mesenchymal transition but intraoperative radiotherapy impairs this effect by activating the radiation-induced bystander effect

Katarzyna Kulcenty,Mateusz Wichtowski,Igor Piotrowski,Joanna Wróblewska,Wiktoria Maria Suchorska,Dawid Murawa,Karolina Zaleska

doi:10.1038/s41598-019-44412-y

Abstract

Wound fluids (WF) are believed to play a role in the local recurrences by inducing an inflammatory process in scar tissue area. Given that most local relapse in primary breast cancer patients occur within the scar tissue area, researchers have investigated whether localized radiotherapy, such as intraoperative radiotherapy (IORT), could be more effective than postoperative RT in inhibiting local tumor recurrence. The epithelial-mesenchymal transition (EMT) program plays a critical role in promoting metastasis in epithelium-derived carcinoma. Given this background the main aim of the present study was to determine the mechanisms by which IORT decreases the tumorigenic potential of WF. We assumed that postoperative fluids from patients would activate the radiation-induced bystander effect (RIBE) in treated cells, thus altering the tumor microenvironment. To confirm this hypothesis, WF collected from patients after breast conserving surgery (BCS) alone, after BCS followed by IORT treatment or WF from BCS patients together with RIBE medium were incubated with MCF7 and MDA-MB-468 cells. Changes in the CSC phenotype, in EMT program and potential to migrate were performed to determine the possible role of WF on the migration of breast cancer cells. Our findings show that wound fluids stimulate the CSC phenotype and EMT program in breast cancer cell lines. This effect was partially abrogated when the cells were incubated in wound fluids collected from patients after breast-conserving surgery followed by IORT. Additionally, we confirmed the role of radiation-induced bystander effect in altering the properties of the WF to induce the CSC phenotype and EMT program.

Highlights

Breast-conserving surgery (BCS) followed by fractionated external beam radiation therapy (EBRT) is a standard treatment approach for early breast cancer
We first analyzed the changes in the cancer stem cell (CSC) phenotype of breast cancer cell lines after incubation with 3 different groups of Wound fluids (WF): (1) RT-WF, defined as WF collected from patients after breast conserving surgery (BCS) plus intraoperative radiotherapy (IORT) treatment, (2) WF, defined as WF collected from patients after BCS alone, and (3) WF + radiation-induced bystander effect (RIBE), defined as WF collected from patients after surgery alone with the addition of the RIBE medium
We did not see any changes in ALDH activity in MDA-MB-468 cell line, probably due to very high ALDH activity in CTR cells (95% of ALDH positive cells). These results show that postoperative WF stimulates a CSC phenotype in MCF7 and MDA-MB-468 cell lines, and that this effect in partially abrogated in both cell lines in WF collected from patients who underwent IORT (RT-WF) and in cases in which WF-treated cells were incubated in the RIBE medium

Summary

Introduction

Breast-conserving surgery (BCS) followed by fractionated external beam radiation therapy (EBRT) is a standard treatment approach for early breast cancer. Recent findings indicate that the EMT process confers mesenchymal cells stem cell features that enable carcinoma cells to metastasize at secondary sites[19] Given this background, the aim of the present study was to examine the effect of WF obtained from patients after BCS alone (WF group) and after BCS plus IORT (RT-WF group) 48 hours after surgery on cancer stem cell (CSC) phenotype and induction of the EMT program in two histopathologically different breast cancer cell lines (luminal A MCF7 and basal MDA-MB-468). Our previously published data indicated the changes of biological activity between surgical fluids from BCS patients and BCS patients followed by IORT treatment and the composition of those fluids Based on these data we hypothesized that this effect might be in part caused by RIBE factors secreted by cells irradiated by IORT to tumor bed[20]. We believe that the present findings will contribute both to a better understanding of the role of wound fluids in tumor progression and to the impact of non-targeted radiation effects on epithelial to mesenchymal process

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