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Epigenetic changes are heritable modifications that do not directly affect the DNA sequence. In cancer cells, the maintenance of a stable epigenetic profile can be crucial to support survival and proliferation, and said profile can differ significantly from that of healthy cells. The epigenetic profile of a cancer cell can be modulated by several factors, including metabolites. Recently, sphingolipids have emerged as novel modulators of epigenetic changes. Ceramide and sphingosine 1-phosphate have become well known in cancer due to activating anti-tumour and pro-tumour signalling pathways, respectively, and they have recently been shown to also induce several epigenetic modifications connected to cancer growth. Additionally, acellular factors in the tumour microenvironment, such as hypoxia and acidosis, are now recognised as crucial in promoting aggressiveness through several mechanisms, including epigenetic modifications. Here, we review the existing literature on sphingolipids, cancer, and epigenetic changes, with a focus on the interaction between these elements and components of the chemical tumour microenvironment.

Cancer-induced bone pain (CIBP) is a common and devastating symptom with limited treatment options in patients, significantly affecting their quality of life. The use of rodent models is the most common approach to uncovering the mechanisms underlying CIBP; however, the translation of results to the clinic may be hindered because the assessment of pain-related behavior is often based exclusively on reflexive-based methods, which are only partially indicative of relevant pain in patients. To improve the accuracy and strength of the preclinical, experimental model of CIBP in rodents, we used a battery of multimodal behavioral tests that were also aimed at identifying rodent-specific behavioral components by using a home-cage monitoring assay (HCM). Rats of all sexes received an injection with either heat-deactivated (sham-group) or potent mammary gland carcinoma Walker 256 cells into the tibia. By integrating multimodal datasets, we assessed pain-related behavioral trajectories of the CIBP-phenotype, including evoked and non-evoked based assays and HCM. Using principal component analysis (PCA), we discovered sex-specific differences in establishing the CIBP-phenotype, which occurred earlier (and differently) in males. Additionally, HCM phenotyping revealed the occurrence of sensory-affective states manifested by mechanical hypersensitivity in sham when housed with a tumor-bearing cagemate (CIBP) of the same sex. This multimodal battery allows for an in-depth characterization of the CIBP-phenotype under social aspects in rats. The detailed, sex-specific, and rat-specific social phenotyping of CIBP enabled by PCA provides the basis for mechanism-driven studies to ensure robustness and generalizability of results and provide information for targeted drug development in the future.

Osteosarcoma is considered the most common bone tumor affecting children and young adults. The standard of care is chemotherapy; however, the onset of drug resistance still jeopardizes osteosarcoma patients, thus making it necessary to conduct a thorough investigation of the possible mechanisms behind this phenomenon. In the last decades, metabolic rewiring of cancer cells has been proposed as a cause of chemotherapy resistance. Our aim was to compare the mitochondrial phenotype of sensitive osteosarcoma cells (HOS and MG-63) versus their clones when continuously exposed to doxorubicin (resistant cells) and identify alterations exploitable for pharmacological approaches to overcome chemotherapy resistance. Compared with sensitive cells, doxorubicin-resistant clones showed sustained viability with less oxygen-dependent metabolisms, and significantly reduced mitochondrial membrane potential, mitochondrial mass, and ROS production. In addition, we found reduced expression of TFAM gene generally associated with mitochondrial biogenesis. Finally, combined treatment of resistant osteosarcoma cells with doxorubicin and quercetin, a known inducer of mitochondrial biogenesis, re-sensitizes the doxorubicin effect in resistant cells. Despite further investigations being needed, these results pave the way for the use of mitochondrial inducers as a promising strategy to re-sensitize doxorubicin cytotoxicity in patients who do not respond to therapy or reduce doxorubicin side effects.

