Targeting the hERG1 and ß1 integrin complex for cancer treatment.

INTRODUCTION: Despite great advances, novel therapeutic targets and strategies are still needed, in particular for some carcinomas in the metastatic stage (breast cancer, colorectal cancer, pancreatic ductal adenocarcinoma and the clear cell renal carcinoma). Ion channels may be considered good cancer biomarkers and targets for antineoplastic therapy. These concepts are particularly relevant considering the hERG1 potassium channel as a novel target for antineoplastic therapy. AREAS COVERED: A great deal of evidence demonstrates that hERG1 is aberrantly expressed in human cancers, in particular in aggressive carcinomas. A relevant cornerstone was the discovery that, in cancer cells, the channel is present in a very peculiar conformation, strictly bound to the ß1 subunit of integrin receptors. The hERG1/ß1 integrin complex does not occur in the heart. Starting from this evidence, we developed a novel single chain bispecific antibody (scDb-hERG1-ß1), which specifically targets the hERG1/ß1 integrin complex and exerts antineoplastic effects in preclinical experiments. EXPERT OPINION: Since hERG1 blockade cannot be pursued for antineoplastic therapy due to the severe cardiac toxic effects (ventricular arrhythmias) that many hERG1 blockers exert, different strategies must be identified to specifically target hERG1 in cancer. The targeting of the hERG1/ß1 integrin complex through the bispecific antibody scDb-hERG1-ß1 can overcome such hindrances.

Integrins regulate hERG1 dynamics by girdin-dependent Gαi3: signaling and modeling in cancer cells.

The hERG1 potassium channel is aberrantly over expressed in tumors and regulates the cancer cell response to integrin-dependent adhesion. We unravel a novel signaling pathway by which integrin engagement by the ECM protein fibronectin promotes hERG1 translocation to the plasma membrane and its association with ß1 integrins, by activating girdin-dependent Gαi3 proteins and protein kinase B (Akt). By sequestering hERG1, ß1 integrins make it avoid Rab5-mediated endocytosis, where unbound channels are degraded. The cycle of hERG1 expression determines the resting potential (Vrest) oscillations and drives the cortical f-actin dynamics and thus cell motility. To interpret the slow biphasic kinetics of hERG1/ß1 integrin interplay, we developed a mathematical model based on a generic balanced inactivation-like module. Integrin-mediated cell adhesion triggers two contrary responses: a rapid stimulation of hERG1/ß1 complex formation, followed by a slow inhibition which restores the initial condition. The protracted hERG1/ß1 integrin cycle determines the slow time course and cyclic behavior of cell migration in cancer cells.

Expression of the ether-a-gò-gò-related gene 1 channel during B and T lymphocyte development: role in BCR and TCR signaling.

The functional relevance of K+ and Ca2+ ion channels in the "Store Operated Calcium Entry" (SOCE) during B and T lymphocyte activation is well proven. However, their role in the process of T- and B- cell development and selection is still poorly defined. In this scenario, our aim was to characterize the expression of the ether à-go-go-related gene 1 (ERG1) and KV1.3 K+ channels during the early stages of mouse lymphopoiesis and analyze how they affect Ca2+signaling, or other signaling pathways, known to mediate selection and differentiation processes of lymphoid clones. We provide here evidence that the mouse (m)ERG1 is expressed in primary lymphoid organs, bone marrow (BM), and thymus of C57BL/6 and SV129 mice. This expression is particularly evident in the BM during the developmental stages of B cells, before the positive selection (large and small PreB). mERG1 is also expressed in all thymic subsets of both strains, when lymphocyte positive and negative selection occurs. Partially overlapping results were obtained for KV1.3 expression. mERG1 and KV1.3 were expressed at significantly higher levels in B-cell precursors of mice developing an experimental autoimmune encephalomyelitis (EAE). The pharmacological blockage of ERG1 channels with E4031 produced a significant reduction in intracellular Ca2+ after lymphocyte stimulation in the CD4+ and double-positive T-cell precursors' subsets. This suggests that ERG1 might contribute to maintaining the electrochemical gradient responsible for driving Ca2+ entry, during T-cell receptor signaling which sustains lymphocyte selection checkpoints. Such role mirrors that performed by the shaker-type KV1.3 potassium channel during the activation process of mature lymphocytes. No effects on Ca2+ signaling were observed either in B-cell precursors after blocking KV1.3 with PSORA-4. In the BM, the pharmacological blockage of ERG1 channels produced an increase in ERK phosphorylation, suggesting an effect of ERG1 in regulating B-lymphocyte precursor clones' proliferation and checkpoint escape. Overall, our results suggest a novel physiological function of ERG1 in the processes of differentiation and selection of lymphoid precursors, paving the way to further studies aimed at defining the expression and role of ERG1 channels in immune-based pathologies in addition to that during lymphocyte neoplastic transformation.

