Heart rate variability and vasovagal tone index in brachycephalic dogs.

Brachycephalic breeds of dogs, most of which show signs of the brachycephalic syndrome may have greater parasympathetic stimulation than other breeds, leading to higher values of heart rate variability and vagal tone index. The aim of this study was to establish a computerized electrocardiographic study and an assessment of the vagus sympathetic balance through heart rate variability and vagal tone index of five brachycephalic breeds compared to mesocephalic dogs. Sixty dogs were used, divided into groups made up of Boxers, English Bulldogs, French Bulldogs, Pugs, Shih-Tzu and no defined breed mesocephalic dogs. Statistical analysis was carried out using the Shapiro-Wilk test, Kruskal-Wallis and Dunn's test or ANOVA and Bonferroni (p<0.05). In the evaluation of vagal sympathetic balance among all the dogs, there was a negative correlation between heart rate and HRV 10RR (r = - 0.7678; p < 0.0001), HRV 20RR (r = - 0.8548, p < 0.0001) and VVTI (r = - 0.2770; p = 0.0321). It can therefore be concluded that the dog's breed and morphology did not alter its electrocardiographic parameters or heart rate variability. The vagal tone index, which in other studies differed in brachycephalic dogs, showed no difference when compared separately in brachycephalic breeds.

Small-angle X-ray and neutron scattering applied to lipid-based nanoparticles: Recent advancements across different length scales.

Lipid-based nanoparticles (LNPs), ranging from nanovesicles to non-lamellar assemblies, have gained significant attention in recent years, as versatile carriers for delivering drugs, vaccines, and nutrients. Small-angle scattering methods, employing X-rays (SAXS) or neutrons (SANS), represent unique tools to unveil structure, dynamics, and interactions of such particles on different length scales, spanning from the nano to the molecular scale. This review explores the state-of-the-art on scattering methods applied to unveil the structure of lipid-based nanoparticles and their interactions with drugs and bioactive molecules, to inform their rational design and formulation for medical applications. We will focus on complementary information accessible with X-rays or neutrons, ranging from insights on the structure and colloidal processes at a nanoscale level (SAXS) to details on the lipid organization and molecular interactions of LNPs (SANS). In addition, we will review new opportunities offered by Time-resolved (TR)-SAXS and -SANS for the investigation of dynamic processes involving LNPs. These span from real-time monitoring of LNPs structural evolution in response to endogenous or external stimuli (TR-SANS), to the investigation of the kinetics of lipid diffusion and exchange upon interaction with biomolecules (TR-SANS). Finally, we will spotlight novel combinations of SAXS and SANS with complementary on-line techniques, recently enabled at Large Scale Facilities for X-rays and neutrons. This emerging technology enables synchronized multi-method investigation, offering exciting opportunities for the simultaneous characterization of the structure and chemical or mechanical properties of LNPs.

Tumor Burden Dictates the Neoantigen Features Required to Generate an Effective Cancer Vaccine.

Tumor neoantigens (nAg) represent a promising target for cancer immunotherapy. The identification of nAgs that can generate T-cell responses and have therapeutic activity has been challenging. Here, we sought to unravel the features of nAgs required to induce tumor rejection. We selected clinically validated Great Ape-derived adenoviral vectors (GAd) as a nAg delivery system for differing numbers and combinations of nAgs. We assessed their immunogenicity and efficacy in murine models of low to high disease burden, comparing multi-epitope versus mono-epitope vaccines. We demonstrated that the breadth of immune response is critical for vaccine efficacy and having multiple immunogenic nAgs encoded in a single vaccine improves efficacy. The contribution of each single neoantigen was examined, leading to the identification of 2 nAgs able to induce CD8+ T cell-mediated tumor rejection. They were both active as individual nAgs in a setting of prophylactic vaccination, although to different extents. However, the efficacy of these single nAgs was lost in a setting of therapeutic vaccination in tumor-bearing mice. The presence of CD4+ T-cell help restored the efficacy for only the most expressed of the two nAgs, demonstrating a key role for CD4+ T cells in sustaining CD8+ T-cell responses and the necessity of an efficient recognition of the targeted epitopes on cancer cells by CD8+ T cells for an effective antitumor response. This study provides insight into understanding the determinants of nAgs relevant for effective treatment and highlights features that could contribute to more effective antitumor vaccines. See related Spotlight by Slingluff Jr, p. 382.

A Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer (MRI-CEST) Method for the Detection of Water Cycling across Cellular Membranes.

Water cycling across the membrane transporters is considered a hallmark of cellular metabolism and it could be of high diagnostic relevance in the characterization of tumors and other diseases. The method relies on the response of intracellular proton exchanging molecules to the presence of extracellular Gd-based contrast agents (GBCAs). Paramagnetic GBCAs enhances the relaxation rate of water molecules in the extracellular compartment and, through membrane exchange, the relaxation enhancement is transferred to intracellular molecules. The effect is detected at the MRI-CEST (Magnetic Resonance Imaging - Chemical Exchange Saturation Transfer) signal of intracellular proton exchanging molecules. The magnitude of the change in the CEST response reports on water cycling across the membrane. The method has been tested on Red Blood Cells and on orthotopic murine models of breast cancer with different degree of malignancy (4T1, TS/A and 168FARN). The distribution of voxels reporting on membrane permeability fits well with the cells' aggressiveness and acts as an early reporter to monitor therapeutic treatments.

FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy.

Skeletal muscle atrophy is a hallmark of cachexia, a wasting condition typical of chronic pathologies, that still represents an unmet medical need. Bone morphogenetic protein (BMP)-Smad1/5/8 signaling alterations are emerging drivers of muscle catabolism, hence, characterizing these perturbations is pivotal to develop therapeutic approaches. We identified two promoters of "BMP resistance" in cancer cachexia, specifically the BMP scavenger erythroferrone (ERFE) and the intracellular inhibitor FKBP12. ERFE is upregulated in cachectic cancer patients' muscle biopsies and in murine cachexia models, where its expression is driven by STAT3. Moreover, the knock down of Erfe or Fkbp12 reduces muscle wasting in cachectic mice. To bypass the BMP resistance mediated by ERFE and release the brake on the signaling, we targeted FKBP12 with low-dose FK506. FK506 restores BMP-Smad1/5/8 signaling, rescuing myotube atrophy by inducing protein synthesis. In cachectic tumor-bearing mice, FK506 prevents muscle and body weight loss and protects from neuromuscular junction alteration, suggesting therapeutic potential for targeting the ERFE-FKBP12 axis.

Particle profiling of EV-lipoprotein mixtures by AFM nanomechanical imaging.

The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.

Cystine/glutamate antiporter xCT deficiency reduces metastasis without impairing immune system function in breast cancer mouse models.

BACKGROUND: The upregulation of antioxidant mechanisms is a common occurrence in cancer cells, as they strive to maintain balanced redox state and prevent oxidative damage. This includes the upregulation of the cystine/glutamate antiporter xCT, which plays a crucial role in protecting cancer cells from oxidative stress. Consequently, targeting xCT has become an attractive strategy for cancer treatment. However, xCT is also expressed by several types of immune cells where it has a role in proliferation and effector functions. In light of these observations, a comprehensive understanding of the specific role of xCT in the initiation and progression of cancer, as well as its potential impact on the immune system within the tumor microenvironment and the anti-tumor response, require further investigation. METHODS: We generated xCTnull BALB/c mice to investigate the role of xCT in the immune system and xCTnull/Erbb2-transgenic BALB-neuT mice to study the role of xCT in a mammary cancer-prone model. We also used mammary cancer cells derived from BALB-neuT/xCTnull mice and xCTKO 4T1 cells to test the contribution of xCT to malignant properties in vitro and in vivo. RESULTS: xCT depletion in BALB-neuT/xCTnull mice does not alter autochthonous tumor initiation, but tumor cells isolated from these mice display proliferation and redox balance defects in vitro. Although xCT disruption sensitizes 4T1 cells to oxidative stress, it does not prevent transplantable tumor growth, but reduces cell migration in vitro and lung metastasis in vivo. This is accompanied by an altered immune cell recruitment in the pre-metastatic niche. Finally, systemic depletion of xCT in host mice does not affect transplantable tumor growth and metastasis nor impair the proper mounting of both humoral and cellular immune responses in vivo. CONCLUSIONS: xCT is dispensable for proper immune system function, thus supporting the safety of xCT targeting in oncology. Nevertheless, xCT is involved in several processes required for the metastatic seeding of mammary cancer cells, thus broadening the scope of xCT-targeting approaches.

Tissue factor as a potential coagulative/vascular marker in relapsing-remitting multiple sclerosis.

Objectives: Recent studies supported coagulation involvement in multiple sclerosis, an inflammatory-demyelinating and degenerative disease of the central nervous system. The main objectives of this observational study were to identify the most specific pro-coagulative/vascular factors for multiple sclerosis pathogenesis and to correlate them with brain hemodynamic abnormalities. Methods: We compared i) serum/plasma levels of complement(C)/coagulation/vascular factors, viral/microbiological assays, fat-soluble vitamins and lymphocyte count among people with multiple sclerosis sampled in a clinical remission (n=30; 23F/7M, 40 ± 8.14 years) or a relapse (n=30; 24F/6M, age 41 ± 10.74 years) and age/sex-matched controls (n=30; 23F/7M, 40 ± 8.38 years); ii) brain hemodynamic metrics at dynamic susceptibility contrast-enhanced 3T-MRI during relapse and remission, and iii) laboratory data with MRI perfusion metrics and clinical features of people with multiple sclerosis. Two models by Partial Least Squares Discriminant Analysis were performed using two groups as input: (1) multiple sclerosis vs. controls, and (2) relapsing vs. remitting multiple sclerosis. Results: Compared to controls, multiple sclerosis patients had a higher Body-Mass-Index, Protein-C and activated-C9; and a lower activated-C4. Levels of Tissue-Factor, Tie-2 and P-Selectin/CD62P were lower in relapse compared to remission and HC, whereas Angiopoietin-I was higher in relapsing vs. remitting multiple sclerosis. A lower number of total lymphocytes was found in relapsing multiple sclerosis vs. remitting multiple sclerosis and controls. Cerebral-Blood-Volume was lower in normal-appearing white matter and left caudatum while Cerebral-Blood-Flow was inferior in bilateral putamen in relapsing versus remitting multiple sclerosis. The mean-transit-time of gadolinium-enhancing lesions negatively correlated with Tissue-Factor. The top-5 discriminating variables for model (1) were: EBV-EBNA-1 IgG, Body-Mass-Index, Protein-C, activated-C4 and Tissue-Factor whereas for model (2) were: Tissue-Factor, Angiopoietin-I, MCHC, Vitamin A and T-CD3. Conclusion: Tissue-factor was one of the top-5 variables in the models discriminating either multiple sclerosis from controls or multiple sclerosis relapse from remission and correlated with mean-transit-time of gadolinium-enhancing lesions. Tissue-factor appears a promising pro-coagulative/vascular biomarker and a possible therapeutic target in relapsing-remitting multiple sclerosis. Clinical trial registration: ClinicalTrials.gov, identifier NCT04380220.

Assessment of BoHV-4-based vector vaccine intranasally administered in a hamster challenge model of lung disease.

Introduction: Bovine herpesvirus 4 (BoHV-4) is a bovine Rhadinovirus not associated with a specific pathological lesion or disease and experimentally employed as a viral vector vaccine. BoHV-4-based vector (BoHV-4-BV) has been shown to be effective in immunizing and protecting several animal species when systemically administrated through intramuscular, subcutaneous, intravenous, or intraperitoneal routes. However, whether BoHV-4-BV affords respiratory disease protection when administered intranasally has never been tested. Methods: In the present study, recombinant BoHV-4, BoHV-4-A-S-ΔRS-HA-ΔTK, was constructed to deliver an expression cassette for the SARS-CoV-2 spike glycoprotein, and its immunogenicity, as well as its capability to transduce cells of the respiratory tract, were tested in mice. The well-established COVID-19/Syrian hamster model was adopted to test the efficacy of intranasally administered BoHV-4-A-S-ΔRS-HA-ΔTK in protecting against a SARS-CoV-2 challenge. Results: The intranasal administration of BoHV-4-A-S-ΔRS-HA-ΔTK elicited protection against SARS-CoV-2, with improved clinical signs, including significant reductions in body weight loss, significant reductions in viral load in the trachea and lungs, and significant reductions in histopathologic lung lesions compared to BoHV-4-A-S-ΔRS-HA-ΔTK administered intramuscularly. Discussion: These results suggested that intranasal immunization with BoHV-4-BV induced protective immunity and that BoHV-4-BV could be a potential vaccine platform for the protection of other animal species against respiratory diseases.

Comparison of Clinical, Biochemical and Histological Features between Adult Celiac Patients with High and Low Anti-Transglutaminase IgA Titer at Diagnosis and Follow-Up.

