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Abstract Background Lung regions excluded from mechanical insufflation are traditionally assumed to be spared from ventilation-associated lung injury. However, preliminary data showed activation of potential mechanisms of injury within these non-ventilated regions (e.g., hypoperfusion, inflammation). Methods In the present study, we hypothesized that non-ventilated lung injury (NVLI) may develop within 24 h of unilateral mechanical ventilation in previously healthy pigs, and we performed extended pathophysiological measures to profile NVLI. We included two experimental groups undergoing exclusion of the left lung from the ventilation with two different tidal volumes (15 vs 7.5 ml/kg) and a control group on bilateral ventilation. Pathophysiological alteration including lung collapse, changes in lung perfusion, lung stress and inflammation were measured. Lung injury was quantified by histological score. Results Histological injury score of the non-ventilated lung is significantly higher than normally expanded lung from control animals. The histological score showed lower intermediate values (but still higher than controls) when the tidal volume distending the ventilated lung was reduced by 50%. Main pathophysiological alterations associated with NVLI were: extensive lung collapse; very low pulmonary perfusion; high inspiratory airways pressure; and higher concentrations of acute-phase inflammatory cytokines IL-6, IL-1β and TNF-α and of Angiopoietin-2 (a marker of endothelial activation) in the broncho-alveolar lavage. Only the last two alterations were mitigated by reducing tidal volume, potentially explaining partial protection. Conclusions Non-ventilated lung injury develops within 24 h of controlled mechanical ventilation due to multiple pathophysiological alterations, which are only partially prevented by low tidal volume.

Abstract We retrospectively investigated in women treated with fulvestrant for HR+/HER2 negative advanced breast cancer clinical, pathological and molecular features associated with long-term benefit from treatment defined as being progression-free at 18 months. Specifically, we analyzed on formalin-fixed paraffin-embedded tumor samples ESR1 and PI3KCA mutations and miRNAs profiles. 59 patients were evaluable (median age of 67 years, range 32–92). 18-month PFS rate was 27%; the lack of visceral metastases significantly predicted the likelihood of being progression-free at 18 months, while PI3KCA mutations, found in 36% of patients, were not associated with 18-month PFS. As of miRNAs, miR-549a, miR-644a, miR-16-5p were negatively while let-7c-5p was positively associated with 18-month PFS. In addition, miR-520d-3p and miR-548g-3p values were significantly lower while miR-603, miR-181a-5p and miR-199a-miR-199b-3p values were significantly higher in patients achieving 18-month PFS. In silico analysis of targets modulated by these two latter groups of miRNAs show that in patients achieving 18-month PFS the Hippo and Wnt signaling pathways were predicted to be upregulated while endocrine resistance was potentially repressed by miR-603, miR-181a-5p and miR-199a-miR-199b-3p. Our results provide additional clues on the molecular mechanisms involved in fulvestrant activity and resistance. Underlying pathways should be further elucidated and confirmed in larger cohorts.

IntroductionThe bone matrix protein osteocalcin (OC), secreted by osteoblasts, displays endocrine effects. We tested the hypothesis that OC modulates parathyroid tumor cell function.MethodsPrimary cell cultures derived from parathyroid adenomas (PAds) and HEK293 cells transiently transfected with the putative OC receptor GPRC6A or the calcium sensing receptor (CASR) were used as experimental models to investigate γ-carboxylated OC (GlaOC) or uncarboxylated OC (GluOC) modulation of intracellular signaling.ResultsIn primary cell cultures derived from PAds, incubation with GlaOC or GluOC modulated intracellular signaling, inhibiting pERK/ERK and increasing active β-catenin levels. GlaOC increased the expression of PTH, CCND1 and CASR, and reduced CDKN1B/p27 and TP73. GluOC stimulated transcription of PTH, and inhibited MEN1 expression. Moreover, GlaOC and GluOC reduced staurosporin-induced caspase 3/7 activity. The putative OC receptor GPRC6A was detected in normal and tumor parathyroids at membrane or cytoplasmic level in cells scattered throughout the parenchyma. In PAds, the membrane expression levels of GPRC6A and its closest homolog CASR positively correlated; GPRC6A protein levels positively correlated with circulating ionized and total calcium, and PTH levels of the patients harboring the analyzed PAds. Using HEK293A transiently transfected with either GPRC6A or CASR, and PAds-derived cells silenced for CASR, we showed that GlaOC and GluOC modulated pERK/ERK and active β-catenin mainly through CASR activation.ConclusionParathyroid gland emerges as a novel target of the bone secreted hormone osteocalcin, which may modulate tumor parathyroid CASR sensitivity and parathyroid cell apoptosis.

