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Abstract Immune cell-derived extracellular vesicles (EV) affect tumor progression and hold promise for therapeutic applications. Eosinophils are major effectors in Th2-related pathologies recently implied in cancer. Here, we evaluated the anti-tumor activities of eosinophil-derived EV following activation with the alarmin IL-33. We demonstrate that IL-33-activated mouse and human eosinophils produce higher quantities of EV with respect to eosinophils stimulated with IL-5. Following incorporation of EV from IL-33-activated eosinophils (Eo33-EV), but not EV from IL-5-treated eosinophils (Eo5-EV), mouse and human tumor cells increased the expression of cyclin-dependent kinase inhibitor (CDKI)-related genes resulting in cell cycle arrest in G0/G1, reduced proliferation and inhibited tumor spheroid formation. Moreover, tumor cells incorporating Eo33-EV acquired an epithelial-like phenotype characterized by E-Cadherin up-regulation, N-Cadherin downregulation, reduced cell elongation and migratory extent in vitro, and impaired capacity to metastasize to lungs when injected in syngeneic mice. RNA sequencing revealed distinct mRNA signatures in Eo33-EV and Eo5-EV with increased presence of tumor suppressor genes and enrichment in pathways related to epithelial phenotypes and negative regulation of cellular processes in Eo33-EV compared to Eo5-EV. Our studies underscore novel IL-33-stimulated anticancer activities of eosinophils through EV-mediated reprogramming of tumor cells opening perspectives on the use of eosinophil-derived EV in cancer therapy.

Abstract We analyze data on $$B\rightarrow K\mu ^+\mu ^-$$ B → K μ + μ - and $$B\rightarrow K^*\mu ^+\mu ^-$$ B → K ∗ μ + μ - decays in the whole dilepton invariant mass spectrum with the aim of disentangling short- vs. long-distance contributions. The sizable long-distance amplitudes from $$c \overline{c}$$ c c ¯ narrow resonances are taken into account by employing a dispersive approach. For each available $$q^2=m^2_{\mu \mu }$$ q 2 = m μ μ 2 bin and each helicity amplitude an independent determination of the Wilson coefficient $$C_9$$ C 9 , describing $$b\rightarrow s\ell ^+\ell ^-$$ b → s ℓ + ℓ - transitions at short distances, is obtained. The consistency of the $$C_9$$ C 9 values thus obtained provides an a posteriori check of the absence of additional, sizable, long-distance contributions. The systematic difference of these values from the Standard Model expectation supports the hypothesis of a non-standard $$b\rightarrow s \mu ^+\mu ^-$$ b → s μ + μ - amplitude of short-distance origin.

The pathophysiology of diverticular disease (DD) is not well outlined. Recent studies performed on the DD human ex vivo model have shown the presence of a predominant transmural oxidative imbalance whose origin remains unknown. Considering the central role of mitochondria in oxidative stress, the present study evaluates their involvement in the alterations of DD clinical phenotypes. Colonic surgical samples of patients with asymptomatic diverticulosis, complicated DD, and controls were analyzed. Electron microscopy, protein expression, and cytofluorimetric analyses were performed to assess the contribution of mitochondrial oxidative stress. Functional muscle activity was tested on cells in response to contractile and relaxant agents. To assess the possibility of reverting oxidative damages, N-acetylcysteine was tested on an in vitro model. Compared with the controls, DD tissues showed a marketed increase in mitochondrial number and fusion accompanied by the altered mitochondrial electron transport chain complexes. In SMCs, the mitochondrial mass increase was accompanied by altered mitochondrial metabolic activity supported by a membrane potential decrease. Ulteriorly, a decrease in antioxidant content and altered contraction–relaxation dynamics reverted by N-acetylcysteine were observed. Therefore, the oxidative stress-driven alterations resulted in mitochondrial impairment. The beneficial effects of antioxidant treatments open new possibilities for tailored therapeutic strategies that have not been tested for this disease.

