Integrin-Linked Kinase Links Integrin Activation to Invadopodia Function and Invasion via the p(T567)-Ezrin/NHERF1/NHE1 Pathway.

Tumor cell invasion depends largely on degradation of the extracellular matrix (ECM) by protease-rich structures called invadopodia, whose formation and activity requires the convergence of signaling pathways engaged in cell adhesion, actin assembly, membrane regulation and ECM proteolysis. It is known that ß1-integrin stimulates invadopodia function through an invadopodial p(T567)-ezrin/NHERF1/NHE1 signal complex that regulates NHE1-driven invadopodia proteolytic activity and invasion. However, the link between ß1-integrin and this signaling complex is unknown. In this study, in metastatic breast (MDA-MB-231) and prostate (PC-3) cancer cells, we report that integrin-linked kinase (ILK) integrates ß1-integrin with this signaling complex to regulate invadopodia activity and invasion. Proximity ligation assay experiments demonstrate that, in invadopodia, ILK associates with ß1-integrin, NHE1 and the scaffold proteins p(T567)-ezrin and NHERF1. Activation of ß1-integrin increased both invasion and invadopodia activity, which were specifically blocked by inhibition of either NHE1 or ILK. We conclude that ILK integrates ß1-integrin with the ECM proteolytic/invasion signal module to induce NHE1-driven invadopodial ECM proteolysis and cell invasion.

Secreted molecules inducing epithelial-to-mesenchymal transition in cancer development.

The epithelial-mesenchymal transition (EMT) is a biologic process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. EMT is involved in embryo development, wound healing, tissue regeneration, organ fibrosis and has also been proposed as the critical mechanism for the acquisition of malignant phenotypes by epithelial cancer cells. These cells have been shown to acquire a mesenchymal phenotype when localized at the invasive front of primary tumours increasing aggressiveness, invasiveness, metastatic potential and resistance to chemotherapy. There is now increasing evidence demonstrating that a crucial role in the development of this process is played by factors secreted by cells of the tumour microenvironment or by the tumour cells themselves. This review summarises the current knowledge of EMT induction in cancer by paracrine or autocrine mechanisms, by exosomes or free proteins and miRNAs.

Iron Metabolism in Cancer Progression.

Iron is indispensable for cell metabolism of both normal and cancer cells. In the latter, several disruptions of its metabolism occur at the steps of tumor initiation, progression and metastasis. Noticeably, cancer cells require a large amount of iron, and exhibit a strong dependence on it for their proliferation. Numerous iron metabolism-related proteins and signaling pathways are altered by iron in malignancies, displaying the pivotal role of iron in cancer. Iron homeostasis is regulated at several levels, from absorption by enterocytes to recycling by macrophages and storage in hepatocytes. Mutations in HFE gene alter iron homeostasis leading to hereditary hemochromatosis and to an increased cancer risk because the accumulation of iron induces oxidative DNA damage and free radical activity. Additionally, the iron capability to modulate immune responses is pivotal in cancer progression. Macrophages show an iron release phenotype and potentially deliver iron to cancer cells, resulting in tumor promotion. Overall, alterations in iron metabolism are among the metabolic and immunological hallmarks of cancer, and further studies are required to dissect how perturbations of this element relate to tumor development and progression.

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

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.

Fluorescent nano- and microparticles for sensing cellular microenvironment: past, present and future applications.

The tumor microenvironment (TME) demonstrates distinct hallmarks, including acidosis, hypoxia, reactive oxygen species (ROS) generation, and altered ion fluxes, which are crucial targets for early cancer biomarker detection, tumor diagnosis, and therapeutic strategies. Various imaging and sensing techniques have been developed and employed in both research and clinical settings to visualize and monitor cellular and TME dynamics. Among these, ratiometric fluorescence-based sensors have emerged as powerful analytical tools, providing precise and sensitive insights into TME and enabling real-time detection and tracking of dynamic changes. In this comprehensive review, we discuss the latest advancements in ratiometric fluorescent probes designed for the optical mapping of pH, oxygen, ROS, ions, and biomarkers within the TME. We elucidate their structural designs and sensing mechanisms as well as their applications in in vitro and in vivo detection. Furthermore, we explore integrated sensing platforms that reveal the spatiotemporal behavior of complex tumor cultures, highlighting the potential of high-resolution imaging techniques combined with computational methods. This review aims to provide a solid foundation for understanding the current state of the art and the future potential of fluorescent nano- and microparticles in the field of cellular microenvironment sensing.

pH-sensing hybrid hydrogels for non-invasive metabolism monitoring in tumor spheroids.

