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Up to approximately 70% of cancer survivors report persistent deficits in memory, attention, speed of information processing, multi-tasking, and mental health functioning, a series of symptoms known as “brain fog.” The severity and duration of such effects can vary depending on age, cancer type, and treatment regimens. In particular, every year, hundreds of thousands of patients worldwide undergo radiotherapy (RT) for primary brain tumors and brain metastases originating from extracranial tumors. Besides its potential benefits in the control of tumor progression, recent studies indicate that RT reprograms the brain tumor microenvironment inducing increased activation of microglia and astrocytes and a consequent general condition of neuroinflammation that in case it becomes chronic could lead to a cognitive decline. Furthermore, radiation can induce endothelium reticulum (ER) stress directly or indirectly by generating reactive oxygen species (ROS) activating compensatory survival signaling pathways in the RT-surviving fraction of healthy neuronal and glial cells. In particular, the anomalous accumulation of misfolding proteins in neuronal cells exposed to radiation as a consequence of excessive activation of unfolded protein response (UPR) could pave the way to neurodegenerative disorders. Moreover, exposure of cells to ionizing radiation was also shown to affect the normal proteasome activity, slowing the degradation rate of misfolded proteins, and further exacerbating ER-stress conditions. This compromises several neuronal functions, with neuronal accumulation of ubiquitinated proteins with a consequent switch from proteasome to immunoproteasome that increases neuroinflammation, a crucial risk factor for neurodegeneration. The etiology of brain fog remains elusive and can arise not only during treatment but can also persist for an extended period after the end of RT. In this review, we will focus on the molecular pathways triggered by radiation therapy affecting cognitive functions and potentially at the origin of so-called “brain fog” symptomatology, with the aim to define novel therapeutic strategies to preserve healthy brain tissue from cognitive decline.

Background and purpose: In this study we want to evaluate the efficacy of yoga practice on dysfunctional stress, inflammation and QOL in breast cancer patients undergoing adjuvant radiotherapy. Patients and methods: Patients with stage 0 to III breast cancer were recruited before starting radiotherapy (XRT) and were randomly assigned to yoga group (YG) two times a week during XRT or control group (CG). Self-report measures of QOL, fatigue and sleep quality, and blood samples were collected at day 1 of treatment, day 15, end of treatment and 1, 3 and 6 months later. Cortisol blood level, IL6, IL10, IL1RA, TNFα and lymphocyte-to-monocyte ratio were analyzed as measures of dysfunctional stress and inflammation. Results: Patients started XRT and yoga classes in October 2019. Due to COVID-19 pandemic we closed the enrollment in March 2020. We analysed 24 patients, 12 YG and 12 CG. The analysis of blood cortisol levels revealed an interaction (p = 0.04) between yoga practice and time, in particular YG had lower cortisol levels at the end of XRT respect to CG (p-adj = 0.02). The analysis of IL-1RA revealed an interaction effect (p = 0.04) suggesting differences between groups at some time points that post-hoc tests were not able to detect. Conclusions: To our knowledge, this is the first study to evaluate the effects of yoga in a cancer population studying inflammation markers, cortisol trend and QOL during and until 6 months after XRT. This study suggests that yoga practice is able to reduce stress and inflammation levels over time. Besides including a larger number of patients to increase the power, future studies should consider other inflammatory or pro inflammatory factors and long-term yoga program to gain more evidence on yoga practice benefits.

Abstract Glioblastoma (GBM) is the most lethal brain tumor in adults. Radiation, together with temozolomide is the standard treatment, but nevertheless, relapse occurs in nearly all cases. Understanding the mechanisms underlying radiation resistance may help to find more effective therapies. After radiation treatment, ATP is released into the tumor microenvironment where it binds and activates purinergic P2 receptors, mainly of the P2X7 subtype. Two main P2X7 splice variants, P2X7A and P2X7B, are expressed in most cell types, where they associate with distinct biochemical and functional responses. GBM cells widely differ for the level of P2X7 isoform expression and accordingly for sensitivity to stimulation with extracellular ATP (eATP). Irradiation causes a dramatic shift in P2X7 isoform expression, with the P2X7A isoform being down- and the P2X7B isoform up-modulated, as well as extensive cell death and overexpression of stemness and senescence markers. Treatment with P2X7 blockers during the post-irradiation recovery potentiated irradiation-dependent cytotoxicity, suggesting that P2X7B activation by eATP generated a trophic/growth-promoting stimulus. Altogether, these data show that P2X7A and B receptor isoform levels are inversely modulated during the post-irradiation recovery phase in GBM cells.

