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.

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.

FLASH radiotherapy: an emerging approach in radiation therapy.

FLASH radiotherapy (RT) is a technique involving the delivery of ultra-high dose rate radiation to the target. FLASH-RT has been shown to reduce radiation-induced toxicity in healthy tissues without compromising the anti-cancer effects of treatment compared to conventional radiation therapy. In the present article, we review the published data on FLASH-RT and discuss the current state of knowledge of this novel approach. We also highlight the technological constraints and complexity of FLASH-RT and describe the physics underlying this modality, particularly how technology supports energy transfer by ionising radiation (e.g., beam on/off sequence, pulse-energy load, intervals). We emphasise that current preclinical experience is mostly based on FLASH electrons and that clinical application of FLASH-RT is very limited. The incorporation of FLASH-RT into routine clinical radiotherapy will require the development of devices capable of producing FLASH photon beams.

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.

Overcoming Resistance to Platinum-Based Drugs in Ovarian Cancer by Salinomycin and Its Derivatives-An In Vitro Study.

Polyether ionophore salinomycin (SAL) and its semi-synthetic derivatives are recognized as very promising anticancer drug candidates due to their activity against various types of cancer cells, including multidrug-resistant populations. Ovarian cancer is the deadliest among gynecologic malignancies, which is connected with the development of chemoresistant forms of the disease in over 70% of patients after initial treatment regimen. Thus, we decided to examine the anticancer properties of SAL and selected SAL derivatives against a series of drug-sensitive (A2780, SK-OV-3) and derived drug-resistant (A2780 CDDP, SK-OV-3 CDDP) ovarian cancer cell lines. Although SAL analogs showed less promising IC50 values than SAL, they were identified as the antitumor agents that significantly overcome the resistance to platinum-based drugs in ovarian cancer, more potent than unmodified SAL and commonly used anticancer drugs-5-fluorouracil, gemcitabine, and cisplatin. Moreover, when compared with SAL used alone, our experiments proved for the first time increased selectivity of SAL-based dual therapy with 5-fluorouracil or gemcitabine, especially towards A2780 cell line. Looking closer at the results, SAL acted synergistically with 5-fluorouracil towards the drug-resistant A2780 cell line. Our results suggest that combinations of SAL with other antineoplastics may become a new therapeutic option for patients with ovarian cancer.

Chondrogenic Differentiation of Pluripotent Stem Cells under Controllable Serum-Free Conditions.

The repair of damaged articular cartilage using currently available implantation techniques is not sufficient for the full recovery of patients. Pluripotent stem cells (iPSC)-based therapies could bring new perspectives in the treatment of joint diseases. A number of protocols of in vitro differentiation of iPSC in chondrocytes for regenerative purposes have been recently described. However, in order to use these cells in clinics, the elimination of animal serum and feeder cells is essential. In our study, a strictly defined and controllable protocol was designed for the differentiation of pluripotent stem cells (BG01V, ND 41658*H, GPCCi001-A) in chondrocyte-like cells in serum- and a feeder cell-free system, using the embryoid bodies step. The extension of the protocol and culture conditions (monolayer versus 3D culture) was also tested after the initial 21 days of chondrogenic differentiation. Promotion of the chondrogenic differentiation in 3D culture via the elevated expression of genes related to chondrogenesis was achieved. Using immunofluorescence and immunohistochemistry staining techniques, the increased deposition of the specific extracellular matrix was indicated. As a result, chondrocyte-like cells in the early stages of their differentiation using pellet culture under fully controlled and defined conditions were obtained.

The Role of MicroRNAs in Early Chondrogenesis of Human Induced Pluripotent Stem Cells (hiPSCs).

Human induced pluripotent stem cells (hiPSCs) play an important role in research regarding regenerative medicine. Particularly, chondrocytes differentiated from hiPSCs seems to be a promising solution for patients suffering from osteoarthritis. We decided to perform chondrogenesis in a three-week monolayer culture. Based on transcriptome analysis, hiPSC-derived chondrocytes (ChiPS) demonstrate the gene expression profile of cells from early chondrogenesis. Chondrogenic progenitors obtained by our group are characterized by significantly high expression of Hox genes, strongly upregulated during limb formation and morphogenesis. There are scanty literature data concerning the role of microRNAs in early chondrogenesis, especially in chondrogenic differentiation of hiPSCs. The main aim of this study was to investigate the microRNA expression profile and to select microRNAs (miRNAs) taking part in early chondrogenesis. Our findings allowed for selection crucial miRNAs engaged in both diminishing pluripotency state and chondrogenic process (inter alia hsa-miR-525-5p, hsa-miR-520c-3p, hsa-miR-628-3p, hsa-miR-196b-star, hsa-miR-629-star, hsa-miR-517b, has-miR-187). These miRNAs regulate early chondrogenic genes such as: HOXD10, HOXA11, RARB, SEMA3C. These results were confirmed by RT-qPCR analysis. This work contributes to a better understanding of the role of miRNAs directly involved in chondrogenic differentiation of hiPSCs. These data may result in the establishment of a more efficient protocol of obtaining chondrocyte-like cells from hiPSCs.