We assessed the impact of DNA damage response and repair (DDR) biomarker expressions in 222 node-positive early breast cancer (BC) patients from a previous Phase III GOIM 9902 trial of adjuvant taxanes. At a median follow-up of 64 months, the original study showed no disease-free survival (DFS) or overall survival (OS) differences with the addition of docetaxel (D) to epirubicine-cyclophosphamide (EC). Immunohistochemistry was employed to assess the expression of DDR phosphoproteins (pATM, pATR, pCHK1, γH2AX, pRPA32, and pWEE1) in tumor tissue, and their association with clinical outcomes was evaluated through the Cox elastic net model. Over an extended follow-up of 234 months, we confirmed no significant differences in DFS or OS between patients treated with EC and those receiving D → EC. A DDR risk score, inversely driven by ATM and ATR expression, emerged as an independent prognostic factor for both DFS (HR = 0.41, p < 0.0001) and OS (HR = 0.61, p = 0.046). Further validation in a public adjuvant BC cohort was possible only for ATM, confirming its protective role. Overall, our findings confirm the potential role of the DDR pathway in BC prognostication and in shaping treatment strategies advocating for an integrated approach, combining molecular markers with clinical–pathological factors.

A phylogenetic conservation analysis of Trop-2 across vertebrate species showed a high degree of sequence conservation, permitting to explore multiple models as pre-clinical benchmarks. Sequence divergence and incomplete conservation of expression patterns were observed in mouse and rat. Primate Trop-2 sequences were found to be 95%–100% identical to the human sequence. Comparative three-dimension primate Trop-2 structures were obtained with AlphaFold and homology modeling. This revealed high structure conservation of Trop-2 (0.66 ProMod3 GMQE, 0.80–0.86 ± 0.05 QMEANDisCo scores), with conservative amino acid changes at variant sites. Primate TACSTD2/TROP2 cDNAs were cloned and transfectants for individual ORF were shown to be efficiently recognized by humanized anti-Trop-2 monoclonal antibodies (Hu2G10, Hu2EF). Immunohistochemistry analysis of Macaca mulatta (rhesus monkey) tissues showed Trop-2 expression patterns that closely followed those in human tissues. This led us to test Trop-2 targeting in vivo in Macaca fascicularis (cynomolgus monkey). Intravenously injected Hu2G10 and Hu2EF were well tolerated from 5 to 10 mg/kg. Neither neurological, respiratory, digestive, urinary symptoms, nor biochemical or hematological toxicities were detected during 28-day observation. Blood serum pharmacokinetic (PK) studies were conducted utilizing anti-idiotypic antibodies in capture-ELISA assays. Hu2G10 (t1/2 = 6.5 days) and Hu2EF (t1/2 = 5.5 days) were stable in plasma, and were detectable in the circulation up to 3 weeks after the infusion. These findings validate primates as reliable models for Hu2G10 and Hu2EF toxicity and PK, and support the use of these antibodies as next-generation anti-Trop-2 immunotherapy tools.

Dapagliflozin, a sodium-glucose co-transporter-2 inhibitor and semaglutide, a glucagon-like peptide 1 receptor agonist, have both demonstrated efficacy in glycemic control, reducing blood pressure, body weight, risk of renal and heart failure in type 2 diabetes mellitus. In this observational, real-world, study we aimed to investigate the efficacy of the combination therapy with those two agents over glycemic control. We thus obtained the data of 1335 patients with type 2 diabetes followed by 11 Diabetes centers in Lombardia, Italy. A group of 443 patients was treated with dapagliflozin alone, the other group of 892 patients was treated with the combination therapy of dapagliflozin plus oral semaglutide. We analyzed changes in glycated hemoglobin from baseline to 6 months of follow-up, as well as changes in fasting glycemia, body weight, body mass index, systolic and diastolic pressure, heart rate, creatinine, estimated glomerular filtration rate and albuminuria. Both groups of patients showed an improvement of glycometabolic control after 6 months of treatment; indeed, the treatment with dapagliflozin plus oral semaglutide showed a reduction of glycated hemoglobin of 1.2% as compared to the 0.5% reduction observed in the dapagliflozin alone group. Significant changes were observed in body mass index, fasting plasmatic glucose, blood pressure, total cholesterol, LDL and albumin to creatinine ratio, with a high rate (55%) of near-normalization of glycated hemoglobin. Our real world data confirmed the potential of the oral combination therapy dapagliflozin with semaglutide in inducing pharmacological remission of type 2 diabetes mellitus.

