Natural killer (NK) cells represent a promising tool for cancer immunotherapy; however, their efficacy against solid tumors is severely limited by the hostile tumor microenvironment (TME). This review provides a comprehensive overview of the physical, molecular, and metabolic barriers that drive NK cell dysfunction and immune evasion, emphasizing the physical challenge posed by extracellular matrix (ECM) density, which restricts infiltration. Beyond structural barriers, we examine the role of immunosuppressive cytokines (e.g., TGF-β) and immune checkpoint upregulation, both of which directly inhibit NK cell activation. Furthermore, NK cell signaling and cytotoxicity are profoundly affected by metabolic stressors such as hypoxia and acidosis, which act synergistically with the accumulation of immunosuppressive metabolites, including adenosine. These factors impair antitumor activity through multiple mechanisms, particularly the shedding of activating ligands. To investigate these complex interactions, we evaluate the advantages and disadvantages of different three-dimensional (3D) preclinical platforms, including tumor spheroids and Organ-on-Chip technologies, highlighting their distinct characteristics. Rather than advocating for a single technology, we emphasize that each model offers unique advantages for studying specific physical, chemical, and cellular components of the TME. Ultimately, leveraging the capabilities of these advanced 3D platforms is essential for deciphering microenvironmental barriers and unlocking the full therapeutic potential of NK cells against solid tumors.

The limited penetration of chemotherapeutic agents into solid tumors remains a major obstacle to effective cancer treatment and is strongly influenced by the extracellular matrix (ECM). Three-dimensional (3D) tumor spheroids derived from primary tumor cells (PTCs) represent a valuable in vitro model to study how ECM composition and organization regulate drug distribution and cytotoxicity with accurate physiological relevance. By recapitulating key features of the tumor microenvironment, including endogenous collagen deposition and diffusion-limited drug accessibility, these models enable mechanistic investigation of microenvironment-driven drug resistance. Here, we describe a set of integrated protocols to evaluate the impact of ECM remodeling on chemotherapeutic response in primary breast tumor spheroids. Spheroids are generated from isolated primary tumor cells and subjected to controlled enzymatic degradation of collagen-rich ECM using ultrapure recombinant collagenases. As a model chemotherapeutic agent, doxorubicin is employed thanks to its widespread clinical use, well-characterized cytotoxic mechanism, and intrinsic fluorescence, which allows direct visualization of drug uptake and spatial distribution by confocal microscopy. In parallel, drug-induced cytotoxicity is quantified using a luminescent 3D viability assay. Together, these protocols provide a reproducible and accessible platform for investigating ECM-mediated barriers to drug delivery and toxicity in 3D tumor models. This framework facilitates the implementation of physiologically relevant assays to study drug efficacy and therapeutic resistance in solid tumors. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of breast cancer, isolation of tumor primary cells, and generation 3D spheroids Support Protocol: Culture and storage of PTCs Basic Protocol 2: Evaluation of collagen expression and enzymatic degradation in primary tumor spheroids by confocal microscopy Basic Protocol 3: Enzymatic degradation of the endogenous matrix collagen in primary tumor spheroids using recombinant collagenases Basic Protocol 4: Assessment of doxorubicin uptake in spheroids by confocal microscopy Basic Protocol 5: Assessment of cell viability in doxorubicin-treated spheroids using CellTiter-Glo 3D assay.

