Proteomics: A new dimension to decode small cell lung cancer.

Small cell lung cancer (SCLC) is a recalcitrant malignancy. Conquering it will require deep insight into its biology. In this issue of Cell, Liu and colleagues describe proteomic and phosphoproteomic landscapes of resected SCLC tumors and illustrate the potential of this knowledge to identify new SCLC vulnerabilities.

Cigarette Smoke Enhances the Malignant Phenotype of Esophageal Adenocarcinoma Cells by Disrupting a Repressive Regulatory Interaction Between miR-145 and LOXL2.

Although linked to esophageal carcinogenesis, the mechanisms by which cigarette smoke mediates initiation and progression of esophageal adenocarcinomas (EAC) have not been fully elucidated. In this study, immortalized esophageal epithelial cells and EAC cells (EACCs) were cultured with or without cigarette smoke condensate (CSC) under relevant exposure conditions. Endogenous levels of microRNA (miR)-145 and lysyl-likeoxidase 2 (LOXL2) were inversely correlated in EAC lines/tumors compared with that in immortalized cells/normal mucosa. The CSC repressed miR-145 and upregulated LOXL2 in immortalized esophageal epithelial cells and EACCs. Knockdown or constitutive overexpression of miR-145 activated or depleted LOXL2, respectively, which enhanced or reduced proliferation, invasion, and tumorigenicity of EACC, respectively. LOXL2 was identified as a novel target of miR-145 as well as a negative regulator of this miR in EAC lines/Barrett's epithelia. Mechanistically, CSC induced recruitment of SP1 to the LOXL2 promoter; LOXL2 upregulation coincided with LOXL2 enrichment and concomitant reduction of H3K4me3 levels within the promoter of miR143HG (host gene for miR-145). Mithramycin downregulated LOXL2 and restored miR-145 expression in EACC and abrogated LOXL2-mediated repression of miR-145 by CSC. These findings implicate cigarette smoke in the pathogenesis of EAC and demonstrate that oncogenic miR-145-LOXL2 axis dysregulation is potentially druggable for the treatment and possible prevention of these malignancies.

High-Precision Mapping of Membrane Proteins on Synaptic Vesicles using Spectrally Encoded Super-Resolution Imaging.

The spatial resolution of single-molecule localization microscopy is limited by the photon number of a single switching event because of the difficulty of correlating switching events dispersed in time. Here we overcome this limitation by developing a new class of photoswitching semiconducting polymer dots (Pdots) with structured and highly dispersed single-particle spectra. We imaged the Pdots at the first and the second vibronic emission peaks and used the ratio of peak intensities as a spectral coding. By correlating switching events using the spectral coding and performing 4-9 frame binning, we achieved a 2-3 fold experimental resolution improvement versus conventional superresolution imaging. We applied this method to count and map SV2 and proton ATPase proteins on synaptic vesicles (SVs). The results reveal that these proteins are trafficked and organized with high precision, showing unprecedented level of detail about the composition and structure of SVs.

Joint-Predominant Rheumatic Complications of Immune Checkpoint Inhibitor Therapy in Patients with Thymic Epithelial Tumors.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced cancers. However, activation of the immune system can occasionally cause life-threatening toxicity involving critical organs. Induction of immune-mediated toxicity is a significant concern for patients with thymic epithelial tumors (TETs) due to defects in immune tolerance. An increased risk of skeletal and cardiac muscle inflammation following treatment with ICIs is well recognized in patients with advanced TETs. However, uncommon musculoskeletal and rheumatic complications can also occur. The cases presented in this report highlight the spectrum of presentation of immune-mediated, joint-predominant musculoskeletal adverse events in patients with advanced TETs treated with ICIs, including polymyalgia rheumatica-like illness and inflammatory arthritis.

Multimode Time-Resolved Superresolution Microscopy Revealing Chain Packing and Anisotropic Single Carrier Transport in Conjugated Polymer Nanowires.

Here, we developed a novel, multimode superresolution method to perform full-scale structural mapping and measure the energy landscape for single carrier transport along conjugated polymer nanowires. Through quenching of the local emission, the motion of a single photogenerated hole was tracked using blinking-assisted localization microscopy. Then, utilizing binding and unbinding dynamics of quenchers onto the nanowires, local emission spectra were collected sequentially and assembled to create a superresolution map of emission sites throughout the structure. The hole polaron trajectories were overlaid with the superresolution maps to correlate structures with charge transport properties. Using this method, we compared the efficiency of inter- and intrachain hole transport inside the nanowires and for the first time directly measured the depth of carrier traps originated from torsional disorder and chemical defects.

Improving the Accuracy of Pdot-Based Continuous Glucose Monitoring by Using External Ratiometric Calibration.