Cardiac safety assessment of a novel recombinant bispecific antibody targeting the ether-à-go-go related gene 1 (hERG1)-ß1 integrin macromolecular complex.

Introduction: In the last decades, mounting evidence has pointed out the human ether-á-go-go-related gene (hERG1) potassium channel as a novel biomarker in human cancers. However, hERG1 sustains the cardiac repolarizing current IKr and its blockade can induce a prolonged QT interval at the ECG, which increases the risk of life-threatening arrhythmias. This represents a major hindrance for targeting hERG1 for antineoplastic therapeutic purposes. Based on our discovery that hERG1 resides in a macromolecular complex with the ß1 subunit of integrin adhesion receptors only in tumors, and not in the heart, we generated (and patented WO2019/015936) a novel engineered, single chain, bispecific antibody in the format of a diabody (scDb-hERG1-ß1). This antibody has been proven to target with high affinity the hERG1/ß1 integrin complex and to exert a good antineoplastic activity in preclinical mouse models. Methods: In the present study, we evaluated the cardiac safety of the scDb-hERG1-ß1, determining the action potential duration (APD) of human cardiomyocytes, either atrial (from valve-disease patients) or ventricular (from aortic stenosis patients). Cardiac cells were incubated in vitro with i) the scDb-hERG1-ß1, ii) the full length anti-hERG1 monoclonal antibody (mAb-hERG1) and iii) its single chain Fragment variable derivative (scFv-hERG1), from which the scDb-hERG1-ß1 was assembled. All the tests were performed before and after treatment with the specific hERG1 blocker E4031. In addition, we have performed preliminary experiments, analyzing the effects of the scDb-hERG1/ß1 in vivo measuring the QT interval length of the surface ECG after its injection intravenously in farm-pigs. Results: The scDb-hERG1-ß1 did not produce any lengthening of APD compared to control (vehicle) conditions, either in atrial or ventricular cardiomyocytes, whereas both the hERG1-mAb and the scFv-hERG1 produced a significant APD prolongation. The addition of E4031 further prolonged APD. The scDb-hERG1-ß1 did not produce any alterations of the QT (and QTc) interval values, once injected intravenously in farm pigs. Discussion: Overall, the above evidences plead for the cardiac safety of the scDb-hERG1-ß1, suggesting that an application of this antibody for anti-cancer therapy will be untainted by cardiotoxicity.

High throughput clone screening on overexpressed hERG1 and Kv1.3 potassium channels using ion channel reader (ICR) label free technology.