Alzheimer’s disease (AD) is a neurodegenerative disorder first described more than one century ago. Over this time, many features of the disease have been discovered and, consequently, many different approaches in the diagnosis and treatment of AD have been developed. A major assumption has guided research on AD in the past: this fatal form of cognitive decline is believed to have a pathogenic basis in the deposition of amyloid beta (Aβ) aggregates throughout the brain. Consequently, a main goal of AD therapy is to reduce Aβ load, and several monoclonal antibodies targeting amyloid are among the most recent approaches to AD treatment. However, the effectiveness of these drugs is limited, as they cannot block the progression of the disease; they only slow it down in certain conditions. Many other causative factors are known to promote the development of the disease, with immune system involvement being the most investigated. Indeed, it has been well documented that the microglial response enhances the deposition of other altered proteins, such as Tau, and induces a neurotoxic microenvironment that promotes neuronal loss. In this scenario, the interaction between microglia and astrocytes is known to accelerate pathogenic processes, and a possible role for peripheral T lymphocytes in AD pathology has also been described. An interesting hypothesis is that immune cells driving chronic inflammation might worsen AD progression and, therefore, could represent a target for treatment strategies in this disease. Thus, this review article aims to summarise the role of brain and peripheral immune molecules and cells in AD. Also, immune-based treatments for AD are described, including those targeting microglia and T cells.

BACKGROUND:Tumorigenesis is a complex and dynamic process in which the tumor microenvironment (TME) plays a central role. In solid tumors, the TME contributes to key mechanisms of tumor progression, including metastasis, immune evasion, and resistance to therapies. One major challenge in preclinical cancer research is the development of reliable three-dimensional (3D) in vitro models, which more accurately replicate the in vivo tumor architecture and microenvironmental conditions, such as hypoxia and extracellular matrix (ECM) organization. However, reproducing functional vascular networks and neo-angiogenesis within these models remains a key challenge. METHODS:In this study, an advanced 3D tumor model, referred to as angiotumoroids, was developed by co-culturing primary murine breast tumor cells (PTCs) with species-specific adipose-derived microvascular fragments (MVFs). Angiotumoroids were characterized using scanning electron microscopy and immunostaining, and angiogenesis was evaluated through collagen gel sprouting assays. High-resolution proteomic profiling was conducted, focusing on signatures associated with angiogenesis, extracellular matrix (ECM) composition, and tissue remodeling. Additionally, the response and internalization to anticancer drug treatments were evaluated. RESULTS:MVFs are successfully integrated in angiotumoroids, resulting in the formation of vasculature-like structures and demonstrating robust structural organization with dynamic modulation of matrix metalloproteinase 9. Formation of neovasculature was visualized through sprouting and branching, driven by both direct PTC-MVF interactions and PTC-conditioned media, highlighting the roles of juxtacrine and paracrine signaling. Proteomic profiling revealed distinct expression patterns associated with angiogenesis, ECM components (including collagen types I and IV), and active ECM remodeling with elevated MMP expression. Additionally, angiotumoroids showed increased expression of ATP-binding cassette (ABC) transporters, particularly ABCB1 (P-glycoprotein), suggesting potential mechanisms of drug efflux. Functionally, angiotumoroids demonstrated reduced sensitivity to doxorubicin compared to PTC spheroids, maintaining structural integrity and higher cell viability post-treatment. Time-course analysis revealed preferential doxorubicin accumulation in MVF-enriched regions, as confirmed by colocalization with CD31, indicating a spatially regulated distribution of the drug mediated by the vascular compartment. CONCLUSIONS:Collectively, these findings establish angiotumoroids as a robust and physiologically relevant in vitro model for studying tumor vascularization, ECM dynamics, and therapeutic response. This platform holds significant promise for predictive cancer research and preclinical drug screening, bridging the gap between traditional in vitro systems and in vivo models.

