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 FP-warhead, 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 gel-based ABPP combined with LC-MS/MS-based target verification.

CONCLUSIONS:

Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

• Authors: Niina Aaltonen, Prosanta K Singha, Hermina Jakupović, Thomas Wirth, Haritha Samaranayake, Sanna Pasonen-Seppänen, Kirsi Rilla, Markku Varjosalo, Laura E Edgington-Mitchell, Paulina Kasperkiewicz, Marcin Drag, Sara Kälvälä, Eemeli Moisio, Juha R Savinainen, Jarmo T Laitinen
• Journal: Biological procedures online (Biol Proced Online) Vol. 22 Pg. 6 ( 2020) ISSN: 1480-9222 [Print] England
• PMID: 32190011 (Publication Type: Journal Article)
• Copyright: © The Author(s) 2020.