BACKGROUND: The anti-programmed cell death-1 (PD-1) and anti-programmed cell death ligand-1 (PD-L1) immunotherapies have shown exceptional activity in many cancers. However, these immunotherapies can also result in diverse adverse cutaneous eruptions that need to be better characterized for ongoing management. The objective was to provide clinical and histopathologic descriptions of the variety of cutaneous adverse events seen in patients who received anti-PD-1/PD-L1 treatment and discuss their management. METHODS: Patients with advanced cancers in clinical trials at University of California Los Angeles (UCLA), receiving anti-PD-1/PD-L1 treatment between 2012 and 2016 who developed cutaneous eruptions and were evaluated in the dermatology clinic were included in this retrospective case series study. A total of 16 patients were included in this study; of these, five were treated with pembrolizumab alone, two with avelumab alone, eight with nivolumab plus ipilimumab and one with nivolumab plus T-Vec. Of these 16 patients, eight had received systemic chemotherapy, six had received radiotherapy, and one had received trememlimumab prior to the immunotherapies described in this study. RESULTS: Cutaneous eruptions occurred at variable times, from week 1 to 88, with a median of 11.5 weeks; the morphologies included lichenoid, bullous, psoriasiform, macular, morbiliform appearances, and alopecia which were confirmed histopathologically in several of the cases. All cutaneous immune-related adverse events were either grade 1 or 2. Ten patients were treated with topical corticosteroids, and one also received NBUVB. Four patients eventually required systemic steroids. Three required discontinuation of their anti-PD-1/PD-L1 therapy secondary to the cutaneous eruptions. CONCLUSIONS: There are several different types of adverse cutaneous morphologies that may be seen with administration of PD-1 and PD-L1 inhibitors. Identifying the patterns of eruption may assist in prompt treatment. Most eruptions could be managed with topical corticosteroids and without discontinuation of the systemic treatment.

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument.

Exosomes, nanosized membrane-bound vesicles released by cells, play roles in cell signaling, immunology, virology, and oncology. Their study, however, has been hampered by difficulty in isolation and quantification due to their size and the complexity of biological samples. Conventional approaches to improved isolation require specialized equipment and extensive sample preparation time. Therefore, isolation and detection methods of exosomes will benefit biological and clinical studies. Here, we report a microfluidic platform for inline exosome isolation and fluorescent detection using inertial manipulation of antibody-coated exosome capture beads from biological fluids.

Metastatic non-small cell lung cancer (NSCLC) is a highly fatal and immunogenic malignancy. Although the immune system is known to recognize these tumor cells, one mechanism by which NSCLC can evade the immune system is via overexpression of programmed cell death ligand 1 (PD-L1). Recent clinical trials of PD-1 and PD-L1 inhibitors have returned promising clinical responses. Important for personalizing therapy, patients with higher intensity staining for PD-L1 on tumor biopsies responded better. Thus, there has been interest in using PD-L1 tumor expression as a criterion for patient selection. Currently available methods of screening involve invasive tumor biopsy, followed by histological grading of PD-L1 levels. Biopsies have a high risk of complications, and only allow sampling from limited tumor sections, which may not reflect overall tumor heterogeneity. Circulating tumor cell (CTC) PD-L1 levels could aid in screening patients, and could supplement tissue PD-L1 biopsy results by testing PD-L1 expression from disseminated tumor sites. Towards establishing CTCs as a screening tool, we developed a protocol to isolate CTCs at high purity and immunostain for PD-L1. Monitoring of PD-L1 expression on CTCs could be an additional biomarker for precision medicine that may help in determining response to immunotherapies.

Evaluation of pleural fluids for metastatic cells is a key component of diagnostic cytopathology. However, a large background of smaller leukocytes and/or erythrocytes can make accurate diagnosis difficult and reduce specificity in identification of mutations of interest for targeted anti-cancer therapies. Here, we describe an automated microfluidic system (Centrifuge Chip) which employs microscale vortices for the size-based isolation and concentration of cancer cells and mesothelial cells from a background of blood cells. We are able to process non-diluted pleural fluids at 6 mL/min and enrich target cells significantly over the background; we achieved improved purity in all patient samples analyzed. The resulting isolated and viable cells are readily available for immunostaining, cytological analysis, and detection of gene mutations. To demonstrate the utility towards aiding companion diagnostics, we also show improved detection accuracy of KRAS gene mutations in lung cancer cells processed using the Centrifuge Chip, leading to an increase in the area under the curve (AUC) of the receiver operating characteristic from 0.90 to 0.99. The Centrifuge Chip allows for rapid concentration and processing of large volumes of bodily fluid samples for improved cytological diagnosis and purification of cells of interest for genetic testing, which will be helpful for enhancing diagnostic accuracy.

