Senescence in head and neck squamous cell carcinoma: relationship between senescence-associated secretory phenotype (SASP) mRNA expression level and clinicopathological features / Senescencia en el carcinoma de células escamosas de cabeza y cuello: relación entre el nivel de expresión del ARNm del fenotipo secretor asociado a la senescencia (SASP) y las características clínico-patológicas

Background: Cellular senescence is a state characterized by cell-cycle arrest and apoptotic resistance. Senescence in cancer may be induced by oncogenes or therapy. While cellular senescence might play an important role in protection against cancer development, elevated and uncontrolled senescent cells accumulation may promote carcinogenesis by secreting a collection of pro-inflammatory factors, collectively termed the senescence-associated secretory phenotype (SASP). Material and methods: We determined the gene expression at mRNA level of selected cellular senescence markers (p16 and LMNB1) and SASP factors (IL-6, IL-1b, CXCL-1 and TNF-α) in 72 cancerous tissues and 64 normal tissues obtained from patients with head and neck squamous cell carcinoma (HNSCC) and correlated this data with patients’ clinical follow-up. Results: Our results indicate higher levels of selected SASP factors in cancerous compared to normal tissues. We presented the relationship between SASP factors expression at the transcript level and the progression of the disease. Moreover, we proposed CXCL1 as a candidate biomarker differentiating normal tissues from cancerous ones and IL1b expression as a molecular factor related to increased TNM stage. Conclusion: Our primary study indicates that SASP expression may be associated with some clinicopathological features. However, a more detailed study is needed to present specific role of senescence-related mechanism and SASPs especially in tumor therapy response and in relation to the patient’s immune system condition.(AU)

Dynamic interactions in the tumor niche: how the cross-talk between CAFs and the tumor microenvironment impacts resistance to therapy.

The tumor microenvironment (TME) is a complex ecosystem of cells, signaling molecules, and extracellular matrix components that profoundly influence cancer progression. Among the key players in the TME, cancer-associated fibroblasts (CAFs) have gained increasing attention for their diverse and influential roles. CAFs are activated fibroblasts found abundantly within the TME of various cancer types. CAFs contribute significantly to tumor progression by promoting angiogenesis, remodeling the extracellular matrix, and modulating immune cell infiltration. In order to influence the microenvironment, CAFs engage in cross-talk with immune cells, cancer cells, and other stromal components through paracrine signaling and direct cell-cell interactions. This cross-talk can result in immunosuppression, tumor cell proliferation, and epithelial-mesenchymal transition, contributing to disease progression. Emerging evidence suggests that CAFs play a crucial role in therapy resistance, including resistance to chemotherapy and radiotherapy. CAFs can modulate the tumor response to treatment by secreting factors that promote drug efflux, enhance DNA repair mechanisms, and suppress apoptosis pathways. This paper aims to understand the multifaceted functions of CAFs within the TME, discusses cross-talk between CAFs with other TME cells, and sheds light on the contibution of CAFs to therapy resistance. Targeting CAFs or disrupting their cross-talk with other cells holds promise for overcoming drug resistance and improving the treatment efficacy of various cancer types.

Navigating challenges: optimising methods for primary cell culture isolation.

Primary cell lines are invaluable for exploring cancer biology and investigating novel treatments. Despite their numerous advantages, primary cultures are laborious to obtain and maintain in culture. Hence, established cell lines are still more common. This study aimed to evaluate a range of techniques for isolating primary breast cancer cultures, employing distinct enzymatic compositions, incubation durations, and mechanical approaches, including filtration. Out of several protocols, we opted for a highly effective method (Method 5) that gave rise to a primary cell culture (BC160). This method combines mechanical disaggregation and enzymatic digestion with hyaluronidase and collagenase. Moreover, the paper addresses common issues in isolating primary cultures, shedding light on the struggle against fibroblasts overgrowing cancer cell populations. To make primary cell lines a preferred model, it is essential to elaborate and categorise isolation methods, develop approaches to separate heterogeneous cultures and investigate factors influencing the establishment of primary cell lines.

Senescence in head and neck squamous cell carcinoma: relationship between senescence-associated secretory phenotype (SASP) mRNA expression level and clinicopathological features.

BACKGROUND: Cellular senescence is a state characterized by cell-cycle arrest and apoptotic resistance. Senescence in cancer may be induced by oncogenes or therapy. While cellular senescence might play an important role in protection against cancer development, elevated and uncontrolled senescent cells accumulation may promote carcinogenesis by secreting a collection of pro-inflammatory factors, collectively termed the senescence-associated secretory phenotype (SASP). MATERIAL AND METHODS: We determined the gene expression at mRNA level of selected cellular senescence markers (p16 and LMNB1) and SASP factors (IL-6, IL-1b, CXCL-1 and TNF-α) in 72 cancerous tissues and 64 normal tissues obtained from patients with head and neck squamous cell carcinoma (HNSCC) and correlated this data with patients' clinical follow-up. RESULTS: Our results indicate higher levels of selected SASP factors in cancerous compared to normal tissues. We presented the relationship between SASP factors expression at the transcript level and the progression of the disease. Moreover, we proposed CXCL1 as a candidate biomarker differentiating normal tissues from cancerous ones and IL1b expression as a molecular factor related to increased TNM stage. CONCLUSION: Our primary study indicates that SASP expression may be associated with some clinicopathological features. However, a more detailed study is needed to present specific role of senescence-related mechanism and SASPs especially in tumor therapy response and in relation to the patient's immune system condition.

Primary cancer-associated fibroblasts exhibit high heterogeneity among breast cancer subtypes.

Background: Cancer-associated fibroblasts (CAFs) are a diverse subset of cells, that is recently gaining in popularity and have the potential to become a new target for breast cancer (BC) therapy; however, broader research is required to understand their mechanisms and interactions with breast cancer cells. The goal of the study was to isolate CAFs from breast cancer tumour and characterise isolated cell lines. We concentrated on numerous CAF biomarkers that would enable their differentiation. Materials and methods: Flow cytometry, immunofluorescence, and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were used to phenotype the primary CAFs. Results/Conclusions: According to our findings, there was no significant pattern in the classification of cancer-associated fibroblasts. The results of biomarkers expression were heterogeneous, thus no specific subtypes were identified. Furthermore, a comparison of cancer-associated fibroblasts derived from different BC subtypes (luminal A and B, triple-negative, HER2 positive) did not reveal any clear trend of expression.

Biological heterogeneity of primary cancer-associated fibroblasts determines the breast cancer microenvironment.

Cancer-associated fibroblasts are a highly heterogeneous group of cells whose phenotypes and gene alterations are still under deep investigation. As a part of tumor microenvironment, they are the focus of a growing number of studies. Cancer-associated fibroblasts might become a new target of breast cancer therapy, but still more tests and analyses are needed to understand mechanisms and interactions between them and breast cancer cells. The study aimed to isolate cancer associated fibroblasts from breast cancer tissue and to phenotype the isolated cell lines. We focused on various cancer-associated fibroblast characteristic biomarkers and those that might differentiate various cancer-associated fibroblasts' subtypes. Patients with a histological diagnosis of invasive breast cancer (diameter ≤15 mm) and qualified for primary surgical treatment were enrolled in the study. Cell lines were isolated from breast cancer biopsy. For the phenotyping, we used flow cytometry, immunofluorescence and RT-qPCR analysis. Based on our study, there was no indication of a clear pattern in the cancer-associated fibroblasts' classification. Results of cancer-associated fibroblasts expression were highly heterogeneous, and specific subtypes were not defined. Moreover, comparing cancer-associated fibroblasts divided into groups based on BC subtypes from which they were isolated also did not allow to notice of any clear pattern of expressions. In the future, a higher number of analyzed cancer-associated fibroblast cell lines should be investigated to find expression schemes.

Cellular Damage in the Target and Out-Of-Field Peripheral Organs during VMAT SBRT Prostate Radiotherapy: An In Vitro Phantom-Based Study.