The extracellular matrix (ECM) modulates cell behavior, shape, and viability as well as mechanical properties. In recent years, ECM disregulation and aberrant remodeling has gained considerable attention in cancer targeting and prevention since it may stimulate tumorigenesis and metastasis. Here, we developed an in vitro model that aims at mimicking the in vivo tumor microenvironment by recapitulating the interactions between osteosarcoma (OS) cells and ECM with respect to cancer progression. We long-term cultured 3D OS spheroids made of metastatic or non-metastatic OS cells mixed with mesenchymal stromal cells (MSCs); confirmed the deposition of ECM proteins such as Type I collagen, Type III collagen, and fibronectin by the stromal component at the interface between tumor cells and MSCs; and found that ECM secretion is inhibited by a neutralizing anti-IL-6 antibody, suggesting a new role of this cytokine in OS ECM deposition. Most importantly, we showed that the cytotoxic effect of doxorubicin is reduced by the presence of Type I collagen. We thus conclude that ECM protein deposition is crucial for modelling and studying drug response. Our results also suggest that targeting ECM proteins might improve the outcome of a subset of chemoresistant tumors.

Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, the outcome is unsatisfactory, and multidrug resistance (MDR) is a pivotal process in OS treatment failure. OS-derived extracellular vesicles (EVs) promote drug resistance to chemotherapy and target therapy through different mechanisms. The aim of this study was to identify subpopulations of osteosarcoma-EVs by Fourier transform infrared spectroscopy (FT-IR) to define a specific spectral signature for sensitive and multidrug-resistant OS-derived EVs. EVs were isolated from sensitive and MDR OS cells as well as from mesenchymal stem cells by differential centrifugation and ultracentrifugation. EVs size, morphology and protein expression were characterized. FT-IR/ATR of EVs spectra were acquired in the region of 400-4000 cm -1 (resolution 4 cm -1 , 128 scans). The FT-IR spectra obtained were consistently different in the EVs compared to cells from which they originate. A specific spectral signature, characterized by a shift and a new band (1601 cm -1 ), permitted to clearly distinguish EVs isolated by sensitive and multidrug-resistant OS cells. Our data suggest that FT-IR spectroscopy allows to characterize and define a specific spectral signature for sensitive and MDR OS-derived EVs.

Osteosarcoma is the most frequent primary malignant bone tumour with an impressive tendency to metastasise. Highly proliferative tumour cells release a remarkable amount of protons into the extracellular space that activates the NF-kB inflammatory pathway in adjacent stromal cells. In this study, we further validated the correlation between tumour glycolysis/acidosis and its role in metastases. In patients, at diagnosis, we found high circulating levels of inflammatory mediators (IL6, IL8 and miR-136-5p-containing extracellular vesicles). IL6 serum levels significantly correlated with disease-free survival and 18 F-FDG PET/CT uptake, an indirect measurement of tumour glycolysis and, hence, of acidosis. In vivo subcutaneous and orthotopic models, co-injected with mesenchymal stromal (MSC) and osteosarcoma cells, formed an acidic tumour microenvironment (mean pH 6.86, as assessed by in vivo MRI-CEST pH imaging). In these xenografts, we enlightened the expression of both IL6 and the NF-kB complex subunit in stromal cells infiltrating the tumour acidic area. The co-injection with MSC also significantly increased lung metastases. Finally, by using 3D microfluidic models, we directly showed the promotion of osteosarcoma invasiveness by acidosis via IL6 and MSC. In conclusion, osteosarcoma-associated MSC react to intratumoural acidosis by triggering an inflammatory response that, in turn, promotes tumour invasiveness at the primary site toward metastasis development.

Bone cancer, both primary and metastatic, is characterized by a low survival rate. Currently, available models lack in mimicking the complexity of bone, of cancer, and of their microenvironment, leading to poor predictivity. Three-dimensional technologies can help address this need, by developing predictive models that can recapitulate the conditions for cancer development and progression. Among the existing tools to obtain suitable 3D models of bone cancer, 3D printing and bioprinting appear very promising, as they enable combining cells, biomolecules, and biomaterials into organized and complex structures that can reproduce the main characteristic of bone. The challenge is to recapitulate a bone-like microenvironment for analysis of stromal-cancer cell interactions and biological mechanics leading to tumor progression. In this review, existing approaches to obtain in vitro 3D-printed and -bioprinted bone models are discussed, with a focus on the role of biomaterials selection in determining the behavior of the models and its degree of customization. To obtain a reliable 3D bone model, the evaluation of different polymeric matrices and the inclusion of ceramic fillers is of paramount importance, as they help reproduce the behavior of both normal and cancer cells in the bone microenvironment. Open challenges and future perspectives are discussed to solve existing shortcomings and to pave the way for potential development strategies.