Pharmacological studies aimed at the development of newly synthesized drugs directed against ion channels (as well as genetic studies of ion channel mutations) involve the development and use of transfected cells. However, the identification of the best clone, in terms of transfection efficiency, is often a time consuming procedure when performed through traditional methods such as manual patch-clamp. On the other hand, the use of other faster techniques, such as for example the IF, are not informative on the effective biological functionality of the transfected ion channel(s). In the present work, we used the high throughput automated ion channel reader (ICR) technology (ICR8000 Aurora Biomed Inc.) that combine atomic absorption spectroscopy with a patented microsampling process to accurately measure ion flux in cell-based screening assays. This technology indeed helped us to evaluate the transfection efficiency of hERG1 and hKv1.3 channels respectively on the HEK-293 and CHO cellular models. Moreover, as proof of the validity of this innovative method, we have corroborated these data with the functional characterization of the potassium currents carried out by the same clones through patch-clamp recordings. The results obtained in our study are promising and represent a valid methodological strategy to screen a large number of clones simultaneously and to pharmacologically evaluate their functionality within an extremely faster timeframe.

Corrigendum to "Transcriptomic data of Bevacizumab-adapted colorectal adenocarcinoma cells HCT-116" [Data In Brief, Volume 48, (Available online 17 March 2023) 1-9/Article 109069].

[This corrects the article DOI: 10.1016/j.dib.2023.109069.].

Immunosuppressive effects of new thiophene-based KV1.3 inhibitors.

Voltage-gated potassium channel KV1.3 inhibitors have been shown to be effective in preventing T-cell proliferation and activation by affecting intracellular Ca2+ homeostasis. Here, we present the structure-activity relationship, KV1.3 inhibition, and immunosuppressive effects of new thiophene-based KV1.3 inhibitors with nanomolar potency on K+ current in T-lymphocytes and KV1.3 inhibition on Ltk- cells. The new KV1.3 inhibitor trans-18 inhibited KV1.3 -mediated current in phytohemagglutinin (PHA)-activated T-lymphocytes with an IC50 value of 26.1 nM and in mammalian Ltk- cells with an IC50 value of 230 nM. The KV1.3 inhibitor trans-18 also had nanomolar potency against KV1.3 in Xenopus laevis oocytes (IC50 = 136 nM). The novel thiophene-based KV1.3 inhibitors impaired intracellular Ca2+ signaling as well as T-cell activation, proliferation, and colony formation.

Evaluation of RAS Mutational Status in Liquid Biopsy to Monitor Disease Progression in Metastatic Colorectal Cancer Patients.

In this study we evaluated both~ K- and N-RAS mutations in plasma samples from patients with metastatic colorectal cancer by means of the BEAMing technology, and we assessed their diagnostic performance compared to RAS analyses performed on tissue. The sensitivity of BEAMing in identifying KRAS mutations was of 89.5%, with a fair specificity. The agreement with tissue analysis was moderate. The sensitivity for NRAS was high with a good specificity, and the agreement between tissue analysis and BEAMing was fair. Interestingly, significantly higher mutant allele fraction (MAF) levels were detected in patients with G2 tumors, liver metastases, and in those who did not receive surgery. NRAS MAF level was significantly higher in patients with mucinous adenocarcinoma and for those with lung metastases. A sharp increase in the MAF values was observed in patients who moved towards disease progression. More strikingly, molecular progression always anticipated the radiological one in these patients. These observations pave the way to the possibility of using liquid biopsy to monitor patients during treatment, and to enable oncologists to anticipate interventions compared to radiological analyses. This will allow time to be saved and ensure a better management of metastatic patients in the near future.