Fucoidan Exerts Anticancer Effects Against Head and Neck Squamous Cell Carcinoma In Vitro.

Fucoidans have been reported to exert anticancer effects with simultaneous low toxicity against healthy tissue. That correlation was observed in several cancer models, however, it has never been investigated in head and neck cancer before. To magnify the efficacy of conventional therapy, the administration of agents like fucoidan could be beneficial. The aim of this study was to evaluate the anticancer effect of Fucus vesiculosus (FV) extract alone and with co-administration of cisplatin in head and neck squamous cell carcinoma (HNSCC) in vitro. MTT assay results revealed an FV-induced inhibition of proliferation in all tested cell lines (H103, FaDu, KB). Flow cytometric cell cycle analysis showed an FV-induced, dose-dependent arrest in either S/G2 phase (H103, FaDu) or G1 arrest (KB). Furthermore, a dose-dependent gain in apoptotic fraction was observed. Western blot analysis confirmed the induction of apoptosis. A significant dose-dependent increase in reactive oxygen species (ROS) production was revealed in the H103 cell line, while FaDu cells remained unresponsive. On the contrary, an HPV-positive cell line, KB, demonstrated a dose-dependent decrease in ROS synthesis. Moreover, fucoidan enhanced the response to cisplatin (synergistic effect) in all cell lines with the HPV-positive one (KB) being the most sensitive. These results have been confirmed by flow-cytometric apoptosis analysis. In conclusion, we confirmed that fucoidan exhibits anticancer properties against HNSCC, which are manifested by the induction of apoptosis, regulation of ROS production, cell cycle arrest, and inhibition of proliferation.

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.

Modified methods for efficiently differentiating human embryonic stem cells into chondrocyte-like cells.

INTRODUCTION: Human articular cartilage has a poor regenerative capacity. This often results in the serious joint disease- osteoarthritis (OA) that is characterized by cartilage degradation. An inability to self-repair provided extensive studies on AC regeneration. The cell-based cartilage tissue engineering is a promising approach for cartilage regeneration. So far, numerous cell types have been reported to show chondrogenic potential, among others human embryonic stem cells (hESCs). MATERIALS AND METHODS: However, the currently used methods for directed differentiation of human ESCs into chondrocyte-like cells via embryoid body (EB) formation, micromass culture (MC) and pellet culture (PC) are not highly efficient and require further improvement. In the present study, these three methods for hESCs differentiation into chondrocyte-like cells in the presence of chondrogenic medium supplemented with diverse combination of growth factors (GFs) were evaluated and modified. RESULTS: The protocols established here allow highly efficient, simple and inexpensive production of a large number of chondrocyte-like cells suitable for transplantation into the sites of cartilage injury. The most crucial issue is the selection of appropriate GFs in defined concentration. The obtained stem-derived cells reveal the presence of chondrogenic markers such as type II collagen, Sox6 and Sox9 as well as the lack or significantly lower level of pluripotency markers including Nanog and Oct3/4. DISCUSSION: The most efficient method is the differentiation throughout embryoid bodies. In turn, chondrogenic differentiation via pellet culture is the most promising method for implementation on clinical scale. The most useful GFs are TGF-ß1, -3 and BMP-2 that possess the most chondrogenic potential. These methods can also be used to obtain chondrocyte-like cells from differentiating induced pluripotent stem cells (iPSCs).

Carcinogenesis Induced by Low-dose Radiation.

BACKGROUND: Although the effects of high dose radiation on human cells and tissues are relatively well defined, there is no consensus regarding the effects of low and very low radiation doses on the organism. Ionizing radiation has been shown to induce gene mutations and chromosome aberrations which are known to be involved in the process of carcinogenesis. The induction of secondary cancers is a challenging long-term side effect in oncologic patients treated with radiation. Medical sources of radiation like intensity modulated radiotherapy used in cancer treatment and computed tomography used in diagnostics, deliver very low doses of radiation to large volumes of healthy tissue, which might contribute to increased cancer rates in long surviving patients and in the general population. Research shows that because of the phenomena characteristic for low dose radiation the risk of cancer induction from exposure of healthy tissues to low dose radiation can be greater than the risk calculated from linear no-threshold model. Epidemiological data collected from radiation workers and atomic bomb survivors confirms that exposure to low dose radiation can contribute to increased cancer risk and also that the risk might correlate with the age at exposure. CONCLUSIONS: Understanding the molecular mechanisms of response to low dose radiation is crucial for the proper evaluation of risks and benefits that stem from these exposures and should be considered in the radiotherapy treatment planning and in determining the allowed occupational exposures.