Comet assay provides an easy solution to estimate DNA damage in single cells through microscopy assessment. It is widely used in the analysis of genotoxic damages induced by radiotherapy or chemotherapeutic agents. DNA damage is quantified at the single-cell level by computing the displacement between the genetic material within the nucleus, typically called “comet head”, and the genetic material in the surrounding part of the cell, considered as the “comet tail”. Today, the number of works based on Comet Assay analyses is really impressive. In this work, besides revising the solutions available to obtain reproducible and reliable quantitative data, we developed an easy-to-use tool named CometAnalyser. It is designed for the analysis of both fluorescent and silver-stained wide-field microscopy images and allows to automatically segment and classify the comets, besides extracting Tail Moment and several other intensity/morphological features for performing statistical analysis. CometAnalyser is an open-source deep-learning tool. It works with Windows, Macintosh, and UNIX-based systems. Source code, standalone versions, user manual, sample images, video tutorial and further documentation are freely available at: https://sourceforge.net/p/cometanalyser.

Abstract Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.

Neuropathic pain (NP) is a chronic condition resulting from damaged pain-signaling pathways. It is a debilitating disorder that affects up to 10% of the world’s population. Although opioid analgesics are effective in reducing pain, they present severe risks; so, there is a pressing need for non-opioid pain-relieving drugs. One potential alternative is represented by sigma-1 receptor (S1R) antagonists due to their promising analgesic effects. Here, we report the synthesis and biological evaluation of a series of S1R antagonists based on a 2-aryl-4-aminobutanol scaffold. After assessing affinity toward the S1R and selectivity over the sigma-2 receptor (S2R), we evaluated the agonist/antagonist profile of the compounds by investigating their effects on nerve growth factor-induced neurite outgrowth and aquaporin-mediated water permeability in the presence and absence of oxidative stress. (R/S)-RC-752 emerged as the most interesting compound for S1R affinity (Ki S1R = 6.2 ± 0.9) and functional antagonist activity. Furthermore, it showed no cytotoxic effect in two normal human cell lines or in an in vivo zebrafish model and was stable after incubation in mouse plasma. (R/S)-RC-752 was then evaluated in two animal models of NP: the formalin test and the spinal nerve ligation model. The results clearly demonstrated that compound (R/S)-RC-752 effectively alleviated pain in both animal models, thus providing the proof of concept of its efficacy as an antinociceptive agent.

Abstract Background Recent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease. However, deeper understanding of the molecular mechanisms underlying the abscopal effect are required to best benefit a larger proportion of patients with metastasis. Several groups including ours, reported the involvement of wild-type (wt) p53 in radiation-induced abscopal effects, however very little is known on the role of wtp53 dependent molecular mechanisms. Methods We investigated through in vivo and in vitro approaches how wtp53 orchestrates radiation-induced abscopal effects. Wtp53 bearing (A549) and p53-null (H1299) NSCLC lines were xenotransplanted in nude mice, and cultured in 2D monolayers and 3D tumor spheroids. Extracellular vesicles (EVs) were isolated from medium cell culture by ultracentrifugation protocol followed by Nanoparticle Tracking Analysis. Gene expression was evaluated by RT-Real Time, digital qRT-PCR, and dot blot technique. Protein levels were determined by immunohistochemistry, confocal anlysis, western blot techniques, and immunoassay. Results We demonstrated that single high-dose irradiation (20 Gy) induces significant tumor growth inhibition in contralateral non-irradiated (NIR) A549 xenograft tumors but not in NIR p53-null H1299 or p53-silenced A549 (A549sh/p53) xenografts. We further demonstrates that irradiation of A549 cells in vitro induces a senescence-associated secretory phenotype (SASP) producing extracellular vesicles (EVs) expressing CD63 and carrying DNA:RNA hybrids and LINE-1 retrotransposon. IR-A549 EVs also hamper the colony-forming capability of recipient NIR A549 cells, induce senescent phenotype, nuclear expression of DNA:RNA hybrids, and M1 macrophage polarization. Conclusions In our models, we demonstrate that high radiation dose in wtp53 tumors induce the onset of SASP and secretion of CD63+ EVs loaded with DNA:RNA hybrids and LINE-1 retrotransposons that convey senescence messages out of the irradiation field triggering abscopal effect in NIR tumors.

