Photoimmunotherapy (PIT) is a novel therapy for cancer treatment. (1) PIT is induced by an antibody-photon absorber conjugate that, when exposed to near infrared light (NIR), induces tumor cell necrosis. Previously, we have demonstrated that NIR PIT is an oxygen dependent process (2). Early experience with NIR PIT demonstrates that while the tumor may become necrotic, its size does not immediately change and so it can be difficult to assess early treatment effectiveness. We explored the use of 13C-hyperpolarized MRS before and after NIR-PIT to better understand the early metabolic changes in this treatment. [1,4-13C2] Fumarate is a 13C labeled tracer for MRS, which can be used to identify tissue necrosis. Fumarate only distributes in the extracellular space where there is a low level of fumarate hydratase (FH). However, in necrosis, FH leaks out of the damaged cell and converts fumarate to malate which is detected on MRS. In contrast 13C pyruvate is readily transported across the membrane and converted to lactate by lactose dehydrogenase (LDH), a process that generally occurs intracellularly but can also occur extracellularly.A431 tumor bearing mice were assigned to 4 groups (n=4 each) including non-treated and treated 13C Pyruvate and 13C Fumarate in separate cohorts. ; Dynamic 13C spectra were obtained from each group after hyperpolarization and injection in animals (see details below). Samples of [1-13C] pyruvic acid (30 μL) and [1,4-13C] fumaric acid (30 μL 2.5M in DMSO) containing 15 mM of OX063 and 2.5 mM of the gadolinium chelate ProHance were polarized at 3.35 T and 1.4 K in the Hypersense DNP polarizer. Each hyperpolarized sample was rapidly dissolved in 4.5 mL of dissolution buffer and injected (12 μL/g body weight) intravenously. Hyperpolarized 13C MRI studies were performed on a 3T scanner using a 17-mm home-built 13C solenoid coil placed inside a saddle coil. The 13C spectra were acquired every second with a spectral width of 3330 Hz, repetition time of 1000ms and flip angle of 10°. Photoacoustic imaging was performed by using the Vevo LAZR (VisualSonics) 21-MHz linear-array transducer system. Photoacoustic mode was then initialized and switched to the oxy/hemo mode to measure sO2 with the following parameters: transducer, LZ-250; depth, 20.00 mm; width, 23.04 mm; wavelength, 750 and 850 nm, total hemoglobin concentration threshold (Hbt), 20 arbitrary units (au); acquisition, sO2/Hbt.Real time sO2 mapping showed rapid decreases in local oxygenation suggesting oxygen consumption and conversion to singlet oxygen that mediated membrane damage during PIT. Histologic analysis confirmed immediate cell damage after PIT. Tumor growth curves showed the strong anti-tumor effect of PIT. Dynamic spectra of 13C labeled pyruvate and fumarate showed that the lactate-to-pyruvate ratio was unchanged after PIT, while the malate-to-fumarate ratio increased 5 fold in the PIT treated group. Since pyruvate is a tracer that can be easily taken up into the cell via monocarboxylate transporter, its conversion to lactate is independent of membrane damage. Fumarate, on the other hand is restricted to the extracellular space and is converted to malate at a higher rate only when FH leaks from necrotic cells, as occurs in early PIT. Thus, 13C fumarate hyperpolarized MRS demonstrates the early effects of NIR PIT-induced necrosis.Using MRS after injection of hyperpolarized substrates, we demonstrate that the malate-to -fumarate ratio increased 5 fold in NIR PIT treated tumors whereas the lactate to pyruvate ratio was unchanged. The conversion of fumarate to malate supports a necrotic cell death mechanism for PIT. 13C labeled fumarate is thus, a promising tracer for the early detection of PIT mediated cell death.