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A crucial challenge in medicine is choosing which drug (or combination) will be the most advantageous for a particular patient. Usually, drug response rates differ substantially, and the reasons for this response unpredictability remain ambiguous. Consequently, it is central to classify features that contribute to the observed drug response variability. Pancreatic cancer is one of the deadliest cancers with limited therapeutic achievements due to the massive presence of stroma that generates an environment that enables tumor growth, metastasis, and drug resistance. To understand the cancer-stroma cross talk within the tumor microenvironment and to develop personalized adjuvant therapies, there is a necessity for effective approaches that offer measurable data to monitor the effect of drugs at the single-cell level. Here, we develop a computational approach, based on cell imaging, that quantifies the cellular cross talk between pancreatic tumor cells (L3.6pl or AsPC1) and pancreatic stellate cells (PSCs), coordinating their kinetics in presence of the chemotherapeutic agent gemcitabine. We report significant heterogeneity in the organization of cellular interactions in response to the drug. For L3.6pl cells, gemcitabine sensibly decreases stroma-stroma interactions but increases stroma-cancer interactions, overall enhancing motility and crowding. In the AsPC1 case, gemcitabine promotes the interactions among tumor cells, but it does not affect stroma-cancer interplay, possibly suggesting a milder effect of the drug on cell dynamics.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Gemcitabina , Comunicación Celular , Línea Celular Tumoral , Microambiente TumoralRESUMEN
Invited for the cover of this issue are Anil Chandra, Lorettaâ L. delâ Mercato and co-workers at the Institute of Nanotechnology of National Research Council and the University of Salento. The image depicts how negatively charged pH-sensitive pyranine (HPTS) molecules were successfully immobilized on silica microparticles (SMPs) without compromising the molecules' pH sensitivity. These resulting sensors can be used to measure pH in vitro and in vivo due to the cytocompatibility of HPTS molecules and SMPs. Read the full text of the article at 10.1002/chem.202101568.
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Arilsulfonatos , Dióxido de Silicio , Colorantes Fluorescentes , Humanos , Concentración de Iones de HidrógenoRESUMEN
Pyranine (HPTS) is a remarkably interesting pH-sensitive dye that has been used for plenty of applications. Its high quantum yield and extremely sensitive ratiometric fluorescence against pH change makes it a very favorable for pH-sensing applications and the development of pH nano-/microsensors. However, its strong negative charge and lack of easily modifiable functional groups makes it difficult to use with charged substrates such as silica. This study reports a methodology for noncovalent HPTS immobilization on silica microparticles that considers the retention of pH sensitivity as well as the long-term stability of the pH microsensors. The study emphasizes the importance of surface charge for governing the sensitivity of the immobilized HPTS dye molecules on silica microparticles. The importance of the immobilization methodology, which preserves the sensitivity and stability of the microsensors, is also assessed.
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Colorantes Fluorescentes , Dióxido de Silicio , Arilsulfonatos , Concentración de Iones de Hidrógeno , Espectrometría de FluorescenciaRESUMEN
The tumour microenvironment (TME) strongly influences tumorigenesis and metastasis. Two of the most characterized properties of the TME are acidosis and hypoxia, both of which are considered hallmarks of tumours as well as critical factors in response to anticancer treatments. Currently, various imaging approaches exist to measure acidosis and hypoxia in the TME, including magnetic resonance imaging (MRI), positron emission tomography and optical imaging. In this review, we will focus on the latest fluorescent-based methods for optical sensing of cell metabolism and MRI as diagnostic imaging tools applied both in vitro and in vivo. The primary emphasis will be on describing the current and future uses of systems that can measure intra- and extra-cellular pH and oxygen changes at high spatial and temporal resolution. In addition, the suitability of these approaches for mapping tumour heterogeneity, and assessing response or failure to therapeutics will also be covered.