Dendritic cell (DC) activation and antigen presentation are critical for efficient priming of T cell responses. Here, we study how lentiviral vectors (LVs) deliver antigen and activate DCs to generate T cell immunization in vivo. We report that antigenic proteins delivered in vector particles via pseudotransduction were sufficient to stimulate an antigen-specific immune response. The delivery of the viral genome encoding the antigen increased the magnitude of this response in vivo but was irrelevant in vitro. Activation of DCs by LVs was independent of MyD88, TRIF, and MAVS, ruling out an involvement of Toll-like receptor or RIG-I-like receptor signaling. Cellular DNA packaged in LV preparations induced DC activation by the host STING (stimulator of interferon genes) and cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase) pathway. Envelope-mediated viral fusion also activated DCs in a phosphoinositide 3-kinase-dependent but STING-independent process. Pseudotransduction, transduction, viral fusion, and delivery of cellular DNA collaborate to make the DC-targeted LV preparation an effective immunogen.

Micro-scale biological tools that have allowed probing of individual cells--from the genetic, to proteomic, to phenotypic level--have revealed important contributions of single cells to direct normal and diseased body processes. In analyzing single cells, sample heterogeneity between and within specific cell types drives the need for high-throughput and quantitative measurement of cellular parameters. In recent years, high-throughput single-cell analysis platforms have revealed rare genetic subpopulations in growing tumors, begun to uncover the mechanisms of antibiotic resistance in bacteria, and described the cell-to-cell variations in stem cell differentiation and immune cell response to activation by pathogens. This review surveys these recent technologies, presenting their strengths and contributions to the field, and identifies needs still unmet toward the development of high-throughput single-cell analysis tools to benefit life science research and clinical diagnostics.

Light-sheet fluorescence microscopy (LSFM) serves to advance developmental research and regenerative medicine. Coupled with the paralleled advances in fluorescence-friendly tissue clearing technique, our cardiac LSFM enables dual-sided illumination to rapidly uncover the architecture of murine hearts over 10 by 10 by 10 mm³ in volume; thereby allowing for localizing progenitor differentiation to the cardiomyocyte lineage and AAV9-mediated expression of exogenous transmembrane potassium channels with high contrast and resolution. Without the steps of stitching image columns, pivoting the light-sheet and sectioning the heart mechanically, we establish a holistic strategy for 3-dimentional reconstruction of the "digital murine heart" to assess aberrant cardiac structures as well as the spatial distribution of the cardiac lineages in neonates and ion-channels in adults.

During mammalian embryogenesis, de novo hematopoiesis occurs transiently in multiple anatomical sites including the yolk sac, dorsal aorta, and heart tube. A long-unanswered question is whether these local transient hematopoietic mechanisms are essential for embryonic growth. Here, we show that endocardial hematopoiesis is critical for cardiac valve remodeling as a source of tissue macrophages. Colony formation assay from explanted heart tubes and genetic lineage tracing with the endocardial specific Nfatc1-Cre mouse revealed that hemogenic endocardium is a de novo source of tissue macrophages in the endocardial cushion, the primordium of the cardiac valves. Surface marker characterization, gene expression profiling, and ex vivo phagocytosis assay revealed that the endocardially derived cardiac tissue macrophages play a phagocytic and antigen presenting role. Indeed, genetic ablation of endocardially derived macrophages caused severe valve malformation. Together, these data suggest that transient hemogenic activity in the endocardium is indispensable for the valvular tissue remodeling in the heart.

Tissue clearing methods promise to provide exquisite three-dimensional imaging information; however, there is a need for simplified methods for lower resource settings and for non-fluorescence based phenotyping to enable light microscopic imaging modalities. Here we describe the simplified CLARITY method (SCM) for tissue clearing that preserves epitopes of interest. We imaged the resulting tissues using light sheet microscopy to generate rapid 3D reconstructions of entire tissues and organs. In addition, to enable clearing and 3D tissue imaging with light microscopy methods, we developed a colorimetric, non-fluorescent method for specifically labeling cleared tissues based on horseradish peroxidase conversion of diaminobenzidine to a colored insoluble product. The methods we describe here are portable and can be accomplished at low cost, and can allow light microscopic imaging of cleared tissues, thus enabling tissue clearing and imaging in a wide variety of settings.