Exploring the Role of HtrA Family Genes in Cancer: A Systematic Review.

PURPOSE: HtrA1, HtrA2, HtrA3 and HtrA4 appear to be involved in the development of pathologies such as cancer. This systematic review reports the results of a literature search performed to compare the expression of HtrA family genes and proteins in cancer versus non-cancer tissues and cell lines, assess relationships between HtrA expression and cancer clinical features in cancer, and analyse the molecular mechanism, by which HtrA family affects cancer. METHODS: The literature search was conducted according to the PRISMA statement among four databases (PubMed, Web of Science, Embase and Scopus). RESULTS: A total of 38 articles met the inclusion criteria and involved the expression of HtrA family members and concerned the effect of HtrA expression on cancer and metastasis development or on the factor that influences it. Additionally, 31 reports were retrieved manually. Most articles highlighted that HtrA1 and HtrA3 exhibited tumour suppressor activity, while HtrA2 was associated with tumour growth and metastasis. There were too few studies to clearly define the role of the HtrA4 protease in tumours. CONCLUSION: Although the expression of serine proteases of the HtrA family was dependent on tumour type, stage and the presence of metastases, most articles indicated that HtrA1 and HtrA3 expression in tumours was downregulated compared with healthy tissue or cell lines. The expression of HtrA2 was completely study dependent. The limited number of studies on HtrA4 expression made it impossible to draw conclusions about differences in expression between healthy and tumour tissue. The conclusions drawn from the study suggest that HtrA1 and HtrA3 act as tumour suppressors.

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.

CLIC1 plasma concentration is associated with lymph node metastases in oral squamous cell carcinoma.

BACKGROUND: Previous studies have shown that the chloride intracellular channel 1 (CLIC1) protein is overexpressed in oral squamous cell carcinoma (OSCC) and nasopharyngeal carcinoma. Patients with these diseases had significantly higher CLIC1 plasma levels than healthy controls. OBJECTIVES: To determine the plasma concentration of CLIC1 in patients with OSCC and laryngeal squamous cell carcinoma (LSCC). MATERIAL AND METHODS: We collected blood samples from patients diagnosed with OSCC (n = 13) and LSCC (n = 7), as well as from healthy controls (n = 8). The blood samples were centrifuged to obtain plasma and stored at -80°C. The CLIC1 plasma concentration was determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: The mean CLIC1 plasma concentration was higher in the OSCC group than in the LSCC and control groups. Patients with OSCC and nodal metastases had significantly higher CLIC1 plasma concentration levels than nonmetastatic patients (p < 0.0001; Tukey's multiple comparisons test) and controls (p = 0.0004). The CLIC1 concentration correlated significantly with the presence of nodal spread (p = 0.0003; Spearman's r = 0.8613) and overall TNM staging (p = 0.0167; Spearman's r = 0.6620). No differences in CLIC1 plasma levels were observed between the LSCC and control groups. The CLIC1 plasma concentration was not associated with age, sex, tumor stage, or tumor grade. CONCLUSIONS: There were no differences in CLIC1 plasma concentration between healthy controls and patients with LSCC. However, our findings suggest that the presence of this protein in plasma may be associated with lymphatic metastasis in patients with OSCC. More research is needed to confirm this possible association.

Gene expression profile of hiPSC-derived cells differentiated with growth factors, forskolin and conditioned medium from human adrenocortical cell line.

BACKGROUND: Adrenocortical carcinoma (ACC) affects approx. 2 in 1,000,000 individuals in the USA, and is more common in females than males. Adrenocortical carcinoma often presents with severe symptoms, such as abdominal pain, high blood pressure, acne, hair overgrowth, and voice deepening. OBJECTIVES: Research on ACC constitutes a large body of published data. There is an increased need for easy access to ACC-derived biological material. Moreover, there are limited numbers of human cell lines available. For this reason, we attempted to differentiate human induced pluripotent stem cells (hiPSCs) into adrenocortical-like cells to establish a new functional cell line. MATERIAL AND METHODS: We conducted a long-term differentiation process (35 and 70 days) in the presence of growth factors (GFs), forskolin and conditioned medium collected from the human adrenal carcinoma (HAC15) cell line. Then, we analyzed the gene expression profile of the differentiated cells. RESULTS: The obtained cells possess features characteristic of all 3 primary germ layers. Interestingly, the differentiated cells demonstrated an extremely high level of gene expression for those involved in endocrine processes, namely glycoprotein hormones, alpha polypeptide (CGA), insulin receptor substrate 4 (IRS4), and pancreatic progenitor cell differentiation and proliferation factor-like protein (PPDPFL). CONCLUSIONS: The results of the study indicate that we obtained progenitors derived from endoderm with some characteristics of pancreatic-like cells. The endodermal derivative differentiation is a very challenging and complicated process; thus, the results presented in this study deserve closer consideration.

