In this work, we investigated the feasibility of using ³¹P spectroscopic imaging to evaluate the early effects of photodynamic therapy (PDT) on tumor metabolism. PDT is an emerging cancer treatment modality [1], which relies on a light-activated drug that generates cytotoxic oxygen radicals upon local illumination. The primary biological actions are 1) direct cell death and 2) vascular shutdown. While PDT has proven clinically successful in a range of superficial cancer types, there is a need for early evaluation methods of treatment outcome. Moreover, development of new PDT drugs and optimization of treatment protocols requires techniques to assess PDT efficacy. Since mitochondria are a known target of many photosensitizers, PDT may affect tissue energy metabolism, resulting in lower adenosine triphosphate (ATP) levels. It has been shown that ³¹P MRS allows rapid detection of PDT-induced changes in phosphorus metabolites, but only with surface coil localization [2]. Here, we assessed the feasibility of using ³¹P spectroscopic imaging to evaluate the early effects of PDT on tumor metabolism in a spatially resolved manner.Eight Balb/C mice with s.c. CT26 colon carcinoma tumors were examined when tumors were 258 +/- 71 mm³. PDT-treated mice (n=6) received an i.v. injection of the photosensitizer Bremachlorin, followed 6 h later by 10 min irradiation with a 655 nm laser at 200 mW/cm². Light was delivered inside the scanner with a custom-built optic setup, creating a Ø10 mm parallel beam, which was aimed onto the tumor (Figure 1). Controls (n=2) received no photosensitizer or light. MRI was performed with a 9.4 T small animal scanner (Bruker BioSpec), using a volume coil for Tx/Rx of proton signals and transmission of ³¹P RF pulses. A ³¹P surface coil was positioned on the tumor for signal reception. T2w anatomical reference images were first acquired. Next, a ³¹P Chemical Shift Imaging (CSI) baseline scan was obtained in 40 min, in a 5 mm central axial tumor slice. A series of five 10 min Image Selected In vivo Spectroscopy (ISIS) scans was acquired in an as large as possible single voxel within the tumor, to detect immediate changes. PDT was performed during the 2^nd ISIS scan. Finally, another CSI scan was acquired. 24h later, single ISIS and CSI scans were obtained. Afterwards, tumors were excised and stored for histological analysis. For both ISIS and CSI, a 1.2 ms sinc-shaped excitation pulse was used, spectral BW = 5000 Hz, γ-ATP was put on resonance, and local shimming was performed. ISIS-specific parameters: 6.25 ms adiabatic hyperbolic secant inversion pulses, FA = 90°, TR = 2 s. CSI specific parameters: 24 mm FOV, 8x8 matrix, Hanning weighted k-space acquisition, FA = 35°, TR = 366 ms. ³¹P spectra were analyzed in jMRUI. The AMARES algorithm [3] was used to fit the following peaks: inorganic phosphate (P_i), γ-ATP (doublet), α-ATP (double), β-ATP (triplet), phosphocreatine, and phosphomonoesters (PME). Metabolite levels were expressed relative to the total ³¹P signal in the spectrum.For all mice, the baseline ISIS tumor spectrum contained a distinct P_i peak (Figure 2). PME and γ-, β-, and α-ATP could generally also be distinguished, but with lower SNR. In contrast to controls, PDT resulted in a significant increase in P_­i within 20 minutes after treatment (Figure 3). Moreover, ATP levels decreased after PDT treatment, although this did not reach statistical significance. The observed changes can be attributed to decreased mitochondrial production of ATP, while P­_i probably accumulated as the end-product of sustained ATP consumption. Metabolite maps with 3 mm spatial resolution could be calculated from CSI data, which allowed clear spatial and spectral distinction between muscle and tumor tissue (Figure 4). PDT-induced increases in P_i were observed in CSI measurements, homogeneously within the tumor, while P_i levels did not change in controls (Figure 5). We are currently performing extensive histological validation. Based on histological findings from previous studies with the same treatment protocol, treated tumors are generally entirely non-viable at 24h after PDT [4]. This is in line with our current observations of a severely impaired tumor energetic status.Using ³¹P MRS, we have shown that changes in tumor energy metabolism can be detected within tens of minutes after PDT in our animal model. Moreover, ³¹P spectroscopic imaging allows for detection of spatial heterogeneity in treatment response, which was not observed with the current treatment protocol, however. This ³¹P MRS method can be useful for mechanistic PDT research, optimization of PDT drugs or treatment protocols, but might also be clinically valuable for early prediction of treatment outcome.