Hereditary cancer syndromes caused by germline mutations account for 5–10% of all cancers. The finding of a genetic mutation could have far-reaching consequences for pharmaceutical therapy, personalized prevention strategies, and cascade testing. According to the National Comprehensive Cancer Network’s (NCCN) and the Italian Association of Medical Oncology (AIOM) guidelines, unaffected family members should be tested only if the affected one is unavailable. This article explores whether germline genetic testing may be offered to high-risk families for hereditary cancer even if a living affected relative is missing. A retrospective study was carried out on 103 healthy subjects tested from 2017 to 2023. We enrolled all subjects with at least two first- or second-degree relatives affected by breast, ovarian, pancreatic, gastric, prostate, or colorectal cancer. All subjects were tested by Next Generation Sequencing (NGS) multi-gene panel of 27 cancer-associated genes. In the study population, 5 (about 5%) pathogenic/likely pathogenic variants (PVs/LPVs) were found, while 40 (42%) had a Variant of Uncertain Significance (VUS). This study highlights the importance of genetic testing for individuals with a strong family history of hereditary malignancies. This approach would allow women who tested positive to receive tailored treatment and prevention strategies based on their personal mutation status.

The use of anthracycline derivatives was approved for the treatment of a broad spectrum of human tumors (i.e., breast cancer). The need to test these drugs on cancer models has pushed the basic research to apply many types of in vitro assays, and, among them, the study of anthracycline-induced apoptosis was mainly based on the application of flow cytometry protocols. However, the chemical structure of anthracycline derivatives gives them a strong autofluorescence effect that must be considered when flow cytometry is used. Unfortunately, the guidelines on the analysis of anthracycline effects through flow cytometry are lacking. Therefore, in this study, we optimized the flow cytometry detection of doxorubicin and epirubicin-treated breast cancer cells. Their autofluorescence was assessed both by using conventional and imaging flow cytometry; we found that all the channels excited by the 488 nm laser were affected. Anthracycline-induced apoptosis was then measured via flow cytometry using the optimized setting. Consequently, we established a set of recommendations that enable the development of optimized flow cytometry settings when the in vitro assays of anthracycline effects are analyzed, with the final aim to reveal a new perspective on the use of those in vitro tests for the further implementation of precision medicine strategies in cancer.

Disruptions to the ER-Golgi network can lead to neurodevelopmental disorders, though our understanding of these Golgipathies remains incomplete. Here Lauri, Tartaglia and colleagues show that ARF3 mutations cause a rare pediatric neurological disorder and perform detailed molecular characterization in fish.

BackgroundIn triple negative breast cancer patients treated with neoadjuvant chemotherapy, residual disease at surgery is the most relevant unfavorable prognostic factor. Current guidelines consider the use of adjuvant capecitabine, based on the results of the randomized CREATE-X study, carried out in Asian patients and including a small subset of triple negative tumors. Thus far, evidence on Caucasian patients is limited, and no real-world data are available.MethodsWe carried out a multicenter, observational study, involving 44 oncologic centres. Triple negative breast cancer patients with residual disease, treated with adjuvant capecitabine from January 2017 through June 2021, were recruited. We primarily focused on treatment tolerability, with toxicity being reported as potential cause of treatment discontinuation. Secondarily, we assessed effectiveness in the overall study population and in a subset having a minimum follow-up of 2 years.ResultsOverall, 270 patients were retrospectively identified. The 50.4% of the patients had residual node positive disease, 7.8% and 81.9% had large or G3 residual tumor, respectively, and 80.4% a Ki-67 >20%. Toxicity-related treatment discontinuation was observed only in 10.4% of the patients. In the whole population, at a median follow-up of 15 months, 2-year disease-free survival was 62%, 2 and 3-year overall survival 84.0% and 76.2%, respectively. In 129 patients with a median follow-up of 25 months, 2-year disease-free survival was 43.4%, 2 and 3-year overall survival 78.0% and 70.8%, respectively. Six or more cycles of capecitabine were associated with more favourable outcomes compared with less than six cycles.ConclusionThe CaRe study shows an unexpectedly good tolerance of adjuvant capecitabine in a real-world setting, although effectiveness appears to be lower than that observed in the CREATE-X study. Methodological differences between the two studies impose significant limits to comparability concerning effectiveness, and strongly invite further research.

Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.

GIM 13-AMBRA is a longitudinal cohort study aimed at describing therapeutic strategies and the relative outcome parameters in 939 HER2-ve MBC patients. Taxanes–based regimens, or taxanes + targeted agents, mainly Bevacizumab, were the preferred first choice in both Luminal (30.2%) and TNBC (33.3%) patients. The median PFS1 was 12.5 months (95% CI 16.79–19.64), without any significant difference according to subtypes, while the median Time to first Treatment Change (TTC1) was significantly lower in TNBC patients (7.7 months—95% CI 5.7–9.2) in comparison to Luminal A (13.2 months, 95% CI 11.7–15.1) and Luminal B patients (11.8 months, 95% CI 10.3–12.8). PFS2 was significantly shorter in TNBC patients (5.5 months, 95% CI 4.3–6.5 vs. Luminal A—9.4, 95% CI 8.1–10.7, and Luminal B—7.7 95% CI 6.8–8.2, F-Ratio 4.30, p = 0.014). TTC2 was significantly lower in patients with TNBC than in those with the other two subtypes. The median OS1 was 35.2 months (95% CI 30.8–37.4) for Luminal A patients, which was significantly higher than that for both Luminal B (28.9 months, 95% CI 26.2–31.2) and TNBC (18.5 months, 95% CI 16–20.1, F-ratio 7.44, p = 0.0006). The GIM 13—AMBRA study is one of the largest collections ever published in Italy and provides useful results in terms of time outcomes for first, second, and further lines of treatment in HER2- MBC patients.

Selinexor, an oral Selective Inhibitor of Nuclear Export, targets Exportin 1 (XPO1, also termed CRM1). Non-clinical studies support combining selinexor with proteasome inhibitors (PIs) and corticosteroids to overcome resistance in relapsed/refractory multiple myeloma (RRMM). We conducted a phase I dose-escalation trial of twice-weekly selinexor in combination with carfilzomib and dexamethasone (SKd) to determine maximum tolerated dose in patients with RRMM (N = 21), with an expansion cohort to assess activity in carfilzomib-refractory disease and identify a recommended phase II dose (RP2D). During dose escalation, there was one dose-limiting toxicity (cardiac failure). The RP2D of twice-weekly SKd was selinexor 60 mg, carfilzomib 20/27 mg/m2 and dexamethasone 20 mg. The most common grade 3/4 treatment-emergent adverse events included thrombocytopenia (71%), anaemia (33%), lymphopenia (33%), neutropenia (33%) and infections (24%). Rates of ≥minimal response, ≥partial response and very good partial response were 71%, 48% and 14%, respectively; similar response outcomes were observed for dual-class refractory (PI and immunomodulatory drug)/quad-exposed (carfilzomib, bortezomib, lenalidomide and pomalidomide) patients (n = 17), and patients refractory to carfilzomib in last line of therapy (n = 13). Median progression-free survival was 3·7 months, and overall survival was 22·4 months in the overall population. SKd was tolerable and re-established disease control in RRMM patients, including carfilzomib-refractory patients. Registered at ClinicalTrials.gov (NCT02199665).

