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Molecular radiotherapy using177Lu-DOTATATE is a most effective treatment for somatostatin receptor–expressing neuroendocrine tumors. Despite its frequent and successful use in the clinic, little or no radiobiologic considerations are made at the time of treatment planning or delivery. On positive uptake on octreotide-based PET/SPECT imaging, treatment is usually administered as a standard dose and number of cycles without adjustment for peptide uptake, dosimetry, or radiobiologic and DNA damage effects in the tumor. Here, we visualized and quantified the extent of DNA damage response after177Lu-DOTATATE therapy using SPECT imaging with111In-anti-γH2AX-TAT. This work was a proof-of-principle study of this in vivo noninvasive biodosimeter with β-emitting therapeutic radiopharmaceuticals.Methods:Six cell lines were exposed to external-beam radiotherapy (EBRT) or177Lu-DOTATATE, after which the number of γH2AX foci and the clonogenic survival were measured. Mice bearing CA20948 somatostatin receptor–positive tumor xenografts were treated with177Lu-DOTATATE or sham-treated and coinjected with111In-anti-γH2AX-TAT,111In-IgG-TAT control, or vehicle.Results:Clonogenic survival after external-beam radiotherapy was cell-line–specific, indicating varying levels of intrinsic radiosensitivity. Regarding in vitro cell lines treated with177Lu-DOTATATE, clonogenic survival decreased and γH2AX foci increased for cells expressing high levels of somatostatin receptor subtype 2. Ex vivo measurements revealed a partial correlation between177Lu-DOTATATE uptake and γH2AX focus induction between different regions of CA20948 xenograft tumors, suggesting that different parts of the tumor may react differentially to177Lu-DOTATATE irradiation.Conclusion:111In-anti-γH2AX-TAT allows monitoring of DNA damage after177Lu-DOTATATE therapy and reveals heterogeneous damage responses.

Purpose:Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used an111In-radiolabelled form of LF with the PA/LF system for non-invasive in vivo imaging of MMP activity in tumour tissue by single photon emission computed tomography (SPECT). Methods:MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484 and MCF7). Uptake of111In-radiolabelled PA-L1,111In-PA-WTK563Cor111In-LFE687A(a catalytically inactive LF mutant) in tumour and normal tissues was measured using SPECT/CT imaging in vivo. Results:Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080>MDA-MB-231>B8484>MCF7). PA-L1-mediated delivery of111In-LFE687Awas demonstrated, and corroborated using confocal microscopy with fluorescently labelled LFE687A. Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of111In-PA-L1 in MDA-MB-231 tumour xenografts (5.7±0.9%ID/g) at 3 h post intravenous administration.111In-LFE687A was selectively delivered to MMP-positive MDA-MB-231 tumour tissue by MMP-activatable PA-L1 (5.98±0.62%ID/g), but not by furin cleavable PA-WT (1.05±0.21%ID/g), or a non-cleavable PA variant control, PA-U7 (2.74 ± 0.24%ID/g). Conclusion:Taken together, our results indicate that radiolabelled forms of mutated anthrax lethal toxin hold promise for non-invasive imaging of MMP activity in tumour tissue.