IntroductionChronic lung allograft dysfunction (CLAD) is the main cause of the reduced survival of lung transplanted (LTx) patients. The possible role of immune checkpoint molecules in establishing tolerance has been scarcely investigated in the setting of lung transplantation.MethodsWe conducted a retrospective, observational pilot study on a consecutive series of transbronchial cryobiopsies (TCB) obtained from 24 patients during LTx follow-up focusing on PD-1, one of the most investigated immune checkpoint molecules.ResultsResults showed that PD-1-expressing T lymphocytes were present in all TCB with a histological diagnosis of acute rejection (AR; 9/9), but not in most (11/15) of the TCB not resulting in a diagnosis of AR (p=0.0006). Notably, the presence of PD-1-expressing T lymphocytes in TCB resulted in a 10-times higher risk of developing chronic lung allograft dysfunction (CLAD), the main cause of the reduced survival of lung transplanted patients, thus being associated with a clearly worst clinical outcome.DiscussionResults of this pilot study indicate a central role of PD-1 in the development of AR and its evolution towards CLAD and suggest that the evaluation of PD-1-expressing lymphocytes in TCB could offer a prognostic advantage in monitoring the onset of AR in patients who underwent lung transplantation.

Background: Breast cancer with osteoclast-like stromal giant cells (OSGC) is an exceedingly rare morphological pattern of invasive breast carcinoma. The tumor immune microenvironment (TIME) of these tumors is populated by OSGC, which resemble osteoclasts and show a histiocytic-like immunophenotype. Their role in breast cancer is unknown. The osteoclast maturation in the bone is regulated by the expression of cytokines that are also present in the TIME of tumors and in breast cancer tumor-associated macrophages (TAMs). TAMs-mediated anti-tumor immune pathways are regulated by miRNAs akin to osteoclast homeostasis. Here, we sought to characterize the different cellular compartments of breast cancers with OSGC and investigate the similarities of OSGC with tumor and TIME in terms of morphology, protein, and miRNA expression, specifically emphasizing on monocytic signatures.Methods and Results: Six breast cancers with OSGC were included. Tumor-infiltrating lymphocytes (TILs) and TAMs were separately quantified. The different cellular populations (i.e., normal epithelium, cancer cells, and OSGC) were isolated from tissue sections by laser-assisted microdissection. After RNA purification, 752 miRNAs were analyzed using a TaqMan Advanced miRNA Low-Density Array for all samples. Differentially expressed miRNAs were identified by computing the fold change (log2Ratio) using the Kolmogorov-Smirnov test and p values were corrected for multiple comparisons using the false discovery rate (FDR) approach. As a similarity analysis among samples, we used the Pearson test. The association between pairs of variables was investigated using Fisher exact test. Classical and non-classical monocyte miRNA signatures were finally applied. All OSGC displayed CD68 expression, TILs (range, 45–85%) and high TAMs (range, 35–75%). Regarding the global miRNAs profile, OSGC was more similar to cancer cells than to non-neoplastic ones. Shared deregulation of miR-143-3p, miR-195-5p, miR-181a-5p, and miR-181b-5p was observed between OSGC and cancer cells. The monocyte-associated miR-29a-3p and miR-21-3p were dysregulated in OSGCs compared with non-neoplastic or breast cancer tissues.Conclusion: Breast cancers with OSGC have an activated TIME. Shared epigenetic events occur during the ontogenesis of breast cancer cells and OSGC but the innumophenotype and miRNA profiles of the different cellular compartmens suggest that OSGC likely belong to the spectrum of M2 TAMs.

Abstract Triple negative breast cancer (TNBC) accounts for about a fifth of all breast cancers and includes a diverse group of cancers. The heterogeneity of TNBC and the lack of target receptors on the cell surface make it difficult to develop specific therapeutic treatments. These aspects cause the high negative prognosis of patients with this type of tumor. The analysis of the molecular profiles of TNBC samples has allowed a better characterization of this tumor, supporting the search for new reliable diagnostic markers. To this end, we have developed a bioinformatic approach to integrate networks of genes differentially expressed in basal breast cancer compared to healthy tissues, with miRNAs able to regulate their expression. We studied the role of these miRNAs in TNBC subtype cell lines. We therefore identified two miRNAs, namely miR-135b and miR-365, with a central role in regulating the altered functional pathways in basal breast cancer. These two miRNAs are differentially expressed in human TNBC immunohistochemistry-selected tissues, and their modulation has been shown to play a role in the proliferation of tumor control and its migratory and invasive capacity in TNBC subtype cell lines. From the perspective of personalized medicine, we managed to modulate the expression of the two miRNAs in organotypic cultures, suggesting their possible use as diagnostic and therapeutic molecules. miR-135b and miR-365 have a key role in TNBC, controlling proliferation and invasion. Their detection could be helpful in TNBC diagnosis, while their modulation could become a new therapeutic tool for TNBC.

