SOX2 promotes a cancer stem cell-like phenotype and local spreading in oral squamous cell carcinoma.

Emerging evidence shows that oral squamous cell carcinoma (OSCC) invasiveness can be attributed to a small subpopulation of cancer stem cells (CSCs) in the bulk of the tumor. However, the presence of CSCs in the OSCC close resection margins is still poorly unexplored. Here, we found that BMI1, CD44, SOX2, OCT4, UBE2C, CXCR4 CSCs marker genes are significantly upregulated, while IGF1-R, KLF4, ALDH1A1, CD133, FAM3C are downregulated in the tumor core vs healthy mucosa of 24 patients with OSCC. Among these, SOX2 appears also upregulated in the tumor close margin vs healthy mucosa and this significantly correlates with tumor size and lymph node compromise. In vitro analyses in CAL27 and SCC15 tongue squamous cell carcinoma cell lines, show that SOX2 transient knockdown i) promotes the mesenchymal-to-epithelial transition, ii) smooths the invasiveness, iii) attenuates the 3D tumor sphere-forming capacity, and iv) partially increases the sensitivity to cisplatin treatment. Overall, our study highlights that the OSCC close margins can retain CSC-specific markers. Notably, SOX2 may represent a useful CSCs marker to predict a more aggressive phenotype and a suitable target to prevent local invasiveness.

Iron affects the sphere-forming ability of ovarian cancer cells in non-adherent culture conditions.

Introduction: Detachment from the extracellular matrix (ECM) is the first step of the metastatic cascade. It is a regulated process involving interaction between tumor cells and tumor microenvironment (TME). Iron is a key micronutrient within the TME. Here, we explored the role of iron in the ability of ovarian cancer cells to successfully detach from the ECM. Methods: HEY and PEO1 ovarian cancer cells were grown in 3D conditions. To mimic an iron rich TME, culture media were supplemented with 100 µM Fe3+. Cell mortality was evaluated by cytofluorimetric assay. The invasive potential of tumor spheroids was performed in Matrigel and documented with images and time-lapses. Iron metabolism was assessed by analyzing the expression of CD71 and FtH1, and by quantifying the intracellular labile iron pool (LIP) through Calcein-AM cytofluorimetric assay. Ferroptosis was assessed by quantifying mitochondrial reactive oxygen species (ROS) and lipid peroxidation through MitoSOX and BODIPY-C11 cytofluorimetric assays, respectively. Ferroptosis markers GPX4 and VDAC2 were measured by Western blot. FtH1 knockdown was performed by using siRNA. Results: To generate spheroids, HEY and PEO1 cells prevent LIP accumulation by upregulating FtH1. 3D HEY moderately increases FtH1, and LIP is only slightly reduced. 3D PEO1upregulate FtH1 and LIP results significantly diminished. HEY tumor spheroids prevent iron import downregulating CD71, while PEO1 cells strongly enhance it. Intracellular ROS drop down during the 2D to 3D transition in both cell lines, but more significantly in PEO1 cells. Upon iron supplementation, PEO1 cells continue to enhance CD71 and FtH1 without accumulating the LIP and ROS and do not undergo ferroptosis. HEY, instead, accumulate LIP, undergo ferroptosis and attenuate their sphere-forming ability and invasiveness. FtH1 knockdown significantly reduces the generation of PEO1 tumor spheroids, although without sensitizing them to ferroptosis. Discussion: Iron metabolism reprogramming is a key event in the tumor spheroid generation of ovarian cancer cells. An iron-rich environment impairs the sphere-forming ability and causes cell death only in ferroptosis sensitive cells. A better understanding of ferroptosis sensitivity could be useful to develop effective treatments to kill ECM-detached ovarian cancer cells.

Iron-mediated oxidative stress induces PD-L1 expression via activation of c-Myc in lung adenocarcinoma.