Combination Therapy with a Bispecific Antibody Targeting the hERG1/ß1 Integrin Complex and Gemcitabine in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) represents an unmet medical need. Difficult/late diagnosis as well as the poor efficacy and high toxicity of chemotherapeutic drugs result in dismal prognosis. With the aim of improving the treatment outcome of PDAC, we tested the effect of combining Gemcitabine with a novel single chain bispecific antibody (scDb) targeting the cancer-specific hERG1/ß1 integrin complex. First, using the scDb (scDb-hERG1-ß1) in immunohistochemistry (IHC), Western blot (WB) analysis and immunofluorescence (IF), we confirmed the presence of the hERG1/ß1 integrin complex in primary PDAC samples and PDAC cell lines. Combining Gemcitabine with scDb-hERG1-ß1 improved its cytotoxicity on all PDAC cells tested in vitro. We also tested the combination treatment in vivo, using an orthotopic xenograft mouse model involving ultrasound-guided injection of PDAC cells. We first demonstrated good penetration of the scDb-hERG1-ß1 conjugated with indocyanine green (ICG) into tumour masses by photoacoustic (PA) imaging. Next, we tested the effects of the combination at either therapeutic or sub-optimal doses of Gemcitabine (25 or 5 mg/kg, respectively). The combination of scDb-hERG1-ß1 and sub-optimal doses of Gemcitabine reduced the tumour masses to the same extent as the therapeutic doses of Gemcitabine administrated alone; yielded increased survival; and was accompanied by minimised side effects (toxicity). These data pave the way for a novel therapeutic approach to PDAC, based on the combination of low doses of a chemotherapeutic drug (to minimize adverse side effects and the onset of resistance) and the novel scDb-hERG1-ß1 targeting the hERG1/ß1 integrin complex as neoantigen.

Combined Inhibition of Smoothened and the DNA Damage Checkpoint WEE1 Exerts Antitumor Activity in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is characterized by resistance to chemotherapy and a poor prognosis. Therefore, treatments that can effectively suppress tumor growth are urgently needed. Aberrant activation of hedgehog (HH) signaling has been implicated in several cancers, including those of the hepatobiliary tract. However, the role of HH signaling in intrahepatic CCA (iCCA) has not been completely elucidated. In this study, we addressed the function of the main transducer Smoothened (SMO) and the transcription factors (TFs) GLI1 and GLI2 in iCCA. In addition, we evaluated the potential benefits of the combined inhibition of SMO and the DNA damage kinase WEE1. Transcriptomic analysis of 152 human iCCA samples showed increased expression of GLI1, GLI2, and Patched 1 (PTCH1) in tumor tissues compared with nontumor tissues. Genetic silencing of SMO, GLI1, and GLI2 inhibited the growth, survival, invasiveness, and self-renewal of iCCA cells. Pharmacologic inhibition of SMO reduced iCCA growth and viability in vitro, by inducing double-strand break DNA damage, leading to mitotic arrest and apoptotic cell death. Importantly, SMO inhibition resulted in the activation of the G2-M checkpoint and DNA damage kinase WEE1, increasing the vulnerability to WEE1 inhibition. Hence, the combination of MRT-92 with the WEE1 inhibitor AZD-1775 showed increased antitumor activity in vitro and in iCCA xenografts compared with single treatments. These data indicate that combined inhibition of SMO and WEE1 reduces tumor burden and may represent a strategy for the clinical development of novel therapeutic approaches in iCCA.

The role of potassium channels in tumours of the gastrointestinal tract: a focus on the human ether-à-go-go related gene 1 channels.

Potassium channels are often dysregulated in tumours of the gastrointestinal (GI) tract. Among them, the voltage-dependent channel KV 11.1, also known as human ether-à-go-go related gene 1 (hERG1), is frequently expressed in tumours and precancerous lesions of the GI tract. In precancerous lesions, hERG1 behaves as a progression factor, contributing to identifying those patients whose lesions can progress towards true cancers. In advanced cancers, such as colorectal and pancreatic cancer, a high hERG1 expression represents a negative prognostic factor, contributing to identifying high risk patients. The only exception is represented by neuroendocrine cancers of both the ileum and the pancreas, where hERG1 represents a positive prognostic factor for survival. In GI tumours, hERG1 can function either as a true channel, allowing outward potassium ion flux and membrane repolarisation, or in a non-canonical, non-conductive way. This occurs because, in cancer, hERG1 forms complexes with different plasma membrane and cytosolic proteins, instead of classical accessory subunits. In particular, hERG1 forms a complex with the ß1 subunit of integrin receptors: the hERG1-ß1 complex. Growth and chemokine receptors, small GTPases, phosphoinositide 3-kinase, as well as other ion transporters or channels, are also recruited in the hERG1-ß1 complex. The formation of multiprotein channel complexes represents an emerging mechanism allowing functional channel networking in both excitable and non-excitable cells. hERG1 represents a prototype of how multiprotein complexes operate in tumours, that is, giving rise to signalling hubs which can transmit and modulate signals arising from the tumour microenvironment, hence contributing to tumour progression and malignancy.