Abstract Despite the promising results achieved so far in long-term survival after lung transplantation (LuTx), airway complications (ACs) still arise in the post-operative period. Early diagnosis and prompt treatment of ACs play a critical role in preventing their onset. Specifically, large bronchi ischemia has been recognized as a triggering factor for ACs. Autofluorescence bronchoscopy, which was first introduced for early cancer diagnosis, displays ischemic mucosae as red spots, while normal vascularized mucosae appear in green. The aim of this study is to investigate whether a significant correlation exists between ACs and the red/green (RG) ratio detected on scheduled autofluorescence bronchoscopy up to 1 year after LuTx. This prospective, observational, single-center cohort study initially considered patients who underwent LuTx between July 2014 and February 2016. All patients underwent concomitant white-light and autofluorescence bronchoscopy at baseline (immediately after LuTx), on POD7, POD14, POD21, POD28, POD45, 3 months, 6 months, and 1 year after LuTx. An autofluorescence image of the first bronchial carina distal to the anastomosis was captured and analyzed using histograms for red and green pixels; the R/G ratio was then recorded. Potential ACs were classified according according to the presence of a white-light following the MDS (macroscopic aspect, diameter and suture) criteria. The authors assessed the association between the R/G ratio and the ACs occurrence using a generalized estimating equations model. Thirty-one patients met the inclusion criteria and were therefore selected. Out of a total of 53 bronchial anastomoses, 8 developed complications (late bronchial stenosis), with an average onset time of 201 days after LuTx. ACs showed a similar baseline covariate value when compared to anastomoses that involved no complication. Generalized estimating equations regression indicated a clear association over time between the R/G ratio and the rise of complications (p = 0.023). The authors observed a significant correlation between post-anastomotic stenosis and the delayed decrease of the R/G ratio. Preliminary outcomes suggest that autofluorescence bronchoscopy may be an effective and manageable diagnostic tool, proving complementary to other instruments for early diagnosis of ACs after LuTx. Further research is needed to confirm and detail preliminary findings.

Long non-coding RNAs (lncRNAs) are an important class of epigenetic regulators involved in both physiological processes and cancer development. Preliminary evidence suggested that lncRNAs could act as accurate prognostic and diagnostic biomarkers. Parathyroid cancer is a rare endocrine neoplasia, whose management represents a clinical challenge due to the lack of accurate molecular biomarkers. Our previous findings showed that human parathyroid tumors are characterized by a different lncRNAs signature, suggesting heterogeneity through the different histotypes. Particularly, we found that the lncRNA BC200/BCYRN1 could represent a candidate biomarker for parathyroid carcinomas (PCas). Here we aimed to extend our preliminary data evaluating whether BC200 could be an accurate non-invasive biomarker of PCas to support the clinical management of patients affected by parathyroid tumors at diagnosis, prognosis and follow-up. To provide a non-invasive point-of-care for parathyroid carcinoma diagnosis and follow-up, we analyzed BC200 expression in patients’ serum through digital PCR. Our results show that BC200 counts are higher in serum from patients harboring PCa (n=4) compared to patients with parathyroid adenoma (PAd; n=27). Further, in PAd patients circulating BC200 levels are positively correlated with serum total calcium. Then, we found that BC200 is overexpressed in metastatic PCas (n=4) compared to non-metastatic ones (n=9). Finally, the lncRNA expression in PCa patients’ serum drops are reduced after parathyroidectomy, suggesting its possible use in the post-operative setting for patients follow-up. Overall, these findings extend the knowledge on BC200 in parathyroid tumors, supporting its role as a useful biomarker for management of PCa.