Continuous Gradient Pore Scaffolds (CGPS) represent a promising strategy in tissue engineering to mimic the hierarchical structures of human tissues. CGPS provide a suitable microenvironment for cell growth while ensuring mechanical support during tissue regeneration due to a gradual transition in geometry, density, and porosity. In this study, poly-L-lactic acid (PLLA) scaffolds were produced via Thermally Induced Phase Separation (TIPS), an exchange-driven technique that enables customized morphologies in terms of pore size and distribution by tuning the thermal path. Heat transfer phenomena were analyzed using MATLAB to predict the temperature-time profiles and evaluate the thermal driving force as a key factor in pore gradient generation. To validate the model, scaffolds were produced in four configurations by changing the presence of a polytetrafluoroethylene (PTFE) thermal resistance during two cooling steps (thermostatic baths), starting from a ternary solution of PLLA (4 wt%), 1,4-dioxane/deionized water (87/13 wt%). Morphological, quantitative, and calorimetric analyses confirmed that both cooling steps influence pore size. In particular, the first bath affects pore dimensions, whereas the quench influences pore gradient generation. Samples quenched without thermal insulation showed a gradient from border (126 ± 51 μm) to center (184 ± 60 μm). Those with PTFE insulator exhibited a more homogeneous morphology (225 ± 90 μm at the border and 244 ± 112 μm at the center). These results suggest that heat-transfer tuning is an effective strategy to produce customized CGPS. This strategy overcomes the difficulty of controlling scaffold morphology and producing porous and well-interconnected networks, essential features for tissue regeneration.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that presents with heterogeneous clinical and pathological features, necessitating improved biomarkers for accurate diagnosis and patient stratification. In this study, we applied a data-independent acquisition-based proteomics workflow to cerebrospinal fluid (CSF) samples from 138 individuals, including AD patients with high (Aβ+/tau+) or normal (Aβ+/tau-) CSF tau levels, and non-AD controls. Analysis using an Astral mass spectrometer enabled unprecedented proteome depth, identifying 2661 proteins with high data completeness. Comparative proteomic profiling revealed distinct protein signatures for Aβ+/tau+ and Aβ+/tau- subtypes. These findings were validated using an independent internal cohort and further corroborated with publicly available datasets from larger external AD cohorts, demonstrating the robustness and reproducibility of our results. Using machine learning, we identified a panel of 15 protein classifiers that accurately distinguished the two AD subtypes and controls across datasets. Notably, several of these proteins were elevated in the preclinical stage, underscoring their potential utility for early diagnosis and stratification. Together, our results demonstrate the power of data-independent acquisition proteomics on the Astral platform, combined with machine learning, to uncover subtype-specific biomarkers of AD and support the development of personalized diagnostic strategies.

Solid tumors have a complex extracellular matrix (ECM) that significantly affects tumor behavior and response to therapy. Understanding the ECM's role is crucial for advancing cancer research and treatment. This study established an in vitro model using primary cells isolated from a rat breast tumor to generate three-dimensional spheroids. Monolayer cells and spheroid cultures exhibited different protein expression patterns, with primary tumor spheroids presenting an increased level of ECM-related proteins and a more complex extracellular environment. Furthermore, spheroids produce endogenous collagen type I matrix, which is the main component of the tumoral ECM. This matrix is arranged predominantly around the 3D structure, mimicking the conditions of solid tumors. Treatments with recombinant collagenases class II (acting on the linear collagen region) and class I (acting on the 3D-helix region) completely degrade collagen within the spheroid structure. Collagenase pretreatment enhances the accessibility of the anticancer drug doxorubicin to penetrate the core of spheroids and sensitize them to doxorubicin-induced cytotoxicity. Our findings highlight the importance of overcoming drug resistance in breast cancer by targeting the ECM and proposing a novel strategy for improving therapeutic outcomes in solid tumors. By employing a three-dimensional spheroid model, with an endogenous ECM, we can offer more relevant insights into tumor biology and treatment responses.

Vibrio is a genus of ubiquitous aquatic bacteria that includes numerous pathogenic species. Their remarkable genomic plasticity and rapid evolution make them of particular interest from both clinical and ecological perspectives. Successful infection by Vibrio species often relies on multiple virulence factors, including secreted enzymes. Here, we report the characterization of a novel environmental Vibrio strain isolated from a wild octopus that developed fulminant septicaemia accompanied by widespread soft tissue lysis. These severe symptoms prompted a detailed investigation into the bacterium’s identity and enzymatic profile, focused on proteases as potential virulence factors. Multi-locus sequence analysis placed the isolate within the Harveyi clade but revealed no perfect match to known strains, supporting its designation as a novel strain. Biochemical assays demonstrated strong proteolytic—including collagenolytic—activity, which makes this strain a promising source of enzymes for biotechnological applications.