Continuous glucose monitoring (CGM) allows type I and II diabetes patients to track changes in their glucose levels, allowing detection of impending hypoglycemia or hyperglycemia. Polymer dots (Pdots) are candidates for use in implanted CGM systems due to their exceptional brightness, photostability, sensitivity, and biocompatibility. However, Pdot glucose transducers are oxygen-dependent, and changes in tissue oxygen levels affect their measurement accuracy. Here, we describe an external ratiometric calibration method that corrects for changes in tissue oxygen levels to improve measurement accuracy. This method uses the ratio of oxygen concentrations inside and outside the Pdot glucose transducer as an indicator of glucose concentration to correct for signal deviations caused by tissue oxygen fluctuations. A second oxygen-sensitive Pdot that is not conjugated with glucose oxidase is used to measure the oxygen concentration outside the Pdot glucose transducer. We describe the theoretical basis for this approach and demonstrate its effectiveness experimentally in a subcutaneous mouse implant model. This external ratiometric system achieves higher accuracy glucose measurements than previous Pdot-based CGM systems and comparable accuracy to current commercial CGM systems, demonstrating the utility of the external ratiometric calibration strategy.

High-Throughput Counting and Superresolution Mapping of Tetraspanins on Exosomes Using a Single-Molecule Sensitive Flow Technique and Transistor-like Semiconducting Polymer Dots.

A method for high-throughput counting and superresolution mapping of surface proteins on exosomes is described. The method combines a single-molecule sensitive flow technique and an adaptive superresolution imaging method. Exosomes stained with membrane dye and dye-conjugated antibodies were analyzed using a microfluidic platform at a flow rate of 100 exosome s-1 to determine size and protein copy number. Superresolution mapping was performed with exosomes labeled with novel transistor-like, semiconducting polymer dots (Pdots), which exhibit spontaneous blinking with <5 nm localization error and a broad range of optical-adjustable duty cycles. Based on the copy numbers extracted from the flow analysis, the switch-on frequency of the Pdots were finely adjusted so that structures of hundreds of exosomes were obtained within five minutes. The high throughput and high sensitivity of this method offer clear advantages for characterization of exosomes and similar biological vesicles.

Reversible Ratiometric NADH Sensing Using Semiconducting Polymer Dots.

Reduced nicotinamide adenine dinucleotide (NADH) is a key coenzyme in living cells due to its role as an electron carrier in redox reactions, and its concentration is an important indicator of cell metabolic state. Abnormal NADH levels are associated with age-related metabolic diseases and neurodegenerative disorders, creating a demand for a simple, rapid analytical method for point-of-care NADH sensing. Here we develop a series of NADH-sensitive semiconducting polymer dots (Pdots) as nanoprobes for NADH measurement, and test their performance in vitro and in vivo. NADH sensing is based on electron transfer from semiconducting polymer chains in the Pdot to NADH upon UV excitation, quenching Pdot fluorescence emission. In polyfluorene-based Pdots, this mechanism resulted in an on-off NADH sensor; in DPA-CNPPV Pdots, UV excitation resulted in NADH-sensitive emission at two wavelengths, enabling ratiometric detection. Ratiometric NADH detection using DPA-CNPPV Pdots exhibits high sensitivity (3.1 µM limit of detection), excellent selectivity versus other analytes, reversibility, and a fast response (less than 5 s). We demonstrate applications of the ratiometric NADH-sensing Pdots including smartphone-based NADH imaging for point-of-care use.

Monitoring Metabolites Using an NAD(P)H-sensitive Polymer Dot and a Metabolite-Specific Enzyme.

We introduce an NAD(P)H-sensitive polymer dot (Pdot) biosensor for point-of-care monitoring of metabolites. The Pdot is combined with a metabolite-specific NAD(P)H-dependent enzyme that catalyzes the oxidation of the metabolite, generating NAD(P)H. Upon UV illumination, the NAD(P)H quenches the fluorescence emission of Pdot at 627 nm via electron transfer, and also fluoresces at 458 nm, resulting in a shift from red to blue emission at higher NAD(P)H concentrations. Metabolite concentration is quantified ratiometrically-based on the ratio of blue-to-red channel emission intensities, with a digital camera-with high sensitivity and specificity. We demonstrate phenylalanine biosensing in human plasma for a phenylketonuria screening test, quantifying several other disease-related metabolites (lactate, glucose, glutamate, and ß-hydroxybutyrate), and a paper-based assay with smartphore imaging for point-of-care use.

Dual-Mode Superresolution Imaging Using Charge Transfer Dynamics in Semiconducting Polymer Dots.

In a conjugated polymer-based single-particle heterojunction, stochastic fluctuations of the photogenerated hole population lead to spontaneous fluorescence switching. We found that 405 nm irradiation can induce charge recombination and activate the single-particle emission. Based on these phenomena, we developed a novel class of semiconducting polymer dots that can operate in two superresolution imaging modes. The spontaneous switching mode offers efficient imaging of large areas, with <10 nm localization precision, while the photoactivation/deactivation mode offers slower imaging, with further improved localization precision (ca. 1 nm), showing advantages in resolving small structures that require high spatial resolution. Superresolution imaging of microtubules and clathrin-coated pits was demonstrated, under both modes. The excellent localization precision and versatile imaging options provided by these nanoparticles offer clear advantages for imaging of various biological systems.