“…Calibration was performed at the beginning of each experiment and used to measure, by resolving the equation of the fit calibration curve, the pH read out of sensors located in the Ch/Pec hydrogel under evaluation. The linear pH sensitivity of the pH sensors was in accordance with our previous work [ 54 ], where it was even demonstrated the good reversibility and stability over time of the microparticles, making them highly suitable for spatio-temporal acidification analyses in biological systems. Fig.…”
Section: Resultssupporting
confidence: 88%
“…We fabricated a pH-sensing hybrid platform for the spatio-temporal extracellular pH mapping in 3D cell culture by embedding recently devised fluorescent ratiometric pH sensors, based on silica (SiO 2 ) microparticles functionalized with fluorescein isothiocyanate (FITC), as pH indicator probe, and rhodamine B isothiocyanate (RBITC), as reference dye [ 54 ], within our optimized Ch/Pec hydrogel system [ 17 , 18 ]. The fabrication procedure is shown in Fig.…”
Section: Resultsmentioning
confidence: 99%
“…According to the optical properties of the selected dyes, the pH indicator dye (FITC, green) showed increased fluorescence under increasing pH values [ 52 , 58 ], while the fluorescence intensity of the reference dye (RBITC, red) was not altered. CLSM images were then processed with a customized algorithm to automatically extract positions and intensity ratio (I FITC /I RBITC ) of the sensors for each pH value [ 54 ]. Next, the values were plotted as function of pH, displaying a linear pH-dependent calibration curve ( Fig.…”
“…Calibration was performed at the beginning of each experiment and used to measure, by resolving the equation of the fit calibration curve, the pH read out of sensors located in the Ch/Pec hydrogel under evaluation. The linear pH sensitivity of the pH sensors was in accordance with our previous work [ 54 ], where it was even demonstrated the good reversibility and stability over time of the microparticles, making them highly suitable for spatio-temporal acidification analyses in biological systems. Fig.…”
Section: Resultssupporting
confidence: 88%
“…We fabricated a pH-sensing hybrid platform for the spatio-temporal extracellular pH mapping in 3D cell culture by embedding recently devised fluorescent ratiometric pH sensors, based on silica (SiO 2 ) microparticles functionalized with fluorescein isothiocyanate (FITC), as pH indicator probe, and rhodamine B isothiocyanate (RBITC), as reference dye [ 54 ], within our optimized Ch/Pec hydrogel system [ 17 , 18 ]. The fabrication procedure is shown in Fig.…”
Section: Resultsmentioning
confidence: 99%
“…According to the optical properties of the selected dyes, the pH indicator dye (FITC, green) showed increased fluorescence under increasing pH values [ 52 , 58 ], while the fluorescence intensity of the reference dye (RBITC, red) was not altered. CLSM images were then processed with a customized algorithm to automatically extract positions and intensity ratio (I FITC /I RBITC ) of the sensors for each pH value [ 54 ]. Next, the values were plotted as function of pH, displaying a linear pH-dependent calibration curve ( Fig.…”
“…Notably, these nanosized electrospun fiber scaffolds provide a large surface-to-volume ratio, which is known to enhance key cellular functions, including adhesion, proliferation, and differentiation. − Moreover, their nanofibrous structure mimics the native extracellular matrix (ECM), which plays a pivotal role in cell polarity as well as in cell-to-cell/matrix interaction. − Representative CLSM images of the pH-sensing nanofibers are shown in Figures d–f. Each pH sensor microparticle is clearly detectable thanks to the fluorescein 5(6)-isothiocyanate, FITC (Figure d), and Rhodamine B isothiocyanate, RBITC (Figure e), dye molecules covalently linked with APTES to the surface of silica (SiO 2 ) microparticles. , The FITC and RBITC fluorophores act as pH indicator and reference dyes, respectively, to enable ratiometric measurements of pH, which is a more robust and reliable way of fluorescence sensing because it compensates for fluctuation in fluorescence intensities due to the utilization of two distinct emission wavelengths. − Thanks to the surrounding polymeric matrix, the pH sensors remain stably immobilized into the lumen of the nanofibers during imaging, making pH-sensing ratiometric hybrid nanofibers an ideal biomaterial scaffold for monitoring local microenvironment proton changes in a fast and noninvasive way, with high spatial control and resolution. The pH-sensing nanofibers were used to culture pancreatic cancer cells (AsPC-1) and pancreatic stellate cancer associated fibroblasts (CAFs) and to monitor extracellular pH changes via time lapse CLSM acquisitions for 6 h with time intervals of 10 min (Figure g).…”
Section: Resultsmentioning
confidence: 99%
“…Each pH sensor microparticle is clearly detectable thanks to the fluorescein 5(6)-isothiocyanate, FITC ( Figure 2 d), and Rhodamine B isothiocyanate, RBITC ( Figure 2 e), dye molecules covalently linked with APTES to the surface of silica (SiO 2 ) microparticles. 56 , 57 The FITC and RBITC fluorophores act as pH indicator and reference dyes, respectively, to enable ratiometric measurements of pH, which is a more robust and reliable way of fluorescence sensing because it compensates for fluctuation in fluorescence intensities due to the utilization of two distinct emission wavelengths. 58 − 61 Thanks to the surrounding polymeric matrix, the pH sensors remain stably immobilized into the lumen of the nanofibers during imaging, making pH-sensing ratiometric hybrid nanofibers an ideal biomaterial scaffold for monitoring local microenvironment proton changes in a fast and noninvasive way, with high spatial control and resolution.…”
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 singlecell metabolic flux analysis.
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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