BackgroundUnilateral ligation of the pulmonary artery (UPAL) induces bilateral lung injury in pigs undergoing controlled mechanical ventilation. Possible mechanisms include redistribution of ventilation toward the non-ligated lung and hypoperfusion of the ligated lung. The addition of 5% CO2 to the inspiratory gas (FiCO2) prevents the injury, but it is not clear whether lung protection is a direct effect of CO2 inhalation or it is mediated by plasmatic hypercapnia. This study aims to compare the effects and mechanisms of FiCO2vs. hypercapnia induced by low tidal volume ventilation or instrumental dead space.MethodsHealthy pigs underwent left UPAL and were allocated for 48 h to the following: Volume-controlled ventilation (VCV) with VT 10 ml/kg (injury, n = 6); VCV plus 5% FiCO2 (FiCO2, n = 7); VCV with VT 6 ml/kg (low VT, n = 6); VCV plus additional circuit dead space (instrumental VD, n = 6). Histological score, regional compliance, wet-to-dry ratio, and inflammatory infiltrate were assessed to evaluate lung injury at the end of the study. To investigate the mechanisms of protection, we quantified the redistribution of ventilation to the non-ligated lung, as the ratio between the percentage of tidal volume to the right and to the left lung (VTRIGHT/LEFT), and the hypoperfusion of the ligated lung as the percentage of blood flow reaching the left lung (PerfusionLEFT).ResultsIn the left ligated lung, injury was prevented only in the FiCO2 group, as indicated by lower histological score, higher regional compliance, lower wet-to-dry ratio and lower density of inflammatory cells compared to other groups. For the right lung, the histological score was lower both in the FiCO2 and in the low VT groups, but the other measures of injury showed lower intensity only in the FiCO2 group. VTRIGHT/LEFT was lower and PerfusionLEFT was higher in the FiCO2 group compared to other groups.ConclusionIn a model of UPAL, inhaled CO2 but not hypercapnia grants bilateral lung protection. Mechanisms of protection include reduced overdistension of the non-ligated and increased perfusion of the ligated lung.

Abstract The outbreak of COVID-19 and SARS-CoV-2 infection is spreading worldwide as the first coronavirus pandemic. The clinical picture is variable but flu-like symptoms are common with bilateral interstitial pneumonia being the most frightening presentation. No specific therapies nor vaccine have been developed to date and the only way to limit the virus diffusion is by modifying one’s lifestyle limiting social life and following strict hygienic precautions. No data is available on the risk of COVID-19 and its outcomes in celiac disease (CeD). The restrictions applied to counter COVID-19 can impact on CeD treatment and gluten-free dieting, the only available therapy for CeD. With the present manuscript, we aim to support gastroenterologists and nutritionists in the management of CeD patients in the new pandemic scenario, being conscious that availability and local situations are extremely various.

Changes in metabolism are a hallmark of cancer, but molecular signatures of altered bioenergetics to aid in clinical decision-making do not currently exist. We recently identified a group of human tumors with constitutively reduced expression of the mitochondrial structural protein, Mic60, also called mitofilin or inner membrane mitochondrial protein (IMMT). These Mic60-low tumors exhibit severe loss of mitochondrial fitness, paradoxically accompanied by increased metastatic propensity and upregulation of a unique transcriptome of Interferon (IFN) signaling and Senescence-Associated Secretory Phenotype (SASP). Here, we show that an optimized, 11-gene signature of Mic60-low tumors is differentially expressed in multiple malignancies, compared to normal tissues, and correlates with poor patient outcome. When analyzed in three independent patient cohorts of pancreatic ductal adenocarcinoma (PDAC), the Mic60-low gene signature was associated with aggressive disease variants, local inflammation, FOLFIRINOX failure and shortened survival, independently of age, gender, or stage. Therefore, the 11-gene Mic60-low signature may provide an easily accessible molecular tool to stratify patient risk in PDAC and potentially other malignancies.

Chronic lung allograft dysfunction (CLAD) is the main cause of poor survival and low quality of life of lung transplanted patients. Several studies have addressed the role of dendritic cells, macrophages, T cells, donor specific as well as anti-HLA antibodies, and interleukins in CLAD, but the expression and function of immune checkpoint molecules has not yet been analyzed, especially in the two CLAD subtypes: BOS (bronchiolitis obliterans syndrome) and RAS (restrictive allograft syndrome). To shed light on this topic, we conducted an observational study on eight consecutive grafts explanted from patients who received lung re-transplantation for CLAD. The expression of a panel of immune molecules (PD1/CD279, PDL1/CD274, CTLA4/CD152, CD4, CD8, hFoxp3, TIGIT, TOX, B-Cell-Specific Activator Protein) was analyzed by immunohistochemistry in these grafts and in six control lungs. Results showed that RAS compared to BOS grafts were characterized by 1) the inversion of the CD4/CD8 ratio; 2) a higher percentage of T lymphocytes expressing the PD-1, PD-L1, and CTLA4 checkpoint molecules; and 3) a significant reduction of exhausted PD-1-expressing T lymphocytes (PD-1pos/TOXpos) and of exhausted Treg (PD-1pos/FOXP3pos) T lymphocytes. Results herein, although being based on a limited number of cases, suggest a role for checkpoint molecules in the development of graft rejection and offer a possible immunological explanation for the worst prognosis of RAS. Our data, which will need to be validated in ampler cohorts of patients, raise the possibility that the evaluation of immune checkpoints during follow-up offers a prognostic advantage in monitoring the onset of rejection, and suggest that the use of compounds that modulate the function of checkpoint molecules could be evaluated in the management of chronic rejection in LTx patients.