The constant increase in cancer incidence and mortality pushes biomedical research towards the development of in vitro 3D systems able to faithfully reproduce and effectively probe the tumor microenvironment. Cancer cells interact with this complex and dynamic architecture, leading to peculiar tumor-associated phenomena, such as acidic pH conditions, rigid extracellular matrix, altered vasculature, hypoxic condition. Acidification of extracellular pH, in particular, is a well-known feature of solid tumors, correlated to cancer initiation, progression, and resistance to therapies. Monitoring local pH variations, non-invasively, during cancer growth and in response to drug treatment becomes extremely important for understanding cancer mechanisms. Here, we describe a simple and reliable pH-sensing hybrid system, based on a thermoresponsive hydrogel embedding optical pH sensors, that we specifically apply for non-invasive and accurate metabolism monitoring in colorectal cancer (CRC) spheroids. First, the physico-chemical properties of the hybrid sensing platform, in terms of stability, rheological and mechanical properties, morphology and pH sensitivity, were fully characterized. Then, the proton gradient distribution in the spheroids proximity, in the presence or absence of drug treatment, was quantified over time by time lapse confocal light scanning microscopy and automated segmentation pipeline, highlighting the effects of the drug treatment in the extracellular pH. In particular, in the treated CRC spheroids the acidification of the microenvironment resulted faster and more pronounced over time. Moreover, a pH gradient distribution was detected in the untreated spheroids, with more acidic values in proximity of the spheroids, resembling the cell metabolic features observed in vivo in the tumor microenvironment. These findings promise to shed light on mechanisms of regulation of proton exchanges by cellular metabolism being essential for the study of solid tumors in 3D in vitro models and the development of personalized medicine approaches.

Probing Single-Cell Fermentation Fluxes and Exchange Networks via pH-Sensing Hybrid Nanofibers.

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 single-cell metabolic flux analysis.

NIR ICG-Enhanced Fluorescence: A Quantitative Evaluation of Bowel Microperfusion and Its Relation to Central Perfusion in Colorectal Surgery.

BACKGROUND: To date, no standardized protocols nor a quantitative assessment of the near-infrared fluorescence angiography with indocyanine green (NIR-ICG) are available. The aim of this study was to evaluate the timing of fluorescence as a reproducible parameter and its efficacy in predicting anastomotic leakage (AL) in colorectal surgery. METHODS: A consecutive cohort of 108 patients undergoing minimally invasive elective procedures for colorectal cancer was prospectively enrolled. The difference between macro and microperfusion (ΔT) was obtained by calculating the timing of fluorescence at the level of iliac artery division and colonic wall, respectively. RESULTS: Subjects with a ΔT ≥ 15.5± 0.5 s had a higher tendency to develop an AL (p < 0.01). The ΔT/heart rate interaction was found to predict AL with an odds ratio of 1.02 (p < 0.01); a cut-off threshold of 832 was identified (sensitivity 0.86, specificity 0.77). Perfusion parameters were also associated with a faster bowel motility resumption and a reduced length of hospital stay. CONCLUSIONS: The analysis of the timing of fluorescence provides a quantitative, easy evaluation of tissue perfusion. A ΔT/HR interaction ≥832 may be used as a real-time parameter to guide surgical decision making in colorectal surgery.

A pH-sensor scaffold for mapping spatiotemporal gradients in three-dimensional in vitro tumour models.

The detection of extracellular pH at single cell resolution is challenging and requires advanced sensibility. Sensing pH at high spatial and temporal resolution might provide crucial information in understanding the role of pH and its fluctuations in a wide range of physio-pathological cellular processes, including cancer. Here, a method to embed silica-based fluorescent pH sensors into alginate-based three-dimensional (3D) microgels tumour models, coupled with a computational method for fine data analysis, is presented. By means of confocal laser scanning microscopy, live-cell time-lapse imaging of 3D alginate microgels was performed and the extracellular pH metabolic variations were monitored in both in vitro 3D mono- and 3D co-cultures of tumour and stromal pancreatic cells. The results show that the extracellular pH is cell line-specific and time-dependent. Moreover, differences in pH were also detected between 3D monocultures versus 3D co-cultures, thus suggesting the existence of a metabolic crosstalk between tumour and stromal cells. In conclusion, the system has the potential to image multiple live cell types in a 3D environment and to decipher in real-time their pH metabolic interplay under controlled experimental conditions, thus being also a suitable platform for drug screening and personalized medicine.

3-Bromo-Isoxazoline Derivatives Inhibit GAPDH Enzyme in PDAC Cells Triggering Autophagy and Apoptotic Cell Death.

A growing interest in the study of aerobic glycolysis as a key pathway for cancer-cell energetic metabolism, favouring tumour progression and invasion, has led to consider GAPDH as an effective drug target to specifically hit cancer cells. In this study, we have investigated a panel of 3-bromo-isoxazoline derivatives based on previously identified inhibitors of Plasmodium falciparum GAPDH (PfGAPDH). The compounds are active, to a different extent, as inhibitors of human-recombinant GAPDH. They showed an antiproliferative effect on pancreatic ductal-adenocarcinoma cells (PDAC) and pancreatic-cancer stem cells (CSCs), and among them two promising compounds were selected to be tested in vivo. Interestingly, these compounds were not effective in fibroblasts. The AXP-3019 derivative was able to block PDAC-cell growth in mice xenograft without apparent toxicity. The overall results support the assumption that selective inhibition of the glycolytic pathway, by targeting GAPDH, is an effective therapy for pancreatic cancer and that 3-bromo-isoxazoline derivatives represent a new class of anti-cancer compounds targeting glycolysis.

