High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections – Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

Abstract

Background: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FPwarhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes.Results: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gelbased ABPP combined with LC-MS/MS-based target verification.(Continued on next page)

MFGE8 links absorption of dietary fatty acids with catabolism of enterocyte lipid stores through HNF4γ-dependent transcription of CES enzymes

“…Interestingly, we observed downregulation of several genes coding for hydrolases belonging to the CES1 family of enzymes in Mfge8 KO mice (Figure 1C). We next performed activity-based staining (12) in WT and Mfge8 KO intestinal cryosections using a fluorescently-labeled fluorophosphonate probe (TAMRA-FP) that binds the active confirmation of serine hydrolases. Cryosections from Mfge8 KO mice showed markedly reduced fluorescence as compared with WT controls (Figure 1D) consistent with lower TG hydrolase activity.…”

“…On the following day, tissue sections were washed with PBST 3 times and then incubated with the secondary antibodies (donkey anti rat Alexafluor in 1:100 dilution) for 1 hour, washed, stained with Bodipy 488 (2mg/ml) for 30 minutes followed by mounting in Vectashield (H-1400) DAPI. For staining for active hydrolases, the fixed tissues on slides were preincubated for 20 minutes with assay buffer (50 mM Tris-HCl, pH 7.4; 1 mM EDTA; 100 mM NaCl; 5 mM MgCl2 and 0.1% (w/v) BSA) followed by incubation for 60 min with TAMRA-FP in the assay buffer (0.5 μM final concentration) (12). Slides were then washed 3 times in 0.1M phosphate buffer before mounting with DAPI.…”

Research Advances Through Activity‐Based Lipid Hydrolase Profiling

“…To this day, most groups still report ABPP imaging via confocal fluorescence microscopy in conjunction with a fluorophore as reporter tag. Commonly used fluorophore tags are BODIPY, Cy5 or TAMRA based compounds [30a, 51‐52] . Extensive research in the field of ABPP imaging led to the emergence of high‐resolution fluorescence imaging methods which can be used to visualize global serine hydrolase activity not only in complex tumorous tissue sections but also in healthy regions of mammalian brains [51] .…”

Research Supporting a Pilot Study of Metronomic Dapsone during Glioblastoma Chemoirradiation

“…Of interest, neutrophil agglomerations form “hotspots” of concentrated serine hydrolases within GBs [ 81 ]. These are related to the neutrophil extracellular traps that are associated with GBs and worsen prognosis [ 78 ].…”