Prostate cancer (PCa) is one of the most common cancers in European males. Although therapeutic approaches have changed in recent years, and several new drugs have been approved by the Food and Drug Administration (FDA), androgen deprivation therapy (ADT) remains the standard of care. Currently, PCa represents a clinical and economic burden due to the development of resistance to ADT, paving the way to cancer progression, metastasis, and to long-term side effects induced by ADT and radio-chemotherapeutic regimens. In light of this, a growing number of studies are focusing on the tumor microenvironment (TME) because of its role in supporting tumor growth. Cancer-associated fibroblasts (CAFs) have a central function in the TME because they communicate with prostate cancer cells, altering their metabolism and sensitivity to drugs; hence, targeted therapy against the TME, and, in particular, CAFs, could represent an alternative therapeutic approach to defeat therapy resistance in PCa. In this review, we focus on different CAF origins, subsets, and functions to highlight their potential in future therapeutic strategies for prostate cancer.

Abstract Prostate cancer represents the major cause of cancer-related death in men and patients frequently develop drug-resistance and metastatic disease. Most studies focus on hormone-resistance mechanisms related to androgen receptor mutations or to the acquired property of prostate cancer cells to over-activate signaling pathways. Tumor microenvironment plays a critical role in prostate cancer progression. However, the mechanism involving androgen/androgen receptor signaling in cancer associated fibroblasts and consequences for prostate cancer progression still remains elusive. We now report that prostate cancer associated fibroblasts express a transcriptional-incompetent androgen receptor. Upon androgen challenging, the receptor co-localizes with the scaffold protein filamin A in the extra-nuclear compartment of fibroblasts, thus mediating their migration and invasiveness. Cancer-associated fibroblasts move towards epithelial prostate cancer cells in 2D and 3D cultures, thereby inducing an increase of the prostate cancer organoid size. Androgen enhances both these effects through androgen receptor/filamin A complex assembly in cancer-associated fibroblasts. An androgen receptor-derived stapled peptide, which disrupts the androgen receptor/filamin A complex assembly, abolishes the androgen-dependent migration and invasiveness of cancer associated fibroblasts. Notably, the peptide impairs the androgen-induced invasiveness of CAFs in 2D models and reduces the overall tumor area in androgen-treated 3D co-culture. The androgen receptor in association with β1 integrin and membrane type-matrix metalloproteinase 1 activates a protease cascade triggering extracellular matrix remodeling. The peptide also impairs the androgen activation of this cascade. This study offers a potential new marker, the androgen receptor/filamin A complex, and a new therapeutic approach targeting intracellular pathways activated by the androgen/androgen receptor axis in prostate cancer-associated fibroblasts. Such a strategy, alone or in combination with conventional therapies, may allow a more efficient treatment of prostate cancer.

Abstract 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.

Abstract Background Concurrent chemotherapy and radiation using conventional fractionation is the standard treatment for inoperable, locally advanced non-small-cell lung cancer (NSCLC). We tested accelerated hypofractionated radiotherapy (AHR) and chemotherapy for the treatment of locally advanced NSCLC. Methods Eligible patients with locally advanced NSCLC were treated with induction chemotherapy (cisplatin and docetaxel), followed by AHR using tomotherapy and consolidation chemotherapy. The prescribed doses were 30 Gy/5 daily fractions at the reference isodose (60–70%) to the tumor, and 25 Gy/5 daily fractions to the clinically involved lymph nodes. The primary end-point was response rate (RR); the secondary end-points were acute and late side-effects, local progression-free survival (PFS), metastasis-free survival (MFS) and overall survival (OS). This trial closed before the first planned interim analysis due to poor accrual. Results From January 2009 to January 2012, 17 of the 23 enrolled patients were evaluable. Treatment yielded an overall RR of 82%. Median follow-up was 87 months (range: 6–87), local PFS was 19.8 months (95% CI 9.7 - not reached), MFS was 9.7 months (95% CI 5.8–46.0) and OS was 23 months (95% CI 8.4–48.4). 70% of patients experienced acute G4 neutropenia, 24% G4 leukopenia, 24% G3 paresthesia, 4% G3 cardiac arrythmia, 4% underwent death after chemotherapy. Late toxicity was represented by 24% dyspnea G3. Conclusions AHR combined with chemotherapy is feasible with no severe side-effects, and it appears highly acceptable by patients. Trial registration This study is registered with the EudractCT registration 2008-006525-14. Registered on 9 December 2008.