BackgroundDespite significant improvement in screening programs for cancers of the respiratory district, especially in at-risk subjects, early disease detection is still a major issue. In this scenario, new molecular and non-invasive biomarkers are needed to improve early disease diagnosis.MethodsWe profiled the miRNome in exhaled breath condensate (EBC) and plasma samples from fourteen patients affected by lung AdCa, nine healthy subjects. miRNA signatures were then analyzed in another neoplasia of the respiratory district, i.e. pleural mesothelioma (n = 23) and subjects previously exposed to asbestos were used as controls for this cohort (n = 19). Selected miRNAs were analyzed in purified pulmonary neoplastic or normal epithelial and stromal cell subpopulation from AdCa patients. Finally, the plasmatic miRNA signature was analyzed in a publicly available cohort of NSCLC patients for data validation and in silico analysis was performed with predicted miRNA targets using the multiMiR tool and STRING database.ResultsmiR-597-5p and miR-1260a are significantly over-expressed in EBC from lung AdCa and are associated with AdCa. Similarly, miR-1260a is also up-regulated in the plasma of AdCa patients together with miR-518f-3p and correlates with presence of lung cancer, whereas let-7f-5p is under-expressed. Analysis of these circulating miRNAs in pleural mesothelioma cases confirmed that up-regulation of miR-518f-3p, -597-5p and -1260a, is specific for lung AdCa. Lastly, quantification of the miRNAs in laser-assisted microdissected lung tissues revealed that miR-518f-3p, 597-5p and miR-1260a are predominantly expressed in tumor epithelial cells. Validation analysis confirmed miR-518f-3p as a possible circulating biomarker of NSCLC. In silico analysis of the potentially modulated biological processes by these three miRNAs, shows that tumor bioenergetics are the most affected pathways.ConclusionsOverall, our data suggest a 3-miRNAs signature as a non-invasive and accurate biomarker of lung AdCa. This approach could supplement the current screening approaches for early lung cancer diagnosis.

Background: Mitochondrial functions are exploited in cancer and provide a validated therapeutic target. However, how this process is regulated has remained mostly elusive and the identification of new pathways that control mitochondrial integrity in cancer is an urgent priority. Methods: We studied clinically-annotated patient series of primary and metastatic prostate cancer, representative cases of multiple myeloma (MM) and publicly available genetic databases. Gene regulation studies involved chromatin immunoprecipitation, PCR amplification and Western blotting of conditional Myc-expressing cell lines. Transient or stable gene silencing was used to quantify mitochondrial functions in bioenergetics, outer membrane permeability, Ca2+ homeostasis, redox balance and cell death. Tumorigenicity was assessed by cell proliferation, colony formation and xenograft tumour growth. Findings: We identified Mitochondrial Fission Factor (MFF) as a novel transcriptional target of oncogenic Myc overexpressed in primary and metastatic cancer, compared to normal tissues. Biochemically, MFF isoforms, MFF1 and MFF2 associate with the Voltage-Dependent Anion Channel-1 (VDAC1) at the mitochondrial outer membrane, in vivo. Disruption of this complex by MFF silencing induces general collapse of mitochondrial functions with increased outer membrane permeability, loss of inner membrane potential, Ca2+ unbalance, bioenergetics defects and activation of cell death pathways. In turn, this inhibits tumour cell proliferation, suppresses colony formation and reduces xenograft tumour growth in mice. Interpretation: An MFF-VDAC1 complex is a novel regulator of mitochondrial integrity and actionable therapeutic target in cancer. Keywords: Mitochondria, MFF, Cell death, Tumour metabolism, VDAC1, Cancer therapy

