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.

Empowering personalized medicine: unleashing the potential of patient-derived explants in clinical practice.

In recent decades, significant progress has been made in understanding the molecular characteristics of cancer and its microenvironment, leading to the development of life-saving treatments. However, patients often experience side effects from standard therapies, highlighting the need for personalized medicine. Personalized medicine aims to customize drug therapy and preventive care based on individual patients' specific requirements. The heterogeneity within tumors and among patients necessitates personalized medicine approaches. Patient-derived organoids (PDOs), xenografts (PDXs), and explants (PDEs) have emerged as valuable models for studying tumor behaviour and drug response. This paper aims to summarize the latest advancements in patient-derived explants, focusing on their potential utility in the clinic. Different methods for culturing PDEs, including the free-floating approach, the grid method, and sponge scaffolds, are discussed. These approaches provide opportunities for long-term viability, oxygen and nutrient supply, and maintenance of tissue integrity. Additionally, various solid tumor models using PDEs are highlighted, together with assays to study PDE viability, characteristics, and response to drug treatment.

The Role of Functionalization and Size of Gold Nanoparticles in the Response of MCF-7 Breast Cancer Cells to Ionizing Radiation Comparing 2D and 3D In Vitro Models.

Gold nanoparticles (AuNPs), as an agent enhancing radiosensitivity, play a key role in the potential treatment of breast cancer (BC). Assessing and understanding the kinetics of modern drug delivery systems is a crucial element that allows the implementation of AuNPs in clinical treatment. The main objective of the study was to assess the role of the properties of gold nanoparticles in the response of BC cells to ionizing radiation by comparing 2D and 3D models. In this research, four kinds of AuNPs, different in size and PEG length, were used to sensitize cells to ionizing radiation. The in vitro viability, uptake, and reactive oxygen species generation in cells were investigated in a time- and concentration-dependent manner using 2D and 3D models. Next, after the previous incubation with AuNPs, cells were irradiated with 2 Gy. The assessment of the radiation effect in combination with AuNPs was analyzed using the clonogenic assay and γH2AX level. The study highlights the role of the PEG chain in the efficiency of AuNPs in the process of sensitizing cells to ionizing radiation. The results obtained imply that AuNPs are a promising solution for combined treatment with radiotherapy.

Conditioned Medium - Is it an Undervalued Lab Waste with the Potential for Osteoarthritis Management?

BACKGROUND: The approaches currently used in osteoarthritis (OA) are mainly short-term solutions with unsatisfactory outcomes. Cell-based therapies are still controversial (in terms of the sources of cells and the results) and require strict culture protocol, quality control, and may have side-effects. A distinct population of stromal cells has an interesting secretome composition that is underrated and commonly ends up as biological waste. Their unique properties could be used to improve the existing techniques due to protective and anti-ageing properties. SCOPE OF REVIEW: In this review, we seek to outline the advantages of the use of conditioned media (CM) and exosomes, which render them superior to other cell-based methods, and to summarise current information on the composition of CM and their effect on chondrocytes. MAJOR CONCLUSIONS: CM are obtainable from a variety of mesenchymal stromal cell (MSC) sources, such as adipose tissue, bone marrow and umbilical cord, which is significant to their composition. The components present in CMs include proteins, cytokines, growth factors, chemokines, lipids and ncRNA with a variety of functions. In most in vitro and in vivo studies CM from MSCs had a beneficial effect in enhance processes associated with chondrocyte OA pathomechanism. GENERAL SIGNIFICANCE: This review summarises the information available in the literature on the function of components most commonly detected in MSC-conditioned media, as well as the effect of CM on OA chondrocytes in in vitro culture. It also highlights the need to standardise protocols for obtaining CM, and to conduct clinical trials to transfer the effects obtained in vitro to human subjects.

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.

Gallic Acid-Functionalized, TiO2-Based Nanomaterial-Preparation, Physicochemical and Biological Properties.

Wound healing and skin tissue regeneration remain the most critical challenges faced by medical professionals. Titanium(IV) oxide-based materials were proposed as components of pharmaceutical formulations for the treatment of difficult-to-heal wounds and unsightly scarring. A gallic acid-functionalized TiO2 nanomaterial (TiO2-GA) was obtained using the self-assembly technique and characterized using the following methods: scanning electron microscopy (SEM), transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), X-ray powder diffraction (XRPD), infrared spectroscopy (IR), Raman spectroscopy and thermogravimetry (TG). Additionally, physicochemical and biological tests (DPPH assay, Microtox® acute toxicity test, MTT assay) were performed to assess antioxidant properties as well as to determine the cytotoxicity of the novel material against eukaryotic (MRC-5 pd19 fibroblasts) and prokaryotic (Staphylococcus aureus, Escherichia coli, Candida albicans, Aliivibrio fischeri) cells. To determine the photocytotoxicity of the material, specific tests were carried out with and without exposure to visible light lamps (425 nm). Following the results, the TiO2-GA material could be considered an additive to dressings and rinsing suspensions for the treatment of difficult-to-heal wounds that are at risk of bacterial infections.

