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.

Methodological and Cellular Factors Affecting the Magnitude of Breast Cancer and Normal Cell Radiosensitization Using Gold Nanoparticles.

Purpose: Breast cancer (BC) is the most common malignant tumor in women, which most often originates from the epithelial tissue of the breast gland. One of the most recommended kinds of treatment is radiotherapy (RT), but irradiation (IR) can affect not only the cancer tumor but also the healthy tissue around it. Au nanoparticles (AuNPs) were proposed as a radiosensitizing agent for RT which would allow for lower radiation doses, reducing the negative radiation effects on healthy tissues. The main objective of the study is to assess the dependence on the radiosensitivity of BC (MDA-MB-231) and normal mammary gland epithelial cells (MCF12A) to ionizing radiation, caused by functionalized AuNPs under diverse conditions. Methods: The viability, uptake, reactive oxygen species induction, and mitochondrial membrane potential in cells were analyzed applying a time and concentration-dependent manner. After different incubation times with AuNPs, cells were exposed to 2 Gy. The determination of radiation effect in combination with AuNPs was investigated using the clonogenic assay, p53, and γH2AX level, as well as, Annexin V staining. Results: Our results highlighted the strong need for assessing the experimental conditions' optimization before the AuNPs will be implemented with IR. Moreover, results indicated that AuNPs did not act universally in cells. Conclusion: AuNPs could be a promising tool as a radiotherapy sensitizing agent, but it should be specified and deeply investigated under what conditions it will be applied taking into consideration not only AuNPs modifications but also the model and experimental conditions.

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.

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.

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 Combination of Liposomes and Metallic Nanoparticles as Multifunctional Nanostructures in the Therapy and Medical Imaging-A Review.

Nanotechnology has introduced a new quality and has definitely developed the possibilities of treating and diagnosing various diseases. One of the scientists' interests is liposomes and metallic nanoparticles (LipoMNPs)-the combination of which has introduced new properties and applications. However, the field of creating hybrid nanostructures consisting of liposomes and metallic nanoparticles is relatively little understood. The purpose of this review was to compile the latest reports in the field of treatment and medical imaging using of LipoMNPs. The authors focused on presenting this issue in the direction of improving the used conventional treatment and imaging methods. Most of all, the nature of bio-interactions between nanostructures and cells is not sufficiently taken into account. As a result, overcoming the existing limitations in the implementation of such solutions in the clinic is difficult. We concluded that hybrid nanostructures are used in a very wide range, especially in the treatment of cancer and magnetic resonance imaging. There were also solutions that combine treatments with simultaneous imaging, creating a theragnostic approach. In the future, researchers should focus on the description of the biological interactions and the long-term effects of the nanostructures to use LipoMNPs in the treatment of patients.

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.

Currently used in clinical practice beam rate changes have no significant effect on the reduction of clonogenic capacity of PNT1A cells in vitro.

BACKGROUND: Due to the lack of selectivity of ionizing radiation between normal and cancer cells, it is important to improve the existing radiation patterns. Lowering the risk of cancer recurrence and comfort during treatment are priorities in radiotherapy. MATERIALS AND METHODS: In the experiment we used dose verification to determine the irradiation time calculated by a treatment planning system for 6XFFF and 10XFFF beams. Cells cultured under standard conditions were irradiated with a dose of 2 Gy at different beam rates 400 MU/min, 600 MU/min, 800 MU/min, 1000 MU/min, 1400 MU/min, 1600 MU/min and 2400 MU/min using 6XFFF, 10XFFF and 6XFF beams. RESULTS: The experiment was aimed at comparing the biological response of normal prostate cells after clinically applied radiation patterns. No statistically significant differences in the cellular response were observed. The wide range of beam rates as well as the beam profiles did not significantly affect cell proliferation. CONCLUSIONS: High beam rates, without significantly affecting the clonogenic capacity of cells, have an impact on the quality of patient's treatment. With the increasing beam rate the irradiation time is shortened, which has an important impact on patients' health. This experiment can have a practical significance.

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.

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.