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Tania Mariastella Caputo,1,* Angela Maria Cusano,2,* Sofia Principe,1 Paola Cicatiello,1 Giorgia Celetti,1 Anna Aliberti,1 Alberto Micco,2 Menotti Ruvo,3 Maria Tagliamonte,4 Concetta Ragone,4 Michele Minopoli,5 Maria Vincenza Carriero,5 Luigi Buonaguro,4 Andrea Cusano1,2 1Optoelectronics Group, Department of Engineering, University of Sannio, Palazzo Dell’ Aquila Bosco Lucarelli, Benevento, Italy; 2CeRICTscrl Regional Center Information Communication Technology, Palazzo Ex Poste, Benevento, Italy; 3Institute of Biostructure and Bioimaging, National Research Council, Naples, Italy; 4Innovative Immunological Models Unit, Istituto Nazionale Tumori - IRCCS - “Fond G. Pascale”, Naples, Italy; 5Neoplastic Progression Unit, Istituto Nazionale Tumori IRCCS “Fondazione G. Pascale”, Naples, Italy*These authors contributed equally to this workCorrespondence: Andrea Cusano; Anna Aliberti, Palazzo Dell’Aquila Bosco Lucarelli, Corso Garibaldi 107, Benevento, I-82100, Italy, Email a.cusano@unisannio.it; anna.aliberti@unisannio.itIntroduction: Currently, conventional treatments of hepatocellular carcinoma (HCC) are not selective enough for tumor tissue and lead to multidrug resistance and drug toxicity. Although sorafenib (SOR) is the standard first-line systemic therapy approved for the clinical treatment of HCC, its poor aqueous solubility and rapid clearance result in low absorption efficiency and severely limit its use for local treatment.Methods: Herein, we present the synthesis of biodegradable polymeric Poly (D, L-Lactide-co-glycolide) (PLGA) particles loaded with SOR (PS) by emulsion-solvent evaporation process. The particles are carefully characterized focusing on particle size, surface charge, morphology, drug loading content, encapsulation efficiency, in vitro stability, drug release behaviour and tested on HepG2 cells. Additionally, PLGA particles have been coupled on side emitting optical fibers (seOF) integrated in a microfluidic device for light-triggered local release.Results: PS have a size of 248 nm, tunable surface charge and a uniform and spherical shape without aggregation. PS shows encapsulation efficiency of 89.7% and the highest drug loading (8.9%) between the SOR-loaded PLGA formulations. Treating HepG2 cells with PS containing SOR at 7.5 μM their viability is dampened to 40%, 30% and 17% after 48, 129 and 168 hours of incubation, respectively.Conclusion: The high PS stability, their sustained release profile and the rapid cellular uptake corroborate the enhanced cytotoxicity effect on HepG2. With the prospect of developing biomedical tools to control the spatial and temporal release of drugs, we successfully demonstrated the potentiality of seOF for light-triggered local release of the carriers. Our prototypical system paves the way to new devices integrating microfluidics, optical fibers, and advanced carriers capable to deliver minimally invasive locoregional cancer treatments.Graphical Abstract: Keywords: poly(lactic-co-glycolic acid), sorafenib, emulsion solvent evaporation technique, hepatocellular carcinoma, optical fiber, microfluidics

Objective: To evaluate the relative importance of ethnicity and socioeconomic deprivation in determining the likelihood and prevalence of placentally derived composite of adverse pregnancy outcomes (CAPO) and composite of severe adverse pregnancy outcomes (CAPO‐S). Methods: This was a single‐center retrospective cohort study of data obtained in a tertiary maternity unit. Data regarding ethnicity and socioeconomic deprivation (as measured with indices of multiple deprivation) were collected for 13 165 singleton pregnancies screened routinely in the first trimester for pre‐eclampsia using the Fetal Medicine Foundation combined risk‐assessment algorithm. CAPO was defined as the presence of one or more interrelated outcomes associated with placental dysfunction, namely, hypertensive disorders of pregnancy, preterm birth, birth weight ≤ 10th centile and stillbirth. CAPO‐S was defined as the presence of one or more of the following: hypertensive disorders of pregnancy at < 37 + 0 weeks, preterm birth at < 34 + 0 weeks, birth weight ≤ 5th centile and stillbirth at < 37 + 0 weeks. Results: The prevalence of CAPO was 16.3% in white women, 29.3% in black women and 29.3% in South Asian women. However, half (51.7%) of all CAPO cases occurred in white women. There was a strong interaction between ethnicity and socioeconomic deprivation, with a correlation coefficient of −0.223. Both ethnicity and socioeconomic deprivation influenced the prevalence of CAPO and CAPO‐S, with the contribution of ethnicity being the strongest. Conclusions: Black and Asian ethnicity, as well as socioeconomic deprivation, influence the prevalence of placenta‐mediated adverse pregnancy outcomes. Despite this, most adverse pregnancy outcomes occur in white women, who represent the majority of the population and are also affected by socioeconomic deprivation. For these reasons, inclusion of socioeconomic deprivation should be considered in early pregnancy risk assessment for placenta‐mediated CAPO. © 2024 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology. This article's abstract has been translated into Spanish and Chinese. Follow the links from the abstract to view the translations. [ABSTRACT FROM AUTHOR]