Abstract Integrase Defective Lentiviral Vectors (IDLVs) represent an attractive vaccine platform for delivering HIV-1 antigens, given their ability to induce specific and persistent immune responses in both mice and non-human primates (NHPs). Recent advances in HIV-1 immunogen design demonstrated that native-like HIV-1 Envelope (Env) trimers that mimic the structure of virion-associated Env induce neutralization breadth in rabbits and macaques. Here, we describe the development of an IDLV-based HIV-1 vaccine expressing either soluble ConSOSL.UFO.664 or membrane-tethered ConSOSL.UFO.750 native-like Env immunogens with enhanced bNAb epitopes exposure. We show that IDLV can be pseudotyped with properly folded membrane-tethered native-like UFO.750 trimers. After a single IDLV injection in BALB/c mice, IDLV-UFO.750 induced a faster humoral kinetic as well as higher levels of anti-Env IgG compared to IDLV-UFO.664. IDLV-UFO.750 vaccinated cynomolgus macaques developed unusually long-lasting anti-Env IgG antibodies, as underlined by their remarkable half-life both after priming and boost with IDLV. After boosting with recombinant ConM SOSIP.v7 protein, two animals developed neutralization activity against the autologous tier 1B ConS virus mediated by V1/V2 and V3 glycan sites responses. By combining the possibility to display stabilized trimeric Env on the vector particles with the ability to induce sustained humoral responses, IDLVs represent an appropriate strategy for delivering rationally designed antigens to progress towards an effective HIV-1 vaccine.

The pathophysiology of diverticular disease (DD) is not well outlined. Recent studies performed on the DD human ex vivo model have shown the presence of a predominant transmural oxidative imbalance whose origin remains unknown. Considering the central role of mitochondria in oxidative stress, the present study evaluates their involvement in the alterations of DD clinical phenotypes. Colonic surgical samples of patients with asymptomatic diverticulosis, complicated DD, and controls were analyzed. Electron microscopy, protein expression, and cytofluorimetric analyses were performed to assess the contribution of mitochondrial oxidative stress. Functional muscle activity was tested on cells in response to contractile and relaxant agents. To assess the possibility of reverting oxidative damages, N-acetylcysteine was tested on an in vitro model. Compared with the controls, DD tissues showed a marketed increase in mitochondrial number and fusion accompanied by the altered mitochondrial electron transport chain complexes. In SMCs, the mitochondrial mass increase was accompanied by altered mitochondrial metabolic activity supported by a membrane potential decrease. Ulteriorly, a decrease in antioxidant content and altered contraction–relaxation dynamics reverted by N-acetylcysteine were observed. Therefore, the oxidative stress-driven alterations resulted in mitochondrial impairment. The beneficial effects of antioxidant treatments open new possibilities for tailored therapeutic strategies that have not been tested for this disease. [ABSTRACT FROM AUTHOR]

Simple Summary: This study evaluates DDR biomarkers in 222 node-positive early breast cancer patients from a Phase III trial on adjuvant taxanes. Over a 234-month follow-up period, no differences in DFS or OS were observed between treatment groups. However, a DDR risk score, influenced by ATM and ATR expression, proved to be an independent prognostic factor for both DFS and OS. Validation in a public cohort confirmed ATM's protective role. These findings highlight the importance of DDR pathways in breast cancer prognostication and support, integrating molecular markers with clinical–pathological factors to inform treatment strategies. We assessed the impact of DNA damage response and repair (DDR) biomarker expressions in 222 node-positive early breast cancer (BC) patients from a previous Phase III GOIM 9902 trial of adjuvant taxanes. At a median follow-up of 64 months, the original study showed no disease-free survival (DFS) or overall survival (OS) differences with the addition of docetaxel (D) to epirubicine-cyclophosphamide (EC). Immunohistochemistry was employed to assess the expression of DDR phosphoproteins (pATM, pATR, pCHK1, γH2AX, pRPA32, and pWEE1) in tumor tissue, and their association with clinical outcomes was evaluated through the Cox elastic net model. Over an extended follow-up of 234 months, we confirmed no significant differences in DFS or OS between patients treated with EC and those receiving D → EC. A DDR risk score, inversely driven by ATM and ATR expression, emerged as an independent prognostic factor for both DFS (HR = 0.41, p < 0.0001) and OS (HR = 0.61, p = 0.046). Further validation in a public adjuvant BC cohort was possible only for ATM, confirming its protective role. Overall, our findings confirm the potential role of the DDR pathway in BC prognostication and in shaping treatment strategies advocating for an integrated approach, combining molecular markers with clinical–pathological factors. [ABSTRACT FROM AUTHOR]

