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Different pathological conditions, including viral infections and cancer, can have a massive impact on the endoplasmic reticulum (ER), causing severe damage to the cell and exacerbating the disease. In particular, coronavirus infections, including SARS coronavirus-2 (SARS-CoV-2), responsible for COVID-19, cause ER stress as a consequence of the enormous amounts of viral glycoproteins synthesized, the perturbation of ER homeostasis and the modification of ER membranes. Therefore, ER has a central role in the viral life cycle, thus representing one of the Achilles' heels on which to focus therapeutic intervention. On the other hand, prolonged ER stress has been demonstrated to promote many pro-tumoral attributes in cancer cells, having a key role in tumor growth, metastasis and response to therapies. In this report, adopting a repurposing approach of approved drugs, we identified the antiplatelet agent ticlopidine as an interferent of the unfolded protein response (UPR) via sigma receptors (SRs) modulation. The promising results obtained suggest the potential use of ticlopidine to counteract ER stress induced by viral infections, such as COVID-19, and cancer.

Rationale : Caloric restriction improves the efficacy of anti-cancer therapy. This effect is largely dependent on the increase of the extracellular ATP concentration in the tumor microenvironment (TME). Pathways for ATP release triggered by nutrient deprivation are largely unknown. Methods: The extracellular ATP (eATP) concentration was in vivo measured in the tumor microenvironment of B16F10-inoculated C57Bl/6 mice with the pmeLuc probe. Alternatively, the pmeLuc-TG-mouse was used. Caloric restriction was in vivo induced with hydroxycitrate (HC). B16F10 melanoma cells or CT26 colon carcinoma cells were in vitro exposed to serum starvation to mimic nutrient deprivation. Energy metabolism was monitored by Seahorse. Microparticle release was measured by ultracentrifugation and by Nanosight. Results : Nutrient deprivation increases eATP release despite the dramatic inhibition of intracellular energy synthesis. Under these conditions oxidative phosphorylation was dramatically impaired, mitochondria fragmented and glycolysis and lactic acid release were enhanced. Nutrient deprivation stimulated a P2X7-dependent release of ATP-loaded, mitochondria-containing, microparticles as well as of naked mitochondria. Conclusions : Nutrient deprivation promotes a striking accumulation of eATP paralleled by a large release of ATP-laden microparticles and of naked mitochondria. This is likely to be a main mechanism driving the accumulation of eATP into the TME., Competing Interests: Competing Interests: FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a biotech Company involved in the development of anti-P2X7 antibodies, and a consultant with Axxam SpA. The other authors declare no conflict of interest., (© The author(s).)

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility., (© 2021. The Author(s).)

Cancer is a complex disease in which both genetic defects and microenvironmental components contribute to the development, progression, and metastasization of disease, representing major hurdles in the identification of more effective and safer treatment regimens for patients. Three-dimensional (3D) models are changing the paradigm of preclinical cancer research as they more closely resemble the complex tissue environment and architecture found in clinical tumors than in bidimensional (2D) cell cultures. Among 3D models, spheroids and organoids represent the most versatile and promising models in that they are capable of recapitulating the heterogeneity and pathophysiology of human cancers and of filling the gap between conventional 2D in vitro testing and animal models. Such 3D systems represent a powerful tool for studying cancer biology, enabling us to model the dynamic evolution of neoplastic disease from the early stages to metastatic dissemination and the interactions with the microenvironment. Spheroids and organoids have recently been used in the field of drug discovery and personalized medicine. The combined use of 3D models could potentially improve the robustness and reliability of preclinical research data, reducing the need for animal testing and favoring their transition to clinical practice. In this review, we summarize the recent advances in the use of these 3D systems for cancer modeling, focusing on their innovative translational applications, looking at future challenges, and comparing them with most widely used animal models.