The Induced Pluripotent Stem Cells in Articular Cartilage Regeneration and Disease Modelling: Are We Ready for Their Clinical Use?

The development of induced pluripotent stem cells has brought unlimited possibilities to the field of regenerative medicine. This could be ideal for treating osteoarthritis and other skeletal diseases, because the current procedures tend to be short-term solutions. The usage of induced pluripotent stem cells in the cell-based regeneration of cartilage damages could replace or improve on the current techniques. The patient's specific non-invasive collection of tissue for reprogramming purposes could also create a platform for drug screening and disease modelling for an overview of distinct skeletal abnormalities. In this review, we seek to summarise the latest achievements in the chondrogenic differentiation of pluripotent stem cells for regenerative purposes and disease modelling.

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.

The m6A RNA Modification Quantity and mRNA Expression Level of RNA Methylation-Related Genes in Head and Neck Squamous Cell Carcinoma Cell Lines and Patients.

RNA methylation at the nitrogen sixth of adenosine (m6A, N6-methyladenosine) is the most abundant RNA modification which plays a crucial role in all RNA metabolic aspects. Recently, m6A modification has been assigned to mediate the biological processes of cancer cells, but their significance in HNSCC development is still poorly described. Thus, the main aim of this study was to globally quantify m6A modification by the mass spectrometry approach and determine the mRNA expression level of selected m6A RNA methyltransferase (METTL3), demethylase (FTO), and m6A readers (YTHDF2, YTHDC2) in 45 HNSCC patients and 4 cell lines (FaDu, Detroit 562, A-253 and SCC-15) using qPCR. In the results, we have not observed differences in the global amount of m6A modification and the mRNA level of the selected genes between the cancerous and paired-matched histopathologically unchanged tissues from 45 HNSCC patients. However, we have found a positive correlation between selected RNA methylation machinery genes expression and m6A abundance on total RNA and characterized the transcript level of those genes in the HNSCC cell lines. Moreover, the lack of global m6A differences between cancerous and histopathologically unchanged tissues suggests that m6A alterations in specific RNA sites may specifically influence HNSCC tumorigenesis.

Future Perspectives of Proton Therapy in Minimizing the Toxicity of Breast Cancer Radiotherapy.

The toxicity of radiotherapy is a key issue when analyzing the eligibility criteria for patients with breast cancer. In order to obtain better results, proton therapy is proposed because of the more favorable distribution of the dose in the patient's body compared with photon radiotherapy. Scientific groups have conducted extensive research into the improved efficacy and lower toxicity of proton therapy for breast cancer. Unfortunately, there is no complete insight into the potential reasons and prospects for avoiding undesirable results. Cardiotoxicity is considered challenging; however, researchers have not presented any realistic prospects for preventing them. We compared the clinical evidence collected over the last 20 years, providing the rationale for the consideration of proton therapy as an effective solution to reduce cardiotoxicity. We analyzed the parameters of the dose distribution (mean dose, Dmax, V5, and V20) in organs at risk, such as the heart, blood vessels, and lungs, using the following two irradiation techniques: whole breast irradiation and accelerated partial breast irradiation. Moreover, we presented the possible causes of side effects, taking into account biological and technical issues. Finally, we collected potential improvements in higher quality predictions of toxic cardiac effects, like biomarkers, and model-based approaches to give the full background of this complex issue.

The involvement of small heat shock protein in chemoresistance in ovarian cancer - in vitro study.