The application of extracellular vesicles (EVs) as therapeutics or nanocarriers in cell-free therapies necessitates meticulous evaluations of different features, including their identity, bioactivity, batch-to-batch reproducibility, and stability. Given the inherent heterogeneity in EV preparations, this assessment demands sensitive functional assays to provide key quality control metrics, complementing established methods to ensure that EV preparations meet the required functionality and quality standards. Here, we introduce the detectEV assay, an enzymatic-based approach for assessing EV luminal cargo bioactivity and membrane integrity. This method is fast, cost-effective, and quantifiable through enzymatic units. Utilizing microalgae-derived EVs, known as nanoalgosomes, as model systems, we optimised the assay parameters and validated its sensitivity and specificity in quantifying the enzymatic activity of esterases within the EV lumen while also evaluating EV membrane integrity. Compared to conventional methods that assess physicochemical features of EVs, our single-step analysis efficiently detects batch-to-batch variations by evaluating changes in luminal cargo bioactivity and integrity across various EV samples, including differences under distinct storage conditions and following diverse isolation and exogenous loading methods, all using small sample sizes. The detectEV assay's application to various human-derived EV types demonstrated its versatility and potential universality. Additionally, the assay effectively predicted EV functionality, such as the antioxidant activity of different nanoalgosome batches. Our findings underscore the detectEV assay's utility in comprehensive characterization of EV functionality and integrity, enhancing batch-to-batch reproducibility and facilitating their therapeutic applications.

Alzheimer’s disease (AD) represents the most common form of dementia and affects million people worldwide, with a high social burden and considerable economic costs. AD diagnosis benefits from a well-established panel of laboratory tests that allow ruling-in patients, along with FDG and amyloid PET imaging tools. The main laboratory tests used to identify AD patients are Aβ40, Aβ42, the Aβ42/Aβ40 ratio, phosphorylated Tau 181 (pTau181) and total Tau (tTau). Although they are measured preferentially in the cerebrospinal fluid (CSF), some evidence about the possibility for blood-based determination to enter clinical practice is growing up. Unfortunately, CSF biomarkers for AD and, even more, the blood-based ones, present a few flaws, and twenty years of research in this field did not overcome these pitfalls. The tale even worsens when the issue of treating AD is addressed due to the lack of effective strategies despite the many decades of attempts by pharmaceutic industries and scientists. Amyloid-based drugs failed to stop the disease, and no neuroinflammation-based drugs have been demonstrated to work so far. Hence, only symptomatic therapy is available, with no disease-modifying treatment on hand. Such a desolate situation fully justifies the active search for novel biomarkers to be used as reliable tests for AD diagnosis and molecular targets for treating patients. Recently, a novel group of molecules has been identified to be used for AD diagnosis and follow-up, the nuclei acid-based biomarkers. Nucleic acid-based biomarkers are a composite group of extracellular molecules consisting of DNA and RNA alone or in combination with other molecules, including proteins. This review article reports the main findings from the studies carried out on these biomarkers during AD, and highlights their advantages and limitations.