Introduction: The PD-1/PD-L1 axis is hijacked by lung adenocarcinoma (LUAD) cells to escape immune surveillance. PD-L1 expression in LUAD is affected, among others, by the metabolic trafficking between tumor cells and the tumor microenvironment (TME). Methods: Correlation between PD-L1 expression and iron content within the TME was established on FFPE LUAD tissue samples. The effects of an iron rich microenvironment on PD-L1 mRNA and protein levels were assessed in vitro in H460 and A549 LUAD by using qPCR, western blot and flow citometry. c-Myc knockdown was performed to validate the role of this transcription factor on PD-L1 expression. The effects of iron-induced PD-L1 on T cell immune function was assessed by quantifying IFN-γ release in a co-colture system. TCGA dataset was used to analyse the correlation between PD-L1 and CD71 mRNA expression in LUAD patients. Results: In this study, we highlight a significant correlation between iron density within the TME and PD-L1 expression in 16 LUAD tissue specimens. In agreement, we show that a more pronounced innate iron-addicted phenotype, indicated by a higher transferrin receptor CD71 levels, significantly correlates with higher PD-L1 mRNA expression levels in LUAD dataset obtained from TCGA database. In vitro, we demonstrate that the addition of Fe3+ within the culture media promotes the significant overexpression of PD-L1 in A549 and H460 LUAD cells, through the modulation of its gene transcription mediated by c-Myc. The effects of iron lean on its redox activity since PD-L1 up-regulation is counteracted by treatment with the antioxidant compound trolox. When LUAD cells are co-cultured with CD3/CD28-stimulated T cells in an iron-rich culture condition, PD-L1 up-regulation causes the inhibition of T-lymphocytes activity, as demonstrated by the significant reduction of IFN-γ release. Discussion: Overall, in this study we demonstrate that iron abundance within the TME may enhance PD-L1 expression in LUAD and, thus, open the way for the identification of possible combinatorial strategies that take into account the iron levels within the TME to improve the outcomes of LUAD patients treated with anti-PD-1/PD-L1-based therapies.

Pre- and in-hospital anticoagulation therapy in coronavirus disease 2019 patients: a propensity-matched analysis of in-hospital outcomes.

AIMS: To estimate if chronic anticoagulant (CAC) treatment is associated with morbidity and mortality outcomes of patients hospitalized for SARS-CoV-2 infection. METHODS: In this European multicentric cohort study, we included 1186 patients of whom 144 were on CAC (12.1%) with positive coronavirus disease 2019 testing between 1 February and 30 July 2020. The average treatment effect (ATE) analysis with a propensity score-matching (PSM) algorithm was used to estimate the impact of CAC on the primary outcomes defined as in-hospital death, major and minor bleeding events, cardiovascular complications (CCI), and acute kidney injury (AKI). We also investigated if different dosages of in-hospital heparin were associated with in-hospital survival. RESULTS: In unadjusted populations, primary outcomes were significantly higher among CAC patients compared with non-CAC patients: all-cause death (35% vs. 18% P < 0.001), major and minor bleeding (14% vs. 8% P = 0.026; 25% vs. 17% P = 0.014), CCI (27% vs. 14% P < 0.001), and AKI (42% vs. 19% P < 0.001). In ATE analysis with PSM, there was no significant association between CAC and primary outcomes except for an increased incidence of AKI (ATE +10.2%, 95% confidence interval 0.3-20.1%, P = 0.044). Conversely, in-hospital heparin, regardless of dose, was associated with a significantly higher survival compared with no anticoagulation. CONCLUSIONS: The use of CAC was not associated with the primary outcomes except for the increase in AKI. However, in the adjusted survival analysis, any dose of in-hospital anticoagulation was associated with significantly higher survival compared with no anticoagulation.

A Rare Evolution to Pneumopericardium in Patient with COVID-19 Pneumonia Treated with High Flow Nasal Cannula.

Infection with severe acute respiratory syndrome coronavirus 2 causes coronavirus disease 2019 (COVID-19) which was revealed an official pandemic by the World Health Organization on 11 March 2020. The current pandemic, the third of this decade, is the worst in terms of suffering and deaths related. COVID-19 represents an unprecedented challenge for medical communities and patients around the world. High-resolution computed tomography of the chest (HRCT) is a fundamental tool in both management and diagnosis of the disease. Imaging plays an essential role in the diagnosis of all the manifestations of the disease and its complications and the correct use and interpretation of imaging tests are essential. Pneumomediastinum has been reported rarely in COVID-19 patients. We were one of the first groups to share our experiences in uncommon parenchymal complications of COVID-19 with spontaneous pneumothorax and pneumomediastinum, but also with new-onset bronchiectasis and cysts. A finding of pneumopericardium is also unusual. We hereby report a rare case of spontaneous pneumopericardium in a patient with COVID-19 pneumonia treated only with a high-flow nasal cannula (HFNC).

Usefulness of the HACOR score in predicting success of CPAP in COVID-19-related hypoxemia.

