Breast cancer (BC) is the most common cancer in women, and is a heterogeneous disease, which has complicated its treatment and study (1). Based on the expression of specific receptors, BC is clinically divided into estrogen receptor (ER)-positive BC (the most common and diverse group), progesterone receptor (PR)-positive BC and human epidermal growth factor receptor 2 (HER2; also known as ERBB2)-positive BC, or in the absence of the three receptors, triple-negative BC (TNBC) (2).

Breast-conserving therapy (BCT) includes a wide local excision followed by adjuvant radiotherapy to the whole breast, and frequently, an additional boost to the tumor bed, including intraoperative radiotherapy (IORT) as one of the more novel strategies (3–6). The rationale for local dose escalation originates in the fact that after BCT, up to 90% of (at least first) local recurrences in the breast occur in the same quadrant as the primary cancer (7,8). The largest evidence for boost IORT preceding whole breast irradiation exists for intraoperative electron radiotherapy (IOERT) with single doses of ~10 Gy. In any risk constellation, local recurrence rates are among the lowest reported thus far, as well as those of long-term follow-up analyses (9). Apart from the mere dose augmentation effect and the high topographical precision in delivery, immediate irradiation during surgery has been hypothesized to exhibit an effect on the tumor microenvironment, abrogating the proliferative cascade induced by surgical wound healing (10–13). Published data have suggested that the wound healing process subsequent to surgery alters the area surrounding the original tumor and around the scar, and that the modified microenvironment is more favorable for the tumor to recur. Local recurrence after surgery is particularly common in tumors characterized by HER2 overexpression (14). It has been shown that wound fluids contain growth factors inducing the proliferation of HER2-positive BCs (15). Radiotherapy is not the only treatment that affects cell survival, surgery also has an impact on the tumor microenvironment. Belletti et al demonstrated the stimulatory effect of post-surgical drainage fluids on BC cells in fluids harvested from a group of patients after IORT treatment and from patients after breast-conserving surgery. It was shown that wound fluids from conservative surgery (without IORT) could stimulate the proliferation, migration and invasion of the BC cell lines, while fluids collected after IORT demonstrated different properties (16).

It has been proposed that cancer cells displaying the stem-like phenotype play a critical role in local recurrence, invasion and metastasis, as well as in radio- and chemoresistance (17–21). Al-Hajj et al identified a population of cluster of differentiation (CD)44⁺/CD24^−/low cells from human breast tumors, which are more tumorigenic than other cell populations (22). In vivo experiments have shown that CD44⁺/CD24^−/low cells were able to generate tumors in NOD/SCID mice. Although a statistically significant association between the clinical behavior and the frequency of these cell populations was not observed, tumors with distant metastases exhibited a higher fraction of the CD44⁺/CD24^−/low phenotype (23).

The presence of high aldehyde dehydrogenase 1 (ALDH1) activity is also associated with stem cell properties, with resistance to chemotherapy and a worse prognosis (24–26). Previous studies found that ALDH is involved in intracellular retinoic acid production correlated with metastasis, tumor grade, and HER2 and Ki-67 status (26–28). Dysregulation of signal transducer and activator of transcription 3 (STAT3) is sufficient for neoplastic transformation, and a previous study showed that constitutively active forms are able to promote malignant transformation in fibroblasts and tumor formation in mice (29). It has also been demonstrated that ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of BC cells express higher levels of phosphorylated STAT3 (30). The pharmacological targeting on STAT3 can suppress ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ cells in vitro and in vivo.

The aim of the present study was to determine the effect of surgical wound fluids from IOERT treatment (RT-WF) compared with wound fluids from conservative-breast surgery only (WF) on the CD44⁺/CD24^−/low phenotype and ALDH1 activity in a range of different BC cell lines. The results obtained will enable a better understanding of the mechanism of recurrence and metastasis following BC surgery.