Hypo-fractionated stereotactic body radiation therapy (SBRT) is an effective treatment for prostate cancer (PCa). Although many studies have investigated the effects of SBRT on the prostate and adjacent organs, little is known about the effects further out-of-field. The aim of this study was to investigate, both in vitro and in a quasi-humanoid phantom, the biological effects (using a dose-scaling approach) of radiation in the out-of-field peripheral organs delivered by 6 MV volumetric modulated arc therapy (VMAT) SBRT in a prostate cancer model. Healthy prostate cells were irradiated in a phantom at locations corresponding to the prostate, intestine, lung, thyroid, and brain. Seven 10 Gy fractions of VMAT SBRT were delivered to the target in a single session without intermission (scaled-up method). Radiochromic films were used to measure the doses. The radiobiological response was assessed by measuring DNA breaks, the cell survival fraction, and differences in gene expression profile. Our results showed a strong, multiparametric radiobiological response of the cells in the prostate. Outside of the radiation field, the highest doses were observed in the intestine and lung. A small increase (not statistically significant) in DNA damage and cell death was observed in the intestines. Several gene groups (cell cycle, DNA replication) were depleted in the lung and thyroid (DNA replication, endocytosis), but further analysis revealed no changes in the relevant biological processes. This study provides extensive evidence of the types and extent of radiobiological responses during VMAT SBRT in a prostate cancer model. Additional research is needed to determine whether the radiobiological effects observed in the peripheral organs are validated in a clinical context.

Nontarget and Out-of-Field Doses from Electron Beam Radiotherapy.

In clinical radiotherapy, the most important aspects are the dose distribution in the target volume and healthy organs, including out-of-field doses in the body. Compared to photon beam radiation, dose distribution in electron beam radiotherapy has received much less attention, mainly due to the limited range of electrons in tissues. However, given the growing use of electron intraoperative radiotherapy and FLASH, further study is needed. Therefore, in this study, we determined out-of-field doses from an electron beam in a phantom model using two dosimetric detectors (diode E and cylindrical Farmer-type ionizing chamber) for electron energies of 6 MeV, 9 MeV and 12 MeV. We found a clear decrease in out-of-field doses as the distance from the field edge and depth increased. The out-of-field doses measured with the diode E were lower than those measured with the Farmer-type ionization chamber at each depth and for each electron energy level. The out-of-field doses increased when higher energy megavoltage electron beams were used (except for 9 MeV). The out-of-field doses at shallow depths (1 or 2 cm) declined rapidly up to a distance of 3 cm from the field edge. This study provides valuable data on the deposition of radiation energy from electron beams outside the irradiation field.

Influence of Specific Treatment Parameters on Nontarget and Out-of-Field Doses in a Phantom Model of Prostate SBRT with CyberKnife and TrueBeam.

The aim of the study was to determine the influence of a key treatment plan and beam parameters on overall dose distribution and on doses in organs laying in further distance from the target during prostate SBRT. Multiple representative treatment plans (n = 12) for TrueBeam and CyberKnife were prepared and evaluated. Nontarget doses were measured with anionization chamber, in a quasi-humanoid phantom at four sites corresponding to the intestines, right lung, thyroid, and head. The following parameters were modified: radiotherapy technique, presence or not of a flattening filter, degree of modulation, and use or not of jaw tracking function for TrueBeam and beam orientation set-up, optimization techniques, and number of MUs for CyberKnife. After usual optimization doses in intestines (near the target) were 0.73% and 0.76%, in head (farthest from target) 0.05% and 0.19% for TrueBeam and CyberKnife, respectively. For TrueBeam the highest peripheral (head, thyroid, lung) doses occurred for the VMAT with the flattening filter while the lowest for 3DCRT. For CyberKnife the highest doses were for gantry with caudal direction beams blocked (gantry close to OARs) while the lowest was the low modulated VOLO optimization technique. The easiest method to reduce peripheral doses was to combine FFF with jaw tracking and reducing monitor units at TrueBeam and to avoid gantry position close to OARs together with reduction of monitor units at CyberKnife, respectively. The presented strategies allowed to significantly reduce out-of-field and nontarget doses during prostate radiotherapy delivered with TrueBeam and CyberKnife. A different approach was required to reduce peripheral doses because of the difference in dose delivery techniques: non-coplanar using CyberKnife and coplanar using TrueBeam, respectively.

Development of a quasi-humanoid phantom to perform dosimetric and radiobiological measurements for out-of-field doses from external beam radiation therapy.