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.

BackgroundThe presence of hypoxic cells in high-grade glioma (HGG) is one of major reasons for failure of local tumour control with radiotherapy (RT). The use of hyperbaric oxygen therapy (HBO) could help to overcome the problem of oxygen deficiency in poorly oxygenated regions of the tumour. We propose an innovative approach to improve the efficacy of hypofractionated stereotactic radiotherapy (HSRT) after HBO (HBO-RT) for the treatment of recurrent HGG (rHGG) and herein report the results of an ad interim analysis.MethodsWe enrolled a preliminary cohort of 9 adult patients (aged >18 years) with a diagnosis of rHGG. HSRT was administered in daily 5-Gy fractions for 3-5 consecutive days a week. Each fraction was delivered up to maximum of 60 minutes after HBO.ResultsMedian follow-up from re-irradiation was 11.6 months (range: 3.2-11.6 months). The disease control rate (DCR) 3 months after HBO-RT was 55.5% (5 patients). Median progression-free survival (mPFS) for all patients was 5.2 months (95%CI: 1.34-NE), while 3-month and 6-month PFS was 55.5% (95%CI: 20.4-80.4) and 27.7% (95%CI: 4.4-59.1), respectively. Median overall survival (mOS) of HBO-RT was 10.7 months (95% CI: 7.7-NE). No acute or late neurologic toxicity >grade (G)2 was observed in 88.88% of patients. One patient developed G3 radionecrosis.ConclusionsHSRT delivered after HBO appears to be effective for the treatment of rHGG, it could represent an alternative, with low toxicity, to systemic therapies for patients who cannot or refuse to undergo such treatments.Clinical Trial Registrationwww.ClinicalTrials.gov, identifier NCT 03411408.

Despite the fact that significant advances in treatment of common cancers have been achieved over the years, orphan tumors still represent an important unmet medical need. Due to their complex multifactorial origin and limited number of cases, such pathologies often have very limited treatment options and poor prognosis. In the search for new anticancer agents, our group recently identified RC-106, a Sigma receptor modulator endowed with proteasome inhibition activity. This compound showed antiproliferative activity toward different cancer cell lines, among them glioblastoma (GB) and multiple myeloma (MM), two currently unmet medical conditions. In this work, we directed our efforts toward the exploration of chemical space around RC-106 to identify new active compounds potentially useful in cancer treatment. Thanks to a combinatorial approach, we prepared 41 derivatives of the compound and evaluated their cytotoxic potential against MM and GB. Three novel potential anticancer agents have been identified.

Despite the gradual decrease in incidence, gastric cancer is still the third leading cause of cancer death worldwide. Although chemotherapy enhances overall survival and quality of life in advanced disease, the median overall survival is < 12 months. In recent years, the human epidermal growth factor receptor (ErbB) family has been extensively investigated in gastric cancer. The ErbB family is composed of four closely-related members: ErbB-1 (HER1 or epidermal growth factor receptor, EGFR), ErbB-2 (HER2), ErbB-3 (HER3), and ErbB-4 (HER4), all of which play a critical role in regulating cell growth, proliferation and migration of tumors. It is well known that gastric cancer overexpresses HER in a heterogeneous pattern, especially EGFR, and HER2. HER3 is another important member of the ErbB family that preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway. Furthermore, its heterodimerization with HER2 seems fundamental for steering HER2-overexpressing breast cancer tumor growth. Less is known about the impact of HER4 on gastric cancer. Improved survival from the use of trastuzumab has paved the way for ErbB receptor family-targeted treatments in gastric cancer. However, unlike trastuzumab, ErbB receptor-targeted drugs have not consistently maintained the encouraging results obtained in preclinical and early clinical trials. This may be attributable to the intrinsic heterogeneity of gastric cancer and/or to the lack of standardized test quality for established biomarkers used to evaluate these biological targets. This review presents an overview of the most recent clinical studies on agents targeting the ErbB family in gastric cancer.