The present study demonstrated that post-operative fluids harvested from patients who underwent breast-conserving surgery alone and from patients who received IOERT affect the stem cell phenotype in human BC cell lines. It is well established that surgery modifies the microenvironment, and that primary tumor removal may stimulate or accelerate metastatic spread. This phenomenon is known as tumor cell dormancy (33) and is likely to be important in treatment failure. Metastatic disease usually develops within 5 years of primary tumor treatment and more than half of patients succumb due to it (34). The wound healing process is a typical consequence of surgery, and it is currently believed that accompanying inflammation may contribute to aggressiveness and treatment resistance (35). The dormancy theory explains that molecular events associated with healing play a pivotal role as a ‘start signal’ to proliferation and the stimulation of angiogenesis (34). It has been shown that post-surgery wound fluids taken from BC patients can stimulate proliferation, metastasis and invasion (15,16). The aforementioned results support the clinical observations and proves that surgery is a perturbing factor and a cause of treatment failure. Notably, the complete inhibition of proliferation and the delayed occurrence of metastases has been achieved in murine mammary studies using systemic treatment prior to or following surgery (36).

Inflammatory factors and changes in the microenvironment have been identified as modifying agents to tumor stem cells (37). CSCs exhibit the ability to self-renew, migrate and invade into surrounding tissues or distant organs, and are associated with radio- and chemoresistance. Due to these properties, CSCs have been proposed as attractive targets and a number of studies have been conducted to identify subpopulations of the cells responsible for treatment failure and tumor progression. Breast CSCs were first described by Al-Hajj et al (22) and identified as a CD44⁺/CD24^−/low high tumorigenic population. In vivo experiments showed that CD44⁺/CD24^−/low cells were able to generate tumors in NOD/SCID mice. Numerous other studies confirmed these results in BC (23,38,39) and various other human tumor types (40). Generally, tumorigenicity and the stemness phenotype of these putative cells have been demonstrated by in vitro clonogenicity and in vivo tumorigenicity. A paradoxical phenomenon of tumor stem cells in BC exists, as there is no statistically significant association in the clinical behavior and the frequency of these cell populations (23,36), however, tumors that develop distant metastases indeed exhibit a higher fraction of CD44⁺/CD24^−/low phenotype cells (23,41).

In order to better understand the effect of surgical wound fluids on the putative stem cell population in BC, 8 different cell lines were examined in the present study. All models used in the study differed not only by the histological subtype, but also by the expression of the CD44 and CD24 subpopulation (Fig. 2). Basal-like cell lines expressed more CD44⁺/CD24^−/low population cells as opposed to the luminal type, and according to literature data and clinical observations, these cells are associated with a worse prognosis (42). Belletti et al (16) observed changes in the proteomic profile between cells incubated in surgical fluids harvested from patients who underwent surgery alone and from those patients who received intraoperative radiotherapy. The fluids taken from the patients that received intraoperative radiotherapy showed reduced expression in several molecules linked to tumor growth and motility, including HGF and leptin, which can explain the different biological activities between the two groups of surgical fluids (16). It was therefore reasonable to hypothesize that surgical fluids can induce a putative stem cell population. Indeed, post-operative fluids affect the population, but those taken from patients treated using IORT (RT-WF) in the present study decreased the population of CD44⁺/CD24^−/low cells in the basal and luminal groups. The strongest effect was observed in the BT-549 and MDA-MB-468 cell lines, whereas in HER-OE cell lines, identified as poorly equipped with CD44⁺/CD24^−/low, the result was the opposite. In the SK-BR-3 and BT-474 cell lines, the two surgical fluids were each able to stimulate the putative stem population. No effect was observed in the MDA-MB-231 cell line. A decrease in the CD44⁺/CD24^−/low population was compensated for by an increase in the CD44⁺/CD24⁺ population, which is considered to be not as tumorigenic (Fig. 4).