Immune cell-derived extracellular vesicles (EV) affect tumor progression and hold promise for therapeutic applications. Eosinophils are major effectors in Th2-related pathologies recently implied in cancer. Here, we evaluated the anti-tumor activities of eosinophil-derived EV following activation with the alarmin IL-33. We demonstrate that IL-33-activated mouse and human eosinophils produce higher quantities of EV with respect to eosinophils stimulated with IL-5. Following incorporation of EV from IL-33-activated eosinophils (Eo33-EV), but not EV from IL-5-treated eosinophils (Eo5-EV), mouse and human tumor cells increased the expression of cyclin-dependent kinase inhibitor (CDKI)-related genes resulting in cell cycle arrest in G0/G1, reduced proliferation and inhibited tumor spheroid formation. Moreover, tumor cells incorporating Eo33-EV acquired an epithelial-like phenotype characterized by E-Cadherin up-regulation, N-Cadherin downregulation, reduced cell elongation and migratory extent in vitro, and impaired capacity to metastasize to lungs when injected in syngeneic mice. RNA sequencing revealed distinct mRNA signatures in Eo33-EV and Eo5-EV with increased presence of tumor suppressor genes and enrichment in pathways related to epithelial phenotypes and negative regulation of cellular processes in Eo33-EV compared to Eo5-EV. Our studies underscore novel IL-33-stimulated anticancer activities of eosinophils through EV-mediated reprogramming of tumor cells opening perspectives on the use of eosinophil-derived EV in cancer therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: The purpose of this study is to emphasize the importance of genetic testing for healthy individuals with a strong family history of hereditary malignancies. A total of 103 healthy subjects with at least two relatives with cancer were enrolled. By NGS analysis of 27 genes, 5% were found to carry a pathogenic variant in a hereditary cancer susceptibility gene. In the era of personalized medicine, genetic testing of healthy subjects in the absence of a living affected collateral is crucially important for early diagnosis, clinical surveillance and surgical choice. Hereditary cancer syndromes caused by germline mutations account for 5–10% of all cancers. The finding of a genetic mutation could have far-reaching consequences for pharmaceutical therapy, personalized prevention strategies, and cascade testing. According to the National Comprehensive Cancer Network's (NCCN) and the Italian Association of Medical Oncology (AIOM) guidelines, unaffected family members should be tested only if the affected one is unavailable. This article explores whether germline genetic testing may be offered to high-risk families for hereditary cancer even if a living affected relative is missing. A retrospective study was carried out on 103 healthy subjects tested from 2017 to 2023. We enrolled all subjects with at least two first- or second-degree relatives affected by breast, ovarian, pancreatic, gastric, prostate, or colorectal cancer. All subjects were tested by Next Generation Sequencing (NGS) multi-gene panel of 27 cancer-associated genes. In the study population, 5 (about 5%) pathogenic/likely pathogenic variants (PVs/LPVs) were found, while 40 (42%) had a Variant of Uncertain Significance (VUS). This study highlights the importance of genetic testing for individuals with a strong family history of hereditary malignancies. This approach would allow women who tested positive to receive tailored treatment and prevention strategies based on their personal mutation status. [ABSTRACT FROM AUTHOR]

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of reactive oxygen species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes in gravitational force, even though published data on germ cell models are scarce. We previously studied the effects of simulated microgravity (s-microgravity) on a 2D cultured TCam-2 seminoma-derived cell line, considered the only human cell line available to study in vitro mitotically active human male germ cells. In this study, we used a corresponding TCam-2 3D cell culture model that mimics cell–cell contacts in organ tissue to test the possible effects induced by s-microgravity exposure. TCam-2 cell spheroids were cultured for 24 h under unitary gravity (Ctr) or s-microgravity conditions, the latter obtained using a random positioning machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels was observed after RPM exposure. In line with these results, a trend of protein and lipid oxidation increase and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis via transmission electron microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, with GPX1, NCF1, and CYBB being downregulated, whereas NOX4 and HMOX1 are upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated with the observed mitochondria alteration. Altogether, these data demonstrate TCam-2 spheroid sensitivity to acute s-microgravity exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow them to overcome the exposure to altered gravitational force.

