Pancreatic ductal adenocarcinoma (PDAC) is typically characterized by high chemoresistance and metastatic spread, features mainly attributable to cancer stem cells (CSCs). It is of central interest the characterization of CSCs and, in particular, the study of their metabolic features in order to selectively identify their peculiarities for an efficient therapeutic approach. In this study, CSCs have been obtained by culturing different PDAC cell lines with a specific growth medium. Cells were characterized for the typical stem/mesenchymal properties at short-, medium-, and long-term culture. Metabolomics, proteomics, analysis of oxygen consumption rate in live cells, and the effect of the inhibition of lactate transporter on cell proliferation have been performed to delineate the metabolism of CSCs. We show that gradually de-differentiated pancreatic cancer cells progressively increase the expression of both stem and epithelial-to-mesenchymal transition markers, shift their metabolism from a glycolytic to an oxidative one, and lastly gain a quiescent state. These quiescent stem cells are characterized by high chemo-resistance, clonogenic ability, and metastatic potential. Re-differentiation reverts these features, re-activating their proliferative capacity and glycolytic metabolism, which generally correlates with high aggressiveness. These observations add an important piece of knowledge to the comprehension of the biology of CSCs, whose metabolic plasticity could be exploited for the generation of promising and selective therapeutic approaches for PDAC patients.

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.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors; it is often diagnosed at an advanced stage and is hardly treatable. These issues are strictly linked to the absence of early diagnostic markers and the low efficacy of treatment approaches. Recently, the study of the metabolic alterations in cancer cells has opened the way to important findings that can be exploited to generate new potential therapies. Within this scenario, mitochondria represent important organelles within which many essential functions are necessary for cell survival, including some key reactions involved in energy metabolism. These organelles remodel their shape by dividing or fusing themselves in response to cellular needs or stimuli. Interestingly, many authors have shown that mitochondrial dynamic equilibrium is altered in many different tumor types. However, up to now, it is not clear whether PDAC cells preferentially take advantage of fusion or fission processes since some studies reported a wide range of different results. This review described the role of both mitochondria arrangement processes, i.e., fusion and fission events, in PDAC, showing that a preference for mitochondria fragmentation could sustain tumor needs. In addition, we also highlight the importance of considering the metabolic arrangement and mitochondria assessment of cancer stem cells, which represent the most aggressive tumor cell type that has been shown to have distinctive metabolic features to that of differentiated tumor cells.

Human chorionic gonadotropin (hCG) is a hormone that specifically binds to luteinizing hormone receptor (LHR) and exerts several roles, including the support of pregnancy and fetal gonadal steroidogenesis. Since hCG is also expressed by some tumor types, like breast cancer, many efforts have been made to study its role in neoplesia, with some studies showing a cancer-supportive role and others showing a cancer-protective role. A critical examination of the literature highlighted that the in vitro effect of hCG has been tested in the presence of fetal serum, which contains other gonadotropins, in the culture medium. Thus, we hypothesized that the use of serum in the cell culture medium might influence the cell response to the hCG treatment due to the presence of other hormones. Thus, we analyzed the in vitro effect of highly purified hCG on cell proliferation and the activation of the down-stream signal transduction pathway in three breast cancer cell lines, particularly focusing on MCF7, cultured in serum-deprived conditions. Our data show that hCG increases cell proliferation and activates the down-stream target Akt, together with a decrease of the LHR mRNA expression level. Finally, we also tested the differentiation capacity of hCG on MCF7 cancer stem cells (CSCs) and show that it favors the proliferation and differentiation of these cells, thus suggesting that hCG also renders cells more able to colonize and invade the organs.

Glucose homeostasis is a complex process regulated by multiple organs and hormones, with insulin playing a central role. Recent evidence underscores the role of small GTP-binding proteins, particularly Rac1, in regulating insulin secretion and glucose uptake. However, the role of Rac1-regulatory proteins in these processes remains largely unexplored. In this study, we investigated the role of β2-chimaerin, a Rac1-specific GTPase-activating protein (GAP), in glucose homeostasis using whole-body β2-chimaerin knockout mice. Our data revealed that β2-chimaerin deficiency results in improved glucose tolerance and enhanced insulin sensitivity in mice. These metabolic effects were associated with increased insulin-induced AKT phosphorylation in the liver and activation of downstream pathways that regulate gluconeogenesis and glycogen synthesis. We show that insulin activates Rac1 in the liver. However, β2-chimaerin deletion did not significantly alter Rac1 activation in this organ, suggesting that β2-chimaerin regulates insulin signaling via a Rac1-independent mechanism. These findings expand our understanding of Rac1 regulation in glucose metabolism, and identify β2-chimaerin as a novel modulator of hepatic insulin signaling, with potential implications for the development of insulin resistance and diabetes.