Introduction: Despite the increasing financial outlay on cancer research and drug discovery, many advanced cancers remain incurable. One possible strategy for increasing the approval rate of new anticancer drugs for use in clinical practice could be represented by three-dimensional (3D) tumor models on which to perform in vitro drug screening. There is a general consensus among the scientific community that 3D tumor models more closely recapitulate the complexity of tumor tissue architecture and biology than bi-dimensional cell cultures. In a 3D context, cells are connected to each other through tissue junctions and show proliferative and metabolic gradients that resemble the intricate milieu of organs and tumors. Areas covered: The present review focuses on available techniques for generating tumor spheroids and discusses current and future applications in the field of drug discovery. The article is based on literature obtained from PubMed. Expert opinion: Given the relative simplicity of spheroid models with respect to clinical tumors, we must be careful not to overestimate the reliability of their drug-response prediction capacity. The next challenge is to combine our knowledge of co-culture methodologies with high-content imaging and advanced microfluidic technologies to improve the readout and biomimetic potential of spheroid-based models.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

Cancer 3-D spheroids are widely used to test drugs and radiotherapy treatments. These 3-D cell clusters range from tens to hundreds of micrometers in size, with shapes that typically differ from a perfect sphere. Change in spheroid volume is one of the most important parameters for evaluating treatment efficacy, and using light sheet fluorescence microscopes (LSFM), optical sections of samples in that size range can be obtained. However, there remains a lack of validated methods for quantifying the volumes of 3-D multicellular aggregates. Here, we present Reconstruction and Visualization from Multiple Sections (ReViMS), an open-source, user-friendly software for automatically segmenting z-stacks of fluorescence images and estimating the volumes of 3-D multicellular spheroids. To assess the precision and accuracy of the volume estimates obtained with ReViMS, we used several cancer spheroids imaged with LSFM. Both the precision and accuracy were >95%, demonstrating the effectiveness of ReViMS.

Introduction: Although several molecular targets against cancer have been identified, there is a continuous need for new therapeutic strategies. Sigma Receptors (SRs) overexpression has been recently associated with different cancer conditions. Therefore, novel anticancer agents targeting SRs may increase the specificity of therapies, overcoming some of the common drawbacks of conventional chemotherapy. Areas covered: The present review focuses on patent documents disclosing SR modulators with possible application in cancer therapy and diagnosis. The analysis reviews patents of the last two decades (1996-2016); patents were grouped according to target subtypes (S1R, S2R, pan-SRs) and relevant Applicants. The literature was searched through Espacenet, ISI Web, PatentScope and PubMed databases. Expert opinion: The number of patents related to SRs and cancer has increased in the last twenty years, confirming the importance of this receptor family as valuable target against neoplasias. Despite their short history in the cancer scenario, many SR modulators are at pre-clinical stage and one is undergoing a phase II clinical trial. SRs ligands may represent a powerful source of innovative antitumor therapeutics. Further investigation is needed for validating SR modulators as anti-cancer drugs. We strongly hope that this review could stimulate the interest of both Academia and pharmaceutical companies.

Background: Cancer multicellular spheroids are commonly used as 3D tumour models for testing drugs and radiotherapy treatments. The volume plays a key role in analysis of the results. Several methods have been proposed in the literature to compute the spheroid's volume from one 2D microscopy image (i.e. a single projection). However, the literature lacks reviews summarising the different methods available. Furthermore, there are no well-established approaches by which to compare the different methods and determine the best one., Objective: In this work we (a) revise the existing single-image based methods used to estimate the volume of multicellular spheroids, also providing different implementations for classical spherical and ellipsoidal pre-defined models; (b) present an upgrade of a volume estimation software recently proposed, Reconstruction and Visualization from a Single Projection (ReViSP), just validated by using four real spheroids imaged in 3D with a light-sheet microscope; (c) propose a quality assessing approach for single-image based methods, relying on 3D home-made macroscopic synthetic models mimicking the shapes of real multicellular spheroids., Results: Seven image-based methods used to estimate the volume of spheroids were compared using six 3D home-made synthetic models. First, the material used to make the synthetic models was characterised to estimate its density. Then, the ground-truth volume of the 3D models was measured by simply weighing them. The volume instances estimated by the different methods were compared with ground truth. ReViSP attained the best result three times out of six and on average., Conclusions: The results obtained proved that (a) different implementations for the classical spherical and ellipsoidal pre-defined models may lead to very different results; (b) ReViSP is the best single-image based method available today to estimate the volume of multicellular spheroids., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The analysis of the global thiol-disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported. Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3-5 and 10-20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15-30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol-disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0-200 μM concentration range). Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200 μM concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Volume is one of the most important features for the characterization of a tumour on a macroscopic scale. It is often used to assess the effectiveness of care treatments, thus making its correct evaluation a crucial issue for patient care. Similarly, volume is a key feature on a microscopic scale. Multicellular cancer spheroids are 3D tumour models widely employed in pre-clinical studies to test the effects of drugs and radiotherapy treatments. Very few methods have been proposed to estimate the tumour volume arising from a 2D projection of multicellular spheroids, and even fewer have been designed to provide a 3D reconstruction of the tumour shape. In this work, we propose Reconstruction and Visualization from a Single Projection (ReViSP), an automatic method conceived to reconstruct the 3D surface and estimate the volume of single cancer multicellular spheroids, or even of spheroid cultures. As the input parameter ReViSP requires only one 2D projection, which could be a widefield microscope image. We assessed the effectiveness of our method by comparing it with other approaches. To this purpose, we used a new strategy that allowed us to achieve accurate volume measurements based on the analysis of home-made 3D objects, built by mimicking the spheroid morphology. The results confirmed the effectiveness of our method for both 3D reconstruction and volume assessment. ReViSP software is distributed as an open source tool., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Lon is a nuclear-encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up-regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon-silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to β-oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5-fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2-deoxy-d-glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3- and 1.3-fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells., (© FASEB.)