Alzheimer’s disease (AD) is the most common form of dementia, characterized by the accumulation of β-amyloid plaques, tau tangles, neuroinflammation, and synaptic/neuronal loss, the latter being the strongest correlating factor with memory and cognitive impairment. Through an in vitro study on a neurons–astrocytes–microglia (NAM) co-culture system, we analyzed the effects of cerebrospinal fluid (CSF) samples from AD and non-AD patients (other neurodegenerative pathologies). Treatment with CSF from AD patients showed a loss of neurofilaments and spheroids, suggesting the presence of elements including CX3CL1 (soluble form), destabilizing the neurofilaments, cellular adhesion processes, and intercellular contacts. The NAM co-cultures were analyzed in immunofluorescence assays for several markers related to AD, such as through zymography, where the expression of proteolytic enzymes was quantified both in cell extracts and the co-cultures’ conditioned medium (CM). Through qRT-PCR assays, several genes involved in the formation of β-amyloid plaque, in phosphorylation of tau, and in inflammation pathways and MMP expression were investigated.

Tissue engineering (TE) offers promising solutions for regenerative medicine through the use of porous scaffolds and cells, providing a favorable environment for the production of functional three-dimensional (3D) tissues. However, TE strategies have faced physiological limitations with static three-dimensional culture alone, and perfusion bioreactors provide a controlled environment that better mimics native tissue. In this study, we present the optimized geometry of an existing custom-made perfusion bioreactor that utilizes an external airlift circulation loop, essentially a specially structured bubble column designed for the simultaneous allocation of multiple seeded scaffolds. By reducing volumes and materials, the optimized system maintains the same level of reliability and functionality. The study employs computational fluid dynamics (CFD) analysis and a mathematical model to gain insights into fluid flow and oxygen transport, respectively. Therefore, in line with the increasingly recognized trend of device miniaturization, scaling down the initial device would enable high-speed analysis of cellular response in perfused cultures, allowing the study of various morphologies, different cell populations, or different drug treatments. Furthermore, the possibility of creating series and parallel connections between multiple devices, while maintaining dimensions suitable for incubator insertion, demonstrates the potential of this system for testing engineered constructs while simultaneously enabling time and cost reduction compared to existing perfusion devices in the field of Tissue Engineering.

Collagen is a crucial protein in the extracellular matrix (ECM) essential for preserving tissue architecture and supporting crucial cellular functions like proliferation and differentiation. There are twenty-eight identified types of collagen, which are further divided into different subgroups. This protein plays a critical role in regulating tissue homeostasis. However, in solid tumors, the balance can be disrupted, due to an abundance of collagen in the tumor microenvironment, which significantly affects tumor growth, cell invasion, and metastasis. It is important to investigate the specific types of collagens in cancer ECM and their distinct roles in tumor progression to comprehend their unique contribution to tumor behavior. The diverse pathophysiological functions of different collagen types in cancers illustrate collagen’s dual roles, offering potential therapeutic options and serving as prognostic markers.

Chronic wounds, commonly known as ulcers, represent a significant challenge to public health, impacting millions of individuals every year and imposing a significant financial burden on the global health system. Chronic wounds result from the interruption of the natural wound-healing process due to internal and/or external factors, resulting in slow or nonexistent recovery. Conventional medical approaches are often inadequate to deal with chronic wounds, necessitating the exploration of new methods to facilitate rapid and effective healing. In recent years, regenerative medicine and tissue engineering have emerged as promising avenues to encourage tissue regeneration. These approaches aim to achieve anatomical and functional restoration of the affected area through polymeric components, such as scaffolds or hydrogels. This review explores collagen-based biomaterials as potential therapeutic interventions for skin chronic wounds, specifically focusing on infective and diabetic ulcers. Hence, the different approaches described are classified on an action-mechanism basis. Understanding the issues preventing chronic wound healing and identifying effective therapeutic alternatives could indicate the best way to optimize therapeutic units and to promote more direct and efficient healing.