Our understanding of low dose, out-of-field radiation and their radiobiological effects are limited, in part due to the rapid technological advances in external beam radiotherapy, especially for non-coplanar and dynamic techniques. Reliable comparisons of out-of-field doses produced by advanced radiotherapy techniques are difficult due to the limitations of commercially available phantoms. There is a clear need for a functional phantom to accurately measure the dosimetric and radiobiological characteristics of out-of-field doses, which would in turn allow clinicians and medical physicists to optimize treatment parameters. We designed, manufactured, and tested the performance of a quasi-humanoid (Q-H) adult phantom. To test the physics parameters, we used computed tomography (CT) scans of assembled Q-H phantom. Static open field and dynamic techniques were measured both in- and out-of-field with ionization chambers and radiochromic films for two configurations (full solid and with water-filled containers). In the areas simulating soft tissues, lung, and bones, median Hounsfield units and densities were, respectively: 129.8, -738.7, 920.8 HU and 1.110, 0.215, 1.669 g/cm3 . Comparison of the measured to treatment planning systems (TPS) in-field dose values for the sample volumetric arc therapy (VMAT) (6 MV flattening filter-free (FFF)) plan, 96.4% of analyzed points passed the gamma evaluation criteria (L2%/2 mm, threshold (TH) 10%) and less than 1.50% for point dose verification. In the two phantom configurations: full poly(methyl) methacrylate (PMMA) and with water container, the off-axis median doses for open field, relative to the central axis of the beam (CAX) were similar, respectively: 0.900% versus 0.907% (15 cm distance to CAX); 0.096% versus 0.120% (35 cm); 0.018% versus 0.018% (52 cm); 0.009% versus 0.008% (74 cm). For VMAT 6 MV FFF, doses relative the CAX were, respectively: 0.667% (15 cm), 0.062% (35 cm), 0.019% (52 cm), 0.016% (74 cm). The Q-H phantom meets the International Commission on Radiation Units and Measurements (ICRU) and American Association of Physicists in Medicine (AAPM) recommended phantom criteria, providing medical physicists with a reliable, comprehensive system to perform dose calculation and measurements and to assess the impact on radiobiological response and on the risk of secondary tumor induction.

MiRNAs from serum-derived extracellular vesicles as biomarkers for uveal melanoma progression.

Uveal melanoma (UM) is a rare type of malignancy that originates from melanocytes in the choroid, iris and the eye's ciliary body. Biomarkers for early detection and progression of UM, especially the molecular traits governing the development of metastasis, are still not available in clinical practice. One extensively studied components of liquid biopsies are extracellular vesicles. Due to their unique molecular cargo, they can contribute to early cancer development and at the same time carry markers for disease onset and progression. For characterisation of the miRNA profiles present in circulating serum-derived exosomes of patients with diagnosed primary and metastatic UM, we have analyzed the miRNA cargos using next-generation sequencing followed by RT-qPCR validation in a cohort of patients (control n = 20; primary n = 9; metastatic n = 11). Nine miRNAs differentiating these patient groups have been established. We show that hsa-miR-144-5p and hsa-miR-191-5p are the most promising biomarker candidates, allowing the categorization of patients into local and advanced UM. Additionally, the comparison of miRNA expression levels in exosomes derived from UM patients with those derived from healthy donors revealed that hsa-miR-191-5p, -223-3p, -483-5p, -203a has the potential to be used as an early marker for the presence of UM. This pilot study reveals that miRNAs extracted from circulating exosomes could be exploited as potential biomarkers in UM diagnosis and, more importantly, for indicating metastatic spread.

From Donor to the Lab: A Fascinating Journey of Primary Cell Lines.

