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.

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.

Wound fluids collected from patients after IORT treatment activates extrinsic apoptotic pathway in MCF7 breast cancer cell line.

OBJECTIVES: Intraoperative radiotherapy (IORT) relates to irradiation of diseased tissue during the surgery within the tumor bed. The reason for this process is based on the fact that the increase in the radiation dose increases local tumor control. It was shown that postoperative fluids obtained from patients after breast cancer conserving surgery, stimulated motility and invasiveness of tumor cells in vitro. The results obtained from TARGIT clinical trial demonstrated that IORT significantly inhibits the stimulatory effect of wound fluids on tumor cells in vitro. We therefore speculated that wound fluids collected from patients after IORT treatment may induce the apoptosis in breast cancer cell lines and it may be a reason for their lower proliferation rate and potential to metastasis. MATERIAL AND METHODS: Breast cancer MCF7 cell line was incubated with wound fluids collected from patients after conserving breast cancer surgery or surgery followed by IORT for 4 days. Then the expression of markers associated with extrinsic or intrinsic apoptosis pathway was established. RESULTS: Our results clearly indicate activation of extrinsic apoptosis pathway by wound fluids collected from patients after IORT treatment. No changes in apoptotic markers were seen in cells treated with wound fluids collected from patients after the surgery alone. CONCLUSIONS: Thus we confirmed that wound fluids collected from patients after IORT treatment may induce the apoptosis in breast cancer cell lines and it may be a reason for their lower proliferation rate and invasiveness of tumor cells in vitro.

Wound fluids affect miR-21, miR-155 and miR-221 expression in breast cancer cell lines, and this effect is partially abrogated by intraoperative radiation therapy treatment.

Breast cancer is the most common malignant disease occurring in women. Conservative breast cancer surgery followed by radiation therapy is currently the standard treatment for this type of cancer. The majority of metastases occur within the scar, which initiated a series of studies. As a result, clinical trials aimed to assess whether localized radiotherapy, as intraoperative radiotherapy (IORT), may more effective in inhibiting the formation of local recurrence compared with the standard postoperative whole breast radiotherapy. The present study determined the role of postoperative wound fluids (WFs) from patients diagnosed with breast cancer subsequent to breast conserving surgery or breast conserving surgery followed by IORT on the expression of three microRNAs (miRNAs), consisting of miR-21, miR-155 and miR-221, in distinct breast cancer cell lines that represent the general subtypes of breast cancer. It was determined that the miRNAs responsible for breast cancer progression, induction of tumorigenesis and enrichment of the cancer stem cell phenotype, which is responsible for resistance to tumor therapy, were highly upregulated in the human epidermal growth factor receptor 2-positive breast cancer SK-BR-3 cell line following stimulation with WFs. It is worth emphasizing, that those changes were more significant in WFs collected from patients after surgery alone. The BT-549 cell line showed altered expression only of miR-155 following incubation with WFs. Notably, this change was not associated with IORT. Additionally, it was indicated that both WFs and RT-WF strongly downregulated the expression of miR-21, miR-155 and miR-221 in basal/epithelial and luminal subtypes of breast cancer. It was concluded that the present study contributes to an increased understanding of the role of surgical WFs and IORT treatment in the regulation of miRNA expression. This may enable the development of the current knowledge of breast cancer biology subsequent to IORT treatment and substantially to improve the therapy in the future.

Electrochemotherapy in Breast Cancer - Discussion of the Method and Literature Review.

Breast cancer is the most common cause of skin metastases in women. The probability of their occurrence ranges from about 5% in the entire population to as much as 30% in the late stages of the disease. Although rarely life-threatening, they have a major impact on the quality of life of patients with this diagnosis, being the cause of pain, effusion, ulceration, infection, and psychological discomfort. Available methods of treatment, both local and systemic, often fail to provide adequate control of the disease. A particular challenge seems to be the treatment of those patients with cutaneous metastases who, due to the extent of their metastases, are not eligible for resection, in whom the possibility of radiation therapy has already been used, and in whom systemic therapy is ineffective or contraindicated. A new method providing the opportunity for effective treatment is electrochemotherapy (ECT). ECT combines electropulsation of tumor cells (by local application of electric pulses) and administration of antineoplastic drugs such as cisplatin or bleomycin (either intravenous or intratumoral). Several clinical studies have demonstrated that ECT provides safe, efficient, and non-invasive locoregional treatment for chest wall breast cancer recurrence.

Effect of surgical wound fluids after intraoperative electron radiotherapy on the cancer stem cell phenotype in a panel of human breast cancer cell lines.