Abstract Background Lung cancer is still the main cause of cancer death worldwide despite the availability of targeted therapies and immune-checkpoint inhibitors combined with chemotherapy. Cancer cell heterogeneity and primary or acquired resistance mechanisms cause the elusive behaviour of this cancer and new biomarkers and active drugs are urgently needed to overcome these limitations. p65BTK, a novel isoform of the Bruton Tyrosine Kinase may represent a new actionable target in non-small cell lung cancer (NSCLC). Methods p65BTK expression was evaluated by immunohistochemistry in 382 NSCLC patients with complete clinico-pathological records including smoking habit, ALK and EGFR status, and in metastatic lymph nodes of 30 NSCLC patients. NSCLC cell lines mutated for p53 and/or a component of the RAS/MAPK pathway and primary lung cancer-derived cells from Kras/Trp53 null mice were used as a preclinical model. The effects of p65BTK inhibition by BTK Tyrosine Kinase Inhibitors (TKIs) (Ibrutinib, AVL-292, RN486) and first-generation EGFR-TKIs (Gefitinib, Erlotinib) on cell viability were evaluated by MTT. The effects of BTK-TKIs on cell growth and clonogenicity were assessed by crystal violet and colony assays, respectively. Cell toxicity assays were performed to study the effect of the combination of non-toxic concentrations of BTK-TKIs with EGFR-TKIs and standard-of-care (SOC) chemotherapy (Cisplatin, Gemcitabine, Pemetrexed). Results p65BTK was significantly over-expressed in EGFR-wild type (wt) adenocarcinomas (AdC) from non-smoker patients and its expression was also preserved at the metastatic site. p65BTK was also over-expressed in cell lines mutated for KRAS or for a component of the RAS/MAPK pathway and in tumors from Kras/Trp53 null mice. BTK-TKIs were more effective than EGFR-TKIs in decreasing cancer cell viability and significantly impaired cell proliferation and clonogenicity. Moreover, non-toxic doses of BTK-TKIs re-sensitized drug-resistant NSCLC cell lines to both target- and SOC therapy, independently from EGFR/KRAS status. Conclusions p65BTK results as an emerging actionable target in non-smoking EGFR-wt AdC, also at advanced stages of disease. Notably, these patients are not eligible for EGFR-TKIs-based therapy due to a lack of EGFR mutation. The combination of BTK-TKIs with EGFR-TKIs is cytotoxic for EGFR-wt/KRAS-mutant/p53-null tumors and BTK-TKIs re-sensitizes drug-resistant NSCLC to SOC chemotherapy. Therefore, our data suggest that adding BTK-TKIs to SOC chemotherapy and EGFR-targeted therapy may open new avenues for clinical trials in currently untreatable NSCLC.

Background: Cancer cells use specific V-ATPase subunits to activate oncogenic pathways. Therefore, we investigated V-ATPase deregulation in aggressive gliomas and associated signaling. Methods: V-ATPase genes expression and associated pathways were analyzed in different series of glioma available from public databases, as well as in patients' cohort. Activation of pathways was analyzed at gene and protein expression levels. A genetic model of glioma in Drosophila melanogaster and mice with GBM patients-derived orthotopic xenografts were used as in vivo models of disease. Findings: GBM and recurrent gliomas display a specific V-ATPase signature. Such signature resolves the heterogeneous class of IDH-wild type lower-grade gliomas, identifying the patients with worse prognosis independently from clinical and molecular features (p = 0·03, by Cox proportional-hazards model). In vivo, V-ATPase subunits deregulation significantly impacts tumor growth and proliferation. At the molecular level, GBM-like V-ATPase expression correlates with upregulation of Homeobox genes. Interpretation: Our data identify a V-ATPase signature that accompanies glioma aggressiveness and suggest new entry points for glioma stratification and follow-up. Fund: This work was supported by Fondazione Cariplo (2014–1148 to VV), Fondazione IRCCS Ca' Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV). Keywords: V-ATPase, IDHwt/lower-grade glioma, Homeobox genes, Glioma stem cells

Background: The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. Methods: Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. Findings: GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. Interpretation: We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. Fund: This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF). Keywords: V-ATPase, Homeobox genes, Glioma stem cells, Large oncosome

Despite following a gluten-free diet, which is currently the only effective therapy for celiac disease, about 5% of patients can develop serious complications, which in the case of refractory type 2 could evolve towards intestinal lymphoma. In this study, we have identified a set of 15 microRNAs in serum discriminating between the two types of refractory disease. Upregulated miR-770-5p, miR-181b-2-3p, miR-1193, and miR-1226-3p could be useful for the better stratification of patients and the monitoring of disease development, while miR-490-3p was found to be dysregulated in patients with refractory type 1. Finally, by using bioinformatic tools applied to the analysis of the targets of dysregulated microRNAs, we have completed a more precise assessment of their functions. These mainly include the pathway of response to Transforming Growth Factor β cell–cell signaling by Wnt; epigenetic regulation, especially novel networks associated with transcriptional and post-transcriptional alterations; and the well-known inflammatory profiles.