Non-steroidal anti-inflammatory drugs (NSAIDs) have repeatedly shown to be effective in tumor prevention, but important side-effects limit their wide clinical use. Nitric oxide-releasing derivatives (NO-NSAIDs) are a promising class of compounds synthesized by combining a classic NSAID molecule with an NO-releasing moiety to counteract side-effects. These new chemical entities exhibit a significantly higher activity and much lower toxicity with respect to the parental drug. In the present paper, we report the results obtained from in in vitro experimental systems aimed to evaluate the activity and mechanisms of action of the novel NO-releasing aspirin derivative, NCX 4040. The in vitro studies were carried out on a panel of human colon (LoVo, LoVo Dx, WiDr, LRWZ), bladder (HT1376, MCR), and pancreatic (Capan-2, MIA PaCa-2, T3M4) cancer cell lines. With regard to colon cancer, NCX 4040 activity was also investigated in vitro in combination with drugs currently used in clinical practice and was validated in vivo on tumor-bearing mice xenografted with the aforementioned colon cancer cell lines. The in vitro studies showed a high cytotoxic activity of NCX 4040 in all tumor histotypes and demonstrated the pivotal role of the NO component in drug activity. It was also observed that NCX 4040 exerts a pro-apoptotic activity via a mitochondria-dependent pathway. Moreover, the in vivo studies on xenografted mice further confirmed the antitumor efficacy and low toxicity of NCX 4040 in colon cancer and highlighted its role as sensitizing agent of oxaliplatin cytotoxicity.

The cytotoxic activity of the imidazoacridinone C1311 was related to its effect on cell cycle progression in 16 human cell lines from different solid tumour types and leukaemias. A 72-h exposure to C1311 induced a wide range of growth inhibition (IC50 values ranging from 0.0094 to 0.8 microM; median, 0.279 microM), with the highest activity in the 2 gastric cancer cell lines (IC50, 0.0094 and 0.0098 microM). No significant association was found between in vitro sensitivtity to C1311 and doxorubicin or taxol. Moreover, the activity of C1311 was independent of the p53 gene status of the cell line. Twenty-four-hour exposure to C1311 led to a marked increase in the number of cells in the G2M phase in the majority of cell lines, although the extent of such accumulation was independent of the level of drug cytotoxic activity. C1311 did not generally affect the expression level of cyclin B1 or Cdk1 (p34cdc2) proteins. Conversely, when normalised on the basis of the number of cells arrested in the G2M compartment, Cdk1 kinase activity appeared lower than that of untreated cells in the 4 cell lines showing the most pronounced accumulation in G2M. Overall, such data show that C1311 is active against a variety of human tumour cell lines and strongly support further evaluation of the drug in clinical trials.