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Colorantes Fluorescentes/química , Imagen por Resonancia Magnética/métodos , Neoplasias/diagnóstico por imagen , Imagen Óptica/métodos , Microambiente Tumoral , Acidosis , Animales , Humanos , Concentración de Iones de Hidrógeno , Metaloporfirinas/química , Nanoestructuras/química , Neoplasias/irrigación sanguínea , Neoplasias/metabolismo , Neoplasias/patología , Hipoxia Tumoral , Microambiente Tumoral/fisiologíaRESUMEN
Despite numerous advances in the field of tissue engineering and regenerative medicine, monitoring the formation of tissue regeneration and its metabolic variations during culture is still a challenge and mostly limited to bulk volumetric assays. Here, a simple method of adding capsules-based optical sensors in cell-seeded 3D scaffolds is presented and the potential of these sensors to monitor the pH changes in space and time during cell growth is demonstrated. It is shown that the pH decreased over time in the 3D scaffolds, with a more prominent decrease at the edges of the scaffolds. Moreover, the pH change is higher in 3D scaffolds compared to monolayered 2D cell cultures. The results suggest that this system, composed by capsules-based optical sensors and 3D scaffolds with predefined geometry and pore architecture network, can be a suitable platform for monitoring pH variations during 3D cell growth and tissue formation. This is particularly relevant for the investigation of 3D cellular microenvironment alterations occurring both during physiological processes, such as tissue regeneration, and pathological processes, such as cancer evolution.
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Células Madre Mesenquimatosas , Diferenciación Celular , Concentración de Iones de Hidrógeno , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Functional, flexible, and integrated lab-on-chips, based on elastic membranes, are capable of fine response to external stimuli, so to pave the way for many applications as multiplexed sensors for a wide range of chemical, physical and biomedical processes. Here, we report on the use of elastic thin membranes (TMs), integrated with a reaction chamber, to fabricate a membrane-based pressure sensor (MePS) for reaction monitoring. In particular, the TM becomes the key-element in the design of a highly sensitive MePS capable to monitor gaseous species production in dynamic and temporally fast processes with high resolution and reproducibility. Indeed, we demonstrate the use of a functional MePS integrating a 2 µm thick polydimethylsiloxane TM by monitoring the dioxygen evolution resulting from catalytic hydrogen peroxide dismutation. The operation of the membrane, explained using a diffusion-dominated model, is demonstrated on two similar catalytic systems with catalase-like activity, assembled into polyelectrolyte multilayers capsules. The MePS, tested in a range between 2 and 50 Pa, allows detecting a dioxygen variation of the µmol L-1 s-1 order. Due to their structural features, flexibility of integration, and biocompatibility, the MePSs are amenable of future development within advanced lab-on-chips.
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A fundamental issue in biomedical and environmental sciences is the development of sensitive and robust sensors able to probe the analyte of interest, under physiological and pathological conditions or in environmental samples, and with very high spatial resolution. In this work, novel hybrid organic fibers that can effectively report the analyte concentration within the local microenvironment are reported. The nanostructured and flexible wires are prepared by embedding fluorescent pH sensors based on seminaphtho-rhodafluor-1-dextran conjugate. By adjusting capsule/polymer ratio and spinning conditions, the diameter of the fibers and the alignment of the reporting capsules are both tuned. The hybrid wires display excellent stability, high sensitivity, as well as reversible response, and their operation relies on effective diffusional kinetic coupling of the sensing regions and the embedding polymer matrix. These devices are believed to be a powerful new sensing platform for clinical diagnostics, bioassays and environmental monitoring.
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Nanofibras/química , Nanotecnología/métodos , Compuestos Orgánicos/química , Concentración de Iones de Hidrógeno , Iones , Microscopía Confocal , Nanofibras/ultraestructura , Factores de TiempoRESUMEN
On page 6417, L. L. del Mercato, D. Pisignano, and co-workers report a new type of 3D nanostructured pH-sensing organic fiber with embedded ratiometric fluorescent capsules. Upon proton-induced switching, the fibers undergo optical changes that are recorded by fluorescence detectors and correlated to the analyte concentration. The developed electrospinning fabrication approach is facile and versatile and enables the creation of sensitive and highly robust pH-sensing 3D scaffolds for environmental monitoring and biomedical applications, including tissue engineering and wound healing.
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Nanofibras/química , Nanotecnología/métodos , Concentración de Iones de Hidrógeno , Iones , Protones , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
Multicompartment, spherical microcontainers were engineered through a layer-by-layer polyelectrolyte deposition around a fluorescent core while integrating a ruthenium polyoxometalate (Ru4POM), as molecular motor, vis-à-vis its oxygenic, propeller effect, fuelled upon H2O2 decomposition. The resulting chemomechanical system, with average speeds of up to 25â µm s(-1), is amenable for integration into a microfluidic set-up for mixing and displacement of liquids, whereby the propulsion force and the resulting velocity regime can be modulated upon H2O2-controlled addition.
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Potassium cations play many important roles in living organisms, especially in electro-physiology, since they are involved in neurotransmission and muscle contractions. We report the synthesis of a ratiometric fluorescent microsensor for potassium (K+) detection, based on the fluorescent probe ION potassium green 4. Potassium-sensitive fluorescent microparticles were obtained by using silica as the core material. We obtained silica-based microsensors with sizes in the micrometer range, spherical shapes, good monodispersity, optimal selectivity and a sensitivity range of 0 to 40 mM. The microsensors also proved to be non-toxic in cell cultures and suitable for fluorescence imaging, offering new possibilities for non-invasive optical detection, quantification and in situ monitoring of K+ variations in cell culture systems.
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A distinct feature of pancreatic ductal adenocarcinoma (PDAC) is a prominent tumor microenvironment (TME) with remarkable cellular and spatial heterogeneity that meaningfully impacts disease biology and treatment resistance. The dynamic crosstalk between cancer cells and the dense stromal compartment leads to spatially and temporally heterogeneous metabolic alterations, such as acidic pH that contributes to drug resistance in PDAC. Thus, monitoring the extracellular pH metabolic fluctuations within the TME is crucial to predict and to quantify anticancer drug efficacy. Here, a simple and reliable alginate-based 3D PDAC model embedding ratiometric optical pH sensors and cocultures of tumor (AsPC-1) and stromal cells for simultaneously monitoring metabolic pH variations and quantify drug response is presented. By means of time-lapse confocal laser scanning microscopy (CLSM) coupled with a fully automated computational analysis, the extracellular pH metabolic variations are monitored and quantified over time during drug testing with gemcitabine, folfirinox, and paclitaxel, commonly used in PDAC therapy. In particular, the extracellular acidification is more pronounced after drugs treatment, resulting in increased antitumor effect correlated with apoptotic cell death. These findings highlight the importance of studying the influence of cellular metabolic mechanisms on tumor response to therapy in 3D tumor models, this being crucial for the development of personalized medicine approaches.
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Fluorescence imaging allows for noninvasively visualizing and measuring key physiological parameters like pH and dissolved oxygen. In our work, we created two ratiometric fluorescent microsensors designed for accurately tracking dissolved oxygen levels in 3D cell cultures. We developed a simple and cost-effective method to produce hybrid core-shell silica microparticles that are biocompatible and versatile. These sensors incorporate oxygen-sensitive probes (Ru(dpp) or PtOEP) and reference dyes (RBITC or A647 NHS-Ester). SEM analysis confirmed the efficient loading and distribution of the sensing dye on the outer shell. Fluorimetric and CLSM tests demonstrated the sensors' reversibility and high sensitivity to oxygen, even when integrated into 3D scaffolds. Aging and bleaching experiments validated the stability of our hybrid core-shell silica microsensors for 3D monitoring. The Ru(dpp)-RBITC microparticles showed the most promising performance, especially in a pancreatic cancer model using alginate microgels. By employing computational segmentation, we generated 3D oxygen maps during live cell imaging, revealing oxygen gradients in the extracellular matrix and indicating a significant decrease in oxygen level characteristics of solid tumors. Notably, after 12 h, the oxygen concentration dropped to a hypoxic level of PO2 2.7 ± 0.1%.
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The tumor microenvironment (TME) demonstrates distinct hallmarks, including acidosis, hypoxia, reactive oxygen species (ROS) generation, and altered ion fluxes, which are crucial targets for early cancer biomarker detection, tumor diagnosis, and therapeutic strategies. Various imaging and sensing techniques have been developed and employed in both research and clinical settings to visualize and monitor cellular and TME dynamics. Among these, ratiometric fluorescence-based sensors have emerged as powerful analytical tools, providing precise and sensitive insights into TME and enabling real-time detection and tracking of dynamic changes. In this comprehensive review, we discuss the latest advancements in ratiometric fluorescent probes designed for the optical mapping of pH, oxygen, ROS, ions, and biomarkers within the TME. We elucidate their structural designs and sensing mechanisms as well as their applications in in vitro and in vivo detection. Furthermore, we explore integrated sensing platforms that reveal the spatiotemporal behavior of complex tumor cultures, highlighting the potential of high-resolution imaging techniques combined with computational methods. This review aims to provide a solid foundation for understanding the current state of the art and the future potential of fluorescent nano- and microparticles in the field of cellular microenvironment sensing.
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The constant increase in cancer incidence and mortality pushes biomedical research towards the development of in vitro 3D systems able to faithfully reproduce and effectively probe the tumor microenvironment. Cancer cells interact with this complex and dynamic architecture, leading to peculiar tumor-associated phenomena, such as acidic pH conditions, rigid extracellular matrix, altered vasculature, hypoxic condition. Acidification of extracellular pH, in particular, is a well-known feature of solid tumors, correlated to cancer initiation, progression, and resistance to therapies. Monitoring local pH variations, non-invasively, during cancer growth and in response to drug treatment becomes extremely important for understanding cancer mechanisms. Here, we describe a simple and reliable pH-sensing hybrid system, based on a thermoresponsive hydrogel embedding optical pH sensors, that we specifically apply for non-invasive and accurate metabolism monitoring in colorectal cancer (CRC) spheroids. First, the physico-chemical properties of the hybrid sensing platform, in terms of stability, rheological and mechanical properties, morphology and pH sensitivity, were fully characterized. Then, the proton gradient distribution in the spheroids proximity, in the presence or absence of drug treatment, was quantified over time by time lapse confocal light scanning microscopy and automated segmentation pipeline, highlighting the effects of the drug treatment in the extracellular pH. In particular, in the treated CRC spheroids the acidification of the microenvironment resulted faster and more pronounced over time. Moreover, a pH gradient distribution was detected in the untreated spheroids, with more acidic values in proximity of the spheroids, resembling the cell metabolic features observed in vivo in the tumor microenvironment. These findings promise to shed light on mechanisms of regulation of proton exchanges by cellular metabolism being essential for the study of solid tumors in 3D in vitro models and the development of personalized medicine approaches.