Locus Coeruleus Neurons' Firing Pattern Is Regulated by ERG Voltage-Gated K+ Channels.

Locus coeruleus (LC) neurons, with their extensive innervations throughout the brain, control a broad range of physiological processes. Several ion channels have been characterized in LC neurons that control intrinsic membrane properties and excitability. However, ERG (ether-à-go-go-related gene) K+ channels that are particularly important in setting neuronal firing rhythms and automaticity have not as yet been discovered in the LC. Moreover, the neurophysiological and pathophysiological roles of ERG channels in the brain remain unclear despite their expression in several structures. By performing immunohistochemical investigations, we found that ERG-1A, ERG-1B, ERG-2 and ERG-3 are highly expressed in the LC neurons of mice. To examine the functional role of ERG channels, current-clamp recordings were performed on mouse LC neurons in brain slices under visual control. ERG channel blockade by WAY-123,398, a class III anti-arrhythmic agent, increased the spontaneous firing activity and discharge irregularity of LC neurons. Here, we have shown the presence of distinct ERG channel subunits in the LC which play an imperative role in modulating neuronal discharge patterns. Thus, we propose that ERG channels are important players behind the changes in, and/or maintenance of, LC firing patterns that are implicated in the generation of different behaviors and in several disorders.

The Interaction between hERG1 and ß1 Integrins Modulates hERG1 Current in Different Pathological Cell Models.

Ion channels are implicated in various diseases, including cancer, in which they modulate different aspects of cancer progression. In particular, potassium channels are often aberrantly expressed in cancers, a major example being provided by hERG1. The latter is generally complexed with ß1 integrin in tumour cells, and such a molecular complex represents a new druggable hub. The present study focuses on the characterization of the functional consequences of the interaction between hERG1 and ß1 integrins on different substrates over time. To this purpose, we studied the interplay alteration on the plasma membrane through patch clamp techniques in a cellular model consisting of human embryonic kidney (HEK) cells stably transfected with hERG1 and in a cancer cell model consisting of SH-SY5Y neuroblastoma cells, endogenously expressing the channel. Cells were seeded on different substrates known to stimulate ß1 integrins, such as fibronectin (FN) for HEK-hERG1 and laminin (LMN) for SH-SY5Y. In HEK cells stably overexpressing hERG1, we observed a hERG1 current density increase accompanied by Vrest hyperpolarization after cell seeding onto FN. Notably, a similar behaviour was shown by SH-SY5Y neuroblastoma cells plated onto LMN. Interestingly, we did not observe this phenomenon when plating the cells on substrates such as Bovine Serum Albumin (BSA) or Polylysine (PL), thus suggesting a crucial involvement of ECM proteins as well as of ß1 integrin activation.

Prognostic role of hERG1 Potassium Channels in Neuroendocrine Tumours of the Ileum and Pancreas.

hERG1 potassium channels are widely expressed in human cancers of different origins, where they affect several key aspects of cellular behaviour. The present study was designed to evaluate the expression and clinical relevance of hERG1 protein in cancer tissues from patients suffering from neuroendocrine tumours (NETs) of ileal (iNETs) and pancreatic (pNETs) origin, with available clinicopathological history and follow-up. The study was carried out by immunohistochemistry with an anti-hERG1 monoclonal antibody. In a subset of samples, a different antibody directed against the hERG1/ß1 integrin complex was also used. The analysis showed for the first time that hERG1 is expressed in human NETs originating from either the ileum or the pancreas. hERG1 turned out to have a prognostic value in NETs, showing (i) a statistically significant positive impact on OS of patients affected by ileal NETs, regardless the TNM stage; (ii) a statistically significant positive impact on OS of patients affected by aggressive (TNM stage IV) disease, either ileal or pancreatic; (iii) a trend to a negative impact on OS of patients affected by less aggressive (TNM stage I-III) disease, either ileal or pancreatic. Moreover, in order to evaluate whether ERG1 was functionally expressed in a cellular model of pNET, the INS1E rat insulinoma cell line was used, and it emerged that blocking ERG1 with a specific inhibitor of the channel (E4031) turned out in a significant reduction in cell proliferation.