Trop-2 proteolytic processing in cancer cells exposes epitopes that were specifically targeted by the 2G10 antibody. We sought additional recognition of Trop-2 within difficult-to-reach, densely packed tumor sites. Trop-2 deletion mutants were employed in immunization and screening procedures, and these led to the recognition of a novel epitope in the N-terminal region of Trop-2, by the 2EF antibody. The 2EF mAb was shown to bind Trop-2 at cell–cell junctions in MCF-7 breast cancer cells, and in deeply seated sites in prostate cancer, that were inaccessible to benchmark anti-Trop-2 antibodies. The 2EF antibody was shown to inhibit the growth of HT29 colon tumor cells in vitro, with the highest activity at high cell density. In vivo, 2EF showed anticancer activity against SKOv3 ovarian, Colo205, HT29, HCT116 colon and DU-145 prostate tumors, with the highest impact on densely packed tumor sites, whereby 2EF outcompeted benchmark anti-Trop-2 antibodies. Given the different recognition modes of Trop-2 by 2EF and 2G10, we hypothesized the effective interaction of the two mAb in vivo. The 2EF mAb was indeed demonstrated to enhance the activity of 2G10 against tumor xenotransplants, opening novel avenues for Trop-2-targeted therapy. We humanized 2EF by state-of-the-art CDR grafting/re-modeling, yielding the Hu2EF for therapy of Trop-2-expressing tumors in patients.

Summary: Trogocytosis is a cellular process whereby a cell acquires a membrane fragment from a donor cell in a contact-dependent manner allowing for the transfer of surface proteins with functional integrity. It is involved in various biological processes, including cell-cell communication, immune regulation, and response to pathogens and cancer cells, with poorly defined molecular mechanisms. With the exception of eosinophils, trogocytosis has been reported in most immune cells and plays diverse roles in the modulation of anti-tumor immune responses. Here, we report that eosinophils acquire membrane fragments from tumor cells early after contact through the CD11b/CD18 integrin complex. We discuss the impact of trogocytosis in innate immune cells on cancer progression in the context of the evidence that eosinophils can engage in trogocytosis with tumor cells. We also discuss shared and cell-specific mechanisms underlying this process based on in silico modeling and provide a hypothetical molecular model for the stabilization of the immunological synapse operating in granulocytes and possibly other innate immune cells that enables trogocytosis.

RPAP3 is a subunit of the R2TP complex, a co-chaperone of HSP90, with substrate proteins involved in transcription, ribosome biogenesis, DNA repair and cell growth. Here the authors report that deletion of Rpap3 abrogates cell proliferation and homeostasis in mouse intestine, partly through destabilization of PI3K-like kinases, while elevated RPAP3 levels in colorectal tumors are associated with poor prognosis.

Abstract Background In Western countries, ovarian cancer (OC) still represents the leading cause of gynecological cancer-related deaths, despite the remarkable gains in therapeutical options. Novel biomarkers of early diagnosis, prognosis definition and prediction of treatment outcomes are of pivotal importance. Prior studies have shown the potentials of micro-ribonucleic acids (miRNAs) as biomarkers for OC and other cancers. Methods We focused on the prognostic and/or predictive potential of miRNAs in OC by conducting a comprehensive array profiling of miRNA expression levels in ovarian tissue samples from 17 non-neoplastic controls, and 60 tumor samples from OC patients treated at the Regina Elena National Cancer Institute (IRE). A set of 54 miRNAs with differential expression in tumor versus normal samples (T/N-deregulated) was identified in the IRE cohort and validated against data from the Cancer Genoma Atlas (TCGA) related to 563 OC patients and 8 non-neoplastic controls. The prognostic/predictive role of the selected 54 biomarkers was tested in reference to survival endpoints and platinum resistance (P-res). Results In the IRE cohort, downregulation of the 2 miRNA-signature including miR-99a-5p and miR-320a held a negative prognostic relevance, while upregulation of miR-224-5p was predictive of less favorable event free survival (EFS) and P-res. Data from the TCGA showed that downregulation of 5 miRNAs, i.e., miR-150, miR-30d, miR-342, miR-424, and miR-502, was associated with more favorable EFS and overall survival outcomes, while miR-200a upregulation was predictive of P-res. The 9 miRNAs globally identified were all included into a single biologic signature, which was tested in enrichment analysis using predicted/validated miRNA target genes, followed by network representation of the miRNA-mRNA interactions. Conclusions Specific dysregulated microRNA sets in tumor tissue showed predictive/prognostic value in OC, and resulted in a promising biological signature for this disease.