Despite the key role played by androgen receptor (AR) in tumor cell aggressiveness and prostate cancer (PCa) progression, its function in the tumor microenvironment (TME) is still controversial. Increasing studies highlight the crucial role played by TME modulation in treatment outcome and tumor cell spreading. In this context, targeting specific constituents of the TME could be considered an alternative approach to classic treatments directed against cancer cells. Currently, androgen deprivation therapy (ADT) is a routinely adopted strategy in the management of PCa, with initial success, and consecutive fail. A possible justification to this is the fact that ADT aims to target all the transcription/translation-related activities of AR, which are typical of tumor epithelial cells. Less is still known about side effects of ADT on TME. Cancer Associated Fibroblasts (CAFs), for example, express a classic AR, mostly confined in the extra-nuclear portion of the cell. In CAFs ADT exerts a plethora of non-transcriptional effects, depending by the protein partner linked to AR, leading to cell migration, proliferation, and differentiation. In recent years, substantial progress in the structure-function relationships of AR, identification of its binding partners and function of protein complexes including AR have improved our knowledge of its signaling axis. Important AR non-genomic effects and lots of its cytoplasmatic binding partners have been described, pointing out a fine control of AR non-genomic pathways. Accordingly, new AR inhibitors have been designed and are currently under investigation. Prompt development of new approaches to target AR or block recruitment of its signaling effectors, or co-activators, is urgently needed. The present review takes an in-depth look at current literature, furnishing an exhaustive state-of-the-art overview of the non-genomic role of AR in PCa, with particular emphasis on its involvement in TME biology.

Introduction: Pancreatic cancer (PC) is one of the most lethal tumor worldwide, with no prognosis improvement over the past 20-years. The silent progressive nature of this neoplasia hampers the early diagnosis, and the surgical resection of the tumor, thus chemotherapy remains the only available therapeutic option. Sigma receptors (SRs) are a class of receptors proposed as new cancer therapeutic targets due to their over-expression in tumor cells and their involvement in cancer biology. The main localization of these receptors strongly suggests their potential role in ER unfolded protein response (ER-UPR), a condition frequently occurring in several pathological settings, including cancer. Our group has recently identified RC-106, a novel pan-SR modulator with good in vitro antiproliferative activities toward a panel of different cancer cell lines. In the present study, we investigated the in vitro properties and pharmacological profile of RC-106 in PC cell lines with the aim to identify a potential lead candidate for the treatment of this tumor.Methods: Pancreatic cancer cell lines Panc-1, Capan-1, and Capan-2 have been used in all experiments. S1R and TMEM97/S2R expression in PC cell lines was quantified by Real-Time qRT-PCR and Western Blot experiments. MTS assay was used to assess the antiproliferative effect of RC-106. The apoptotic properties of RC-106 was evaluated by TUNEL and caspase activation assays. GRP78/BiP, ATF4, and CHOP was quantified to evaluate ER-UPR. Proteasome activity was investigated by a specific fluorescent-based assay. Scratch wound healing assay was used to asses RC-106 effect on cell migration. In addition, we delineated the in vivo pharmacokinetic profile and pancreas distribution of RC-106 in male CD-1 mice.Results: Panc-1, Capan-1, and Capan-2 express both SRs. RC-106 exerts an antiproliferative and pro-apoptotic effect in all examined cell lines. Cells exposure to RC-106 induces the increase of the expression of ER-UPR related proteins, and the inhibition of proteasome activity. Moreover, RC-106 is able to decrease PC cell lines motility. The in vivo results show that RC-106 is more concentrated in pancreas than plasma.Conclusion: Overall, our data evidenced that the pan-SR modulator RC-106 is an optimal candidate for in vivo studies in animal models of PC.

A Correction to this paper has been published: https://doi.org/10.1038/s41419-021-03483-4

Subcellular spatial location is an essential descriptor of molecules biological function. Presently, super-resolution microscopy techniques enable quantification of subcellular objects distribution in fluorescence images, but they rely on instrumentation, tools and expertise not constituting a default for most of laboratories. We propose a method that allows resolving subcellular structures location by reinforcing each single pixel position with the information from surroundings. Although designed for entry-level laboratory equipment with common resolution powers, our method is independent from imaging device resolution, and thus can benefit also super-resolution microscopy. The approach permits to generate density distribution maps (DDMs) informative of both objects’ absolute location and self-relative displacement, thus practically reducing location uncertainty and increasing the accuracy of signal mapping. This work proves the capability of the DDMs to: (a) improve the informativeness of spatial distributions; (b) empower subcellular molecules distributions analysis; (c) extend their applicability beyond mere spatial object mapping. Finally, the possibility of enhancing or even disclosing latent distributions can concretely speed-up routine, large-scale and follow-up experiments, besides representing a benefit for all spatial distribution studies, independently of the image acquisition resolution. DDMaker, a Software endowed with a user-friendly Graphical User Interface (GUI), is also provided to support users in DDMs creation.