Glioblastoma (GBM) is a highly aggressive tumor of the brain. Despite the efforts, response to current therapies is poor and 2-years survival rate ranging from 6-12%. Here, we evaluated the preclinical efficacy of Metformin (MET) as add-on therapy to Temozolomide (TMZ) and the ability of [18F]FLT (activity of thymidine kinase 1 related to cell proliferation) and [18F]VC701 (translocator protein, TSPO) Positron Emission Tomography (PET) radiotracers to predict tumor response to therapy. Indeed, TSPO is expressed on the outer mitochondrial membrane of activated microglia/macrophages, tumor cells, astrocytes and endothelial cells. TMZ-sensitive (Gli36ΔEGFR-1 and L0627) or -resistant (Gli36ΔEGFR-2) GBM cell lines representative of classical molecular subtype were tested in vitro and in vivo in orthotopic mouse models. Our results indicate that in vitro, MET increased the efficacy of TMZ on TMZ-sensitive and on TMZ-resistant cells by deregulating the balance between pro-survival (bcl2) and pro-apoptotic (bax/bad) Bcl-family members and promoting early apoptosis in both Gli36ΔEGFR-1 and Gli36ΔEGFR-2 cells. In vivo, MET add-on significantly extended the median survival of tumor-bearing mice compared to TMZ-treated ones and reduced the rate of recurrence in the TMZ-sensitive models. PET studies with the cell proliferation radiopharmaceutical [18F]FLT performed at early time during treatment were able to distinguish responder from non-responder to TMZ but not to predict the duration of the effect. On the contrary, [18F]VC701 uptake was reduced only in mice treated with MET plus TMZ and levels of uptake negatively correlated with animals’ survival. Overall, our data showed that MET addition improved TMZ efficacy in GBM preclinical models representative of classical molecular subtype increasing survival time and reducing tumor relapsing rate. Finally, results from PET imaging suggest that the reduction of cell proliferation represents a common mechanism of TMZ and combined treatment, whereas only the last was able to reduce TSPO. This reduction was associated with the duration of treatment response. TSPO-ligand may be used as a complementary molecular imaging marker to predict tumor microenvironment related treatment effects.

Breast cancer tumors originating from mammary luminal epithelial cells are highly heterogeneous. Here, the authors show MYC-driven tumor initiation is reliant on cell reprogramming via an epigenetic program which leads to mammary luminal epithelial cells acquiring basal/stem cell-like properties.

Tumors of the parathyroid glands are the second most common endocrine neoplasia. Epigenetic studies revealed an embryonic signature involved in parathyroid tumorigenesis. Here, we investigated the expression of the stem core genes SOX2, POU5F1/OCT4, and NANOG. Rare cells within normal parathyroid glands expressed POU5F1/OCT4 and NANOG, while SOX2 was undetectable. Nuclear SOX2 expression was detectable in 18% of parathyroid adenomas (PAds, n = 34) involving 5–30% of cells, while OCT4 and NANOG were expressed at the nuclear level in a more consistent subset of PAds involving 15–40% of cells. Most parathyroid carcinomas expressed the core stem genes. SOX2-expressing cells co-expressed parathormone (PTH). In PAds-derived primary cultures, silencing of the tumor suppressor gene MEN1 induced the expression of SOX2, likely through a MEN1/HAR1B/SOX2 axis, while calcium-sensing receptor activation increased SOX2 mRNA levels through YAP1 activation. In addition, inducing nuclear β-catenin accumulation in PAds-derived primary cultures by short-term incubation with lithium chloride (LiCl), SOX2 and POU5F1/OCT4 expression levels increased, while NANOG transcripts were reduced, and LiCl long-term incubation induced an opposite pattern of gene expression. In conclusion, detection of the core stem genes in parathyroid tumors supports their embryogenic signature, which is modulated by crucial genes involved in parathyroid tumorigenesis.

Mitochondria have a controversial role in cancer. Here, the authors demonstrate the reprogramming of a neuronal network of mitochondrial trafficking in tumor cells, and identify Syntaphilin as a key protein that suppresses organelle dynamics thereby blocking chemotaxis and metastasis in mice.

We herein characterize the immunopathological features of two Italian COVID-19 patients who underwent bilateral lung transplantation (bLTx). Removed lungs underwent histopathological evaluation. Gene expression profiling (GEP) for immune-related signatures was performed on lung specimens and SARS-CoV-2-stimulated peripheral blood mononuclear cells (PBMCs). Cytokine levels were measured on lungs, bronchoalveolar lavage fluids and in culture supernatants. Pathological assessment showed extensive lung damage with the pattern of proliferative to fibrotic phases, with diffuse alveolar damage mimicking usual interstitial pneumonia (UIP). Lungs’ GEP revealed overexpression of pathogen recognition receptors, effector cytokines and chemokines, immune activation receptors and of the inflammasome components. Multiplex cytokine analysis confirmed a proinflammatory state, with high levels of monocyte/macrophage chemotactic and activating factors and of IL-6 and TNF-α. A similar profile was observed in SARS-CoV-2-stimulated PBMCs collected 7 days after transplant. The pattern of tissue damage observed in the lungs suggests that this may represent the output of protracted disease, resembling a diffuse UIP-like picture. The molecular immune profiling supports the paradigm of a persistent proinflammatory state and sustained humoral immunity, conditions that are maintained despite the iatrogenic immunosuppression.

