MSACL 2022 Abstract

INTRODUCTION
Elevated levels of the antioxidant glutathione (GSH) are characteristic to many types of tumors. However, the heterogeneity in synthesis kinetics across different cellular microenvironments requires further elucidation and will vastly improve our clinical understanding of this biomarker’s role in cancer proliferation and tumor drug resistance. Mass spectrometry imaging (MSI) offers several advantages over traditional positron emission topography (PET) imaging techniques, including sensitivity and spatial resolution improvements. A challenge in MSI is obtaining organs from dosed replicates over a time course. We therefore utilize a multiple infusion start time (MIST) protocol for dosing with stable isotope labels (SIL) of glycine that can be traced for enrichment measurements simultaneously in a single tissue. We combine this technique with infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI to spatially map the heterogeneity of glutathione biosynthesis across proliferating tissue sections.

METHODS
Mice with 4T1 tumors were injected with three differentially labeled glycine isotopologues, strategically selected to enrich separate GSH isotopologues after metabolic incorporation, using the MIST protocol. The MIST protocol allows for the measurement of specific enrichments from three time points within one organism. Organs (livers, tumors) were harvested and sectioned to 20 µm thickness prior to thaw-mounting on glass slides evenly coated with homoglutathione (hGSH) as a normalization standard. Enrichments were measured and spatially mapped using the IR-MALDESI source coupled to an Exploris 240 orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) to achieve both high resolution (240K at 200 m/z) and mass accuracy (< 3 ppm) of the GSH isotopologue peaks, for mass spectrometry imaging analysis. Enrichments were calculated and visualized using the percent isotope enrichment (PIE) tool in MSiReader.

RESULTS
After preliminary measurements of high (up to ~2% successful enrichment of the total GSH pool per SIL) but homogeneous enrichment in liver tissues using a 60-minute MIST infusion, the timings were adjusted to successfully measure quantifiable signal of SIL enrichment across the 4T1 tumors. Using the PIE tool, areas of high enrichment, and thus high rates of synthesis, were visualized in heatmaps. Heterogeneous regions were compared with histological staining of adjacent tissue sections to determine the function of the heterogeneity of GSH synthesis in different areas of the proliferating tissue. A tumor from a mouse labeled with naturally abundant glycine served as a negative control to check for interferences and confirm enrichment of these peaks specifically due to label incorporation.

DISCUSSION
This novel imaging technique offers potential towards the development of new clinical assays using changes in glutathione synthesis pathways to better characterize tumor tissues. Additionally, by studying the effects of microenvironments on the synthesis of GSH, we may further elucidate its role in imparting chemoresistance to tumor cells. This may provide insight into the adjustment of targeted therapies towards preventing this resistance.