1 Multimodality small animal imaging: registration of functional EPR images with MRI anatomy Supported by grants DAMD (DoD) and P41EB002034(NIBIB) Chad R. Haney, Adrian Parasca, Charles A. Pelizzari, Greg S. Karczmar*, Howard J. Halpern Department of Radiation and Cellular Oncology and *Department of Radiology The University of Chicago

2 In Vivo EPR Imaging – Topic of NIBIB Research Resource (PI: Howard Halpern, MD, PhD) Long term goal - develop EPR imaging techniques which provide functional information that can be of use in designing, delivering, and assessing cancer therapy.

3 Biological imaging to enhance targeting of radiation therapy: oxygen imaging Intensity modulated radiation therapy allows sophisticated control over spatial distribution of radiation dose Areas of hypoxia could be given extra dose if we could identify them

4 Why EPR Imaging? Spectroscopic Imaging: Specific quantitative sensitivity to Oxygen, Temperature, Viscosity, pH, Thiol No water background obscures spectrum of interest (vs MRI) ~600 times stronger coupling to magnetic field, environment (vs MRI) Deep sensitivity at lower frequency (vs optical) Noninvasive (vs probes)

5 EPR in vivo oximetry techniques Localized spectroscopy with implanted particulate probes (Dartmouth) Spectroscopic imaging with stepped fixed gradients, water soluble probes –CW (Chicago, OSU, Aberdeen, L’Aquila) –pulsed (NCI, Chicago) OMRI (NCI, Aberdeen) –dynamic nuclear polarization using EPR spin probes

6 EPR is analogous to NMR: Fix RF frequency, sweep field or fix field, sweep frequency: Zeeman splitting of electron spin energy states in magnetic field

7 EPRI is not identical to MRI: Relaxation times ~10 -6 as long pulsed gradient techniques not applicable FID correspondingly short → demanding of pulsed measurement techniques π/2 pulse ~50 ns long, FID lasts few μs have to introduce spin probe – no endogenous signal frequency ~660 times higher for given field (or, field 660 times lower for given frequency)

8 RF penetration favors lower frequency 250 MHz ~ 6 T MRI, 90 G EPR S/N   N~  1.2 IN LOSSY, CONDUCTIVE TISSUE proton Larmor frequency = 4258 Hz/gauss 42.6 MHz at 1 Tesla electron Larmor frequency = 2.80 MHz/gauss 28 GHz at 1 Tesla ratio meas/calc

9 Continuous wave spectral spatial imaging: each voxel yields a spectrum whose linewidth increases linearly with local oxygen concentration fixed stepped field gradients, swept magnetic field EPR line broadening for current narrow line spin probes: approximately 0.5 mG/torr O 2

10 Line width pO 2 calibration Oxygen dependence of lorentzian line width obtained in a series of homogenous solutions of OX31spin probe

11 Spectral-spatial projection (a) With no gradient, a field sweep integrates over all spatial locations. This is a pure spectral projection. (b) A gradient along the x direction couples the spatial and spectral coordinates (the spectrum is shifted linearly with position). (c) A field sweep now corresponds to a projection along a direction rotated in the spectral-spatial plane. Larger gradients correspond to larger rotation angles. Pure spatial projection would require infinite gradient.

MHz Spectrometer Magnets varying diameter homogeneous field regions (90 G) Small 8 cm diam. Intermediate 15 cm diam. Large 30 cm diam.

13 Mouse Image using OX063 spin probe PC3 human prostate cancer xenograft on nude mouse hind limb

14 Registration of EPR with MRI for anatomically aided analysis Registration based on - Fiducials - Surfaces - Intensity distribution Note high intensity due to poor clearance of spin probe from tumor, and low oxygen tension in same region

15 Early fiducial markers filled with dilute spin probe solution. Problem: need to remove during 4D image to avoid artifacts

16 Immobilization cast, fiducial markers for serial and intermodality registration

17 Alignment of MRI and EPRI (red) fiducial surfaces

18 Manual refinement of initial registration estimate based on fiducials

19 Application: radiation inducible antivascular gene therapy

20 PC3 tumor treated with Ad.CMV.null virus (control) Pre treatment: mean pO2 in tumor 44.6 torr, std 35.1, SEM tumor volume from MRI: mL 4 days post treatment (right): mean pO2 in tumor 28.7 torr, std 29.1, SEM tumor volume from MRI: mL

21 4 days post treatment: mean pO2 in tumor 31.7 torr, std 17.1, SEM tumor volume from MRI: mL PC3 tumor treated with Ad.EGR- TNFα virus + 10 Gy Pre treatment: mean pO2 in tumor 27.3, std 36.1, SEM tumor volume from MRI: mL

22 Conclusions 4D EPR Images can be obtained with ~1 mm spatial resolution and ~1.5mG (~3 torr pO 2 ) spectral resolution Preliminary images of increased and decreased regional oxygenation levels following radiation + adeno-EGR-TNF  anti-vascular therapy have been seen. These images may have potential for biologically- based planning and assessment of radiation therapy Registration of these functional images with anatomic images such as MRI is key to accurate interpretation and to eventual clinical applications

23 Chicago EPRI Lab: Howard Halpern Martyna Elas Colin Mailer Chad Haney Charles Pelizzari Kazuhiro Ichikawa Gene Barth Ben Williams Kang-Hyun Ahn Adrian Parasca VS Subramanian Chicago MRI Lab: Greg Karczmar Jonathan River Xiaobing Fan Marta Zamora EGRF-TNF  radiation therapy: Ralph Weichselbaum Helena Mauceri Michael Beckett Denver EPR Lab: Gareth Eaton Sandra Eaton Richard Quine George Rinard