Optimizing Multi-Photon Fluorescence Microscopy Light Collection from Living Tissue by Non-Contact Total Emission Detection (TEDII)

Farrando Sicilia, Jordi; Fuente Fuente, Carlo

FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

Abstract Background: Antigen processing by dendritic cells (DC) exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials) are similarly processed by these cells has not yet been resolved. Methods: In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II) in mature dendritic cells (mDC) from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by F\uf6rster Resonance Energy Transfer (FRET) measurements, which effectively extends the application of fluorescence microscopy to the molecular level (<100?). Tumor lysates were labelled with Alexa-488, as the donor, and mDC MHC II HLA-DR molecules were labelled with Alexa-546-conjugated IgG, as the acceptor. Results: We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr. Conclusions: The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates

The N2K Consortium. III. Short-Period Planets Orbiting HD 149143 and HD 109749

We report the detection of two short-period planets discovered at Keck Observatory. HD 149143 is a metal-rich G0 IV star with a planet of M sin i = 1.33M_J and an orbital radius of 0.053 AU. The best-fit Keplerian model has an orbital period, P = 4.072 days, semivelocity amplitude, K = 149.6 m s^(-1), and eccentricity, e = 0.016 ± 0.01. The host star is chromospherically inactive and metal-rich, with [Fe/H] = 0.26. Based on the T_(eff) and stellar luminosity, we derive a stellar radius of 1.49 R_☉. Photometric observations of HD 149143 were carried out using the automated photometric telescopes at Fairborn Observatory. HD 149143 is photometrically constant over the radial velocity period to 0.0003 ± 0.0002 mag, supporting the existence of the planetary companion. No transits were detected down to a photometric limit of approximately 0.02%, eliminating transiting planets with a variety of compositions and constraining the orbital inclination to less than 83°. A short-period planet was also detected around HD 109749, a G3 IV star. HD 109749 is chromospherically inactive, with [Fe/H] = 0.25 and a stellar radius of 1.24. The radial velocities for HD 109749 are modeled by a Keplerian with P = 5.24 days and K = 28.7 m s^(-1). The inferred planet mass is M sin i = 0.28M_J and the semimajor axis of this orbit is 0.0635 AU. Photometry of HD 109749 was obtained with the SMARTS consortium telescope, the PROMPT telescope, and by transitsearch.org observers in Adelaide and Pretoria. These observations did not detect a decrement in the brightness of the host star at the predicted ephemeris time, and they constrain the orbital inclination to less than 85° for gas giant planets with radii down to 0.7R_J

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment