Molecular imaging of tumor photoimmunotherapy: Evidence of photosensitized tumor necrosis and hemodynamic changes.

Near-infrared photoimmunotherapy (NIR PIT) employs the photoabsorbing dye IR700 conjugated to antibodies specific for cell surface epidermal growth factor receptor (EGFR). NIR PIT has shown highly selective cytotoxicity in vitro and in vivo. Cell necrosis is thought to be the main mode of cytotoxicity based mainly on in vitro studies. To better understand the acute effects of NIR PIT, molecular imaging studies were performed to assess its cellular and vascular effects. In addition to in vitro studies for cytotoxicity of NIR PIT, the in vivo tumoricidal effects and hemodynamic changes induced by NIR PIT were evaluated by 13C MRI using hyperpolarized [1,4-13C2] fumarate, R2* mapping from T2*-weighted MRI, and photoacoustic imaging. In vitro studies confirmed that NIR PIT resulted in rapid cell death via membrane damage, with evidence for rapid cell expansion followed by membrane rupture. Following NIR PIT, metabolic MRI using hyperpolarized fumarate showed the production of malate in EGFR-expressing A431 tumor xenografts, providing direct evidence for photosensitized tumor necrosis induced by NIR PIT. R2* mapping studies showed temporal changes in oxygenation, with an accompanying increase of deoxyhemoglobin at the start of light exposure followed by a sustained decrease after cessation of light exposure. This result suggests a rapid decrease of blood flow in EGFR-expressing A431 tumor xenografts, which is supported by the results of the photoacoustic imaging experiments. Our findings suggest NIR PIT mediates necrosis and hemodynamic changes in tumors by photosensitized oxidation pathways and that these imaging modalities, once translated, may be useful in monitoring clinical treatment response.

Evaluation of oxygen dependence on in vitro and in vivo cytotoxicity of photoimmunotherapy using IR-700-antibody conjugates.

Photoimmunotherapy (PIT) using the near-infrared-absorbing photosensitizing phthalocyanine dye, IRDye 700DX (IR-700), conjugated with a tumor-targeting antibody such as panitumumab (Pan) has shown efficacy in in vitro studies and several preclinical models in mice with promise for clinical translation. PIT results in rapid necrotic cell death in vitro and tumor shrinkage in vivo. Photochemical studies with the Pan-IR-700 conjugate showed that this agent can support generation of singlet oxygen and also generate reactive oxygen species after exposure to near-infrared (NIR) light. Moreover, in vitro studies using A431 cells, singlet oxygen scavengers abrogated the efficacy of PIT with Pan-IR-700, while oxygen depletion to undetectable levels in the exposure chamber almost completely inhibited the cellular cytotoxicity of PIT. Survival of tumor bearing mice was prolonged in PIT-treated animals but mice whose tumors were made transiently hypoxic prior to PIT had no benefit from the treatment. The results from this study support a central role for molecular oxygen-derived species in cell death caused by PIT.

Tempol-mediated activation of latent iron regulatory protein activity prevents symptoms of neurodegenerative disease in IRP2 knockout mice.

In mammals, two homologous cytosolic regulatory proteins, iron regulatory protein 1 (also known as IRP1 and Aco1) and iron regulatory protein 2 (also known as IRP2 and Ireb2), sense cytosolic iron levels and posttranscriptionally regulate iron metabolism genes, including transferrin receptor 1 (TfR1) and ferritin H and L subunits, by binding to iron-responsive elements (IREs) within target transcripts. Mice that lack IRP2 develop microcytic anemia and neurodegeneration associated with functional cellular iron depletion caused by low TfR1 and high ferritin expression. IRP1 knockout (IRP1(-/-)) animals do not significantly misregulate iron metabolism, partly because IRP1 is an iron-sulfur protein that functions mainly as a cytosolic aconitase in mammalian tissues and IRP2 activity increases to compensate for loss of the IRE binding form of IRP1. The neurodegenerative disease of IRP2(-/-) animals progresses slowly as the animals age. In this study, we fed IRP2(-/-) mice a diet supplemented with a stable nitroxide, Tempol, and showed that the progression of neuromuscular impairment was markedly attenuated. In cell lines derived from IRP2(-/-) animals, and in the cerebellum, brainstem, and forebrain of animals maintained on the Tempol diet, IRP1 was converted from a cytosolic aconitase to an IRE binding protein that stabilized the TfR1 transcript and repressed ferritin synthesis. We suggest that Tempol protected IRP2(-/-) mice by disassembling the cytosolic iron-sulfur cluster of IRP1 and activating IRE binding activity, which stabilized the TfR1 transcript, repressed ferritin synthesis, and partially restored normal cellular iron homeostasis in the brain.

MR assessment of changes of tumor in response to hyperbaric oxygen treatment.

Enhancement of image intensity, using the T1-weighted spoiled gradient-echo (SPGR) sequence, was measured in SCC tumor implanted in the flank of C3H mice while they were subjected to several types of oxygenation challenges inside a hyperbaric chamber designed and constructed to fit in an MRI resonator. The central portions of the tumor gave a positive enhancement, while the periphery showed signal reduction during both normobaric (NBO) and hyperbaric (HBO) oxygen challenges. In the contralateral normal leg, nearly 70% of the region showed a decrease in intensity, and the rest showed a positive enhancement. The positive signal enhancement was markedly greater under HBO compared to NBO. Calculated R1, R2, and M0 maps from multivariate fitting of images acquired by a multislice multiecho (MSME) sequence with variable TR before, during, and after HBO treatment confirm that the source of SPGR signal enhancement in the tumor is associated with shortening of T1.