Viability qPCR, a new tool for Legionella risk management

Background Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. Methods The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. Results In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p < 0.001). Otherwise, no significant differences between methods were found in the rest of the groups. Conclusions The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy

Cancer cell sensitivity to bortezomib is associated with survivin expression and p53 status but not cancer cell types

<p>Abstract</p> <p>Background</p> <p>Survivin is known playing a role in drug resistance. However, its role in bortezomib-mediated inhibition of growth and induction of apoptosis is unclear. There are conflicting reports for the effect of bortezomib on survivin expression, which lacks of a plausible explanation. Methods: In this study, we tested cancer cells with both p53 wild type and mutant/null background for the relationship of bortezomib resistance with survivin expression and p53 status using MTT assay, flow cytometry, DNA fragmentation, caspase activation, western blots and RNAi technology.</p> <p>Results</p> <p>We found that cancer cells with wild type p53 show a low level expression of survivin and are sensitive to treatment with bortezomib, while cancer cells with a mutant or null p53 show a high level expression of survivin and are resistant to bortezomib-mediated apoptosis induction. However, silencing of survivin expression utilizing survivin mRNA-specific siRNA/shRNA in p53 mutant or null cells sensitized cancer cells to bortezomib mediated apoptosis induction, suggesting a role for survivin in bortezomib resistance. We further noted that modulation of survivin expression by bortezomib is dependent on p53 status but independent of cancer cell types. In cancer cells with mutated p53 or p53 null, bortezomib appears to induce survivin expression, while in cancer cells with wild type p53, bortezomib downregulates or shows no significant effect on survivin expression, which is dependent on the drug concentration, cell line and exposure time.</p> <p>Conclusions</p> <p>Our findings, for the first time, unify the current inconsistent findings for bortezomib treatment and survivin expression, and linked the effect of bortezomib on survivin expression, apoptosis induction and bortezomib resistance in the relationship with p53 status, which is independent of cancer cell types. Further mechanistic studies along with this line may impact the optimal clinical application of bortezomib in solid cancer therapeutics.</p

KRAS-mutant non-small cell lung cancer (NSCLC) therapy based on tepotinib and omeprazole combination

Background KRAS-mutant non-small cell lung cancer (NSCLC) shows a relatively low response rate to chemotherapy, immunotherapy and KRAS-G12C selective inhibitors, leading to short median progression-free survival, and overall survival. The MET receptor tyrosine kinase (c-MET), the cognate receptor of hepatocyte growth factor (HGF), was reported to be overexpressed in KRAS-mutant lung cancer cells leading to tumor-growth in anchorage-independent conditions. Methods Cell viability assay and synergy analysis were carried out in native, sotorasib and trametinib-resistant KRAS-mutant NSCLC cell lines. Colony formation assays and Western blot analysis were also performed. RNA isolation from tumors of KRAS-mutant NSCLC patients was performed and KRAS and MET mRNA expression was determined by real-time RT-qPCR. In vivo studies were conducted in NSCLC (NCI-H358) cell-derived tumor xenograft model. Results Our research has shown promising activity of omeprazole, a V-ATPase-driven proton pump inhibitor with potential anti-cancer properties, in combination with the MET inhibitor tepotinib in KRAS-mutant G12C and non-G12C NSCLC cell lines, as well as in G12C inhibitor (AMG510, sotorasib) and MEK inhibitor (trametinib)-resistant cell lines. Moreover, in a xenograft mouse model, combination of omeprazole plus tepotinib caused tumor growth regression. We observed that the combination of these two drugs downregulates phosphorylation of the glycolytic enzyme enolase 1 (ENO1) and the low-density lipoprotein receptor-related protein (LRP) 5/6 in the H358 KRAS G12C cell line, but not in the H358 sotorasib resistant, indicating that the effect of the combination could be independent of ENO1. In addition, we examined the probability of recurrence-free survival and overall survival in 40 early lung adenocarcinoma patients with KRAS G12C mutation stratified by KRAS and MET mRNA levels. Significant differences were observed in recurrence-free survival according to high levels of KRAS mRNA expression. Hazard ratio (HR) of recurrence-free survival was 7.291 (p = 0.014) for high levels of KRAS mRNA expression and 3.742 (p = 0.052) for high MET mRNA expression. Conclusions We posit that the combination of the V-ATPase inhibitor omeprazole plus tepotinib warrants further assessment in KRAS-mutant G12C and non G12C cell lines, including those resistant to the covalent KRAS G12C inhibitors

HMGB1 Expression Levels Correlate with Response to Immunotherapy in Non-Small Cell Lung Cancer

Maria González-Cao,1 Xueting Cai,2 Jilian Wilhelmina Paulina Bracht,3 Xuan Han,2 Yang Yang,2 Carlos Pedraz-Valdunciel,4 Teresa Morán,5 Javier García-Corbacho,6 Andrés Aguilar,1 Reyes Bernabé,7 Pedro De Marchi,8,9 Luciane Sussuchi da Silva,8 Leticia Ferro Leal,8 Rui Manuel Reis,8,10,11 Jordi Codony-Servat,4 Eloisa Jantus-Lewintre,12– 14 Miguel Angel Molina-Vila,4 Peng Cao,2,15 Rafael Rosell1,16 1Translational Cancer Research Unit, Instituto Oncológico Dr Rosell, Dexeus University Hospital, Barcelona, Spain; 2Integrated Traditional Chinese and Western Medicine Department of Affiliated Hospital, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China; 3Amsterdam University Medical Center (UMC), Amsterdam, The Netherlands; 4Laboratory of Oncology, Pangaea Oncology, Quirón Dexeus University Hospital, Barcelona, Spain; 5Medical Oncology Department, Catalan Institute of Oncology (ICO), Germans Trias i Pujol Hospital, Badalona, Spain; 6Medical Oncology Department (Hospital Clinic)/Translational Genomics and Targeted Therapies in Solid Tumors (IDIBAPs), Barcelona, Spain; 7Medical Oncology Department, Hospital Universitario Virgen del Rocío, Sevilla, Spain; 8Molecular Oncology Research Center; Barretos Cancer Hospital, Barretos, Brazil; 9Oncoclinicas, Rio de Janeiro, Brazil; 10Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal; 11ICVS/3b’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal; 12Valencian Community Foundation Principe Felipe Research Center, Laboratory of Molecular Oncology, Valencia, Spain; 13Centro de Investigación Biomédica en Red (CIBERONC), Madrid, Spain; 14Universitat Politècnica de Valencia, Biotechnology Department, Valencia, Spain; 15College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China; 16Laboratory of Molecular Biology, Germans Trias i Pujol Health Sciences Institute and Hospital (IGTP), Badalona, SpainCorrespondence: Rafael Rosell, Laboratory of Molecular Biology, Germans Trias i Pujol Health Sciences Institute and Hospital (IGTP), Camí de les Escoless/n, Badalona, Barcelona, 08916, Spain, Tel +34 930330520, Email [email protected] Peng Cao, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, People’s Republic of China, Tel +86 85608666, Email [email protected]: High-mobility group box 1 protein (HMGB1) is subject to exportin 1 (XPO1)-dependent nuclear export, and it is involved in functions implicated in resistance to immunotherapy. We investigated whether HMGB1 mRNA expression was associated with response to immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC).Patients and Methods: RNA was isolated from pretreatment biopsies of patients with advanced NSCLC treated with ICI. Gene expression analysis of several genes, including HMGB1, was conducted using the NanoString Counter analysis system (PanCancer Immune Profiling Panel). Western blotting analysis and cell viability assays in EGFR and KRAS mutant cell lines were carried out. Evaluation of the antitumoral effect of ICI in combination with XPO1 blocker (selinexor) and trametinib was determined in a murine Lewis lung carcinoma model.Results: HMGB1 mRNA levels in NSCLC patients treated with ICI correlated with progression-free survival (PFS) (median PFS 9.0 versus 18.0 months, P=0.008, hazard ratio=0.30 in high versus low HMGB1). After TNF-α stimulation, HMGB1 accumulates in the cytoplasm of PC9 cells, but this accumulation can be prevented by using selinexor or antiretroviral drugs. Erlotinib or osimertinib with selinexor in EGFR-mutant cells and trametinib plus selinexor in KRAS mutant abolish tumor cell proliferation. Selinexor with a PD-1 inhibitor with or without trametinib abrogates the tumor growth in the murine Lewis lung cancer model.Conclusion: An in-depth exploration of the functions of HMGB1 mRNA and protein is expected to uncover new potential targets and provide a basis for treating metastatic NSCLC in combination with ICI.Keywords: HMGB1, immunotherapy, non-small cell lung cancer, Lewis lung cancer murine model, K-Ras mutation

Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17

Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFα-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment