Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance

Summary

Quantitative 3D oxygen maps of murine tumors were imaged noninvasively using Pulse Electron Paramagnetic Resonance. Ultrasound B-mode and Power Doppler were used for anatomy and vascular structure. Images from both modalities were superimposed enabling multi-parametric tumor analysis.

Abstract

The precise and real-time measurement of oxygen partial pressure (pO₂) brings valuable information in many pathologies, including cancer. Low tumor pO₂ (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO₂ allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO₂ in selected voxels.

Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO₂, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO₂ tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.

Introduction

Comprehending the tumor microenvironment (TME), with its complex spatial and dynamic interactions, brings a fuller understanding of tumor biology. Hypoxia, or low oxygen levels, is the key component of TME and plays a critical role in the development of other life-threatening conditions, including cardiovascular diseases, metabolic disorders such as diabetes, and chronic kidney disease^1,2,3. Tissue oxygenation is a fundamental factor, particularly in the context of cancer, where partial tissue oxygen pressure (pO₂) is correlated with therapy resistance. A pO_2<....

Protocol

Mice were obtained from an approved animal breeding facility and all experiments were conducted in compliance with ethical guidelines (in our case - Permission No. 165/2023, First Local Ethics Committee, Kraków, Poland).

1. Animals and tumor line

NOTE: The mice were housed under standard laboratory conditions: Light/dark: 12 h/12 h, humidity: 60%, temperature: 23 °C. They were provided a standard chow diet with free access to drinking water in community cages.

1. Murine glioblastoma multiforme LN229 cell line culture
   1. Culture LN229 cells in 25 cm² tissue c....

Discussion

There are a few critical steps in the described imaging protocol. First, to register the anatomy images with the oxygen maps, MRI might be a better choice than ultrasound due to better resolution and the ability to provide detailed 3D data¹⁹. Ultrasound with a high-frequency transducer provides excellent resolution and sufficient imaging depth for preclinical studies. Both MRI and US, however, require the use of fiducials to secure the tumor position across different imaging modalities.

Materials

Name	Company	Catalog Number	Comments
aqua pro injectione	Polpharma	1280610	-
ArbuzGUI	O2M Technologies	-	accesible in the github repository
disodium phosphate	POCH S.A.	799280115	-
Dulbecco′s Modified Eagle′s Medium - high glucose	Merck Life Science	D5648	4500 mg/L glucose and L-glutamine
fetal bovine serum	Gibco, Thermo Fisher Scientific	10500064	-
fishing wire	Good Fish	A-55A-035	US position marker - 0.35 mm
Geltrex	Gibco, Thermo Fisher Scientific	A1413302	reduced growth factor basement membrane matrix
ibGUI	O2M Technologies	-	accesible in the github repository
injectio natrii chlorati isotonica	Polpharma	multipe items were used	9 mg/mL
insulin needles 29 G	Becton, Dickinson and Company	multipe items were used	-
Jiva 25	O2M Technologies	-	EPROI
MATLAB	MathWorks	-	version R2021b
penicillin-streptomycin	Merck Life Science	P4333	with 10,000 units penicillin and 10 mg streptomycin/mL
potassium chloride	POCH S.A.	739740114	-
potassium dihydrogen phosphate	POCH S.A.	742020112	-
ProcessGUI	O2M Technologies	-	accesible in the github repository
PTFE tubing	Cole Palmer Instrument Co	06412-11	-
sodium chloride	POCH S.A.	794121116	-
SpecMan4EPR	FEMI Instruments	-	version 3.4 CS 64bit
Surflash I.V. Catheter	Terumo	SR*FF2419	size: 24G x ¾"
tape	3M	multipe items were used	micropore
Trypsin-EDTA	Gibco, Thermo Fisher Scientific	25200072	-
Ultrasonography	Telemed	-	Anatomical US
US gel	KONIX	NUG-0019	-
Vetflurane	Virbac	137317	1000 mg/g
Vevo F2	FujiFilms, Visual Sonics	-	B-mode and Doppler
vinyl polysiloxane dental clay	3M ESPE	multiple items were used	-