INTRODUCTION: In COVID-19 associated hypoxemic acute respiratory failure (ARF) without mandatory indication for urgent endotracheal intubation, a trial of CPAP may be considered. We aimed to evaluate HACOR (heart rate, acidosis, consciousness, oxygenation, respiratory rate) score performance in these patients as predictor of CPAP failure. METHODS: Prospective observational multicentric study (three centers in different countries), including adult patients with SARS-CoV-2 pneumonia admitted to a respiratory intermediate care unit, presenting PaO2/FiO2 < 300 and PaCO2 < 45 mmHg, who received CPAP. One hour after starting CPAP, HACOR was calculated. RESULTS: We enrolled 128 patients, mean age 61,7 years. Mean HACOR at 1 h after starting CPAP was 3,27 ± 3,84 and mean PaO2/FiO2 was 203,30 ± 92,21 mmHg; 35 patients (27,3 %) presented CPAP failure: 29 underwent oro-tracheal intubation and 6 died due to COVID-19 (all having a do-not-intubate order). HACOR accuracy for predicting CPAP failure was 82,03 %, while PaO2/FiO2 accuracy was 81,25 %. CONCLUSION: Although HACOR score had a good diagnostic performance in predicting CPAP failure in COVID-19-related ARF, PaO2/FiO2 has also shown to be a good predictor of failure.

The Role of High Flow Nasal Cannula in COVID-19 Associated Pneumomediastinum and Pneumothorax.

BACKGROUND: Pneumomediastinum, subcutaneous emphysema and pneumothorax are not rarely observed during the COVID-19 pandemic. Such complications can worsen gas exchange and the overall prognosis in critical patients. The aim of this study is to investigate what predisposing factors are related to pneumomediastinum and pneumothorax in SARS-CoV2-Acute Respiratory Distress Syndrome (ARDS), what symptoms may predict a severe and potentially fatal complication and what therapeutical approach may provide a better outcome. METHODS: In this single center cohort study, we recorded data from 45 critically ill COVID-19 patients who developed one or more complicating events among pneumomediastinum, subcutaneous emphysema and pneumothorax. All patients showed ARDS and underwent non-invasive ventilation (NIV) at baseline. Patients with mild to moderate ARDS and pneumomediastinum/pneumothorax (n = 25) received High Flow Nasal Cannula (HFNC), while patients with severe ARDS and pneumomediastinum/pneumothorax underwent HFNC (n = 10) or invasive mechanical ventilation (IMV) (n = 10). RESULTS: Pneumomediastinum/pneumothorax developed in 10.5% of subjects affected by SARS-coV2-ARDS. Dyspnea affected 40% and cough affected 37% of subjects. High resolution computed tomography of the chest showed bilateral diffuse ground glass opacities (GGO) in 100% of subjects. Traction bronchiolectasis, reticulation, crazy paving and distortion were observed in 64%. Furthermore, 36% showed subcutaneous emphysema. Non-severe ARDS cases received HFNC, and 76% patients recovered from pneumomediastinum/pneumothorax over a median follow up of 5 days. Among severe ARDS cases the recovery rate of pneumomediastinum/pneumothorax was 70% with the HFNC approach, and 10% with IMV. CONCLUSION: HFNC is a safe and effective ventilatory approach for critical COVID-19 and has a positive role in associated complications such as pneumomediastinum and pneumothorax.

Daytime alternatives for non-invasive mechanical ventilation in neuromuscular disorders.

Mechanical ventilation in recent years has benefited from the development of new techniques and interfaces. These developments allowed clinicians to offer increasingly personalised therapies with the combination of different complementary techniques for treating respiratory insufficiency in patients with neuromuscular diseases. The mouthpiece ventilation, intermittent abdominal pressure ventilator and the negative pressure ventilation can offer many patients alternative therapy options when ventilation is required for many hours a day. In this non-systematic review, we will highlight the use of alternative methods to non-invasive mechanical ventilation at positive pressure in neuromuscular patients, to ensure the optimal interface for each patient.

Successful intravenous immunoglobulin treatment in severe COVID-19 pneumonia.

We describe a 42-year old woman, admitted to our Department after 15 days of persistence of respiratory failure and treated with infusion of intravenous immunoglobulin with a successful outcome.

Circulating dendritic cells are severely decreased in idiopathic pulmonary fibrosis with a potential value for prognosis prediction.

