Comparison of phosphorescent agents for noninvasive sensing of tumor oxygenation via Cherenkov-excited luminescence imaging

Abstract

Cherenkov emission generated in tissue during radiotherapy can be harnessed for the imaging biochemistry of tissue microenvironments. Cherenkov-excited luminescence scanned imaging (CELSI) provides a way to optically and noninvasively map oxygen-related signals, which is known to correlate to outcomes in radiotherapy. Four candidate phosphorescent reagents PtG4, MM2, Ir(btb)2 ( acac ) , and MitoID were studied for oxygen sensing, testing in a progressive series of (a) in solution, (b) in vitro, and (c) in subcutaneous tumors. In each test, the signal strength and response to oxygen were assessed by phosphorescence intensity and decay lifetime measurement. MM2 showed the most robust response to oxygen changes in solution, followed by PtG4, Ir(btb)2 ( acac ) , and MitoID. However, in PANC-1 cells, their oxygen responses differed with Ir(btb)2 ( acac ) exhibiting the largest phosphorescent intensity change in response to changes in oxygenation, followed by PtG4, MM2, and MitoID. In vivo, it was only possible to utilize Ir(btb)2 ( acac ) and PtG4, with each being used at nanomole levels, to determine signal strength, lifetime, and pO2. Oxygen sensing with CELSI during radiotherapy is feasible and can estimate values from 1 mm regions of tissue when used in the configuration of this study. PtG4 was the most amenable to in vivo sensing on the timescale of external beam LINAC x-rays.

Keywords: Cherenkov radiation; optical imaging; phosphorescence; tumor hypoxia.

Fig. 1

Absorbance and emission spectra for oxygen sensors. The absorbance and fluorescence emission spectra for $\mathrm{Ir}{(\mathrm{btb})}_2(\mathrm{acac})$ (blue), MitoID (red), PtG4 (green), and MM2 (gray).

Fig. 2

Comparison of commercially available oxygen sensors in PANC-1 cells. (a) MM2 (gray), MitoID (red), and $\mathrm{Ir}{(\mathrm{btb})}_2(\mathrm{acac})$ (blue) were incubated with PANC-1 cells overnight. Fluorescence was measured in the presence of ambient oxygen [light gray, MM2, medium gray, MitoID, and dark gray, $\mathrm{Ir}{(\mathrm{btb})}_2(\mathrm{acac})$] and with glucose oxidase catalase enzymatic oxygen scavenging (black). (b) The brightest commercial oxygen sensors were explored at decreasing concentrations (20, 2, 0.2, and $0.02\mu \mathrm{g}/\mathrm{mL}$) to determine the lowest concentration that can be detected. Inset shows fluorescence microscopy of each sensor ($20\mu \mathrm{g}/\mathrm{mL}$) loaded into PANC-1 cells. Excitation and emission were measured at 380 and 660 nm, respectively. The fluorescence intensity represents the average of six experiments $\pm \mathrm{SEM}$.

Fig. 3

Comparison of CEL with phosphorescent oxygen sensors. Increasing concentrations of each sensor indicated by darker shades of each color [0.02, 0.2, 2, and $20\mu \mathrm{g}/\mathrm{mL}$, MitoID (red), MM2 (gray), PtG4 (green), and $\mathrm{Ir}{(\mathrm{btb})}_2(\mathrm{acac})$ (blue)] were analyzed in ambient oxygen or with enzymatic oxygen scavenger glucose oxidase/catalase (black). A 96-well plate with these reagents in (a) solution or (b) loaded into PANC-1 cells was exposed to 6-MV radiation from a LINAC, and the phosphorescence intensity was measured via ICCD.

Fig. 4

CELSI setup and in vivo imaging with PtG4 and $\mathrm{Ir}{(\mathrm{btb})}_2(\mathrm{acac})$. The LINAC gantry head that delivers radiation is placed below the mouse that is positioned on the treatment couch. A mirror above redirects the photons to the ICCD. (a) Lateral view of the mouse depicting passage of radiation sheet in red (b) aerial view as imaged from the ICCD camera. (c) Under anesthesia of isofluorane, 2.5 nmol of each reagent was injected into subcutaneous MDA-MB-231luc-D3H2LN tumors. Maximum intensity projection of PtG4 and of $\mathrm{Ir}{(\mathrm{btb})}_2(\mathrm{acac})$ at $4.26\mu \mathrm{s}$ delay time, respectively. Tumors are indicated with line arrows.

Fig. 5

In vivo lifetime imaging with PtG4 and $\mathrm{Ir}{(\mathrm{btb})}_2(\mathrm{acac})$. Under anesthesia of isofluorane, the animal was imaged at various delay times based on reported lifetimes of each reagent to gain emission lifetime information alive and 30 min after euthanize, when the drop in blood circulation and respiration causes a marked decrease in ${\mathrm{pO}}_2$ values. (a) Comparison of PtG4 phosphorescence intensity at various delay times based on known PtG4 emission lifetime in subcutaneous tumors alive (top) and 30 min after euthanize(bottom). (b) PtG4 emission lifetime maps and box and whiskers plot of emission lifetimes of alive and euthanized mouse. (c) ${\mathrm{pO}}_2$ maps and box and whiskers plot of oxygen levels in alive and euthanized mouse for PtG4. (d) Phosphorescence intensity of tumors injected with $\mathrm{Ir}{(\mathrm{btb})}_2(\mathrm{acac})$ at different delay times informed from the reported $\mathrm{Ir}{(\mathrm{btb})}_2(\mathrm{acac})$ emission lifetime, map of emission lifetime, and ${\mathrm{pO}}_2$ map in euthanized mouse. All lifetime maps and subsequent ${\mathrm{pO}}_2$ maps for both reagents were constructed utilizing data from the $4.26\text{-}\mu \mathrm{s}$ delay time.

Fig. 6

Considerations for CELSI. (a) Cherenkov imaging timing. Experimentally, the gate width is generally set between 5 and 10 times the deoxygenated lifetime (${\tau }_0$) of the reagent of interest. Luminescence is detected at a series of delay times (red blocks) after the Cherenkov emission (blue column) to assess the emission lifetime of phosphorescent agents. The LINAC generates radiation pulses at a repetition rate between 2.7 and 17 ms (600 to $100\mathrm{MU}/\min$). (b) Plot of emission decay curves as a function of quantum yield.