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.

Pancreatic cancer (PC) remains one of the most lethal cancers worldwide. Sigma receptors (SRs) have been proposed as cancer therapeutic targets. Their main localization suggests they play a potential role in ER stress and in the triggering of the unfolded protein response (UPR). Here, we investigated the mechanisms of action of RC-106, a novel pan-SR modulator, to characterize therapeutically exploitable role of SRs in tumors. Two PC cell lines were used in all the experiments. Terminal UPR activation was evaluated by quantifying BiP, ATF4 and CHOP by Real-Time qRT-PCR, Western Blot, immunofluorescence and confocal microscopy. Cell death was studied by flow cytometry. Post-transcriptional gene silencing was performed to study the interactions between SRs and UPR key proteins. RC-106 activated ER stress sensors in a dose- and time-dependent manner. It also induced ROS production accordingly with ATF4 upregulation at the same time reducing cell viability of both cell lines tested. Moreover, RC-106 exerted its effect through the induction of the terminal UPR, as shown by the activation of some of the main transducers of this pathway. Post-transcriptional silencing studies confirmed the connection between SRs and these key proteins. Overall, our data highlighted a key role of SRs in the activation of the terminal UPR pathway, thus indicating pan-SR ligands as candidates for targeting the UPR in pancreatic cancer.

Hypofractionation is currently considered a valid alternative to conventional radiotherapy for the treatment of patients with organ-confined prostate cancer. Recent data have demonstrated that extreme hypofractionation, which involves the use of a high radiation dose per delivered fraction and concomitant reduction of sessions, is a safe and effective treatment, even though its radiobiological rationale is still lacking. The present work aims to investigate the biological basis sustaining this approach and to evaluate the potential of a hypofractionated regimen in combination with androgen deprivation therapy, one of the major standards of care for prostate cancer. Findings show that androgen receptor (AR) modulation, by use of androgens and antiandrogens, has a significant impact on cell survival, especially in hypoxic conditions (4% O2). Subsequent experiments have revealed that AR activity as a transcription factor is involved in the onset of malignant senescence-associated secretory phenotype (SASP) and activation of DNA repair cascade. In particular, we found that AR stimulation in hypoxic conditions promotes the enhanced transcription of ATM gene, the cornerstone kinase of the DNA damage repair genes. Together, these data provide new potential insights to justify the use of androgen deprivation therapy, in particular with second-generation anti-androgens such as enzalutamide, in combination with radiotherapy.

Despite the interest aroused by sigma receptors (SRs) in the area of oncology, their role in tumor biology remains enigmatic. The predominant subcellular localization and main site of activity of SRs are the endoplasmic reticulum (ER). Current literature data, including recent findings on the sigma 2 receptor subtype (S2R) identity, suggest that SRs may play a role as ER stress gatekeepers. Although SR endogenous ligands are still unknown, a wide series of structurally unrelated compounds able to bind SRs have been identified. Currently, the identification of novel antiproliferative molecules acting via SR interaction is a challenging task for both academia and industry, as shown by the fact that novel anticancer drugs targeting SRs are in the preclinical-stage pipeline of pharmaceutical companies (i.e., Anavex Corp. and Accuronix). So far, no clinically available anticancer drugs targeting SRs are still available. The present review focuses literature advancements and provides a state-of-the-art overview of SRs, with emphasis on their involvement in cancer biology and on the role of SR modulators as anticancer agents. Findings from preclinical studies on novel anticancer drugs targeting SRs are presented in brief.

Radiation therapy is one of the most important treatment modalities for thoracic tumors. Despite significant advances in radiation techniques, radiation-induced lung injury (RILI) still occurs in up to 30% of patients undergoing thoracic radiotherapy, and therefore remains the main dose-limiting obstacle. RILI is a potentially lethal clinical complication of radiotherapy that has 2 main stages: an acute stage defined as radiation pneumonitis, and a late stage defined as radiation-induced lung fibrosis. Patients who develop lung fibrosis have a reduced quality of life with progressive and irreversible organ malfunction. Currently, the most effective intervention for the treatment of lung fibrosis is lung transplantation, but the lack of available lungs and transplantation-related complications severely limits the success of this procedure. Over the last few decades, advances have been reported in the use of mesenchymal stem cells (MSCs) for lung tissue repair and regeneration. MSCs not only replace damaged lung epithelial cells but also promote tissue repair through the secretion of anti-inflammatory and anti-fibrotic factors. Here, we present an overview of MSC-based therapy for radiation-induced lung fibrosis, focusing in particular on the molecular mechanisms involved and describing the most recent preclinical and clinical studies carried out in the field.