Objective: Antenatal growth assessment using ultrasound aims to identify small fetuses that are at higher risk of perinatal morbidity and mortality. This study explored whether the association between suboptimal fetal growth and adverse perinatal outcome varies with different definitions of fetal growth restriction (FGR) and different weight charts/standards. Methods: This was a retrospective cohort study of 17 261 singleton non‐anomalous pregnancies at ≥ 24 + 0 weeks' gestation that underwent routine ultrasound at a tertiary referral hospital. Estimated fetal weight (EFW) and Doppler indices were converted into percentiles using a reference standard (INTERGROWTH‐21st (IG‐21)) and various reference charts (Hadlock, Fetal Medicine Foundation (FMF) and Swedish). Test characteristics were assessed using the consensus definition, Society for Maternal–Fetal Medicine (SMFM) definition and Swedish criteria for FGR. Adverse perinatal outcome was defined as perinatal death, admission to the neonatal intensive care unit at term, 5‐min Apgar score < 7 and therapeutic cooling for neonatal encephalopathy. The association between FGR according to each definition and adverse perinatal outcome was compared. Multivariate logistic regression analysis was used to test the strength of association between ultrasound parameters and adverse perinatal outcome. Ultrasound parameters were also tested for correlation. Results: IG‐21, Hadlock and FMF fetal size references classified as growth‐restricted 1.5%, 3.6% and 4.6% of fetuses, respectively, using the consensus definition and 2.9%, 8.8% and 10.6% of fetuses, respectively, using the SMFM definition. The sensitivity of the definition/chart combinations for adverse perinatal outcome varied from 4.4% (consensus definition with IG‐21 charts) to 13.2% (SMFM definition with FMF charts). Specificity varied from 89.4% (SMFM definition with FMF charts) to 98.6% (consensus definition with IG‐21 charts). The consensus definition and Swedish criteria showed the highest specificity, positive predictive value and positive likelihood ratio in detecting adverse outcome, irrespective of the reference chart/standard used. Conversely, the SMFM definition had the highest sensitivity across all investigated growth charts. Low EFW, abnormal mean uterine artery pulsatility index (UtA‐PI) and abnormal cerebroplacental ratio were significantly associated with adverse perinatal outcome and there was a positive correlation between the covariates. Multivariate logistic regression showed that UtA‐PI > 95th percentile and EFW < 5th percentile were the only parameters consistently associated with adverse outcome, irrespective of the definitions or fetal growth chart/standard used. Conclusions: The apparent prevalence of FGR varies according to the definition and fetal size reference chart/standard used. Irrespective of the method of classification, the sensitivity for the identification of adverse perinatal outcome remains low. EFW, UtA‐PI and fetal Doppler parameters are significant predictors of adverse perinatal outcome. As these indices are correlated with one other, a prediction algorithm is advocated to overcome the limitations of using these parameters in isolation. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology. [ABSTRACT FROM AUTHOR]

This article discusses a study that aims to develop an artificial intelligence (AI) algorithm for the automatic classification of ultrasound images of the fetal brain in the first trimester. The algorithm focuses on identifying abnormal sonographic findings of the posterior cranial fossa (PCF), which can be early markers of open spina bifida or Dandy Walker malformation. The study used a dataset of 251 images and achieved promising results, demonstrating the potential for the AI algorithm to support clinicians in detecting major central nervous system anomalies during early pregnancy. The next phase of the study will assess the algorithm's clinical applicability in routine practice. [Extracted from the article]

Objective: To describe the incidence, clinical features and perinatal outcome of late-onset fetal growth restriction (FGR) associated with genetic syndrome or aneuploidy, structural malformation or congenital infection.Methods: This was a retrospective multicenter cohort study of patients who attended one of four tertiary maternity hospitals in Italy. We included consecutive singleton pregnancies between 32 + 0 and 36 + 6 weeks' gestation with either fetal abdominal circumference (AC) or estimated fetal weight < 10th percentile for gestational age or a reduction in AC of > 50 percentiles from the measurement at an ultrasound scan performed between 18 and 32 weeks. The study group consisted of pregnancies with late-onset FGR and a genetic syndrome or aneuploidy, structural malformation or congenital infection (anomalous late-onset FGR). The presence of congenital anomalies was ascertained postnatally in neonates with abnormal findings on antenatal investigation or detected after birth. The control group consisted of pregnancies with structurally and genetically normal fetuses with late-onset FGR. Composite adverse perinatal outcome was defined as the presence of at least one of stillbirth, 5-min Apgar score < 7, admission to the neonatal intensive care unit (NICU), need for respiratory support at birth, neonatal jaundice and neonatal hypoglycemia. The primary aims of the study were to assess the incidence and clinical features of anomalous late-onset FGR, and to compare the perinatal outcome of such cases with that of fetuses with non-anomalous late-onset FGR.Results: Overall, 1246 pregnancies complicated by late-onset FGR were included in the study, of which 120 (9.6%) were allocated to the anomalous late-onset FGR group. Of these, 11 (9.2%) had a genetic syndrome or aneuploidy, 105 (87.5%) had an isolated structural malformation, and four (3.3%) had a congenital infection. The most frequent structural defects associated with late-onset anomalous FGR were genitourinary malformations (28/105 (26.7%)) and limb malformation (21/105 (20.0%)). Compared with the non-anomalous late-onset FGR group, fetuses with anomalous late-onset FGR had an increased incidence of composite adverse perinatal outcome (35.9% vs 58.3%; P < 0.01). Newborns with anomalous, compared to those with non-anomalous, late-onset FGR showed a higher frequency of need for respiratory support at birth (25.8% vs 9.0%; P < 0.01), intubation (10.0% vs 1.1%; P < 0.01), NICU admission (43.3% vs 22.6%; P < 0.01) and longer hospital stay (median, 24 days (range, 4-250 days) vs 11 days (range, 2-59 days); P < 0.01).Conclusions: Most pregnancies complicated by anomalous late-onset FGR have structural malformations rather than genetic abnormality or infection. Fetuses with anomalous late-onset FGR have an increased incidence of complications at birth and NICU admission and a longer hospital stay compared with fetuses with isolated late-onset FGR. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the relationship between Doppler and biometric ultrasound parameters measured at diagnosis and perinatal adverse outcome in a cohort of late-onset growth-restricted (FGR) fetuses.Methods: This was a multicenter retrospective study of data obtained between 2014 and 2019 including non-anomalous singleton pregnancies complicated by late-onset FGR (≥ 32 weeks), which was defined either as abdominal circumference (AC) or estimated fetal weight (EFW) < 10th percentile for gestational age or as reduction of the longitudinal growth of AC by over 50 percentiles compared to ultrasound scan performed between 18 and 32 weeks of gestation. We evaluated the association between sonographic findings at diagnosis of FGR and composite adverse perinatal outcome (CAPO), defined as stillbirth or at least two of the following: obstetric intervention due to intrapartum fetal distress, neonatal acidemia, birth weight < 3rd percentile and transfer to the neonatal intensive care unit (NICU).Results: Overall, 468 cases with complete biometric and umbilical, fetal middle cerebral and uterine artery (UtA) Doppler data were included, of which 53 (11.3%) had CAPO. On logistic regression analysis, only EFW percentile was associated independently with CAPO (P = 0.01) and NICU admission (P < 0.01), while the mean UtA pulsatility index (PI) multiples of the median (MoM) > 95th percentile at diagnosis was associated independently with obstetric intervention due to intrapartum fetal distress (P = 0.01). The model including baseline pregnancy characteristics and the EFW percentile was associated with an area under the receiver-operating-characteristics curve of 0.889 (95% CI, 0.813-0.966) for CAPO (P < 0.001). A cut-off value for EFW corresponding to the 3.95th percentile was found to discriminate between cases with and those without CAPO, yielding a sensitivity of 58.5% (95% CI, 44.1-71.9%), specificity of 69.6% (95% CI, 65.0-74.0%), positive predictive value of 19.8% (95% CI, 13.8-26.8%) and negative predictive value of 92.9% (95% CI, 89.5-95.5%).Conclusions: Retrospective data from a large cohort of late-onset FGR fetuses showed that EFW at diagnosis is the only sonographic parameter associated independently with the occurrence of CAPO, while mean UtA-PI MoM > 95th percentile at diagnosis is associated independently with intrapartum distress leading to obstetric intervention. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology. [ABSTRACT FROM AUTHOR]