In bone sarcomas, extracellular proton accumulation is an intrinsic driver of malignancy. Extracellular acidosis increases stemness, invasion, angiogenesis, metastasis, and resistance to therapy of cancer cells. It reprograms tumour-associated stroma into a protumour phenotype through the release of inflammatory cytokines. It affects bone homeostasis, as extracellular proton accumulation is perceived by acid-sensing ion channels located at the cell membrane of normal bone cells. In bone, acidosis results from the altered glycolytic metabolism of bone cancer cells and the resorption activity of tumour-induced osteoclasts that share the same ecosystem. Proton extrusion activity is mediated by extruders and transporters located at the cell membrane of normal and transformed cells, including vacuolar ATPase and carbonic anhydrase IX, or by the release of highly acidic lysosomes by exocytosis. To date, a number of investigations have focused on the effects of acidosis and its inhibition in bone sarcomas, including studies evaluating the use of photodynamic therapy. In this review, we will discuss the current status of all findings on extracellular acidosis in bone sarcomas, with a specific focus on the characteristics of the bone microenvironment and the acid-targeting therapeutic approaches that are currently being evaluated.

In the tumor microenvironment, mesenchymal stromal cells (MSCs) are key modulators of cancer cell behavior in response to several stimuli. Intratumoral acidosis is a metabolic trait of fast-growing tumors that can induce a pro-tumorigenic phenotype in MSCs through the activation of the NF-κB-mediated inflammatory pathway, driving tumor clonogenicity, invasion, and chemoresistance. Recent studies have indicated that curcumin, a natural ingredient extracted from Curcuma longa , acts as an NF-κB inhibitor with anti-inflammatory properties. In this work, highly proliferating osteosarcoma cells were used to study the ability of curcumin to reduce the supportive effect of MSCs when stimulated by acidosis. Due to the poor solubility of curcumin in biological fluids, we used spherical polymeric nanoparticles as carriers (SPN-curc) to optimize its uptake by MSCs. We showed that SPN-curc inhibited the release of inflammatory cytokines (IL6 and IL8) by acidity-stimulated MSCs at a higher extent than by free curcumin. SPN-curc treatment was also successful in blocking tumor stemness, migration, and invasion that were driven by the secretome of acid-stressed MSCs. Overall, these data encourage the use of lipid-polymeric nanoparticles encapsulating NF-κB inhibitors such as curcumin to treat cancers whose progression is stimulated by an activated mesenchymal stroma.

Acidity is a key player in cancer progression, modelling a microenvironment that prevents immune surveillance and enhances invasiveness, survival, and drug resistance. Here, we demonstrated in spheroids from osteosarcoma cell lines that the exposure to acidosis remarkably caused intracellular lipid droplets accumulation. Lipid accumulation was also detected in sarcoma tissues in close proximity to tumor area that express the acid-related biomarker LAMP2. Acid-induced lipid droplets-accumulation was not functional to a higher energetic request, but rather to cell survival. As a mechanism, we found increased levels of sphingomyelin and secretion of the sphingosine 1-phosphate, and the activation of the associated sphingolipid pathway and the non-canonical NF-ĸB pathway, respectively. Moreover, decreasing sphingosine 1-phosphate levels (S1P) by FTY720 (Fingolimod) impaired acid-induced tumor survival and migration. As a confirmation of the role of S1P in osteosarcoma, we found S1P high circulating levels (30.8 ± 2.5 nmol/mL, n = 17) in the serum of patients. Finally, when we treated osteosarcoma xenografts with FTY720 combined with low-serine/glycine diet, both lipid accumulation (as measured by magnetic resonance imaging) and tumor growth were greatly inhibited. For the first time, this study profiles the lipidomic rearrangement of sarcomas under acidic conditions, suggesting the use of anti-S1P strategies in combination with standard chemotherapy.