The wound healing process after surgery alters the area surrounding the original tumor and around the scar, and the modified microenvironment is more favorable for tumor recurrence. Intraoperative radiotherapy (IORT) is one of the more novel strategies in breast cancer (BC) treatment. Irradiation during surgery has effects on the tumor microenvironment, abrogating the proliferative cascade induced by surgical wound healing. 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 cancer stem cell phenotype in a panel of BC cell lines. Post-operative wound fluids were derived from patients with BC who underwent a tumor resection (quadrantectomy) plus intraoperative electron radiotherapy using a single dose of ≤10 Gy on the tumor bed and surrounding tissues, or from those who underwent a tumor resection without IOERT. Cell lines were incubated with 10% wound fluids, and after 4 days, the cluster of differentiation (CD)44+/CD24-/low phenotype and aldehyde dehydrogenase 1 (ALDH1) activity were determined by flow cytometry. The two types of fluid each affected the CD44+/CD24-/low phenotype. The results varied markedly between each cell line, even for the same histological subtypes. RT-WF decreased the CD44+/CD24-/low populations in the basal-like BT-549 and MDA-MB-468 cell lines, whereas in the luminal type MCF7 cell line, the two fluids inhibited these populations. The HER-OE subtypes harbored a minimal CD44+/CD24-/low population, but the growth of SK-BR-3 was stimulated by the two post-operative fluids. WF exhibited a stronger effect on ALDH1 activity compared with RT-WF. The stimulatory effect was dependent on the histological subtype of the cell line and the strongest dependence was observed in luminal subtypes characterized by low dehydrogenase activity in the control group. The present results enable a better understanding of the mechanism of recurrence and metastases following BC surgery. With respect to histological phenotype, its effect on tumor progression, either local or systemic, strongly suggests the requirement for further research and clinical validation.

miRNA - Therapeutic tool in breast cancer? Where are we now?

OBJECTIVE: The aim of this study was to review the current knowledge about involvement of microRNAs in breast cancer, and their potential in the clinic, published in scientific journals searched in Pubmed/Medline database until March 2014. RESULTS: MicroRNAs (miRNAs) are a family of 21-25 nucleotide small RNAs molecules. Currently, it is well known that miRNA plays a key role in all cellular processes of the organism including tumour initiation and progression. Many studies have shown that circulating miRNAs are attractive, easily detectable tumour biomarkers. Breast cancer is one of the most common cancers in the world. It is clinically established that different subtypes may respond differently to therapies, give metastases and present drug resistance. MicroRNAs have a potential as diagnostic, prognostic and therapeutic tools in breast cancer. CONCLUSION: Molecular knowledge is crucial for choosing the most effective therapy for individual patients. MicroRNAs holds a great potential in anticancer therapy.

Tumour-infiltrating CD11b+ myelomonocytes and response to fractionated irradiation of human squamous cell carcinoma (hSCC) xenografts.

PURPOSE: Bone marrow derived CD11b+ myelomonocytes have been shown to be recruited by the tumour and to promote tumour regrowth after irradiation. Here we investigated in a panel of well characterised hSCC tumour models the number of tumour-infiltrating CD11b+ cells and the association with response to clinically relevant fractionated irradiation. METHODS: Six hSCC tumour models (UT-SCC-5, -14, -15, XF354, FaDu, SAS) xenografted in nude mice were excised after injection of pimonidazole hypoxia marker before irradiation and after 5 and 10 fractions. In parallel, TCD(50) (dose to cure 50% of the tumours) assays were performed to determine the response to 30 fractions within 6 weeks. The TCD(50) values have been previously published [1]. Double staining of CD11b and pimonidazole was performed using immunofluorescence. CD11b+ cells were counted in viable pimonidazole-negative areas (non-hypoxic) and pimonidazole-positive areas (hypoxic) of whole tumour cross-sections. RESULTS: The median number of tumour-infiltrating CD11b+ cells either decreased or remained unchanged after 5 and 10 fractions in most of the tumour models. The density of CD11b+ cells in hypoxic areas was similar or lower than in non-hypoxic regions independently on treatment in majority of the tumour models. After 10 fractions the median CD11b+ cell density was significantly associated with the TCD(50) values after 30 fractions. CONCLUSION: The data from our exploratory study suggest that tumour-infiltrating CD11b+ cells may contribute to local tumour control after fractionated irradiation, which supports to further study their prognostic value and to evaluate specific myelomonocyte targeting strategies to overcome radiation resistance.