The perinatal outcomes of SGA neonates were compared between the SGA fetuses from pregnancies complicated by GDM/DM and those from pregnancies not complicated by GDM/DM. To describe the perinatal outcomes of small-for-gestational-age (SGA) neonates from pregnancies complicated by gestational diabetes (GDM) and diabetes mellitus (DM). [Extracted from the article]

OP05.04: Expert neurosonography versus magnetic resonance imaging: comparison of techniques in detecting cortical abnormalities in a high-risk population A non-consecutive series of cases with a diagnosis of central nervous system (CNS) abnormality at antenatal NSG and MRI, were included in the study. To compare the accuracy of neurosonography (NSG) and magnetic resonance imaging (MRI) in the assessment of the fetal cortex in a high-risk cohort of fetuses. [Extracted from the article]

To develop an artificial intelligence-based algorithm toward the automatic classification of Anterior/Posterior/Transverse Occiput position images acquired by means of transperineal sonography. In our approach, two Convolutional Neural Networks (CNNs) were independently trained to classify Anterior/non-Anterior (CNN SB A/nA sb ) and Posterior/Transverse (CNN SB P/T sb ), respectively. [Extracted from the article]

To evaluate the relationship between fetal Doppler parameters and functional echocardiography in fetuses at risk for late-onset fetal growth restriction (FGR). Conclusions Functional echocardiographic findings on a selected population of fetuses at risk for late-onset FGR and birthweight < 10th percentile demonstrate a relationship between the UA Doppler and left ventricular strain and EF. At sub-analysis performed according to the birthweight, fetuses with birthweight 3 SP rd sp -10 SP th sp percentile and abnormal UA Doppler showed lower LV global longitudinal strain (-18.6 versus -21.9, p = 0.01), LV global radial strain (-31.1 versus -47.5, p = 0.003), LV global circumferential strain (-24.4 versus -31.6, p = 0.03) and EF (52.5 versus 60.2, p = 0.004) compared to those with normal UA Doppler. [Extracted from the article]

Recent data from our group showed that in protracted active phase of labour OA fetuses submitted to Caesarean delivery (CD) appeared to pool in two clusters, one characterised by deflexed head and high station and another featured by high station and no obvious sign of malposition nor malpresentation, as it occurred in the reported case, and OSA/AoP ratio > 1.19 and a OSA/HPD ratio <2.72 showed a 100% and 77.8% sensitivity, respectively, for CD for labour dystocia. On examination head position was left occiput anterior (LOA) and the head station -2 as confirmed by a narrow angle-of progression (AoP) measuring 96.3° (figure 1A) and a long HPD measuring 6.2 cm (figure 1B); caput and moulding were also noted (figure 1C). A 35-year-old nullipara at 41 weeks of gestation was admitted in the active phase of the first stage of labour. [Extracted from the article]

To evaluate whether the cervical sliding sign (CSS) can predict the outcome of induction of labour (IoL). Similar figures have been found for multiparous patients, who showed higher rate of D24 (86,8% vs 17.1%, p = 0,01) and shorter Latency (11 ±7.6 vs 16.7 ±9.1 hours, p < 0.01) and TtD (13.8 ±8.7 vs 19.6 ±9.6 hours, p < 0.001) compared to parous women with no CSS. In nulliparous patients the presence of CSS was associated with an increased frequency of D24 (52.4% vs 24.6%, p < 0.01) and shorter latency (30 ±10.7 vs 20.1 ±10.8 hours, p < 0.01) and TtD (24.9±12.1 vs 30 ±10.7, p = 0.01) compared to nulliparae with no CSS. [Extracted from the article]

Conclusions The perinatal survival and outcome is not dissimilar between anomalous and non-anomalous FGR diagnosed between 32 SP +0 sp -36 SP +6 sp weeks of gestation. To investigate the etiology and perinatal outcome of suspected fetal growth restriction (FGR) diagnosed between 32 SP +0 sp -36 SP +6 sp weeks and associated with structural defects, genetic anomalies or congenital infections. [Extracted from the article]

Conclusions Within a population with suspected FGR between 32 SP +0 sp -36 SP +6 sp weeks, approximately one third of the fetuses fulfil the Delphi criteria for late onset FGR. OC04.10: Incidence and perinatal outcome of late onset fetal growth restriction (FGR) identified within a population of fetuses with suspected FGR diagnosed beyond 32 weeks To evaluate the incidence and the perinatal outcome of late onset fetal growth restriction (FGR) defined according to the Delphi criteria within a population of fetuses with suspected FGR diagnosed 32 SP +0 sp -36 SP +6 sp weeks. [Extracted from the article]

Chondrosarcoma (ChS), a malignant cartilage-producing tumor, is the second most frequently diagnosed osseous sarcoma after osteosarcoma. It represents a very heterogeneous group of malignant chemo- and radiation-resistant neoplasms, accounting for approximately 20% of all bone sarcomas. The majority of ChS patients have a good prognosis after a complete surgical resection, as these tumors grow slowly and rarely metastasize. Conversely, patients with inoperable disease, due to the tumor location, size, or metastases, represent a great clinical challenge. Despite several genetic and epigenetic alterations that have been described in distinct ChS subtypes, very few therapeutic options are currently available for ChS patients. Therefore, new prognostic factors for tumor progression as well as new treatment options have to be explored, especially for patients with unresectable or metastatic disease. Recent studies have shown that a correlation between immune infiltrate composition, tumor aggressiveness, and survival does exist in ChS patients. In addition, the intra-tumor microvessel density has been proven to be associated with aggressive clinical behavior and a high metastatic potential in ChS. This review will provide an insight into the ChS microenvironment, since immunotherapy and antiangiogenic agents are emerging as interesting therapeutic options for ChS patients.

