Development of a protocol for simultaneous study of tumor metabolism and oxygenation in vivo using FLIM and PLIM microscopy
Anastasia D. Komarova 1,2, Leonid N. Bochkarev 3,Vladislav I. Shcheslavskiy 1,4, Marina V. Shirmanova 1;
1 Privolzhskiy Research Medical University, Nizhny Novgorod, Russia
2 Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia
3 G.A. Razuvaev Institute of Organometallic Chemistry of the Russian Academy of Sciences, Nizhny Novgorod, Russia
4 Becker & Hickl GmbH, Berlin, Germany
Abstract
The effect of oxygen on metabolism and heterogeneity hasn’t been fully studied. PLIM (Phosphorescence lifetime imaging) and FLIM (Fluorescence lifetime imaging) allow real time noninvasive assessment of the oxygen and metabolic status.
The purpose was to develop a method for real time simultaneous metabolic and oxygen status analysis of tumor cells in vivo using the FLIM/PLIМ method with a new iridium oxygen sensor.
The studies were performed on CT26 cells in a cell monolayer model, 3D spheroids, and in vivo tumors. Microscopic studies were performed using an LSM880 microscope with a FLIM/PLIM TCSPC module. Oxygen status was assessed by phosphorescence lifetime of the O2-sensitive sensor PIr3 simultaneously with assessment of metabolic status by NAD(P)H autofluorescence (λex=750nm, λem=450–490nm (NAD(P)H), λem=600–740nm (PIr3)).
The parameters for visualization of the PIr3 sensor in cancer cells were selected and a technique for simultaneous FLIM/PLIM was developed and tested on a monolayers, spheroids and in vivo model. The developed technique demonstrated the absence of oxygen and metabolic heterogeneity in the monolayer and a high correlation between metabolic parameters and oxygen levels with variations. In spheroids, the inner layer were more hypoxic than the outer, and metabolism was shifted toward glycolysis. In vivo model had a high intratumoral and intertumoral heterogeneity in oxygen and metabolic statuses. In a tumor, the correlation between metabolic parameters and oxygen content was moderate, which indicated the contribution of factors other than oxygen to the regulation of cell metabolism.
This work was supported by the Russian Science Foundation, project №24-19-00618.
Speaker
Komarova Anastasia Denisovna
1. Privolzhskiy Research Medical University, Nizhny Novgorod, Russia
Russia
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