SHMT2-mediated mitochondrial serine metabolism drives 5-FU resistance by fueling nucleotide biosynthesis.

5-Fluorouracil (5-FU) is a key component of chemotherapy for colorectal cancer (CRC). 5-FU efficacy is established by intracellular levels of folate cofactors and DNA damage repair strategies. However, drug resistance still represents a major challenge. Here, we report that alterations in serine metabolism affect 5-FU sensitivity in in vitro and in vivo CRC models. In particular, 5-FU-resistant CRC cells display a strong serine dependency achieved either by upregulating endogenous serine synthesis or increasing exogenous serine uptake. Importantly, regardless of the serine feeder strategy, serine hydroxymethyltransferase-2 (SHMT2)-driven compartmentalization of one-carbon metabolism inside the mitochondria represents a specific adaptation of resistant cells to support purine biosynthesis and potentiate DNA damage response. Interfering with serine availability or affecting its mitochondrial metabolism revert 5-FU resistance. These data disclose a relevant mechanism of mitochondrial serine use supporting 5-FU resistance in CRC and provide perspectives for therapeutic approaches.

The Transcriptional Landscape of BRAF Wild Type Metastatic Melanoma: A Pilot Study.

Melanoma is a relatively rare disease worldwide; nevertheless, it has a great relevance in some countries, such as in Europe. In order to shed some light upon the transcriptional profile of skin melanoma, we compared the gene expression of six independent tumours (all progressed towards metastatic disease and with wild type BRAF) to the expression profile of non-dysplastic melanocytes (considered as a healthy control) in a pilot study. Paraffin-embedded samples were manually micro-dissected to obtain enriched samples, and then, RNA was extracted and analysed through a microarray-based approach. An exhaustive bioinformatics analysis was performed to identify differentially expressed transcripts between the two groups, as well as enriched functional terms. Overall, 50 up- and 19 downregulated transcripts were found to be significantly changed in the tumour compared to the control tissue. Among the upregulated transcripts, the majority belonged to the immune response group and to the proteasome, while most of the downregulated genes were related to cytosolic ribosomes. A Gene Set Enrichment Analysis (GSEA), along with the RNA-Seq data retrieved from the TCGA/GTEx databases, confirmed the general trend of downregulation affecting cytoribosome proteins. In contrast, transcripts coding for mitoribosome proteins showed the opposite trend.

hERG1 Potassium Channel Expression in Colorectal Adenomas: Comparison with Other Preneoplastic Lesions of the Gastrointestinal Tract.

Preneoplastic lesions represent a useful target for early diagnosis and follow-up of gastrointestinal malignancies. hERG1 channel expression was tested by immunohistochemistry (IHC) in a cohort of colorectal adenoma samples belonging to Italian subjects. Overall, hERG1 was expressed in 56.5% of cases with both high staining intensity and a high percentage of positive cells. Moreover, hERG1 was expressed in a higher percentage of dysplastic adenomas with respect to hyperplastic lesions, and the proportion of positive samples further increased in patients with high-grade dysplasia. Comparing hERG1 expression in other preneoplastic lesions of the GI tract (gastric dysplasia and Barrett's esophagus), it emerged that in all the conditions, hERG1 was expressed with a diffused pattern, throughout the cell, with variable staining intensity within the samples. The highest expression was detected in gastric dysplasia samples and the lowest in Barrett's esophagus at similar levels observed in colorectal adenomas. Our results show that hERG1 is aberrantly expressed in human preneoplastic lesions of the gastrointestinal tract and has a different pattern of expression and role in the different sites. Overall, the detection of hERG1 expression in preneoplastic lesions could represent a novel diagnostic or prognostic marker of progression in the gastrointestinal tract.