Objective: Uteroplacental dysfunction may not only result in pre-eclampsia (PE) but also in preterm birth (PTB), small-for-gestational-age (SGA) birth and stillbirth. The aim of this study is to evaluate the positive predictive value (PPV) of first-trimester combined PE screening for all of these placenta-mediated adverse pregnancy outcomes., Design: Retrospective cohort study., Setting: Tertiary referral maternity unit., Sample: A total of 13 211 singleton pregnancies., Methods: First-trimester combined screening for preterm PE using the Fetal Medicine Foundation (FMF) algorithm., Main Outcomes Measures: Hypertensive disorders of pregnancy (HDP), PTB, SGA birth and stillbirth were combined to assess composite adverse and severe adverse pregnancy outcomes (CAPO and CAPO-S). The PPVs for CAPO and CAPO-S were calculated for women with a combined risk for preterm PE of ≥1 in 50 and ≥1 in 100., Results: First-trimester combined screening identified 2215 women (16.8%) with a risk of ≥1 in 100 for preterm PE. The PPVs for a risk of ≥1 in 100 for CAPO and CAPO-S were 38.8% and 18.2%, respectively. The equivalent PPVs for a risk of ≥1 in 50 were 45.1% and 21.1%, respectively., Conclusions: Women identified at high risk of preterm PE are also at increased risk of other placenta-mediated adverse pregnancy outcomes, such as PTB, SGA birth and stillbirth. Women at high risk for preterm PE after first-trimester screening may benefit from a higher surveillance care pathway, with interventions to mitigate all the adverse outcomes associated with placental dysfunction., (© 2023 The Authors. BJOG: An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd.)

Background: BRAF-mutant melanoma patients benefit from the combinatorial treatments with BRAF and MEK inhibitors. However, acquired drug resistance strongly limits the efficacy of these targeted therapies in time. Recently, many findings have underscored the involvement of microRNAs as main drivers of drug resistance. In this context, we previously identified a subset of oncomiRs strongly up-regulated in drug-resistant melanomas. In this work, we shed light on the molecular role of two as yet poorly characterized oncomiRs, miR-4443 and miR-4488., Methods: Invasion and migration have been determined by wound healing, transwell migration/invasion assays and Real Time Cell Analysis (RTCA) technology. miR-4488 and miR-4443 have been measured by qRT-PCR. Nestin levels have been tested by western blot, confocal immunofluorescence, immunohistochemical and flow cytometry analyses., Results: We demonstrate that the two oncomiRs are responsible for the enhanced migratory and invasive phenotypes, that are a hallmark of drug resistant melanoma cells. Moreover, miR-4443 and miR-4488 promote an aberrant cytoskeletal reorganization witnessed by the increased number of stress fibers and cellular protrusions-like cancer cell invadopodia. Mechanistically, we identified the intermediate filament nestin as a molecular target of both oncomiRs. Finally, we have shown that nestin levels are able to predict response to treatments in melanoma patients., Conclusions: Altogether these findings have profound translational implications in the attempt i) to develop miRNA-targeting therapies to mitigate the metastatic phenotypes of BRAF-mutant melanomas and ii) to identify novel biomarkers able to guide clinical decisions., (© 2023. The Author(s).)

Objective: To evaluate whether routine mid-gestational uterine artery Doppler (UtAD) modifies the risk for preterm pre-eclampsia after first-trimester combined pre-eclampsia screening., Design: Retrospective cohort study., Setting: London Tertiary Hospital., Population: A cohort of 7793 women with singleton pregnancies, first-trimester pre-eclampsia screening using the Fetal Medicine Foundation (FMF) algorithm and UtAD pulsatility index (PI) assessment at the mid-gestation ultrasound., Methods: Pregnancies were divided into four groups: high risk in both trimesters (H 1 H 2 ), high risk in the first but not in the second trimester (H 1 L 2 ), low risk in the first but high risk in the second trimester (L 1 H 2 ) and low risk in both trimesters (L 1 L 2 )., Main Outcome Measures: Small for gestational age (SGA), hypertensive disorders of pregnancy (HDP) and stillbirth., Results: In this cohort, 600 (7.7%) and 620 (7.9%) women were designated as being at high risk in the first and second trimesters, respectively. Preterm pre-eclampsia was more prevalent in the H 1 L 2 group (4.5%) than in women considered at low risk in the first trimester (0.4%, p < 0.0001). The prevalence of preterm pre-eclampsia in the L 1 H 2 group (3.3%) was significantly lower than that in women considered at high risk in the first trimester (7.0%, p = 0.0076), and was higher than that observed in the L 1 L 2 group (0.2%, p < 0.0001). The prevalence of SGA and term HDP followed similar trends., Conclusions: Pre-eclampsia risk after first-trimester FMF pre-eclampsia screening may be stratified through mid-gestational routine UtAD assessment. Pregnancy care should not be de-escalated for low mid-gestational UtAD resistance in women classified as being at high risk in the first trimester. The escalation of care may be justified in women at low risk but with high mid-gestational UtAD resistance., (© 2023 The Authors. BJOG: An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd.)

Gastrointestinal stromal tumors (GISTs) are the most common malignant mesenchymal neoplasms of the gastrointestinal tract. The gold standard for the diagnosis of GISTs is morphologic analysis with an immunohistochemical evaluation plus genomic profiling to assess the mutational status of lesions. The majority of GISTs are driven by gain-of-function mutations in the proto-oncogene c- KIT encoding the tyrosine kinase receptor (TKR) known as KIT and in the platelet-derived growth factor-alpha receptor ( PDGFRA ) genes. Approved therapeutics are orally available as tyrosine kinase inhibitors (TKIs) targeting KIT and/or PDGFRA oncogenic activation. Among these, imatinib has changed the management of patients with unresectable or metastatic GISTs, improving their survival time and delaying disease progression. Nevertheless, the majority of patients with GISTs experience disease progression after 2-3 years of imatinib therapy due to the development of secondary KIT mutations. Today, based on the identification of new driving oncogenic mutations, targeted therapy and precision medicine are regarded as the new frontiers for GISTs. This article reviews the most important mutations in GISTs and highlights their importance in the current understanding and treatment options of GISTs, with an emphasis on the most recent clinical trials.

Pre-eclampsia and fetal growth restriction (FGR) have been long related to primary placental dysfunction, caused by abnormal trophoblast invasion. Nevertheless, emerging evidence has led to a new hypothesis for the origin of pre-eclampsia and FGR. Suboptimal maternal cardiovascular adaptation has been shown to result in uteroplacental hypoperfusion, ultimately leading to placental hypoxic damage with secondary dysfunction. In this review, we summarize current evidence on maternal cardiac hemodynamics in FGR and pre-eclampsia. We also discuss the different approaches for antihypertensive treatment according to the hemodynamic phenotype observed in pre-eclampsia and FGR., (© 2022 Wiley Periodicals LLC.)