Dendritic cells (DCs) accumulate in the lung of patients affected by idiopathic pulmonary fibrosis (IPF). We measured the frequencies of circulating conventional CD1c + and CD141+ cells (namely, cDC2 and cDC1) and of plasmacytoid CD303+ DCs in a cohort of 60 therapy naive IPF patients by flow cytometry. Peripheral levels of reactive oxygen species (ROS) and of pro-inflammatory and Th1/Th2 polarizing cytokines were also analyzed. All blood DC subtypes were significantly reduced in IPF patients in comparison to age- and sex-matched controls, while ROS and interleukin (IL-6) levels were augmented. IL-6 expression increased along with disease severity, according to the gender-age-physiology index, and correlated with the frequency of cDC2. IL-6 and cDC2 were not influenced by anti-fibrotic therapies but were associated with a reduced survival, the latter being an independent predictive biomarker of worse prognosis. Deciphering the role of DCs in IPF might provide information on disease pathogenesis and clinical behavior.

Subclinical impairment of dynamic left ventricular systolic and diastolic function in patients with obstructive sleep apnea and preserved left ventricular ejection fraction.

BACKGROUND: Hypoxia affects myocardial oxygen supply resulting in subclinical cardiac dysfunction in obstructive sleep apnea (OSA) patients, with cardiovascular complications being associated with increased oxidative burst (OB). The aims of our study were to assess left ventricular (LV) dynamic myocardial deformation and diastolic reserve at rest and upon exercise, along with OB determination in this patients subset. METHODS: Conventional echocardiography, Doppler myocardial imaging and LV 2D speckle tracking echocardiography were performed in 55 OSA patients with preserved LV ejection fraction (EF) and 35 age and sex-comparable healthy controls. Peripheral OB levels were evaluated by flow cytometry. RESULTS: Despite comparable LVEF, LV global longitudinal strain (GLS) was significantly reduced in OSA at rest (- 13.4 ± 3.8 vs - 18.4 ± 3.3 in controls, P <  0.001) and at peak exercise (- 15.8 ± 2.6 vs - 23.4 ± 4.3, P <  0.001). Systolic pulmonary artery pressure (sPAP) and E/E' ratios increase during effort were higher in OSA than in controls (ΔsPAP 44.3% ± 6.4 vs 32.3% ± 5.5, P <  0.0001, and ΔE/E' 87.5% ± 3.5 vs 25.4% ± 3.3, P <  0.0001, respectively). The best correlate of E/E' at peak stress was peak exertion capacity (r = - 0.50, P <  0.001). OB was also increased in OSA patients (P = 0.001) but, unlike OSA severity, was not associated with LV diastolic dysfunction. CONCLUSIONS: Evaluation of diastolic function and myocardial deformation during exercise is feasible through stress echocardiography. OSA patients with preserved LVEF show subclinical LV systolic dysfunction, impaired LV systolic and diastolic reserve, reduced exercise tolerance, and increased peripheral levels of OB. Therapy aimed at increasing LV diastolic function reserve might improve the quality of life and exercise tolerability in OSA patients.

First Description of the Hyperpnea-Hypopnea Periodic Breathing in Patients with Interstitial Lung Disease-Obstructive Sleep Apnea: Treatment Implications in a Real-Life Setting.

There is evidence that hypopneas are more common than apneas in obstructive sleep apnea (OSA) related to idiopathic pulmonary fibrosis (IPF). We investigated the frequency distribution of hypopneas in 100 patients with interstitial lung diseases (ILDs) (mean age 69 yrs ± 7.8; 70% males), including 54 IPF cases, screened for OSA by home sleep testing. Fifty age- and sex-matched pure OSA patients were included as controls. In ILD-OSA patients the sleep breathing pattern was characterized by a high prevalence of hypopneas that were preceded by hyperpnea events configuring a sort of periodic pattern. This finding, we arbitrarily defined hyperpnea-hypopnea periodic breathing (HHPB), was likely reflecting a central event and was completely absent in control OSA. Also, the HHPB was highly responsive to oxygen but not to the continuous positive pressure support. Thirty-three ILD-OSA patients (42%) with a HHPB associated with a hypopnea/apnea ratio ≥3 had the best response to oxygen with a median residual AHI of 2.6 (1.8-5.6) vs. 28.3 (20.7-37.8) at baseline (p < 0.0001). ILD-OSA patients with these characteristics were similarly distributed in IPF (54.5%) and no-IPF cases (45.5%), the most of them being affected by moderate-severe OSA (p = 0.027). Future studies addressing the pathogenesis and therapy management of the HHPB should be encouraged in ILD-OSA patients.