John B Little Center for Radiation Sciences

To be able to evaluate new radiopharmaceuticals and optimize diagnostic and therapeutic procedures, relevant animal models are required. The aim of this study was to evaluate the medullary thyroid carcinoma GOT2 animal model by analyzing the biodistribution of 177 Lu-octreotate and 111

Introduction: The kidneys are the dose-limiting organs in some radionuclide therapy regimens. However, the biological impact of internal exposure from radionuclides is still not fully understood. The aim of this study was to examine the effects of dose rate and time after i.v. injection of 177 LuCl 3 on changes in transcriptional patterns in mouse kidney tissue. Methods: To investigate the effect of dose rate, female Balb/c nude mice were i.v. injected with 11, 5.6, 1.6, 0.8, 0.30, and 0 MBq of 177 LuCl 3 , and killed at 3, 6, 24, 48, 168, and 24 hours after injection, respectively. Furthermore, the effect of time after onset of exposure was analysed using mice injected with 0.26, 2.4, and 8.2 MBq of 177 LuCl 3 , and killed at 45, 90, and 140 days after injection. Global transcription patterns of irradiated kidney cortex and medulla were assessed and enriched biological processes were determined from the regulated gene sets using Gene Ontology terms. Results: The average dose rates investigated were 1.6, 0.84, 0.23, 0.11 and 0.028 mGy/min, with an absorbed dose of 0.3 Gy. At 45, 90 and 140 days, the absorbed doses were estimated to 0.3, 3, and 10 Gy. In general, the number of differentially regulated transcripts increased with time after injection, and decreased with absorbed dose for both kidney cortex and medulla. Differentially regulated transcripts were predominantly involved in metabolic and stress response-related processes dependent on dose rate, as well as transcripts associated with metabolic and cellular integrity at later time points. Conclusion: The observed transcriptional response in kidney tissue was diverse due to difference in absorbed dose, dose rate and time after exposure. Nevertheless, several transcripts were significantly regulated in all groups despite differences in exposure parameters, which may indicate potential biomarkers for exposure of kidney tissue.

Background: In cancer radiotherapy, knowledge of normal tissue responses and toxicity risks is essential in order to deliver the highest possible absorbed dose to the tumor while maintaining normal tissue exposure at non-critical levels. However, few studies have investigated normal tissue responses in vivo after 211 At administration. In order to identify molecular biomarkers of ionizing radiation exposure, we investigated genome-wide transcriptional responses to (very) low mean absorbed doses from 211 At in normal mouse tissues. Methods: Female BALB/c nude mice were intravenously injected with 1.7 kBq 211 At and killed after 1 h, 6 h, or 7 days or injected with 105 or 7.5 kBq and killed after 1 and 6 h, respectively. Controls were mock-treated. Total RNA was extracted from tissue samples of kidney cortex and medulla, liver, lungs, and spleen and subjected to microarray analysis. Enriched biological processes were categorized after cellular function based on Gene Ontology terms.

Background
211At-labeled radiopharmaceuticals are potentially useful for tumor therapy. However, a limitation
has been the preferential accumulation of released 211At in the thyroid gland, which
is a critical organ for such therapy. The aim of this study was to determine the effect of
absorbed dose, dose-rate, and time after 211At exposure on genome-wide transcriptional
expression in mouse thyroid gland.
Methods
BALB/c mice were i.v. injected with 1.7, 7.5 or 100 kBq 211At. Animals injected with 1.7 kBq
were killed after 1, 6, or 168 h with mean thyroid absorbed doses of 0.023, 0.32, and 1.8 Gy,
respectively. Animals injected with 7.5 and 100 kBq were killed after 6 and 1 h, respectively;
mean thyroid absorbed dose was 1.4 Gy. Total RNA was extracted from pooled thyroids
and the Illumina RNA microarray platform was used to determine mRNA levels. Differentially
expressed transcripts and enriched GO terms were determined with adjusted p-value
<0.01 and fold change >1.5, and p-value <0.05, respectively.
Results
In total, 1232 differentially expressed transcripts were detected after 211At administration,
demonstrating a profound effect on gene regulation. The number of regulated transcripts
increased with higher initial dose-rate/absorbed dose at 1 or 6 h. However, the number of
regulated transcripts decreased with mean absorbed dose/time after 1.7 kBq 211At administration. Furthermore, similar regulation profiles were seen for groups administered
1.7 kBq. Interestingly, few previously proposed radiation responsive genes were detected
in the present study. Regulation of immunological processes were prevalent at 1, 6, and
168 h after 1.7 kBq administration (0.023, 0.32, 1.8 Gy).

