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.
