Wound Fluid from Breast Cancer Patients Undergoing Intraoperative Radiotherapy Exhibits an Altered Cytokine Profile and Impairs Mesenchymal Stromal Cell Function

Abstract

Intraoperative radiotherapy (IORT) displays an increasingly used treatment option for early breast cancer. It exhibits non-inferiority concerning the risk of recurrence compared to conventional external irradiation (EBRT) in suitable patients with early breast cancer. Since most relapses occur in direct proximity of the former tumor site, the reduction of the risk of local recurrence effected by radiotherapy might partially be due to an alteration of the irradiated tumor bed’s micromilieu. Our aim was to investigate if IORT affects the local micromilieu, especially immune cells with concomitant cytokine profile, and if it has an impact on growth conditions for breast cancer cells as well as mammary mesenchymal stromal cells (MSC), the latter considered as a model of the tumor bed stroma.42 breast cancer patients with breast-conserving surgery were included, of whom 21 received IORT (IORT group) and 21 underwent surgery without IORT (control group). Drainage wound fluid (WF) was collected from both groups 24 h after surgery for flow cytometric analysis of immune cell subset counts and potential apoptosis and for multiplex cytokine analyses (cytokine array and ELISA). It served further as a supplement in cultures of MDA-MB 231 breast cancer cells and mammary MSC for functional analyses, including proliferation, wound healing and migration. Furthermore, the cytokine profile within conditioned media from WF-treated MSC cultures was assessed. Flow cytometric analysis showed no group-related changes of cell count, activation state and apoptosis rates of myeloid, lymphoid leucocytes and regulatory T cells in the WF. Multiplex cytokine analysis of the WF revealed group-related differences in the expression levels of several cytokines, e.g., oncostatin-M, leptin and IL-1β. The application of WF in MDA-MB 231 cultures did not show a group-related difference in proliferation, wound healing and chemotactic migration. However, WF from IORT-treated patients significantly inhibited mammary MSC proliferation, wound healing and migration compared to WF from the control group. The conditioned media collected from WF-treated MSC-cultures also exhibited altered concentrations of VEGF, RANTES and GROα. IORT causes significant changes in the cytokine profile and MSC growth behavior. These changes in the tumor bed could potentially contribute to the beneficial oncological outcome entailed by this technique. The consideration whether this alteration also affects MSC interaction with other stroma components presents a promising gateway for future investigations.

New clinical and biological insights from the international TARGIT-A randomised trial of targeted intraoperative radiotherapy during lumpectomy for breast cancer

“…This long-term outcome is consistent with previous reports and prompts the hypothesis that a single large dose of radiation such as TARGIT-IORT given during the trauma of surgery might possibly have an abscopal effect, i.e., an effect away from the site of irradiation, by influencing the tumour microenvironment or by immunological mechanisms. 14 – 27 , 28 Strange as it may seem, such an abscopal effect appears to give long-term protection against deaths from cardiovascular causes and other cancers. The early separation of lines in the K–M curves that starts soon after randomisation also suggests such a ‘drug-like’ effect, while a separation starting a few years later in the comparison of TARGIT-IORT alone vs. EBRT suggests an effect of avoiding EBRT (Fig.…”

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“…There is no time for tumor repopulation. IORT immediately after the removal of the tumor modifies the cytokines like oncostatin-M, leptin and IL-1β in the wound fluids (38,39). Wound fluids from IORT-treated patients significantly inhibit mammary mesenchymal stromal cells proliferation in comparison to wound fluids of non-irradiated patients.…”

Biological and clinical review of IORT-induced wound fluid in breast cancer patients

“…They found that several cytokines were overexpressed in the luminal A subtype in the IORT-treated group, which may have anti-tumor characteristics (111). In a recent study, Wuhrer et al analyzed seromas collected 24h after breast-conserving surgery (from 42 patients) with and without IORT treatment and observed dramatic changes in populations of immune cells and levels of cytokine (112). None of the investigated subpopulations, such as Treg, T cells, and myeloid cells, showed alteration in their activation states or their counts in cellular fraction analysis of the seroma and blood samples of the patients 24 h after IR treatment compared to control.…”

“…Moreover, analysis of MSCs-CM cultured in 0.5% IORT- and control-seroma and collected after 72h showed significantly decreased RANTES, GRO-α, and VEGF in the IORT-seroma group ( 112 ). To confirm the tumor inhibitory effects of IORT-seroma compared with non-IORT-seroma, we evaluated migration, proliferation, viability, and invasion in three BC cell lines.…”