Plant-derived exosome-like nanovesicles (EPDENs) have recently been isolated and evaluated as potential bioactive nutraceutical biomolecules. It has been hypothesized that EPDENs may exert their activity on mammalian cells through their specific cargo. In this study, we isolated and purified EPDENs from the strawberry juice of Fragaria x ananassa (cv. Romina), a new cultivar characterized by a high content of anthocyanins, folic acid, flavonols, and vitamin C and an elevated antioxidant capacity. Fragaria -derived EPDENs were purified by a series of centrifugation and filtration steps. EPDENs showed size and morphology similar to mammalian extracellular nanovesicles. The internalization of Fragaria -derived EPDENs by human mesenchymal stromal cells (MSCs) did not negatively affect their viability, and the pretreatment of MSCs with Fragaria -derived EPDENs prevented oxidative stress in a dose-dependent manner. This is possibly due to the presence of vitamin C inside the nanovesicle membrane. The analysis of EPDEN cargo also revealed the presence of small RNAs and miRNAs. These findings suggest that Fragaria -derived EPDENs may be considered nanoshuttles contained in food, with potential health-promoting activity.

Microfluidic principles have been widely applied for more than 30 years to solve biological and micro-electromechanical problems. Despite the numerous advantages, microfluidic devices are difficult to manage as their handling comes with several technical challenges. We developed a new portable tool, the microfluidic trainer (MT), that assesses the operator handling skills and that may be used for maintaining or improving the ability to inject fluid in the inlet of microfluidic devices for in vitro cell culture applications. After several tests, we optimized the MT tester cell to reproduce the real technical challenges of a microfluidic device. In addition to an exercise path, we included an overfilling indicator and a correct infilling indicator at the inlet (control path). We manufactured the MT by engraving a 3 mm-high sheet of methacrylate with 60W CO 2 laser plotter to create multiple capillary paths. We validated the device by enrolling 21 volunteers (median age 33) to fill both the MT and a commercial microfluidic device. The success rate obtained with MT significantly correlated with those of a commercial microfluidic culture plate, and its 30 min-continuous use for three times significantly improved the performance. Overall, our data demonstrate that MT is a valid assessment tool of individual performances in using microfluidic devices and may represent a low-cost solution to training, improve or warm up microfluidic handling skills.

Most pathological conditions that cause local or systemic acidosis by overcoming the buffering activities of body fluids overlap with those diseases that are characterized by glucose metabolic disorders, including diabetes mellitus, inflammation, and cancer. This simple observation suggests the existence of a strong relationship between acidosis and insulin metabolism or insulin receptor signaling. In this review, we summarized the current knowledge on the activity of insulin on the induction of acidosis and, vice versa, on the effects of changes of extracellular and intracellular pH on insulin resistance. Insulin influences acidosis by promoting glycolysis. Although with an unclear mechanism, the lowering of pH, in turn, inhibits insulin sensitivity or activity. In addition to ketoacidosis that is frequently associated with diabetes, other important and more complex factors are involved in this delicate feedback mechanism. Among these, in this review we discussed the acid-mediated inhibiting effects on insulin binding affinity to its receptor, on glycolysis, on the recycling of glucose transporters, and on insulin secretion via transforming growth factor β (TGF-β) activity by pancreatic β-cells. Finally, we revised current data available on the mutual interaction between insulin signaling and the activity of ion/proton transporters and pH sensors, and on how acidosis may enhance insulin resistance through the Nuclear Factor kappa B (NF-κB) inflammatory pathway.

Similar to other types of cancer, acidification of tumor microenvironment is an important feature of osteosarcoma, and a major source of cellular stress that triggers cancer aggressiveness, drug resistance, and progression. Among the different effects of low extracellular pH on tumor cells, we have recently found that short-term exposure to acidosis strongly affects gene expression. This alteration might also occur for the most commonly used housekeeping genes (HKG), thereby causing erroneous interpretation of RT-qPCR data. On this basis, by using osteosarcoma cells cultured at different pH values, we aimed to identify the ideal HKG to be considered in studies on tumor-associated acidosis. We verified the stability of 15 commonly used HKG through five algorithms (NormFinder, geNorm, BestKeeper, ΔCT, coefficient of variation) and found that no universal HKG is suitable, since at least four HKG are necessary for proper normalization. Furthermore, according to the acceptable range of values, YWHAZ , GAPDH , GUSB , and 18S rRNA were the most stable reference genes at different pH. Our results will be helpful for future investigations focusing on the effect of altered microenvironment on cancer behavior, particularly on the effectiveness of anticancer therapies in acid conditions.