The expression level of the tumor suppressor p53 is controlled by the E3 ubiquitin ligase MDM2 with a regulatory feedback loop, which allows p53 to upregulate its inhibitor MDM2. In this manuscript we demonstrated that p90RSK binds and phosphorylates MDM2 on serine 166 both in vitro and in vivo by kinase assay, immunoblot, and co-immunoprecipitation assay; this phosphorylation increases the stability of MDM2 which in turn binds p53, ubiquitinating it and promoting its degradation by proteasome. A pharmacological inhibitor of p90RSK, BI-D1870, decreases MDM2 phosphorylation, and restores p53 function, which in turn transcriptionally increases the expression of cell cycle inhibitor p21 and of pro-apoptotic protein Bax and downregulates the anti-apoptotic protein Bcl-2, causing a block of cell proliferation, measured by a BrdU assay and growth curve, and promoting apoptosis, measured by a TUNEL assay. Finally, an immunohistochemistry evaluation of primary thyroid tumors, in which p90RSK is very active, confirms MDM2 stabilization mediated by p90RSK phosphorylation.

Background: Pancreatic ductal adenocarcinoma (PDAC) represents an unmet clinical need due to the very poor prognosis and the lack of effective therapy. Here we investigated the potential of domatinostat (4SC-202), a new class I histone deacetylase (HDAC) inhibitor, currently in clinical development, to sensitize PDAC to first line standard gemcitabine (G)/taxol (T) doublet chemotherapy treatment., Methods: Synergistic anti-tumor effect of the combined treatment was assessed in PANC1, ASPC1 and PANC28 PDAC cell lines in vitro as well as on tumor spheroids and microtissues, by evaluating combination index (CI), apoptosis, clonogenic capability. The data were confirmed in vivo xenograft models of PANC28 and PANC1 cells in athymic mice. Cancer stem cells (CSC) targeting was studied by mRNA and protein expression of CSC markers, by limiting dilution assay, and by flow cytometric and immunofluorescent evaluation of CSC mitochondrial and cellular oxidative stress. Mechanistic role of forkhead box M1 (FOXM1) and downstream targets was evaluated in FOXM1-overexpressing PDAC cells., Results: We showed that domatinostat sensitized in vitro and in vivo models of PDAC to chemotherapeutics commonly used in PDAC patients management and particularly to GT doublet, by targeting CSC compartment through the induction of mitochondrial and cellular oxidative stress. Mechanistically, we showed that domatinostat hampers the expression and function of FOXM1, a transcription factor playing a crucial role in stemness, oxidative stress modulation and DNA repair. Domatinostat reduced FOXM1 protein levels by downregulating mRNA expression and inducing proteasome-mediated protein degradation thus preventing nuclear translocation correlated with a reduction of FOXM1 target genes. Furthermore, by overexpressing FOXM1 in PDAC cells we significantly reduced domatinostat-inducing oxidative mitochondrial and cellular stress and abolished GT sensitization, both in adherent and spheroid cells, confirming FOXM1 crucial role in the mechanisms described. Finally, we found a correlation of FOXM1 expression with poor progression free survival in PDAC chemotherapy-treated patients., Conclusions: Overall, we suggest a novel therapeutic strategy based on domatinostat to improve efficacy and to overcome resistance of commonly used chemotherapeutics in PDAC that warrant further clinical evaluation., (© 2022. The Author(s).)

Several studies have ascertained that uPA and uPAR do participate in tumor progression and metastasis and are involved in cell adhesion, migration, invasion and survival, as well as angiogenesis. Increased levels of uPA and uPAR in tumor tissues, stroma and biological fluids correlate with adverse clinic-pathologic features and poor patient outcomes. After binding to uPAR, uPA activates plasminogen to plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme able to facilitate tumor cell invasion and dissemination to distant sites. Moreover, uPAR activated by uPA regulates most cancer cell activities by interacting with a broad range of cell membrane receptors. These findings make uPA and uPAR not only promising diagnostic and prognostic markers but also attractive targets for developing anticancer therapies. In this review, we debate the uPA/uPAR structure-function relationship as well as give an update on the molecules that interfere with or inhibit uPA/uPAR functions. Additionally, the possible clinical development of these compounds is discussed.

Myxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma and has tendency to metastasize to soft tissues. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and new therapeutic strategies that improve patients' outcomes are expected. In this study, we analyzed by immunohistochemistry the immune cellular components and microvessel density in tumor tissues from patients affected by MLPS. In order to evaluate the effects of primary human MLPS cells on macrophage polarization and, in turn, the ability of macrophages to influence invasiveness of MLPS cells, non-contact and 3D organotypic co-cultures were set up. High grade MLPS tissues were found heavily vascularized, exhibited a CD3, CD4, and CD8 positive T lymphocyte-poor phenotype and were massively infiltrated by CD163 positive M2-like macrophages. Conversely, low grade MLPS tissues were infiltrated by a discrete amount of CD3, CD4, and CD8 positive T lymphocytes and a scarce amount of CD163 positive macrophages. Kaplan-Meier analysis revealed a shorter Progression Free Survival in MLPS patients whose tumor tissues were highly vascularized and heavily infiltrated by CD163 positive macrophages, indicating a clear-cut link between M2-like macrophage abundance and poor prognosis in patients. Moreover, we documented that, in co-culture, soluble factors produced by primary human MLPS cells induce macrophage polarization toward an M2-like phenotype which, in turn, increases MLPS cell capability to spread into extracellular matrix and to cross endothelial monolayers. The identification of M2-like polarization factors secreted by MLPS cells may allow to develop novel targeted therapies counteracting MLPS progression.

