Plasticity and resistance of cancer stem cells as a challenge for innovative anticancer therapies - do we know enough to overcome this?

According to the CSC hypothesis, cancer stem cells are pivotal in initiating, developing, and causing cancer recurrence. Since the identification of CSCs in leukemia, breast cancer, glioblastoma, and colorectal cancer in the 1990s, researchers have actively investigated the origin and biology of CSCs. However, the CSC hypothesis and the role of these cells in tumor development model is still in debate. These cells exhibit distinct surface markers, are capable of self-renewal, demonstrate unrestricted proliferation, and display metabolic adaptation. CSC phenotypic plasticity and the capacity to EMT is strictly connected to the stemness state. CSCs show high resistance to chemotherapy, radiotherapy, and immunotherapy. The plasticity of CSCs is significantly influenced by tumor microenvironment factors, such as hypoxia. Targeting the genetic and epigenetic changes of cancer cells, together with interactions with the tumor microenvironment, presents promising avenues for therapeutic strategies. See also the Graphical abstract(Fig. 1).

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.

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.

Cancer Stem Cells-The Insight into Non-Coding RNAs.

Since their initial identification three decades ago, there has been extensive research regarding cancer stem cells (CSCs). It is important to consider the biology of cancer stem cells with a particular focus on their phenotypic and metabolic plasticity, the most important signaling pathways, and non-coding RNAs (ncRNAs) regulating these cellular entities. Furthermore, the current status of therapeutic approaches against CSCs is an important consideration regarding employing the technology to improve human health. Cancer stem cells have claimed to be one of the most important group of cells for the development of several common cancers as they dictate features, such as resistance to radio- and chemotherapy, metastasis, and secondary tumor formation. Therapies which could target these cells may develop into an effective strategy for tumor eradication and a hope for patients for whom this disease remains uncurable.

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.

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.