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BACKGROUND: To date, no standardized protocols nor a quantitative assessment of the near-infrared fluorescence angiography with indocyanine green (NIR-ICG) are available. The aim of this study was to evaluate the timing of fluorescence as a reproducible parameter and its efficacy in predicting anastomotic leakage (AL) in colorectal surgery. METHODS: A consecutive cohort of 108 patients undergoing minimally invasive elective procedures for colorectal cancer was prospectively enrolled. The difference between macro and microperfusion (ΔT) was obtained by calculating the timing of fluorescence at the level of iliac artery division and colonic wall, respectively. RESULTS: Subjects with a ΔT ≥ 15.5± 0.5 s had a higher tendency to develop an AL (p < 0.01). The ΔT/heart rate interaction was found to predict AL with an odds ratio of 1.02 (p < 0.01); a cut-off threshold of 832 was identified (sensitivity 0.86, specificity 0.77). Perfusion parameters were also associated with a faster bowel motility resumption and a reduced length of hospital stay. CONCLUSIONS: The analysis of the timing of fluorescence provides a quantitative, easy evaluation of tissue perfusion. A ΔT/HR interaction ≥832 may be used as a real-time parameter to guide surgical decision making in colorectal surgery.
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The homeostatic control of their environment is an essential task of living cells. It has been hypothesized that, when microenvironmental pH inhomogeneities are induced by high cellular metabolic activity, diffusing protons act as signaling molecules, driving the establishment of exchange networks sustained by the cell-to-cell shuttling of overflow products such as lactate. Despite their fundamental role, the extent and dynamics of such networks is largely unknown due to the lack of methods in single-cell flux analysis. In this study, we provide direct experimental characterization of such exchange networks. We devise a method to quantify single-cell fermentation fluxes over time by integrating high-resolution pH microenvironment sensing via ratiometric nanofibers with constraint-based inverse modeling. We apply our method to cell cultures with mixed populations of cancer cells and fibroblasts. We find that the proton trafficking underlying bulk acidification is strongly heterogeneous, with maximal single-cell fluxes exceeding typical values by up to 3 orders of magnitude. In addition, a crossover in time from a networked phase sustained by densely connected "hubs" (corresponding to cells with high activity) to a sparse phase dominated by isolated dipolar motifs (i.e., by pairwise cell-to-cell exchanges) is uncovered, which parallels the time course of bulk acidification. Our method addresses issues ranging from the homeostatic function of proton exchange to the metabolic coupling of cells with different energetic demands, allowing for real-time noninvasive single-cell metabolic flux analysis.
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Nanofibras , Protones , Fermentación , Ácido Láctico , Concentración de Iones de HidrógenoRESUMEN
Zein, a corn-derived protein, has a variety of applications ranging from drug delivery to tissue engineering and wound healing. This work aims to develop a biocompatible scaffold for dermal applications based on thermally annealed electrospun propolis-loaded zein nanofibers. Pristine fibers' biocompatibility is determined in vitro. Next, propolis from Melipona quadrifasciata is added to the fibers at different concentrations (5% to 25%), and the scaffolds are studied. The physicochemical properties of zein/propolis precursor dispersions are evaluated and the results are correlated to the fibers' properties. Due to zein's and propolis' very favorable interactions, which are responsible for the increase in the dispersions surface tension, nanometric size ribbon-like fibers ranging from 420 to 575 nm are obtained. The fiber's hydrophobicity is not dependent on propolis concentration and increases with the annealing procedure. Propolis inhibitory concentration (IC50 ) is determined as 61.78 µg mL-1 . When loaded into fibers, propolis is gradually delivered to cells as Balb/3T3 fibroblasts and are able to adhere, grow, and interact with pristine and propolis-loaded fibers, and cytotoxicity is not observed. Therefore, the zein-propolis nanofibers are considered biocompatible and safe. The results are promising and provide prospects for the development of wound-healing nanofiber patches-one of propolis' main applications.
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Nanofibras , Própolis , Zeína , Animales , Própolis/química , Zeína/química , Nanofibras/química , Ingeniería de Tejidos/métodos , Sistemas de Liberación de MedicamentosRESUMEN
The detection of extracellular pH at single cell resolution is challenging and requires advanced sensibility. Sensing pH at high spatial and temporal resolution might provide crucial information in understanding the role of pH and its fluctuations in a wide range of physio-pathological cellular processes, including cancer. Here, a method to embed silica-based fluorescent pH sensors into alginate-based three-dimensional (3D) microgels tumour models, coupled with a computational method for fine data analysis, is presented. By means of confocal laser scanning microscopy, live-cell time-lapse imaging of 3D alginate microgels was performed and the extracellular pH metabolic variations were monitored in both in vitro 3D mono- and 3D co-cultures of tumour and stromal pancreatic cells. The results show that the extracellular pH is cell line-specific and time-dependent. Moreover, differences in pH were also detected between 3D monocultures versus 3D co-cultures, thus suggesting the existence of a metabolic crosstalk between tumour and stromal cells. In conclusion, the system has the potential to image multiple live cell types in a 3D environment and to decipher in real-time their pH metabolic interplay under controlled experimental conditions, thus being also a suitable platform for drug screening and personalized medicine.
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Técnicas Biosensibles , Microgeles , Neoplasias , Alginatos , Humanos , Concentración de Iones de Hidrógeno , Neoplasias/diagnóstico por imagenRESUMEN
pH balance and regulation within organelles are fundamental to cell homeostasis and proliferation. The ability to track pH in cells becomes significantly important to understand these processes in detail. Fluorescent sensors based on micro- and nanoparticles have been applied to measure intracellular pH; however, an accurate methodology to precisely monitor acidification kinetics of organelles in living cells has not been established, limiting the scope of this class of sensors. Here, silica-based fluorescent microparticles were utilized to probe the pH of intracellular organelles in MDA-MB-231 and MCF-7 breast cancer cells. In addition to the robust, ratiometric, trackable, and bioinert pH sensors, we developed a novel dimensionality reduction algorithm to automatically track and screen massive internalization events of pH sensors. We found that the mean acidification time is comparable among the two cell lines (ΔTMCF-7 = 16.3 min; ΔTMDA-MB-231 = 19.5 min); however, MCF-7 cells showed a much broader heterogeneity in comparison to MDA-MB-231 cells. The use of pH sensors and ratiometric imaging of living cells in combination with a novel computational approach allow analysis of thousands of events in a computationally inexpensive and faster way than the standard routes. The reported methodology can potentially be used to monitor pH as well as several other parameters associated with endocytosis.
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Colorantes Fluorescentes , Orgánulos , Homeostasis , Humanos , Concentración de Iones de Hidrógeno , Células MCF-7RESUMEN
Among the strategies to fight cancer, multi-therapeutic approaches are considered as a wise choice to put in place multiple weapons to suppress tumors. In this work, to combine chemotherapeutic effects to magnetic hyperthermia when using biocompatible scaffolds, we have established an electrospinning method to produce nanofibers of polycaprolactone loaded with magnetic nanoparticles as heat mediators to be selectively activated under alternating magnetic field and doxorubicin as a chemotherapeutic drug. Production of the fibers was investigated with iron oxide nanoparticles of peculiar cubic shape (at 15 and 23â¯nm in cube edges) as they provide benchmark heat performance under clinical magnetic hyperthermia conditions. With 23â¯nm nanocubes when included into the fibers, an arrangement in chains was obtained. This linear configuration of magnetic nanoparticles resemble that of the magnetosomes, produced by magnetotactic bacteria, and our magnetic fibers exhibited remarkable heating effects as the magnetosomes. Magnetic fiber scaffolds showed excellent biocompatibility on fibroblast cells when missing the chemotherapeutic agent and when not exposed to magnetic hyperthermia as shown by viability assays. On the contrary, the fibers containing both magnetic nanocubes and doxorubicin showed significant cytotoxic effects on cervical cancer cells following the exposure to magnetic hyperthermia. Notably, these tests were conducted at magnetic hyperthermia field conditions of clinical use. As here shown, on the doxorubicin sensitive cervical cancer cells, the combination of heat damage by magnetic hyperthermia with enhanced diffusion of doxorubicin at therapeutic temperature are responsible for a more effective oncotherapy.