Abstract In metastatic breast cancer (mBC), the change of human epidermal growth factor receptor 2 (HER2) status between primary and metastatic lesions is widely recognized, however clinical implications are unknown. Our study address the question if relevant differences exist between subjects who preserve the HER2 status and those who gain the HER2 positivity when relapsed. Data of patients affected by HER2-positive mBC, treated with pertuzumab and/or trastuzumab-emtansine (T-DM1) in a real-world setting at 45 Italian cancer centers were retrospectively collected and analyzed. From 2003 to 2017, 491 HER2‐positive mBC patients were included. Of these, 102 (20.7%) had been initially diagnosed as HER2-negative early BC. Estrogen and/or progesterone receptor were more expressed in patients with HER2-discordance compared to patients with HER2-concordant status (p

Dapagliflozin has been demonstrated to improve glycemic control, blood pressure, and body weight in type 2 diabetes mellitus (T2D); indeed, it can also reduce the risk of progression to renal failure, of hospitalization for heart failure and of cardiovascular death. We aim to investigate the acute effect of Dapagliflozin on kidney function in the common clinical practice in T2D. This is a study including 1402 patients with T2D recruited from 11 centers in Lombardia, Italy, who were evaluated at baseline and after 6 months of treatment with Dapagliflozin 10 mg per day. The primary outcome of the study was the change in HbA1c, while the secondary outcomes were modification of weight, BMI, systolic and diastolic pressure, creatinine, eGFR and albuminuria status. After 24 weeks of treatment with Dapagliflozin, a reduction in Hb1Ac was observed (−0.6 ± 1.8%) as well as in BMI (−1.5 ± 5.2 kg/m2). Statistically significant changes were also found for systolic and diastolic blood pressure, cholesterol and triglycerides. Interestingly, a statistically significant acute improvement of kidney function was evident. Our analyses confirm the beneficial effects of dapagliflozin after 6 months of therapy, with improvements of glycemic and lipid profiles, blood pressure, BMI. Finally, an acute positive effect on albuminuria and KIDGO classes was observed during a 6 months treatment with dapagliflozin in patients with T2D.

Abstract The 90K protein, also known as Mac-2 BP or LGALS3BP, can activate the immune response in part by increasing major histocompatibility (MHC) class I levels. In studies on a non-immune cell model, the rat FRTL-5 cell line, we observed that transforming growth factor (TGF)-β1, like γ-interferon (IFN), increased 90K levels, despite its immunosuppressive functions and the ability to decrease MHC class I. To explain this paradoxical result, we investigated the mechanisms involved in the TGF-β1 regulation of 90K expression with the aim to demonstrate that TGF-β1 utilizes different molecular pathways to regulate the two genes. We found that TGF-β1 was able to increase the binding of Upstream Stimulatory Factors, USF1 and USF2, to an E-box element, CANNTG, at −1926 to −1921 bp, upstream of the interferon response element (IRE) in the 90K promoter. Thyrotropin (TSH) suppressed constitutive and γ-IFN-induced 90K expression by decreasing USF binding to the E-box. TGF-β1 was able to overcome TSH suppression at the transcriptional level by increasing USF binding to the E-box. We suggest that the ability of TGF-β1 to increase 90K did not result in an increase in MHC class I because of a separate suppressive action of TGF-β1 directly on the MHC class I gene. We propose that the increased levels of 90K may play a role, rather than in immune response, in the context of the TGF-β1-induced changing of the cellular microenvironment that predisposes to cell motility and cancer progression. Consistently, analyzing the publicly available cancer patient data sets cBioPortal, we found that 90K expression directly correlated with TGF-β1 and USFs and that high levels of 90K were significantly associated with increased mortality in patients affected by different types of cancer.