Dynamics and physiological meaning of complexes between ion channels and integrin receptors: the case of Kv11.1.

The cellular functions are regulated by a complex interplay of diffuse and local signals. Studying the latter is challenging, but experimental work in cell physiology has led to recognize that understanding a cell's dynamics requires a deep comprehension of local fluctuations of cytosolic regulators. Macromolecular complexes are major determinants of local signaling. Multienzyme assemblies limit the diffusion restriction to reaction kinetics by direct exchange of metabolites. Likewise, close coupling of ion channels and transporters modulates the ion concentration around a channel mouth or transporter binding site. Extreme signal locality is brought about by conformational coupling between membrane proteins, as is typical of mechanotransduction. A paradigmatic case is integrin-mediated cell adhesion. Sensing the extracellular microenvironment and providing an appropriate response are essential in growth and development and have innumerable pathological implications. The process involves bidirectional signal transduction by complex supramolecular structures that link integrin receptors to ion channels and transporters, growth factor receptors, cytoskeletal elements, and other regulatory elements. The dynamics of such complexes are only beginning to be understood. A thoroughly studied example is the association between integrin receptors and the voltage-gated K+ channels Kv11.1. These channels are widely expressed in early embryos, where their physiological roles are poorly understood and apparently different from the shaping of action potential firing in the adult. Hints about these roles come from studies in cancer cells, where Kv11.1 is often overexpressed and appears to reassume functions it presumably exerts during embryogenesis, such as controlling cell proliferation/differentiation, apoptosis, and migration. Kv11.1 is implicated in these processes through its linking to integrin subunits, which in turn regulates channel expression. Specific cellular functions, such as proliferation and migration, appear to be modulated by distinct conformational states of the channel (e.g., open and closed), whose balance is affected by the link with integrin subunits.

A unique circulating miRNA profile highlights thrombo-inflammation in Behçet's syndrome.

OBJECTIVES: Behçet's syndrome (BS) is a rare systemic vasculitis often complicated by thrombotic events. Given the lack of validated biomarkers, BS diagnosis relies on clinical criteria.In search of novel biomarkers for BS diagnosis, we determined the profile of plasmatic circulating microRNAs (ci-miRNAs) in patients with BS compared with healthy controls (HCs). METHODS: ci-miRNA profile was evaluated by microarray in a screening cohort (16 patients with BS and 18 HCs) and then validated by poly(T) adaptor PCR (PTA-PCR) in a validation cohort (30 patients with BS and 30 HCs). Two disease control groups (30 patients with systemic lupus erythematosus (SLE) and 30 patients with giant cell arteritis (GCA) were also analysed. RESULTS: From the microarray screening, 29 deregulated (differentially expressed (DE)) human ci-miRNAs emerged. A hierarchical cluster analysis indicated that DE ci-miRNAs clearly segregated patients from controls, independently of clinical features. PTA-PCR analysis on the validation cohort confirmed the deregulation of miR-224-5p, miR-206 and miR-653-5p. The combined receiver operating characteristic (ROC) curve analyses showed that such ci-miRNAs discriminate BS from HCs (and BS with active vs inactive disease), as well as BS from patients with SLE and GCA.The functional annotation analyses (FAAs) showed that the most enriched pathways affected by DE ci-miRNAs (ie, cell-matrix interaction, oxidative stress and blood coagulation) are related to thrombo-inflammatory mechanisms. Accordingly, the expression of the three ci-miRNAs from the validation cohort significantly correlated with leucocyte reactive oxygen species production and plasma lipid peroxidation. CONCLUSIONS: The ci-miRNA profile identified in this study may represent a novel, poorly invasive BS biomarker, while suggesting an epigenetic control of BS-related thrombo-inflammation.

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.