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.

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.

Biological response of adrenal carcinoma and melanoma cells to mitotane treatment.

A previous case report described an adrenal incidentaloma initially misdiagnosed as adrenocortical carcinoma (ACC), which was treated with mitotane. The final diagnosis was metastatic melanoma of unknown primary origin. However, the patient developed rapid disease progression after mitotane withdrawal, suggesting a protective role for mitotane in a non-adrenal-derived tumor. The aim of the present study was to determine the biological response of primary melanoma cells obtained from that patient, and that of other established melanoma and ACC cell lines, to mitotane treatment using a proliferation assay, flow cytometry, quantitative PCR and microarrays. Although mitotane inhibited the proliferation of both ACC and melanoma cells, its role in melanoma treatment appears to be limited. Flow cytometry analysis and transcriptomic studies indicated that the ACC cell line was highly responsive to mitotane treatment, while the primary melanoma cells showed a moderate response in vitro. Mitotane modified the activity of several key biological processes, including 'mitotic nuclear division', 'DNA repair', 'angiogenesis' and 'negative regulation of ERK1 and ERK2 cascade'. Mitotane administration led to elevated levels of DNA double-strand breaks, necrosis and apoptosis. The present study provides a comprehensive insight into the biological response of mitotane-treated cells at the molecular level. Notably, the present findings offer new knowledge on the effects of mitotane on ACC and melanoma cells.

Ionizing radiation exposure of stem cell-derived chondrocytes affects their gene and microRNA expression profiles and cytokine production.

Human induced pluripotent stem cells (hiPSCs) can be differentiated into chondrocyte-like cells. However, implantation of these cells is not without risk given that those transplanted cells may one day undergo ionizing radiation (IR) in patients who develop cancer. We aimed to evaluate the effect of IR on chondrocyte-like cells differentiated from hiPSCs by determining their gene and microRNA expression profile and proteomic analysis. Chondrocyte-like cells differentiated from hiPSCs were placed in a purpose-designed phantom to model laryngeal cancer and irradiated with 1, 2, or 3 Gy. High-throughput analyses were performed to determine the gene and microRNA expression profile based on microarrays. The composition of the medium was also analyzed. The following essential biological processes were activated in these hiPSC-derived chondrocytes after IR: "apoptotic process", "cellular response to DNA damage stimulus", and "regulation of programmed cell death". These findings show the microRNAs that are primarily responsible for controlling the genes of the biological processes described above. We also detected changes in the secretion level of specific cytokines. This study demonstrates that IR activates DNA damage response mechanisms in differentiated cells and that the level of activation is a function of the radiation dose.

The Analysis of Inflammation-Related Proteins in a Cargo of Exosomes Derived from the Serum of Uveal Melanoma Patients Reveals Potential Biomarkers of Disease Progression.

BACKGROUND: Uveal melanoma (UM) is the most common intraocular tumour in adults with a poor prognosis and extremely high mortality rate due to the development of metastatic disease. However, despite relatively good knowledge about the histological and genetic risk factors for metastasis development, there is no specific biomarker that would allow early detection of UM progression. Recently, exosomes and their molecular cargo have been widely studied in the search for potential biomarkers in several cancers. The purpose of this study was to analyze the inflammation-related protein cargo of exosomes derived from the serum of primary and metastatic UM patients and healthy donors. METHODS: The exosomes were isolated from the serum of primary and metastatic UM patients and healthy donors. Using multiplex immunoassay technology, we analyzed the concentration of 37 inflammation-related proteins in obtained exosomes. RESULTS: The analysis of protein cargo showed several molecules related to inflammation, such as interferon-gamma, interleukin 2, 22 and 12(p40), Pentraxin-3, TNFSF13B and TNFSF8 which were significantly enriched in metastatic UM exosomes. We showed a significant correlation between the disease stage and the concentration of these inflammation-related proteins from exosomal cargo. CONCLUSIONS: Based on the obtained results, we propose the panel of exosomal proteins for early detection of uveal melanoma progression into metastatic disease.