In adults, celiac disease (CD) diagnosis is based on specific serology (anti-transglutaminase IgA-anti-tTG) and duodenal histology. Evidence is raising the possibility of perform CD diagnosis based only on high anti-tTG titer in children. We aimed to evaluate clinical, histological and biochemical differences between adult patients with high tTG IgA titer (HT) and those with low titer (LT) at CD diagnosis and follow-up. This retrospective study included consecutive adult CD patients divided into two groups: HT (anti-tTG > 10 × ULN) and LT (anti-tTG < 10 × ULN). Clinical, biochemical and histological features were compared between groups at CD diagnosis and at follow-up. A total of 291 patients were included (HT: 47.1%; LT: 52.9%). At CD diagnosis, HT patients showed a non 'classical' presentation (p = 0.04), Marsh 3C (p = 0.005), hypoferritinaemia (p = 0.006) and osteopenia/osteoporosis (p = 0.04) more frequently than LT patients. A total of 216 patients (HT: 48.6%; LT: 51.4%) performed a follow-up after a median Gluten-free diet of 14 months; HT patients had persistent antibodies positivity (p = 0.001) more frequently and GI symptoms (p = 0.04) less frequently than LT patients. In conclusion, HT patients presented severe histological damage more frequently at diagnosis, recovering similarly to LT patients after the start of the Gluten-free diet. At follow-up, anti-tTG persisted positive in HT more frequently compared to LT patients, without differences regarding histological recovery and clinical improvement.

p140Cap inhibits ß-Catenin in the breast cancer stem cell compartment instructing a protective anti-tumor immune response.

The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of ß-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of ß-Catenin depends on its ability to localize in and stabilize the ß-Catenin destruction complex, promoting enhanced ß-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the ß-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response.

Metacognitive interpersonal therapy in borderline personality disorder: Clinical and neuroimaging outcomes from the CLIMAMITHE study-A randomized clinical trial.

Different psychotherapeutic approaches demonstrated their efficacy but the possible neurobiological mechanism underlying the effect of psychotherapy in borderline personality disorder (BPD) patients is poorly investigated. We assessed the effects of metacognitive interpersonal therapy (MIT) on BPD features and other dimensions compared to structured clinical management (SCM). We also assessed changes in amygdala activation by viewing emotional pictures after psychotherapy. One hundred forty-one patients were referred and 78 BPD outpatients were included and randomized to MIT or SCM. Primary outcome was emotional dysregulation assessed with the Difficulties in Emotion Regulation Scale (DERS). We also assessed BPD symptomatology, number of PD criteria, metacognitive abilities, state-psychopathology, depression, impulsiveness, interpersonal functioning, and alexithymia. A subset of 60 patients underwent functional magnetic resonance imaging before and after 1 year of psychotherapy to assess amygdala activation by viewing standardized emotional pictures (secondary outcome). DERS scores decreased in both groups (time effect p < .001). The Cohen's d effect size for change (baseline posttreatment) on DERS was very large (d = 0.84) in MIT, and large (d = 0.76) in SCM. Both groups significantly improved in depressive symptoms, state-psychopathology, alexithymia, and interpersonal functioning. MIT showed larger effect on metacognitive functions than SCM (Time × Group p < .001). Both interventions showed a significant effect on BPD symptomatology although SCM group showed a larger decrease. On the contrary, MIT group showed larger decrease in impulsivity and number of PD criteria. Interestingly, both MIT and SCM modulated amygdala activation in BPD patients. MIT is a valid and effective psychotherapy for BPD with an impact on amygdala activation. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Tissue memory relies on stem cell priming in distal undamaged areas.

Epithelial cells that participated in wound repair elicit a more efficient response to future injuries, which is believed to be locally restricted. Here we show that cell adaptation resulting from a localized tissue damage has a wide spatial impact at a scale not previously appreciated. We demonstrate that a specific stem cell population, distant from the original injury, originates long-lasting wound memory progenitors residing in their own niche. Notably, these distal memory cells have not taken part in the first healing but become intrinsically pre-activated through priming. This cell state, maintained at the chromatin and transcriptional level, leads to an enhanced wound repair that is partially recapitulated through epigenetic perturbation. Importantly wound memory has long-term harmful consequences, exacerbating tumourigenesis. Overall, we show that sub-organ-scale adaptation to injury relies on spatially organized memory-dedicated progenitors, characterized by an actionable cell state that establishes an epigenetic field cancerization and predisposes to tumour onset.

Autoimmune atrophic gastritis and coeliac disease: A case-control study.

BACKGROUND: Autoimmune atrophic gastritis (AAG) is rarely associated with coeliac disease (CD). AIMS: To assess the frequency of AAG-CD association and to compare clinical, biochemical, and histological features of adults affected by both diseases (cases) with AAG controls. METHODS: This case-control study included 9 cases (F55%, median age 47, range 23-59yrs) matched (1:3) by age (±4 yrs) and gender to 27 controls randomly selected from our AAG cohort (2009-2021). The AAG and CD diagnosis was based on internationally agreed criteria. RESULTS: Of 434 AAG patients (median age:62.5yrs, range18-92yrs, F:M ratio=2.2:1),9 had a concomitant diagnosis of CD. The occurrence of AAG-CD association was 2% and 1.65% among AAG/CD cohorts, respectively. Cases were significantly younger than AAG cohort (n = 425, p = 0.002). In 4/9cases, AAG was diagnosed by proactive screening for autoimmune disorders. Autoimmune thyroid disorders were present in 5/9 cases. Cases had a significant higher prevalence of normocytic anaemia than controls (p = 0.004). No significant differences were found between cases and controls concerning clinical and histological features. CONCLUSIONS: AAG-CD association is rare. Gastric and duodenal biopsies might be advisable in young people with normocytic anaemia and associated autoimmune disorders to timely diagnose clinically silent conditions.

Dynamics of humoral and cellular response to three doses of anti-SARS-CoV-2 BNT162b2 vaccine in patients with hematological malignancies and older subjects.

Background: Few data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients. Objective: To investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy. Methods: We measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80-92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D. Results: An increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts. Conclusion: FHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response.

The Role of the Toll-like Receptor 2 and the cGAS-STING Pathways in Breast Cancer: Friends or Foes?

Breast cancer stands as a primary malignancy among women, ranking second in global cancer-related deaths. Despite treatment advancements, many patients progress to metastatic stages, posing a significant therapeutic challenge. Current therapies primarily target cancer cells, overlooking their intricate interactions with the tumor microenvironment (TME) that fuel progression and treatment resistance. Dysregulated innate immunity in breast cancer triggers chronic inflammation, fostering cancer development and therapy resistance. Innate immune pattern recognition receptors (PRRs) have emerged as crucial regulators of the immune response as well as of several immune-mediated or cancer cell-intrinsic mechanisms that either inhibit or promote tumor progression. In particular, several studies showed that the Toll-like receptor 2 (TLR2) and the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathways play a central role in breast cancer progression. In this review, we present a comprehensive overview of the role of TLR2 and STING in breast cancer, and we explore the potential to target these PRRs for drug development. This information will significantly impact the scientific discussion on the use of PRR agonists or inhibitors in cancer therapy, opening up new and promising avenues for breast cancer treatment.

Identification of a miRNA-based non-invasive predictive biomarker of response to target therapy in BRAF-mutant melanoma.

Rationale: Metastatic melanoma is the most aggressive and dangerous form of skin cancer. The introduction of immunotherapy with Immune checkpoint Inhibitors (ICI) and of targeted therapy with BRAF and MEK inhibitors for BRAF mutated melanoma, has greatly improved the clinical outcome of these patients. Nevertheless, response to therapy remains highly variable and the development of drug resistance continues to be a daunting challenge. Within this context there is a need to develop diagnostic tools capable of predicting response or resistance to therapy in order to select the best therapeutic approach. Over the years, accumulating evidence brought to light the role of microRNAs (miRNAs) as disease biomarkers. Methods: In particular, the detection of miRNAs in whole blood or specific blood components such as serum or plasma, allows these molecules to be good candidates for diagnosis, prognosis and for monitoring response to anticancer therapy. In this paper, we evaluated circulating basal levels of 6 previously identified miRNAs in serum samples of 70 BRAF-mutant melanoma patients before starting targeted therapy. Results: Results show that the circulating levels of the oncosuppressor miR-579-3p and of the oncomiR miR-4488 are able to predict progression free survival (PFS) but not overall survival (OS). Most importantly, we observed that the best predictor of disease outcome is represented by the ratio of circulating miR-4488 vs. miR-579-3p (miRatio). Finally, the combination of the Lactate dehydrogenase (LDH) blood levels with the two circulating miRNAs alone or together did not produce any improvement in predicting PFS indicating that miR-579-3p and miR-4488 are independent predictors of PFS as compared to LDH. Conclusions: All together these data underscored the relevance of circulating miRNAs as suitable tools to predict therapy response in melanoma and maybe further developed as companion diagnostics in the clinic.