A study by Segatto et al largely explains how the surgery-wound response can promote a stem-like phenotype in BC cells via the STAT3 signaling pathway (37). A strong stimulation of the ability of mammosphere formation by wound fluid sera was observed in all examined cell lines. The presented results differed slightly. A dependence in the surgical fluid response according to the receptor expression in the cell line and the surgical fluid type used in the examination was observed (WF or RT-WF). Most likely, this effect is caused by the different times of fluid collection. In the present study, WF and RT-WF were collected at 7 days post-surgery by percutaneous aspiration, while in the aforementioned study (37), fluids were collected from drains at 24 h post-surgery. The difference in the time of collection is crucial in fluids, particularly in the panel of IORT-treated cell lines. Based on Belletti's results of the molecular analysis and the knowledge of surgery-induced inflammation (16), we believe that during the 1 week of the healing process, a pool of various molecules was accumulated that was responsible for the stronger effect on the putative stem phenotype.

While the subpopulation of cells with CD44⁺/CD24^−/low expression has the highest invasiveness and metastatic potential in BC, more and more studies indicate that the CD44⁻/CD24⁺ population is a better prognostic marker in BC (43). The tissue microarray and histopathological analysis of patients' tumor samples indicates that CD44⁻/CD24⁺ tumors have the worst prognosis in early invasive BC (43), and the tumor group exhibiting the CD44⁺/CD24^−/low phenotype is considered a favorable prognostic subgroup in BC. Notably, in the present study, in the HER-OE cell lines and the luminal MCF7 cell line, this population was decreased by surgical fluids (Fig. 4). Meyer et al demonstrated that CD24 expression is under dynamic regulation in vitro and in vivo, and that the population of CD44⁻/CD24⁺ cells can interconvert into CD44⁺/CD24^−/low cells and vice versa (44). This phenomenon can be explained as a consequence of loss of CD24 activity during tumor development or treatment.

ALDH activity is also associated with the putative stem cell phenotype. It was previously demonstrated that the expression of ALDH1 in breast tumors was a good predictor of a poor clinical outcome and this high activity was specific to the stemness phenotype (24). In turn, Zhong et al found that ALDH1 was not significantly associated with conventional clinical features such as tumor size or receptor expression, but was associated with breast tumor recurrence (45).

The present study assessed the impact of post-operative fluids on ALDH1 activity. A total of 8 cell lines were used with varied ALDH1 expression and activity that was comparable within the histological subtypes (Fig. 1A). to the best of our knowledge, the study presents the first examination of ALDH1 activity in BC affected by post-operative surgical fluids. Surgical fluids stimulated ALDH1 activity in all cell lines. However, RT-WF demonstrated a less stimulatory effect, which was most noticeable in the cell lines with a moderate ALDH1 level. Basal-like features are much more frequently associated with the incidence of metastasis than other subtypes. The present results showed that, as in the CD44/CD24 phenotype investigation, in this group of tumors, RT-WF inhibited the stimulation potential of fluids after surgery. Therefore, IORT may essentially contribute to treatment success.

The study did not observe that the examined putative breast CSC markers of a CD44⁺/CD24^−/low phenotype and high ALDH1 activity result in the same response under post-operative surgical fluids, indicating that in vitro these markers do not correlate with the fluid response, and that behavior is an independent factor, subsidiary to the histopathological subtype. These results may be an explanation for the lower relapse rates in the TARGIT-A trial for patients treated with immediate IORT using 50-kV orthovoltage (46).

The present study supports the hypothesis that surgery is a perturbing factor and explains how surgical intervention can impact the critical CSC behavior. To the best of our knowledge, this is the first study describing inhibitive mechanisms for IOERT. With respect to histological phenotype, the effect of the treatment on tumor progression, either local or systemic, strongly suggests a requirement for further research and clinical validation.

In summary, surgical wound fluids from the WF and RT-WF groups affected the putative stem cell phenotype, as determined by CD44⁺/CD24^−/low and high ALDH1 activity. Following IOERT, a lower stimulation stem cell phenotype was observed in the RT-WF compared with the WF.