Objectives: To assess whether the additional training with transabdominal ultrasound may improve the accuracy of the transvaginal digital examination in the assessment of the fetal head position during the active stage of labor., Methods: Prospective observational study involving 2 residents in their 1 st year of training in Obstetrics with no prior experience in neither transvaginal digital examination nor ultrasound. Women with term, cephalic presenting fetus and active labor with cervical dilation ≥ 8 cm and ruptured membranes were included. In the preliminary phase of the study, the resident A ("blinded") was assigned to assess the fetal head position by transvaginal digital examination, while the resident B ("unmasked") performed transvaginal digital examination following transabdominal ultrasound, which was considered to be the gold standard to determine the fetal head position. After 50 examinations independently performed by each resident in the training phase, a post-training phase of the study was carried out to compare the accuracy of each resident in the diagnosis of fetal head position by digital assessment. The occiput position was eventually confirmed by ultrasound performed by the main investigator., Results: Over a 6 months period, 90 post-training vaginal examinations were performed by each resident. The number of incorrect diagnoses of head position was higher for the "blinded" resident compared with the "unmasked" resident subjected to the ultrasound training (28/90 or 31.1 % vs 15/90 or 16.7 % p = 0.02). For both residents a wrong diagnosis was more likely with non-OA vs OA fetuses but this difference was statistically significant for the "blinded" Resident (10/20 or 50 % vs 18/70 or 25.7 % p = 0.039) but not for the "unmasked" Resident (5/18 or 27.9 % vs 10/72 or 13.9 % p = 0.16)., Conclusion: The addition of transabdominal ultrasound as a training tool in the determination of the fetal head position during labor seems to improve the accuracy of the transvaginal digital examination in unexperienced residents., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Neutrophil Extracellular Traps (NETs) are net-like structures composed of DNA-histone complexes and proteins released by activated neutrophils. In addition to their key role in the neutrophil innate immune response, NETs are also involved in autoimmune diseases, like systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and in other non-infectious pathological processes, as coagulation disorders, thrombosis, diabetes, atherosclerosis, vasculitis, and cancer. Recently, a large body of evidence indicates that NETs are involved in cancer progression and metastatic dissemination, both in animal models and cancer patients. Interestingly, a close correlation between cancer cell recruitment of neutrophils in the tumor microenvironment (Tumor Associated Neutrophils. TANs) and NET formation has been also observed either in primary tumors and metastatic sites. Moreover, NETs can also catch circulating cancer cells and promote metastasis. Furthermore, it has been reported that wake dormant cancer cells, causing tumor relapse and metastasis. This review will primarily focus on the pro-tumorigenic activity of NETs in tumors highlighting their ability to serve as a potential target for cancer therapy., (Copyright © 2020 Masucci, Minopoli, Del Vecchio and Carriero.)

Advances in the use of targeted and immune therapies have revolutionized the clinical management of melanoma patients, prolonging significantly their overall and progression-free survival. However, both targeted and immune therapies suffer limitations due to genetic mutations and epigenetic modifications, which determine a great heterogeneity and phenotypic plasticity of melanoma cells. Acquired resistance of melanoma patients to inhibitors of BRAF (BRAFi) and MEK (MEKi), which block the mitogen-activated protein kinase (MAPK) pathway, limits their prolonged use. On the other hand, immune checkpoint inhibitors improve the outcomes of patients in only a subset of them and the molecular mechanisms underlying lack of responses are under investigation. There is growing evidence that altered expression levels of microRNAs (miRNA)s induce drug-resistance in tumor cells and that restoring normal expression of dysregulated miRNAs may re-establish drug sensitivity. However, the relationship between specific miRNA signatures and acquired resistance of melanoma to MAPK and immune checkpoint inhibitors is still limited and not fully elucidated. In this review, we provide an updated overview of how miRNAs induce resistance or restore melanoma cell sensitivity to mitogen-activated protein kinase inhibitors (MAPKi) as well as on the relationship existing between miRNAs and immune evasion by melanoma cell resistant to MAPKi.

S‑Adenosyl‑L‑methionine (AdoMet) is the principal methyl donor in transmethylation reactions fundamental to sustaining epigenetic modifications. Over the past decade, AdoMet has been extensively investigated for its anti‑proliferative, pro‑apoptotic and anti‑metastatic roles in several types of human cancer. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide, and is an aggressive type of cancer that is associated with a high recurrence rate, metastasis and poor treatment outcomes. The present study demonstrates, for the first time, to the best of our knowledge, that AdoMet induces cell cycle arrest and inhibits the migratory and invasive ability of two different HNSCC cell lines, oral Cal‑33 and laryngeal JHU‑SCC‑011 cells. In both cell lines, AdoMet attenuated cell cycle progression, decreased the protein level of several cyclins and downregulated the expression of p21 cell cycle inhibitor. Moreover, AdoMet was able to inhibit Cal‑33 and JHU‑SCC‑011 cell migration in a dose‑dependent manner after 24 and 48 h, respectively, and also induced a significant reduction in the cell invasive ability, as demonstrated by Matrigel invasion assay monitored by the xCELLigence RTCA system. Western blot analysis of several migration and invasion markers confirmed the inhibitory effects exerted by AdoMet on these processes and highlighted AKT, β‑catenin and small mothers against decapentaplegic (SMAD) as the main signaling pathways modulated by AdoMet. The present study also demonstrated that the combination of AdoMet and cisplatin synergistically inhibited HNSCC cell migration. Taken together, these findings demonstrate that the physiological compound, AdoMet, affects the motility and extracellular matrix invasive capability in HNSCC. Thus, AdoMet may prove to be a good candidate for future drug development against metastatic cancer.

Chondrosarcomas (CHS) are malignant cartilaginous neoplasms with diverse morphological features, characterized by resistance to chemo- and radiation therapies. In this study, we investigated the role of tumor-associated macrophages (TAM)s in tumor tissues from CHS patients by immunohistochemistry. Three-dimensional organotypic co-cultures were set up in order to evaluate the contribution of primary human CHS cells in driving an M2-like phenotype in monocyte-derived primary macrophages, and the capability of macrophages to promote growth and/or invasiveness of CHS cells. Finally, with an in vivo model of primary CHS cells engrafted in nude mice, we tested the ability of a potent peptide inhibitor of cell migration (Ac-d-Tyr-d-Arg-Aib-d-Arg-NH 2 , denoted RI-3) to reduce recruitment and infiltration of monocytes into CHS neoplastic lesions. We found a significant correlation between alternatively activated M2 macrophages and intratumor microvessel density in both conventional and dedifferentiated CHS human tissues, suggesting a link between TAM abundance and vascularization in CHS. In 3D and non-contact cu-culture models, soluble factors produced by CHS induced a M2-like phenotype in macrophages that, in turn, increased motility, invasion and matrix spreading of CHS cells. Finally, we present evidence that RI-3 successfully prevent both recruitment and infiltration of monocytes into CHS tissues, in nude mice.

Tumor Associated Neutrophils (TANs) are engaged into the tumor microenvironment by cytokines and chemokines, can be distinguished according to their activation and cytokine status and effects on tumor cell growing in N1 and N2 TANs. N1 TANs exert an antitumor activity, by direct or indirect cytotoxicity. N2 TANs stimulate immunosuppression, tumor growth, angiogenesis and metastasis by DNA instability, or by cytokines and chemokines release. In tumor patients, either a high number of TANs and Neutrophil-to-Lymphocyte Ratio (NLR) do correlate with poor prognosis, and, so far, TAN counts and NLR can be regarded as biomarkers. Owing to the pivotal role of TANs in stimulating tumor progression, therapeutic strategies to target TANs have been suggested, and two major approaches have been proposed: (a) targeting the CXCL-8/CXCR-1/CXCR-2 axis, thereby blocking TANs or (b) targeting substances produced by polymorpho-nuclear cells that promote tumor growth. Many studies have been accomplished either in vitro and in animal models, whereas clinical studies are restrained, presently, due to the risk of inducing immunosuppression. In this review, we deeply discuss the anti-tumorigenic or pro-tumorigenic activity of TANs. In particular, TANs relevance in tumor prognosis and in vitro therapeutic strategies are widely described. On-going clinical trials, aimed to inhibit neutrophil recruitment into the tumor are also accurately debated., (Copyright © 2019 Masucci, Minopoli and Carriero.)

Background: The biological behavior of epithelial ovarian cancer (EOC) is unique since EOC cells metastasize early to the peritoneum. Thereby, new anti-target agents designed to block trans-coelomic dissemination of EOC cells may be useful as anti-metastatic drugs. The Urokinase Plasminogen Activator Receptor (uPAR) is overexpressed in EOC tissues, and its truncated forms released in sera and/or ascitic fluid are associated with poor prognosis and unfavorable clinical outcome. We documented that uPAR triggers intra-abdominal dissemination of EOC cells through the interaction of its 84-95 sequence with the Formyl Peptide Receptor type 1 (FPR1), even as short linear peptide Ser-Arg-Ser-Arg-Tyr (SRSRY). While the pro-metastatic role of uPAR is well documented, little information regarding the expression and role of FPR1 in EOC is currently available., Methods: Expression levels of uPAR and FPR1 in EOC cells and tissues were assessed by immunofluorescence, Western blot, or immunohystochemistry. Cell adhesion to extra-cellular matrix proteins and mesothelium as well as mesothelium invasion kinetics by EOC cells were monitored using the xCELLigence technology or assessed by measuring cell-associated fluorescence. Cell internalization of FPR1 was identified on multiple z-series by confocal microscopy. Data from in vitro assays were analysed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons. Tissue microarray data were analyzed with the Pearson's Chi-square (χ 2 ) test., Results: Co-expression of uPAR and FPR1 by SKOV-3 and primary EOC cells confers a marked adhesion to vitronectin. The extent of cell adhesion decreases to basal level by pre-exposure to anti-uPAR84-95 Abs, or to the RI-3 peptide, blocking the uPAR84-95/FPR1 interaction. Furthermore, EOC cells exposed to RI-3 or desensitized with an excess of SRSRY, fail to adhere also to mesothelial cell monolayers, losing the ability to cross them. Finally, primary and metastatic EOC tissues express a high level of FPR1., Conclusions: Our findings identify for the first time FPR1 as a potential biomarker of aggressive EOC and suggests that inhibitors of the uPAR84-95/FPR1 crosstalk may be useful for the treatment of metastatic EOC.

The interaction between the short 88Ser-Arg-Ser-Arg-Tyr92 sequence of the urokinase receptor (uPAR) and the formyl peptide receptor type 1 (FPR1) elicits cell migration. We generated the Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH2 (RI-3) peptide which inhibits the uPAR/FPR1 interaction, reducing migration of FPR1 expressing cells toward N-formyl-methionyl-leucyl-phenylalanine (fMLF) and Ser-Arg-Ser-Arg-Tyr (SRSRY) peptides. To understand the structural basis of the RI-3 inhibitory effects, the FPR1/fMLF, FPR1/SRSRY and FPR1/RI-3 complexes were modeled and analyzed, focusing on the binding pocket of FPR1 and the interaction between the amino acids that signal to the FPR1 C-terminal loop. We found that RI-3 shares the same binding site of fMLF and SRSRY on FPR1. However, while fMLF and SRSRY display the same agonist activation signature (i.e. the series of contacts that transmit the conformational transition throughout the complex), translating binding into signaling, RI-3 does not interact with the activation region of FPR1 and hence does not activate signaling. Indeed, fluorescein-conjugated RI-3 prevents either fMLF and SRSRY uptake on FPR1 without triggering FPR1 internalization and cell motility in the absence of any stimulus. Collectively, our data show that RI-3 is a true FPR1 antagonist and suggest a pharmacophore model useful for development of compounds that selectively inhibit the uPAR-triggered, FPR1-mediated cell migration.

Background: Molecular markers for prostate cancer (PCa) are required to improve the early definition of patient outcomes. Atypically large extracellular vesicles (EVs), referred as "Large Oncosomes" (LO), have been identified in highly migratory and invasive PCa cells. We recently developed and characterized the DU145R80 subline, selected from parental DU145 cells as resistant to inhibitors of mevalonate pathway. DU145R80 showed different proteomic profile compared to parental DU145 cells, along with altered cytoskeleton dynamics and a more aggressive phenotype., Methods: Immunofluorescence staining and western blotting were used to identify blebbing and EVs protein cargo. EVs, purified by gradient ultra-centrifugations, were analyzed by tunable resistive pulse sensing and multi-parametric flow cytometry approach coupled with high-resolution imaging technologies. LO functional effects were tested in vitro by adhesion and invasion assays and in vivo xenograft model in nude mice. Xenograft and patient tumor tissues were analyzed by immunohistochemistry., Results: We found spontaneous blebbing and increased shedding of LO from DU145R80 compared to DU145 cells. LO from DU145R80, compared to those from DU145, carried increased amounts of key-molecules involved in PCa progression including integrin alpha V (αV-integrin). By incubating DU145 cells with DU145R80-derived LO we demonstrated that αV-integrin on LO surface was functionally involved in the increased adhesion and invasion of recipient cells, via AKT. Indeed either the pre-incubation of LO with an αV-integrin blocking antibody, or a specific AKT inhibition in recipient cells are able to revert the LO-induced functional effects. Moreover, DU145R80-derived LO also increased DU145 tumor engraftment in a mice model. Finally, we identified αV-integrin positive LO-like structures in tumor xenografts as well as in PCa patient tissues. Increased αV-integrin tumor expression correlated with high Gleason score and lymph node status., Conclusions: Overall, this study is the first to demonstrate the critical role of αV-integrin positive LO in PCa aggressive features, adding new insights in biological function of these large EVs and suggesting their potential use as PCa prognostic markers.

Disseminating Cancer Stem Cells (CSCs) initiate growth in specific niches of the host tissues, the cellular and molecular components of which sustain signaling pathways that support their survival, self-renewal dormancy and reactivation. In the metastatic niche, tumor cells may enter in a dormant state to survive and, consequently, the metastasis can remain latent for years. Despite the clinical importance of metastatic latency, little is known about what induces CSCs to enter a dormant state and what allows them to remain viable for years in this state. CSCs exhibit genetic, epigenetic and cellular adaptations that confer resistance to classical therapeutic approaches. The identification of potential CSC targets is complicated by the fact that CSCs may arise as a consequence of their relationship with the local microenvironment into the metastatic niches. Indeed, microenvironment modulates the capability of CSCs to evade the innate immune response and survive. Some new therapeutic options that include drugs targeting microenvironment components are achieving encouraging results in reducing the number of CSCs in tumors and/or overcoming their resistance in preclinical studies. This review will focus on specific CSC features with an emphasis on the role of tumor microenvironment in supporting metastatic dissemination of CSCs. In addition, it sheds light on potential microenvironment-targeted therapies aimed to counteract seeding and survival of CSCs in the metastatic niche.