Immunotherapy against the Cystine/Glutamate Antiporter xCT Improves the Efficacy of APR-246 in Preclinical Breast Cancer Models.

Breast cancer is the most frequent cancer in women. Despite recent clinical advances, new therapeutic approaches are still required. The cystine-glutamate antiporter xCT, encoded by the SLC7A11 gene, which imports cystine in exchange with glutamate, is a potentially new target for breast cancer therapy, being involved in tumor cell redox balance and resistance to therapies. xCT expression is regulated by the oncosuppressor p53, which is mutated in many breast cancers. Indeed, mutant p53 (mut-p53) can induce xCT post-transcriptional down modulation, rendering mut-p53 tumors susceptible to oxidative damage. Interestingly, the drug APR-246, developed to restore the wild-type function of p53 in tumors harboring its mutation, alters the cell redox balance in a p53-independent way, possibly rendering the cells more sensitive to xCT inhibition. Here, we propose a combinatorial treatment based on xCT immunetargeting and APR-246 treatment as a strategy for tackling breast cancer. We demonstrate that combining the inhibition of xCT with the APR-246 drug significantly decreased breast cancer cell viability in vitro and induced apoptosis and affected cancer stem cells' self-renewal compared to the single treatments. Moreover, the immunetargeting of xCT through DNA vaccination in combination with APR-246 treatment synergistically hinders tumor progression and prevents lung metastasis formation in vivo. These effects can be mediated by the production of anti-xCT antibodies that are able to induce the antibody dependent cellular cytotoxicity of tumor cells. Overall, we demonstrate that DNA vaccination against xCT can synergize with APR-246 treatment and enhance its therapeutic effect. Thus, APR-246 treatment in combination with xCT immunetargeting may open new perspectives in the management of breast cancer.

A Novel PSMA-Targeted Probe for NIRF-Guided Surgery and Photodynamic Therapy: Synthesis and Preclinical Validation.

A total of 20% to 50% of prostate cancer (PCa) patients leave the surgery room with positive tumour margins. The intraoperative combination of fluorescence guided surgery (FGS) and photodynamic therapy (PDT) may be very helpful for improving tumour margin delineation and cancer therapy. PSMA is a transmembrane protein overexpressed in 90−100% of PCa cells. The goal of this work is the development of a PSMA-targeted Near InfraRed Fluorescent probe to offer the surgeon a valuable intraoperative tool for allowing a complete tumour removal, implemented with the possibility of using PDT to kill the eventual not resected cancer cells. PSMA-617 binding motif was conjugated to IRDye700DX-NHS and the conjugation did not affect the photophysical characteristics of the fluorophore. The affinity of IRDye700DX-PSMA-617 towards PCa cells followed the order of their PSMA expression, i.e., PC3-PIP > LNCaP > PC3, PC3-FLU. NIRF imaging showed a significant PC3-PIP tumour uptake after the injection of 1 or 5 nmol with a maximum tumour-to-muscle ratio (ca. 60) observed for both doses 24 h post-injection. Importantly, urine, healthy prostate, and the bladder were not fluorescent at 24 h post-injection. Flow cytometry and confocal images highlighted a co-localization of PSMA+ cells with IRDye700DX-PSMA uptake. Very interestingly, ex vivo analysis on a tumour specimen highlighted a significant PSMA expression by tumour-associated macrophages, likely attributable to extracellular vesicles secreted by the PSMA(+) tumour cells. FGS proved that IRDye700DX-PSMA was able to easily delineate tumour margins. PDT experiments showed a concentration-dependent decrease in cell viability (from 75% at 10 nM to 12% at 500 nM), whereas controls did not show any cytotoxicity. PC3-PIP tumour-bearing mice subjected to photodynamic therapy showed a delayed tumour growth. In conclusion, a novel PSMA-targeted NIRF dye with dual imaging-PDT capabilities was synthesized and displayed superior specificity compared to other small PSMA targeted molecules.