Holistic Characterization of a Salmonella Typhimurium Infection Model Using Integrated Molecular Imaging.

A more complete and holistic view on host-microbe interactions is needed to understand the physiological and cellular barriers that affect the efficacy of drug treatments and allow the discovery and development of new therapeutics. Here, we developed a multimodal imaging approach combining histopathology with mass spectrometry imaging (MSI) and same section imaging mass cytometry (IMC) to study the effects of Salmonella Typhimurium infection in the liver of a mouse model using the S. Typhimurium strains SL3261 and SL1344. This approach enables correlation of tissue morphology and specific cell phenotypes with molecular images of tissue metabolism. IMC revealed a marked increase in immune cell markers and localization in immune aggregates in infected tissues. A correlative computational method (network analysis) was deployed to find metabolic features associated with infection and revealed metabolic clusters of acetyl carnitines, as well as phosphatidylcholine and phosphatidylethanolamine plasmalogen species, which could be associated with pro-inflammatory immune cell types. By developing an IMC marker for the detection of Salmonella LPS, we were further able to identify and characterize those cell types which contained S. Typhimurium.

High-Dose Cholecalciferol Booster Therapy is Associated with a Reduced Risk of Mortality in Patients with COVID-19: A Cross-Sectional Multi-Centre Observational Study.

The worldwide pandemic of 2019 novel coronavirus disease (COVID-19) has posed the most substantial and severe public health issue for several generations, and therapeutic options have not yet been optimised. Vitamin D (in its "parent" form, cholecalciferol) has been proposed in the pharmacological management of COVID-19 by various sources. We aimed to determine whether COVID-19 mortality was affected by serum 25-hydroxyvitamin D (25(OH)D) levels, vitamin D status, or cholecalciferol therapy, and to elucidate any other predictors of COVID-19 mortality. Patients hospitalised with COVID-19 were opportunistically recruited from three UK hospitals, and their data were collected retrospectively. Logistic regression was used to determine any relationships between COVID-19 mortality and potential predictors, including 25(OH)D levels and cholecalciferol booster therapy. A total of 986 participants with COVID-19 were studied, of whom 151 (16.0%) received cholecalciferol booster therapy. In the primary cohort of 444 patients, cholecalciferol booster therapy was associated with a reduced risk of COVID-19 mortality, following adjustment for potential confounders (ORadj 0.13, 95% CI 0.05-0.35, p < 0.001). This finding was replicated in a validation cohort of 541 patients (ORadj 0.38, 95% CI 0.17-0.84, p = 0.018). In this observational study, treatment with cholecalciferol booster therapy, regardless of baseline serum 25(OH)D levels, appears to be associated with a reduced risk of mortality in acute in-patients admitted with COVID-19. Further work with large population studies needs to be carried out to determine adequate serum 25(OH)D levels, as well as multi-dose clinical trials of cholecalciferol therapy to assess maximum efficacy.

Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 µm spatial resolution. Protein markers of proliferation (Ki-67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development.

The brain-penetrant clinical ATM inhibitor AZD1390 radiosensitizes and improves survival of preclinical brain tumor models.

Poor survival rates of patients with tumors arising from or disseminating into the brain are attributed to an inability to excise all tumor tissue (if operable), a lack of blood-brain barrier (BBB) penetration of chemotherapies/targeted agents, and an intrinsic tumor radio-/chemo-resistance. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR. We report the primary pharmacology of the clinical-grade, exquisitely potent (cell IC50, 0.78 nM), highly selective [>10,000-fold over kinases within the same phosphatidylinositol 3-kinase-related kinase (PIKK) family], orally bioavailable ATM inhibitor AZD1390 specifically optimized for BBB penetration confirmed in cynomolgus monkey brain positron emission tomography (PET) imaging of microdosed 11C-labeled AZD1390 (Kp,uu, 0.33). AZD1390 blocks ATM-dependent DDR pathway activity and combines with radiation to induce G2 cell cycle phase accumulation, micronuclei, and apoptosis. AZD1390 radiosensitizes glioma and lung cancer cell lines, with p53 mutant glioma cells generally being more radiosensitized than wild type. In in vivo syngeneic and patient-derived glioma as well as orthotopic lung-brain metastatic models, AZD1390 dosed in combination with daily fractions of IR (whole-brain or stereotactic radiotherapy) significantly induced tumor regressions and increased animal survival compared to IR treatment alone. We established a pharmacokinetic-pharmacodynamic-efficacy relationship by correlating free brain concentrations, tumor phospho-ATM/phospho-Rad50 inhibition, apoptotic biomarker (cleaved caspase-3) induction, tumor regression, and survival. On the basis of the data presented here, AZD1390 is now in early clinical development for use as a radiosensitizer in central nervous system malignancies.