Melanoma is a molecularly heterogeneous disease with many genetic mutations and altered signaling pathways. Activating mutations in the BRAF oncogene are observed in approximately 50% of cutaneous melanomas and the use of BRAF inhibitor (BRAFi) compounds has been reported to improve the outcome of patients with BRAF-mutated metastatic melanoma. However, the majority of these patients develop resistance within 6-8 months following the initiation of BRAFi treatment. In this study, we examined the possible use of the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, ABT-888 (veliparib), as a novel molecule that may be successfully employed in the treatment of BRAFi-resistant melanoma cells. Sensitive and resistant to BRAFi dabrafenib A375 cells were exposed to increasing concentrations of ABT-888. Cell viability and apoptosis were assessed by MTT assay and Annexin V-FITC analysis, respectively. The cell migratory and invasive ability was investigated using the xCELLigence technology and Boyden chamber assays, respectively. ABT-888 was found to reduce cell viability and exhibited pro-apoptotic activity in melanoma cell lines, independently from the BRAF/NRAS mutation status, in a dose-dependent manner, with the maximal effect being reached in the 25-50 µM concentration range. Moreover, ABT-888 promoted apoptosis in both the sensitive and resistant A375 cells, suggesting that ABT-888 may be useful in the treatment of BRAFi-resistant subsets of melanoma cells. Finally, in accordance with the involvement of PARP1 in actin cytoskeletal machinery, we found that the cytoskeletal organization, motility and invasive capability of both the A375 and A375R cells decreased upon exposure to 5 µM ABT-888 for 24 h. On the whole, the findings of this study highlight the pivotal role of PARP1 in the migration and invasion of melanoma cells, suggesting that ABT-888 may indeed be effective, not only as a pro-apoptotic drug for use in the treatment of BRAFi-resistant melanoma cells, but also in suppressing their migratory and invasive activities.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration. We and others have previously documented that the uPAR(84-95) sequence, interacts with the formyl peptide receptors (FPR)s, henceforth inducing cell migration of several cell lines, including leukocytes, and the synthetic shorter peptide (Ser 88 -Arg-Ser-Arg-Tyr 92 , SRSRY) retains chemotactic activity in vitro and in vivo. Recently, we have developed the head-to-tail cyclic analog [SRSRY], a new potent and stable inhibitor of monocyte trafficking. This prompted us to develop novel cyclic and linear analogs of [SRSRY] with the aim to broaden the knowledge about structure-activity relationships of peptide [SRSRY]. Herein we report their synthesis, effects on cell migration, conformational and docking analyses which served to envisage a new pharmacophore model for inhibitors of FPR1-triggered cell migration., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: Accumulating evidence demonstrates that the Urokinase Receptor (uPAR) regulates tumor cell migration through its assembly in composite regulatory units with transmembrane receptors, and uPAR 88-92 is the minimal sequence required to induce cell motility through the Formyl Peptide Receptor type 1 (FPR1). Both uPAR and FPR1 are involved in melanoma tumor progression, suggesting that they may be targeted for therapeutic purposes. In this study, the role of the uPAR-FPR1 cross-talk to sustain melanoma cell ability to invade extracellular matrix and cross endothelial barriers is investigated. Also, the possibility that inhibition of the uPAR mediated FPR1-dependent signaling may prevent matrix invasion and transendothelial migration of melanoma cells was investigated., Methods: Expression levels of uPAR and FPR1 were assessed by immunocytochemistry, Western Blot and qRT-PCR. Cell migration was investigated by Boyden chamber and wound-healing assays. Migration and invasion kinetics, trans-endothelial migration and proliferation of melanoma cells were monitored in real time using the xCELLigence technology. The agonist-triggered FPR1 internalization was visualized by confocal microscope. Cell adhesion to endothelium was determined by fluorometer measurement of cell-associated fluorescence or identified on multiple z-series by laser confocal microscopy. The 3D-organotypic models were set up by seeding melanoma cells onto collagen I matrices embedded dermal fibroblasts. Data were analyzed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons., Results: We found that the co-expression of uPAR and FPR1 confers to A375 and M14 melanoma cells a clear-cut capability to move towards chemotactic gradients, to cross extracellular matrix and endothelial monolayers. FPR1 activity is required, as cell migration and invasion were abrogated by receptor desensitization. Finally, melanoma cell ability to move toward chemotactic gradients, invade matrigel or fibroblast-embedded collagen matrices and cross endothelial monolayers are prevented by anti-uPAR 84-95 antibodies or by the RI-3 peptide which we have previously shown to inhibit the uPAR 84-95 /FPR1 interaction., Conclusions: Collectively, our findings identify uPAR and FPR1 as relevant effectors of melanoma cell invasiveness and suggest that inhibitors of the uPAR 84-95 /FPR1 cross-talk may be useful for the treatment of metastatic melanoma.

The development of metastases is a multistep process that requires the activation of physiological and biochemical processes that govern migration, invasion and entry of metastatic cells into blood vessels. The urokinase receptor (uPAR) promotes cell migration by interacting with the Formyl Peptide Receptors (FPRs). Since both uPAR and FPR1 are involved in tumor progression, the uPAR-FPR1 interaction is an attractive therapeutic target. We previously described peptide antagonists of the uPAR-FPR1 interaction that inhibited cell migration and angiogenesis. To develop enzyme-resistant analogues, we applied here the Retro-Inverso (RI) approach, whereby the topology of the side chains is maintained by inverting the sequence of the peptide and the chirality of all residues. Molecular dynamics suggests that peptide RI-3 adopts the turn structure typical of uPAR-FPR1 antagonists. Accordingly, RI-3 is a nanomolar competitor of N-formyl-Met-Leu-Phe for binding to FPR1 and inhibits migration, invasion, trans-endothelial migration of sarcoma cells and VEGF-triggered endothelial tube formation. When sarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density, circulating tumor cells and pulmonary metastases were significantly reduced in animals treated daily with 6 mg/Kg RI-3 as compared to animals treated with vehicle only. Thus, RI-3 represents a promising lead for anti-metastatic drugs.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88-92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC50 value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.