Primary cancer cell lines are ex vivo cell cultures originating from resected tissues during biopsies and surgeries. Primary cell cultures are objects of intense research due to their high impact on molecular biology and oncology advancement. Initially, the patient-derived specimen must be subjected to dissociation and isolation. Techniques for tumour dissociation are usually reliant on the organisation of connecting tissue. The most common methods include enzymatic digestion (with collagenase, dispase, and DNase), chemical treatment (with ethylene diamine tetraacetic acid and ethylene glycol tetraacetic acid), or mechanical disaggregation to obtain a uniform cell population. Cells isolated from the tissue specimen are cultured as a monolayer or three-dimensional culture, in the form of multicellular spheroids, scaffold-based cultures (i.e., organoids), or matrix-embedded cultures. Every primary cell line must be characterised to identify its origin, purity, and significant features. The process of characterisation should include different assays utilising specific (extra- and intracellular) markers. The most frequently used approaches comprise immunohistochemistry, immunocytochemistry, western blot, flow cytometry, real-time polymerase chain reaction, karyotyping, confocal microscopy, and next-generation sequencing. The growing body of evidence indicates the validity of the usage of primary cancer cell lines in the formulation of novel anti-cancer treatments and their contribution to drug development.

miRNAs as Biomarkers for Diagnosing and Predicting Survival of Head and Neck Squamous Cell Carcinoma Patients.

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer worldwide. These tumors originate from epithelial cells of the upper aerodigestive tract. HNSCC tumors in different regions can have significantly different molecular characteristics. While many microRNAs (miRNAs) have been found to be involved in the regulation of the carcinogenesis and pathogenesis of HNSCC, new HNSCC related miRNAs are still being discovered. The aim of this study was to explore potential miRNA biomarkers that can be used to diagnose HNSCC and prognose survival of HNSCC patients. For this purpose, we chose a panel of 12 miRNAs: miR-146a-5p, miR-449a, miR-126-5p, miR-34a-5p, miR-34b-5p, miR-34c-5p, miR-217-5p, miR-378c, miR-6510-3p, miR-96-5p, miR-149-5p, and miR-133a-5p. Expression of these miRNAs was measured in tumor tissue and neighboring healthy tissue collected from patients diagnosed with HNSCC (n = 79) in either the oral cavity, oropharynx, or larynx. We observed a pattern of differentially expressed miRNAs at each of these cancer locations. Our study showed that some of these miRNAs, separately or in combination, could serve as biomarkers distinguishing between healthy and tumor tissue, and their expression correlated with patients' overall survival.

The Role of Gold Nanorods in the Response of Prostate Cancer and Normal Prostate Cells to Ionizing Radiation-In Vitro Model.

To increase the efficiency of therapy via enhancing its selectivity, the usage of gold nanorods (GNR) as a factor sensitizing cancer cells to radiation was proposed. Due to gold nanoparticles' characteristics, the smaller doses of radiation would be sufficient in the treatment, protecting the healthy tissue around the tumor. The aim of this study was to investigate the effect of gold nanorods on cancer and normal prostate cells and the role of nanorods in the cell response to ionizing radiation. The effect was evaluated by measuring the toxicity, cell cycle, cell granularity, reactive oxygen species (ROS) level, and survival fractions. Nanorods showed a strong toxicity dependent on the concentration and incubation time toward all used cell lines. A slight effect of nanorods on the cycle distribution was observed. The results demonstrated that the administration of nanorods at higher concentrations resulted in an increased level of generated radicals. The results of cellular proliferation after irradiation are ambiguous; however, there are noticeable differences after the application of nanorods before irradiation. The obtained results lead to the conclusion that nanorods affect the physiology of both normal and cancer cells. Nanorods might become a potential tool used to increase the effectiveness of radiation treatment.

Interplay between inflammation and cancer.

Tumor-promoting inflammation is one of the hallmarks of cancer. It has been shown that cancer development is strongly influenced by both chronic and acute inflammation process. Progress in research on inflammation revealed a connection between inflammatory processes and neoplastic transformation, the progression of tumour, and the development of metastases and recurrences. Moreover, the tumour invasive procedures (both surgery and biopsy) affect the remaining tumour cells by increasing their survival, proliferation and migration. One of the concepts explaining this phenomena is an induction of a wound healing response. While in normal tissue it is necessary for tissue repair, in tumour tissue, induction of adaptive and innate immune response related to wound healing, stimulates tumour cell survival, angiogenesis and extravasation of circulating tumour cells. It has become evident that certain types of immune response and immune cells can promote tumour progression more than others. In this review, we focus on current knowledge on carcinogenesis and promotion of cancer growth induced by inflammatory processes.