Ovarian cancer is the most deadly gynecologic malignancy worldwide. Although the primary response to chemotherapy is high, the majority of patients will develop resistance against applied treatment. In this study, we focused on resistance to cisplatin, a first-line drug used for the treatment of ovarian cancer. The mechanism of the resistance development process is widely described, but there is a lack of information about the involvement of members of small heat shock proteins (HSPs) and their transport via exosomes. In this study, we used two cell lines: A2780 and SKOV3, and their cisplatin-resistance variants: A2780 CDDP and SKOV3 CDDP. We have shown that the expression of three small HSPs (HSPB5, HSPB6, and HSPB8) in cisplatin-resistant cell lines differs from their sensitive counterparts. Further, we isolated exosomes and determined the small HSPs in their cargo. In A2780 WT we observed a low amount of HSPB5 and HSPB6. We did not observe the expression of small HSPs in the SKOV3 cell line in both sensitive and resistant variants. Our data suggest the involvement of small HSPs in drug resistance of ovarian cancer and their presence is not related to exosomal transport. Analysis of the biological consequences of the imbalance of small HSPs expression in cisplatin resistance needs further investigation.

DHMEQ enhances the cytotoxic effect of cisplatin and carboplatin in ovarian cancer cell lines.

Ovarian cancer (OvCa) is one of the most lethal gynaecological malignancies. It is diagnosed mostly in advanced stages. Due to a lack of appropriate early detection markers and non-ambiguous symptoms, the five-year survival rate is significantly reduced. Despite a primary good response to platinum-based therapy, approximately 70% of patients will develop a chemoresistance phenotype. The activation of the NF-κB signalling pathway plays a crucial role in this process. It is responsible for increasing cell viability, cell cycle progression and induces growth and migration of neoplastic cells. A few independent studies have yet suggested a high correlation between activation of NF-κB and poor outcome in OvCa patients. Thus, developing inhibitors of the NF-κB pathway has become a new target of cancer therapies. One of the promising compounds is DHMEQ (dehydroxymethylepoxyquinomicin). Our preliminary studies indicated that DHMEQ combined with cisplatin (CDDP) or carboplatin (CBP) enhanced apoptosis in the A2780 cell line and caused cell cycle arrest in the G2/M phase in the SKOV3 cell line, but not in the normal cell line MRC-5 pd19. Moreover, the combination of those agents caused decreased motility of cells, especially with the CBP. However, the invasion of cells was not changed significantly. The analysis of drug interactions using CompuSyn software has revealed that observed effect of the doses used in the study was antagonistic, but the DRI guidelines and in vitro observation of biological response indicate that a combination of DHMEQ with CDDP or CBP could be a novel proposal in ovarian cancer treatment.

Head and Neck Squamous Cell Carcinoma: Epigenetic Landscape.

Head and neck squamous carcinoma (HNSCC) constitutes the sixth most prevalent cancer worldwide. The molecular pathogenesis of HNSCC includes disorders in cell cycle, intercellular signaling, proliferation, squamous cell differentiation and apoptosis. In addition to the genetic mutations, changes in HNSCC are also characterized by the accumulation of epigenetic alterations such as DNA methylation, histone modifications, non-coding RNA activity and RNA methylation. In fact, some of them may promote cancer formation and progression by controlling the gene expression machinery, hence, they could be used as biomarkers in the clinical surveillance of HNSCC or as targets for therapeutic strategies. In this review, we focus on the current knowledge regarding epigenetic modifications observed in HNSCC and its predictive value for cancer development.

Radiobiological models in prediction of radiation cardiotoxicity.

Coronary disease induced by previous radiotherapy is the most common cause of death among patients treated with radiotherapy for cancer. Risk factors that may affect the frequency and intensity of radiotherapy's cardiac toxicity are primarily the radiation dose and the volume of the heart exposed to radiation. The prolonged survival time of patients after radiotherapy, but also the intensive development of modern radiotherapy techniques results in the necessity of precise estimation of both tumor control probability, and the risk of normal tissue damage, thus the models describing the probability of complications in normal tissues have also been developed. The response from the cardiovascular system to high-dose radiation is known and associated with a pro-inflammatory response. However, the effect of low doses may be completely different because it induces an anti-inflammatory response. Also, there is no unambiguous answer to the question of whether RICD is a deterministic effect. Moreover, there is a lack of literature data on the use of known radiobiological models to assess the risk of cardiovascular complications. The models described are general and concerns any healthy tissue. Therefore, when planning treatment for patients, particular attention should be paid to the dose and area of ​​the heart to be irradiated.

MicroRNA Profiling During Neural Differentiation of Induced Pluripotent Stem Cells.