Background: Melanoma remains largely resistant to currently available chemotherapy, and new strategies have been proposed to flank standardized therapeutic protocols in an effort to improve efficacy. Such an approach requires good knowledge of the mechanisms involved in the resistance and survival of melanoma cells. In this context, the SLUG gene has recently been characterized as a major regulator of melanocytes and melanoma cell survival. Methods: We tested the hypothesis that an oligonucleotide-based short interfering RNA (siRNA) directed against the SLUG gene increases the susceptibility of melanoma cells to drugs such as cisplatin and fotemustine, which are frequently used to treat this cancer. Results: It was found that SLUG siRNA increased cisplatin-induced cell death and rendered the drug active in vitro at half its plasmatic peak concentration. Such activity was correlated with an upregulation of the pro-apoptotic gene, PUMA. Furthermore, SLUG siRNA increased the capacity of fotemustine to elicit cell death and induced p21WAF1 upregulation, resulting in cell cycle arrest. Interestingly, this pathway did not require functional p53. Conclusion: These findings suggest that SLUG siRNA enhances the efficacy of two of the most widely used drugs to treat melanoma.

To investigate the cellular/molecular basis of the activity of a novel lipophilic camptothecin, gimatecan (ST1481), against slowly proliferating cells, we performed a comparative study of topotecan and gimatecan in human bladder cancer models (HT1376 and MCR). Gimatecan was significantly more effective than topotecan in inhibiting the growth of HT1376 tumor, thus reflecting anti proliferative potency. In both HT1376 and MCR cells, gimatecan caused a persistent S-phase arrest, indicating an efficient DNA damage checkpoint. This response was consistent with a cytostatic effect, because no evidence of apoptosis was detected. In contrast to gimatecan, topotecan at equitoxic concentrations caused an early and persistent downregulation of topoisomerase I. Modulation of protein level could not be solely ascribed to the proteasome-mediated degradation of the enzyme because the proteasome inhibitor PS341 sensitized MCR but not HT1376 cells to camptothecins, suggesting alternative mechanisms of drug-induced topoisomerase I downregulation. Indeed, the two camptothecins caused a differential inhibition of topoisomerase I transcription, which is more marked in topotecan-treated cells. The HT1376 model was more sensitive to this immediate decrease of mRNA level. Our data document a marked antitumor activity of gimatecan against a bladder carcinoma model. A limited downregulation of topoisomerase I by gimatecan provides additional insights into the cellular basis of drug potency.

Cell invasiveness quantification is of paramount importance in cancer research and is often evaluated in vitro through scratch wound healing assays that determine the rate at which a population of cells fills a gap created in a confluent 2D culture. The quantification of the results of this experiment, however, lacks standardization and is often highly time consuming and user dependent. To overcome these limitations, we have developed AIM (Automatic Invasiveness Measure), a freely-available software tool for the automatic quantification of the cell-free region in scratch wound healing assays. This study will completely describe AIM and will show its equivalence to three analysis methods commonly used for the quantification of the scratch area and the measure of true wound extension. Furthermore, the analysis time and the dependency of the results of these techniques on the structure of the time course (total duration and number of points) will be studied. To the best of our knowledge, AIM is the first entirely-automated analysis method for scratch wound healing assays and represents a significant improvement of this technique both in terms of results’ quality and reliability.

The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (S)-11 and (R)-9, in in vitro and in vivo experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). In vitro experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progression-related markers were evaluated by western blot. In vivo experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (R)-9 and (S)-11 exhibited a higher cytotoxic effect compared to that of the reference compound ((R)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (R)-9 was higher than that of (S)-11 in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (S)-11 and (R)-9 inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. In vivo studies on the toxicological profile of (R)-9 did not reveal the presence of adverse events. Furthermore, (R)-9 inhibited tumor growth in various in vivo models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our in vitro results highlight the antitumor activity of the two novel molecules (R)-9 and (S)-11, making them a potentially attractive option for the treatment of CRPC.