Surgical Wound Fluids from Patients with Breast Cancer Reveal Similarities in the Biological Response Induced by Intraoperative Radiation Therapy and the Radiation-Induced Bystander Effect-Transcriptomic Approach.

In patients with breast cancer who undergo breast-conserving surgery (BCS), more than 90% of local recurrences occur in the same quadrant as the primary cancer. Surgical wound fluids (SWF) are believed to play a role in this process by inducing an inflammatory process in the scar tissue area. Despite strong clinical data demonstrating the benefits of intraoperative radiotherapy (IORT), the biological basis underlying this process remains poorly understood. Ionizing radiation (IR) directly affects cells by damaging DNA, thereby altering the cell phenotype. IR directly affects cancer cells and also influences unirradiated cells located nearby, a phenomenon known as the radiation-induced bystander effect (RIBE), significantly modifying the tumor microenvironment. We hypothesized that SWF obtained from patients after BCS and IORT would induce a radiobiological response (due to RIBE) in unirradiated cells, thereby modifying their phenotype. To confirm this hypothesis, breast cancer cells were incubated with SWF collected from patients after BCS: (1) without IORT (wound fluid (WF) group), (2) with IORT (radiotherapy wound fluid (RT-WF) group), and (3) WF with conditioned medium from irradiated cells (WF+RIBE group) and then subjected to microarray analysis. We performed gene set enrichment analysis to determine the biological processes present in these cells. This analysis showed that the RT-WF and WF+RIBE groups shared common biological processes, including the enhancement of processes involved in cell-cycle regulation, DNA repair, and oxidative phosphorylation. The WF group was characterized by overrepresentation of pathways involved in the INF-α and INF-γ response, inflammatory response, and the IL6 JAK/STAT3 signaling pathway. These findings show that MDA-MB-468 cells stimulated with surgical wound fluids obtained from patients who underwent BCS plus IORT and from cells stimulated with SWF plus RIBE share common biological processes. This confirms the role of the radiation-induced bystander effect in altering the biological properties of wound fluids.

The Composition of Surgical Wound Fluids from Breast Cancer Patients is Affected by Intraoperative Radiotherapy Treatment and Depends on the Molecular Subtype of Breast Cancer.

Invasive oncological procedures affect the remaining tumor cells by increasing their survival, proliferation, and migration through the induction of wound healing response. The phenomena of local relapse after breast-conserving surgery (BCS) has resulted in a series of research and clinical trials with the aim of assessing whether localized intraoperative radiotherapy (IORT), may be beneficial in inhibiting local recurrences. Therefore, it is essential to assess the impact of intraoperative radiotherapy in modulating the immunological response and wound healing process. Thus, we decided to perform a quantitative analysis of the composition of surgical wound fluids (SWF) in two groups of breast cancer (BC) patients: those treated with BCS followed by IORT, and those who underwent BCS alone. We found that several cytokines, which are believed to have anti-tumor properties, were highly expressed in the luminal A breast cancer subtype in the IORT treatment group. Interestingly, we also found significant differences between IORT patients with tumors of different molecular subtypes. Based on these findings, we hypothesized that IORT treatment might be beneficial in changing the tumor bed microenvironment, making it less favorable for tumor recurrence due to decreased concentration of tumor-facilitating cytokines, especially in the luminal A subtype of BC.

Superparamagnetic iron oxide nanoparticles (SPIONs) as a multifunctional tool in various cancer therapies.

Over the past two decades nanotechnology has become an important part of novel medical research. Researchers have made great progress in developing nanotechnology applications used for detecting and treating oncological diseases. Recently, many research groups have focused on the use of superparamagnetic iron oxide nanoparticles (SPIONs) in cancer treatment. Due to the range of therapeutic properties and possibilities of various modifications, SPIONs are a promising and multifunctional tool in various cancer therapies and may help to overcome the limitations of conventional therapies. Moreover, it is still necessary to develop new methods of treatment with expected properties, such as lower toxicity, long-lasting effectiveness and higher selectivity. Analyzing the literature data, we found that currently SPIONs are used in the transport of drugs, immunotherapy and hyperthermia. The main aim of this review is to present various cancer treatment therapies utilizing SPIONs, the importance of the experiments carried out by research groups and further perspectives in the nanotechnological use of SPIONs.

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.