MicroRNAs (miRNA) play an essential role in the regulation of gene expression and influence signaling networks responsible for several cellular processes like differentiation of pluripotent stem cells. Despite several studies on the neurogenesis process, no global analysis of microRNA expression during differentiation of induced pluripotent stem cells (iPSC) to neuronal stem cells (NSC) has been done. Therefore, we compared the profile of microRNA expression in iPSC lines and in NSC lines derived from them, using microarray-based analysis. Two different protocols for NSC formation were used: Direct and two-step via neural rosette formation. We confirmed the new associations of previously described miRNAs in regulation of NSC differentiation from iPSC. We discovered upregulation of miR-10 family, miR-30 family and miR-9 family and downregulation of miR-302 and miR-515 family expression. Moreover, we showed that miR-10 family play a crucial role in the negative regulation of genes expression belonging to signaling pathways involved in neural differentiation: WNT signaling pathway, focal adhesion, and signaling pathways regulating pluripotency of stem cells.

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.

hTERT promoter methylation status in peripheral blood leukocytes as a molecular marker of head and neck cancer progression.

Cancer cells, including head and neck cancer cell carcinoma (HNSCC), are characterized by an increased telomerase activity. This enzymatic complex is active in approximately 80-90% of all malignancies, and is regulated by various factors, including methylation status of hTERT gene promoter. hTERT methylation pattern has been thoroughly studied so far. It was proved that hTERT is aberrantly methylated in tumor tissue versus healthy counterparts. However, such effect has not yet been investigated in PBLs (peripheral blood leukocytes) of cancer patients. The aim of this study was to analyze the hTERT gene promoter methylation status in blood leukocytes. DNA was extracted from PBL of 92 patients with histologically diagnosed HNSCC and 53 healthy controls. Methylation status of whole hTERT promoter fragment with independent analysis of each 19 CpG sites was performed using bisulfide conversion technique followed by sequencing of PCR products. Not significant (p = 0.0532) differences in the general frequency of hTERT CpG sites methylation were detected between patients and healthy controls. However, it was discovered that some of analyzed positions (CpG islands: 1 [p = 0.0235], 5 [p = 0.0462], 8 [p = 0.0343]) are significantly more often methylated in HNSCC patients than in controls. The opposite finding was observed in case of CpG position 2 (p = 0.0210). Furthermore, closer analysis of single CpG positions revealed differences in methylation status dependent on anatomical site and TNM classification. To conclude, hTERT promoter methylation status (general or single CpG sites) would be considered as a molecular markers of HNSCC diagnostics.

hTERT gene knockdown enhances response to radio- and chemotherapy in head and neck cancer cell lines through a DNA damage pathway modification.

The aim of the study was to analyze the effect of hTERT gene knockdown in HNSCC cells by using novel in vitro models of head and neck cancer (HNSCC), as well as improving its personalized therapy. To obtain the most efficient knockdown siRNA, shRNA-bearing lentiviral vectors were used. The efficiency of hTERT silencing was verified with qPCR, Western blot, and immunofluorescence staining. Subsequently, the type of cell death and DNA repair mechanism induction after hTERT knockdown was assessed with the same methods, followed by flow cytometry. The effect of a combined treatment with hTERT gene knockdown on Double-Strand Breaks levels was also evaluated by flow cytometry. Results showed that the designed siRNAs and shRNAs were effective in hTERT knockdown in HNSCC cells. Depending on a cell line, hTERT knockdown led to a cell cycle arrest either in phase G1 or phase S/G2. Induction of apoptosis after hTERT downregulation with siRNA was observed. Additionally, hTERT targeting with lentiviruses, followed by cytostatics administration, led to induction of apoptosis. Interestingly, an increase in Double-Strand Breaks accompanied by activation of the main DNA repair mechanism, NER, was also observed. Altogether, we conclude that hTERT knockdown significantly contributes to the efficacy of HNSCC treatment.

Expression of Pluripotency Genes in Chondrocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies.

hTERT C250T promoter mutation and telomere length as a molecular markers of cancer progression in patients with head and neck cancer.

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth leading cause of cancer worldwide, representing over half a million incidents every year. Cancer cells, including HNSCC, are characterized by increased telomerase activity. This enzymatic complex is active in ~90% of all cancer types and is responsible for the lengthening of telomeres. Highly recurrent point mutations in the human telomerase reverse transcriptase (hTERT) promoter have recently been reported in a number of human neoplasms. The aim of the present study was to analyze the prevalence of the hTERT promoter C250T mutation and telomere length in the blood leukocytes of 61 patients with HNSCC and 49 healthy individuals. Quantitative polymerase chain reaction identified the hTERT promoter mutation in 36% of patients with HNSCC. To the best of our knowledge this is first report indicating the presence of shorter telomeres in early stage tumors. In addition, the results suggest that the C250T hTERT promoter mutation and telomere length assessment may serve as important molecular markers of HNSCC progression.