Electrospun hybrid scaffolds composed of synthetic and natural polymers have gained increasing interest in tissue engineering applications over the last decade. In this work, scaffolds composed of polylactic acid electrospun fibers, either treated (P-PLA) or non-treated (PLA) with air-plasma, were coated with high molecular weight chitosan to create a core–shell microfibrous structure. The effective thickness control of the chitosan layer was confirmed by gravimetric, spectroscopic (FTIR-ATR) and morphological (SEM) investigations. The chitosan coating increased the fiber diameter of the microfibrous scaffolds while the tensile mechanical tests, conducted in dry and wet environments, showed a reinforcing action of the coating layer on the scaffolds, in particular when deposited on P-PLA samples. The stability of the Chi coating on both PLA and P-PLA substrates was confirmed by gravimetric analysis, while their mineralization capacity was evaluated though scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) after immersing the scaffolds in simulated body fluids (SBF) at 37 °C for 1 week. Sample biocompatibility was investigated through cell viability assay and SEM analysis on mouse pre-osteoblastic MC3T3-E1 cells grown on scaffolds at different times (1, 7, 14 and 21 days). Finally, Alizarin Red assay and qPCR analysis suggested that the combination of plasma treatment and chitosan coating on PLA electrospun scaffolds influences the osteoblastic differentiation of MC3T3-E1 cells, thus demonstrating the great potential of P-PLA/chitosan hybrid scaffolds for bone tissue engineering applications.

Abstract BACKGROUND A cell operates as an interconnected bioelectrical circuit, utilising electron transfer processes for intracellular communication, with cytochrome c (Cyt c) playing a pivotal role. The redox processes of Cyt c, occurring via electron tunnelling, are essential for its translocation into the cytosol and modulation of its conformation to bind apoptotic protease activating factor 1. This highlights the need for novel technologies capable of interacting with these processes at the atomic scale to control downstream effects and induce apoptosis in cancer cells. METHODS We demonstrate that ‘bio-nanoantennae’, when supplied with an electrical current, enable quantum biological tunnelling for electron transfer (QBET) and facilitate cellular apoptosis in patient-derived IDH wild-type glioblastoma from both the infiltrative tumour margin and proliferative core. The bio-nanoantennae were constructed from gold nanoparticles functionalised with reduced Cyt c and zinc porphyrin as a redox couple. RESULTS Electrical polarisation of these bio-nanoantennae via resonant alternating currents in preclinical glioblastoma cells led to decreased metabolic activity and reduced cell viability by oxidizing Cyt c, thus inducing cellular stress. No significant effect was observed in healthy human astrocyte counterparts. The cytosol localised bio-nanoantennae induced differential gene expression related to ion channels, apoptosis, cancer proliferation and tumour suppression upon activation, in tumour relative to astrocyte cell populations. The bio-nanoantennae were also tested in 3D glioblastoma spheroid models, showing similar effects, and in vivo studies demonstrated a significant reduction in glioblastoma xenograft tumour size. CONCLUSION We propose that bio-nanoantennae modulate the redox state of Cyt c under an electrical field through QBET. To validate this, we investigated the tunnel junction energy and plasmon resonance scattering, and developed a mathematical model to explain the system’s behaviour. This innovative wireless electrical–molecular nanodevice, capable of inducing cancer cell apoptosis, paves the way for further applications of quantum signalling as a new (non-pharmacological) therapeutic paradigm.

Marine environments cover more than 70% of the Earth’s surface and are among the richest and most complex ecosystems. In terms of biodiversity, the ocean represents an important source, still not widely exploited, of bioactive products derived from species of bacteria, plants, and animals. However, global warming, in combination with multiple anthropogenic practices, represents a serious environmental problem that has led to an increase in gelatinous zooplankton, a phenomenon referred to as jellyfish bloom. In recent years, the idea of “sustainable development” has emerged as one of the essential elements of green-economy initiatives; therefore, the marine environment has been re-evaluated and considered an important biological resource. Several bioactive compounds of marine origin are being studied, and among these, marine collagen represents one of the most attractive bio-resources, given its use in various disciplines, such as clinical applications, cosmetics, the food sector, and many other industrial applications. This review aims to provide a current overview of marine collagen applications in the pharmacological and biomedical fields, regenerative medicine, and cell therapy.