Imaging DNA Damage Repair in vivo Following Lu-DOTATATE Therapy

Purpose . Molecular Radiotherapy (MRT) using 177 Lu-DOTATATE is a most effective therapy for the treatment of somatostatin receptor expressing neuroendocrine tumors (NETs). Despite its frequent and successful use in the clinic, little or no radiobiological considerations are taken into account at the time of treatment planning or delivery, and upon positive uptake of octreotide-based PET/SPECT imaging, treatment is usually administered as a standard dose and number of cycles without adjustment for peptide uptake, dosimetry, or radiobiological and DNA damage effects in the tumor. Here, we visualize and quantify the extent of DNA damage response following 177 Lu-DOTATATE therapy using SPECT imaging with 111 In-anti- γ H2AX-TAT. This work is a proof-of-principle study of this in vivo non-invasive biodosimeter with beta-emitting therapeutic radiopharmaceuticals. Methods . Six cell lines were exposed to external beam radiotherapy (EBRT) or 177 Lu-DOTATATE, after which the number of γ H2AX foci and clonogenic survival were measured. Mice bearing CA20948 somatostatin receptor positive tumor xenografts were treated with 177 Lu-DOTATATE or sham-treated, and co-injected with 111 In-anti- γ H2AX-TAT, 111 In-IgG-TAT control, or vehicle. Results . Clonogenic survival following EBRT was cell line specific, indicating varying levels of intrinsic radiosensitivity. In vitro , cell lines treated with 177 Lu-DOTATATE, clonogenic survival decreased and γ H2AX foci increased in cells expressing high levels of somatostatin receptor subtype 2 (SST2). Ex vivo measurements revealed a partial correlation between 177 Lu-DOTATATE uptake and γ H2AX foci induction between different regions of CA20948 xenograft tumors, suggesting different parts of the tumor may react differentially to 177 Lu-DOTATATE irradiation. Conclusion . 111 In-anti- γ H2AX-TAT allows monitoring of DNA damage following 177 Lu-DOTATATE therapy, and reveals heterogeneous damage responses. in vitro resulted in reduced clonogenic survival. Different cell lines responded differently to the same 177 Lu absorbed dose. The same was true for EBRT. Nonetheless, sensitivity to EBRT did not correlate linearly with sensitivity to 177 Lu. DNA double strand break damage was observed in vitro by immunofluorescence, as measured by γ H2AX foci. Kinetics of γ H2AX formation and dissolution was different following 177 Lu exposure and EBRT. It has been shown previously that therapeutic success of ionizing radiation is closely correlated with the induction of DNA double strand break damage, especially with late, unrepaired damage ( 28 ). 177 Lu-DOTATATE causes DNA damage in vivo in tumor tissue, and thus causes expression of γ H2AX. We demonstrate that this induction of γ H2AX following 177 Lu-DOTATATE therapy can be monitored by SPECT imaging with 111 In-anti- γ H2AX- TAT. This allowed us to simultaneously study, in the whole tumor, the relationship between 177 Lu distribution, as a surrogate for uptake of indium-111 in mice that did not receive 177 Lu-DOTATATE. Results were reported as percentage of the injected dose per mL of tissue (%ID/mL). tissues and blood were removed and transferred into pre-weighed counting tubes with radioactivity spectra measured using a HiDex automated gamma counter. Activities for each isotope were calculated from area under curve analysis using GraphPad prism, based upon standard curves of mixtures containing known activities of lutetium-177 and indium-111. All values were decay-corrected to the time of injection, and the percentage of the injected dose per gram (%ID/g) of each sample was calculated. Five animals were used per group. temperature for 1 h. After further washing, excess fluid was removed and a small drop of Vectashield plus DAPI (Vector H-1200) was applied to each section and a coverslip placed on the slide and secured with Covergrip (Biotium 23005). Fluorescence micrographs were acquired on an Andor Dragonfly microscope (Oxford Instruments) using a 40x/1.3 oil lens. Autoradiography was performed on adjacent sections, using a digital autoradiography platform (Cyclone, Perkin Elmer). Micrographs were registered to autoradiographs using a landmark-based MATLAB algorithm developed in-house. Coincidence of anti-γH2AX antib ody associated fluorescence with autoradiography signal was performed on binned co-registered adjacent slides using rigid registration with the JaCOP (Just Another Co-localisation Plugin) in ImageJ.