Extracellular Matrix Composition Modulates the Responsiveness of Differentiated and Stem Pancreatic Cancer Cells to Lipophilic Derivate of Gemcitabine

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance. Methods: We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs. Results: PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters’ expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions. Conclusion: We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment.

Tumor Microenvironment Features and Chemoresistance in Pancreatic Ductal Adenocarcinoma: Insights into Targeting Physicochemical Barriers and Metabolism as Therapeutic Approaches

“…Biological chemoresistance can arise from different mechanisms. Target cells can (a) acquire resistance to drug uptake, (b) reduce their sensitivity to drugs by increasing the expression of anti-apoptotic proteins and activating protective mechanisms such as autophagy [ 63 ], (c) use DNA repair mechanisms to counteract the drug-dependent destruction of the tumor cell DNA [ 64 , 65 ] and (d) recruit more transporters/proteins responsible for drug efflux, thus preventing their action in the cancer cell. In PDAC, both physiological and biological chemoresistance are present and constitute a considerable problem for effective chemotherapy [ 31 ].…”

“…Moreover, the collagen-reducing effects of the anti-hypertensive agent, losartan, resulted in a significant augmentation of nanoparticle penetration towards the target cells [ 70 ]. However, the decreased collagen I content induced a switch in cancer stem cells (CSCs) from slow growing, avascular-type cells to fast-growing, highly autophagic endothelial-like cells, creating a favorable mechanism for tumor progression and chemoresistance to gemcitabine [ 63 , 71 ]. Altogether, these data suggest that the correct manipulation of ECM collagen composition may be able to enhance the accessibility of drugs to tumor tissues and decrease chemoresistance.…”

Promising 3D <em>in vitro</em> models for studying tumour heterogeneity and testing novel therapeutic approaches in pancreatic cancer

“…Furthermore, C18GEM induced an increase in cell death and stimulated protective autophagy in Panc1 and CSCs cultured on 3D conditions. 5 In addition, we produced MiaPaCa-2 pancreatic tumour spheroids that show a critical volume, can be easily manipulated and maintain a vital activity (Figure 2) and gene expression patterns for many days. 5…”

“…Statistical legend: *GEM versus C12GEM or C18GEM and C12GEM versus C18 GEM; + refers to growth on collagen I versus Matrigel for each drug; † CSCs versus parental (P) cells in the two ECMs for each treatment. Adapted from Forciniti et al5…”

Cost-Effective Real-Time Metabolic Profiling of Cancer Cell Lines for Plate-Based Assays

“…Pancreatic ductal adenocarcinoma (PDAC) cell lines AsPC1, MIA-PaCa-2 and PANC1 were a gift from Prof. Anna Trauzold (Christian-Albrecht University, Kiel, Germany), maintained in RPMI (Sigma-Aldrich, R0883) supplemented with 10% FBS, 1% penicillinstreptomycin mixture, 1% GlutaMAX (35050-038, Gibco, Waltham, MA, USA), 1% sodium pyruvate (11360-039, Gibco, Waltham, MA, USA), and used within passage 3-8 [29,30]. Adult mouse myocytes were isolated from Langendorff-perfused mice hearts using a previously published method [31,32].…”