Mitochondria must buffer the risk of proteotoxic stress to preserve bioenergetics, but the role of these mechanisms in disease is poorly understood. Using a proteomics screen, we now show that the mitochondrial unfoldase-peptidase complex ClpXP associates with the oncoprotein survivin and the respiratory chain Complex II subunit succinate dehydrogenase B (SDHB) in mitochondria of tumor cells. Knockdown of ClpXP subunits ClpP or ClpX induces the accumulation of misfolded SDHB, impairing oxidative phosphorylation and ATP production while activating "stress" signals of 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy. Deregulated mitochondrial respiration induced by ClpXP targeting causes oxidative stress, which in turn reduces tumor cell proliferation, suppresses cell motility, and abolishes metastatic dissemination in vivo. ClpP is universally overexpressed in primary and metastatic human cancer, correlating with shortened patient survival. Therefore, tumors exploit ClpXP-directed proteostasis to maintain mitochondrial bioenergetics, buffer oxidative stress, and enable metastatic competence. This pathway may provide a "drugable" therapeutic target in cancer.

The original version of this Article contained an error in the spelling of the author Miriam Gaggianesi, which was incorrectly given as Miriam Giaggianesi. Furthermore, the affiliation details for Gabriella Gaudioso, Valentina Vaira, and Silvano Bosari incorrectly omitted ‘Division of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, 20122, Italy’. Finally, the affiliation details for Alice Turdo, Miriam Gaggianesi, Aurora Chinnici and Elisa Lipari were incorrectly given as ‘Dipartimento di Biotecnologie Mediche e Medicina Legale Sezione di Biochimica Medica, Facoltà di Medicina e Chirurgia, Policlinico “P.Giaccone”, Università di Palermo, Palermo, 90127, Italy’. The correct affiliation is ‘Department of Surgical, Oncological and Stomatological Sciences, University of Palermo, Palermo, 90127, Italy’. These errors have now been corrected in both the PDF and HTML versions of the Article.

Most men diagnosed with prostate cancer will have an indolent and curable disease, whereas approximately 15% of these patients will rapidly progress to a castrate-resistant and metastatic stage with high morbidity and mortality. Therefore, the identification of molecular signature(s) that detect men at risk of progressing disease remains a pressing and still unmet need for these patients. Here, we used an integrated discovery platform combining prostate cancer cell lines, a Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model and clinically-annotated human tissue samples to identify loss of expression of microRNA-34b as consistently associated with prostate cancer relapse. Mechanistically, this was associated with epigenetics silencing of the MIR34B/C locus and increased DNA copy number loss, selectively in androgen-dependent prostate cancer. In turn, loss of miR-34b resulted in downstream deregulation and overexpression of the "stemness" marker, Sox2. These findings identify loss of miR-34b as a robust biomarker for prostate cancer progression in androgen-sensitive tumors, and anticipate a potential role of progenitor/stem cell signaling in this stage of disease.

Lung cancer encompasses a constellation of malignancies with no validated prognostic markers. p16Ink4A expression has been reported in different subtypes of lung cancers; however, its prognostic value is controversial. Here, we sought to investigate the clinical significance of p16Ink4A immunoexpression according to specific staining patterns and its operational implications. A total of 502 tumors, including 277 adenocarcinomas, 84 squamous cell carcinomas, 22 large cell carcinomas, 47 typical carcinoids, 12 atypical carcinoids, 28 large cell neuroendocrine carcinomas, and 32 small cell carcinomas were reviewed and subjected to immunohistochemical analysis for p16Ink4A and Ki67. The spectrum of p16Ink4A expression was annotated for each case as negative, sporadic, focal, or diffuse. Expression at immunohistochemical level showed intra-tumor homogeneity, regardless tumor histotype. Enrichments in cells expressing p16Ink4A were observed from lower- to higher-grade neuroendocrine malignancies, whereas a decrease was seen in poorly and undifferentiated non-neuroendocrine carcinomas. Tumor proliferation indices were higher in neuroendocrine tumors expressing p16Ink4A while non-neuroendocrine malignancies immunoreactive for p16Ink4A showed a decrease in Ki67-positive cells. Quantitative statistical analyses including each histotype and the p16Ink4A status confirmed the independent prognostic role of p16Ink4A expression, being a high-risk indicator in neuroendocrine tumors and a marker of good prognosis in non-neuroendocrine lung malignancies. In this study, we provide circumstantial evidence to suggest that the routinary assessment of p16Ink4A expression using a three-tiered scoring algorithm, even in a small biopsy, may constitute a reliable, reproducible, and cost-effective substrate for a more accurate risk stratification of each individual patient.