Pancreatic ductal adenocarcinoma progression: a complex crosstalk between tumor physico-chemical stimuli and Ca2+ channels

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive malignancy of the exocrine pancreas, characterized by a distinctive microenvironment (TME), where stromal cells excessively produce extracellular matrix components, enhancing tissue stiffness. This condition impacts on tissue vascularization and contributes to the development of an acidic and hypoxic TME, further triggered by the metabolic reprogramming known as the “Warburg effect”, which typically characterizes PDAC cancer cells. This environment promotes cancer progression and selection of more aggressive phenotypes which are strongly exposed to mechanical cues generated by the dense extracellular matrix, sensed through plasma membrane mechanosensors that are able to activate specific intracellular signaling cascades, culminating in gene expression regulation. Among these, Ca2+-permeable channels represent an interesting target regulating intracellular Ca2+ homeostasis, a crucial player in modulating cell behavior and promoting tumor growth. To date, commonly used anti-cancer therapies remain largely ineffective for PDAC, and no approved chemotherapeutic agent has demonstrated improvement in overall survival since gemcitabine’s approval in 1996, underscoring the urgent need for novel therapeutic targets. In this work, we investigated the role of physico-chemical stimuli within the TME in modulating PDAC cell behavior, with a particular focus on acidity, ECM stiffness and the interplay with Ca2+ dynamics. The first part of the project was devoted to unraveling the role of acidity on PDAC progression. PDAC cancer cells were either exposed to short- or long- term acidic (pHe 6.6) environments and next recovered to physiological pHe (7.4), to mimic the metastatic event in which cells move from the acidic core of the tumor, toward the periphery of the mass, characterized by a more physiological pHe. Functional characterization of the models revealed limited growth, adhesion, invasion and viability of cells exposed to short acidic treatment. Long- term acidic exposure instead promoted the selection of acid-resistant clones which acquired migratory and invasive advantages, further enhancing their metastatic potential when recovered to pHe 7.4. The second part of the work was instead centered on investigating the role of physical forces in promoting PDAC progression in both a 2D and a 3D model, in which different rigidities were generated to mimic the healthy soft pancreatic tissue and the stiffer PDAC TME. Enhanced rigidity was accompanied by more aggressive traits of cancer cells, underlying the pivotal role of ECM fibrosis in driving PDAC progression. Ca2+ imaging experiments revealed alterations in Ca2+ homeostasis and dynamics (including SOCE and Ca2+ oscillations), prompting us to further investigate key channels that could be involved in the observed phenotypic alterations. ORAI1 and PIEZO1 were demonstrated to play opposite and balancing roles in our model, with the first involved in inducing cancer progression while the second revealing an inhibitory role. Overall, this project unravels the role of chemical and physical stimuli in the promotion of PDAC progression. Acidic-induced selection as well as mechanical cell exposure, promote the generation of a more aggressive cellular phenotype in vitro, where Ca2+ dynamics and Ca2+ channels play a crucial role in the modulation of cell behavior