Ultra High-plex Spatial Proteogenomic Investigation of Giant Cell Glioblastoma Multiforme Immune Infiltrates Reveals Distinct Protein and RNA Expression Profiles.

A deeper understanding of complex biological processes, including tumor development and immune response, requires ultra high-plex, spatial interrogation of multiple "omes". Here we present the development and implementation of a novel spatial proteogenomic (SPG) assay on the GeoMx Digital Spatial Profiler platform with next-generation sequencing readout that enables ultra high-plex digital quantitation of proteins (>100-plex) and RNA (whole transcriptome, >18,000-plex) from a single formalin-fixed paraffin-embedded (FFPE) sample. This study highlighted the high concordance, R > 0.85 and <15% change in sensitivity between the SPG assay and the single-analyte assays on various cell lines and tissues from human and mouse. Furthermore, we demonstrate that the SPG assay was reproducible across multiple users. When used in conjunction with advanced cellular neighborhood segmentation, distinct immune or tumor RNA and protein targets were spatially resolved within individual cell subpopulations in human colorectal cancer and non-small cell lung cancer. We used the SPG assay to interrogate 23 different glioblastoma multiforme (GBM) samples across four pathologies. The study revealed distinct clustering of both RNA and protein based on pathology and anatomic location. The in-depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared with that of the more common GBM. More importantly, the use of spatial proteogenomics allowed simultaneous interrogation of critical protein posttranslational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods. Significance: We describe ultra high-plex spatial proteogenomics; profiling whole transcriptome and high-plex proteomics on a single FFPE tissue section with spatial resolution. Investigation of gcGBM versus GBM revealed distinct protein and RNA expression profiles.

ß-Catenin signaling dynamics regulate cell fate in differentiating neural stem cells.

Stem cells undergo differentiation in complex and dynamic environments wherein instructive signals fluctuate on various timescales. Thus, cells must be equipped to properly respond to the timing of signals, for example, to distinguish sustained signaling from transient noise. However, how stem cells respond to dynamic variations in differentiation cues is not well characterized. Here, we use optogenetic activation of ß-catenin signaling to probe the dynamic responses of differentiating adult neural stem cells (NSCs). We discover that, while elevated, sustained ß-catenin activation sequentially promotes proliferation and differentiation, transient ß-catenin induces apoptosis. Genetic perturbations revealed that the neurogenic/apoptotic fate switch was mediated through cell-cycle regulation by Growth Arrest and DNA Damage 45 gamma (Gadd45γ). Our results thus reveal a role for ß-catenin dynamics in NSC fate decisions and may suggest a role for signal timing to minimize cell-fate errors, analogous to kinetic proofreading of stem-cell differentiation.

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant.

We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein and a popular marker for photoactivated localization microscopy. We found the excitation light photoactivates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a slow-switching Dronpa variant, rsKame, featuring a V157L amino acid substitution proximal to the chromophore. The increased steric hindrance generated by the substitution reduced the excitation light-induced photoactivation from the dark to fluorescent state. To demonstrate applicability, we paired rsKame with PAmCherry1 in a two-color photoactivated localization microscopy imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We determined the diameter and length of Drp1 helical rings encircling mitochondria during fission and showed that, whereas their lengths along mitochondria were not significantly changed, their diameters decreased significantly. These results suggest support for the twistase model of Drp1 constriction, with potential loss of subunits at the helical ends.