Pancreatic cancer stem cell proliferation is strongly inhibited by diethyldithiocarbamate-copper complex loaded into hyaluronic acid decorated liposomes.

BACKGROUND: Pancreatic cancer stem cells (CSCs) are responsible for resistance to standard therapy, metastatic potential, and disease relapse following treatments. The current therapy for pancreatic ductal adenocarcinoma (PDAC) preferentially targets the more differentiated cancer cell population, leaving CSCs as a cell source for tumor mass formation and recurrence. For this reason, there is an urgent need to improve current therapies and develop novel CSC-targeted therapeutic approaches. METHODS: Hyaluronic acid (HA) decorated liposomes, containing diethyldithiocarbamate­copper (Cu(DDC)2), able to target the specific CSC marker CD44 receptor were prepared by ion gradient technique and fully characterized. Their antiproliferative effect was evaluated on pancreatic CSCs derived from PDAC cell lines or patients. To clarify the mechanism of action of Cu(DDC)2 liposomes, ROS level neutralization assay in the presence of N-acetyl-L-cysteine was performed. RESULTS: Liposomes showed high encapsulation efficiency and Cryo-TEM analysis revealed the presence of Cu(DDC)2 crystals in the aqueous core of liposomes. In vitro test on pancreatic CSCs derived from PDAC cell lines or patients showed high ROS mediated anticancer activity of HA decorated liposomes. The sphere formation capability of CSCs obtained from patients was drastically reduced by liposomal formulations containing Cu(DDC)2. CONCLUSIONS: The obtained results show that the encapsulation of Cu(DDC)2 complex in HA decorated liposomes strongly increases its anti-proliferative activity on pancreatic CSCs. GENERAL SIGNIFICANCE: This paper describes for the first time the use of HA decorated liposomes containing Cu(DDC)2 against pancreatic CSCs and opens the way to the development of nanomedicine based CSC-targeted therapeutic approaches.

Extracellular matrix composition modulates PDAC parenchymal and stem cell plasticity and behavior through the secretome.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Its aggressiveness is driven by an intense fibrotic desmoplastic reaction in which the increasingly collagen I-rich extracellular matrix (ECM) and several cell types, including cancer stem cells (CSCs), create a tumor-supportive environment. However, how ECM composition regulates CSC dynamics and their relationship with the principle parenchymal tumor population to promote early invasive growth is not yet characterized. For this, we utilized a platform of 3D organotypic cultures composed of laminin-rich Matrigel, representative of an early tumor, plus increasing concentrations of collagen I to simulate malignant stroma progression. As ECM collagen I increases, CSCs progress from a rapidly growing, vascular phenotype to a slower growing, avascular phase, while maintaining their endothelial-like gene signatures. This transition is supported autocrinically by the CSCs and paracrinically by the parenchymal cells via their ECM-dependent secretomes. Indeed, when growing on an early tumor ECM, the CSCs are dedicated toward the preparation of a vascular niche by (a) activating their growth program, (b) secreting high levels of proangiogenic factors which stimulate both angiogenesis and vasculogenic mimicry, and (c) overexpressing VEGFR-2, which is activated by VEGF secreted by both the CSC and parenchymal cells. On Matrigel, the more differentiated parenchymal tumor cell population had reduced growth but a high invasive capacity. This concerted high local invasion of parenchymal cells into the CSC-derived vascular network suggests that a symbiotic relationship between the parenchymal cells and the CSCs underlies the initiation and maintenance of early PDAC infiltration and metastasis.

The scaffolding protein NHERF1 sensitizes EGFR-dependent tumor growth, motility and invadopodia function to gefitinib treatment in breast cancer cells.

Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress the epidermal growth factor receptor (EGFR), but are resistant to tyrosine kinase inhibitors (TKIs) and anti-EGFR therapies. Mechanisms suggested for resistance to TKIs include EGFR independence, mutations and alterations in EGFR and in its downstream signalling pathways. Ligand-induced endocytosis and degradation of EGFR play important roles in the downregulation of the EGFR signal suggesting that its activity could be regulated by targeting its trafficking. Evidence in normal cells showing that the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) can associate with EGFR to regulate its trafficking, led us to hypothesize that NHERF1 expression levels could regulate EGFR trafficking and functional expression in TNBC cells and, in this way, modulate its role in progression and response to treatment. We investigated the subcellular localization of NHERF1 and its interaction with EGFR in a metastatic basal like TNBC cell model, MDA-MB­231, and the role of forced NHERF1 overexpression and/or stimulation with EGF on the sensitivity to EGFR specific TKI treatment with gefitinib. Stimulation with EGF induces an interaction of NHERF1 with EGFR to regulate its localization, degradation and function. NHERF1 overexpression is sufficient to drive its interaction with EGFR in non-stimulated conditions, inhibits EGFR degradation and increases its retention time in the plasma membrane. Importantly, NHERF1 overexpression strongly sensitized the cell to the pharmacological inhibition by gefitinib of EGFR-driven growth, motility and invadopodia-dependent ECM proteolysis. The further determination of how the NHERF1­EGFR interaction is regulated may improve our understanding of TNBC resistance to the action of existing anticancer drugs.