Background: 131 I and 211 At are used in nuclear medicine and accumulate in the thyroid gland and may impact normal thyroid function. The aim of this study was to determine transcriptional profile variations, assess the impact on cellular activity, and identify genes with biomarker properties in thyroid tissue after 131 I and 211 At administration in mice. Methods: To further investigate thyroid tissue transcriptional responses to 131 I and 211 At administration, we generated a new transcriptional dataset that includes re-evaluated raw intensity values from our previous 131 I and 211 At studies. Differential transcriptional profiles were identified by comparing treated and mock-treated samples using Nexus Expression 3.0 software. Further data analysis was performed using R/Bioconductor and IPA.

Non-targeted effects can induce responses in tissues that have not been exposed to ionizing radiation. Despite their relevance for risk assessment, few studies have investigated these effects in vivo. In particular, these effects have not been studied in context with thyroid exposure, which can occur e.g. during irradiation of head and neck tumors. To determine the similarity between in-field and out-of-field responses in normal tissue, we used a partial body irradiation setup with female mice where the thyroid region, the thorax and abdomen, or all three regions were irradiated. After 24 h, transcriptional regulation in the kidney cortex, kidney medulla, liver, lungs, spleen, and thyroid was analyzed using microarray technology. Thyroid irradiation resulted in transcriptional regulation in the kidney medulla and liver that resembled regulation upon direct exposure of these tissues regarding both strength of response and associated biological function. The kidney cortex showed fewer similarities between the setups, while the lungs and spleen showed little similarity between in-field and out-of-field responses. Interestingly, effects were generally not found to be additive. Future studies are needed to identify the molecular mechanisms that mediate these systemic effects, so that they may be used as targets to minimize detrimental side effects in radiotherapy. Radiation biology is based on the paradigm that DNA double-strand break induction is the hallmark event upon ionizing radiation (IR) exposure that determines cellular outcome 1. Observations of non-targeted effects in experimental and clinical settings have challenged this paradigm, which has been frequently reviewed (and discussed) in recent years 2–8. Based on the context, non-targeted effects are also termed 'bystander effect' for unirradiated cells in the vicinity of irradiated cells, 'abscopal' or 'out-of-field' effects in external irradiation, or 'long-range bystander effects' or 'systemic effects' when regarding a complex physiological setting 2–8. The question has been raised whether non-targeted effects represent a new paradigm in radiation research or simply the fact of biological complexity 8. It has also been proposed that the frequency of reported clinical abscopal effects may increase due to the increasing use of stereotactic ablative body radiation therapy and hypofractionation techniques 9. While the underlying mechanisms of in vivo abscopal bystander effects remain elusive 3 , it has been reasoned that normal tissue responses in radiotherapy are highly complex and that the degree of non-targeted effects are not only related to the irradiated volume or physical irradiation parameters 8,10. Non-targeted effects are considered relevant for radiation therapy, both for use as a novel therapeutic target and for improvement of risk assessment 4,8,11–16 .

Background: 177 Lu-[DOTA 0 , Tyr 3 ]-octreotate (177 Lu-octreotate) is used for treatment of patients with somatostatin receptor (SSTR) expressing neuroendocrine tumors. However, complete tumor remission is rarely seen, and optimization of treatment protocols is needed. In vitro studies have shown that irradiation can up-regulate the expression of SSTR1, 2 and 5, and increase 177 Lu-octreotate uptake. The aim of the present study was to examine the anti-tumor effect of a 177 Lu-octreotate priming dose followed 24 h later by a second injection of 177 Lu-octreotate compared to a single administration of 177 Lu-octreotate, performed on the human small intestine neuroendocrine tumor cell line, GOT1, transplanted to nude mice. Results: Priming resulted in a 1.9 times higher mean absorbed dose to the tumor tissue per administered activity, together with a reduced mean absorbed dose for kidneys. Priming gave the best overall anti-tumor effects. Magnetic resonance imaging showed no statistically significant difference in tumor response between treatment with and without priming. Gene expression analysis demonstrated effects on cell cycle regulation. Biological processes associated with apoptotic cell death were highly affected in the biodistribution and dosimetry study, via differential regulation of, e.g., APOE, BAX, CDKN1A, and GADD45A. Conclusions: Priming had the best overall anti-tumor effects and also resulted in an increased therapeutic window. Results indicate that potential biomarkers for tumor regrowth may be found in the p53 or JNK signaling pathways. Priming administration is an interesting optimization strategy for 177 Lu-octreotate therapy of neuroendocrine tumors, and further studies should be performed to determine the mechanisms responsible for the reported effects.

Humans are exposed to 131 I in medical diagnostics and treatment but also from nuclear accidents , and better knowledge of the molecular response in thyroid is needed. The aim of the study was to examine the transcriptional response in thyroid tissue 24 h after 131 I administration in rats. The exposure levels were chosen to simulate both the clinical situation and the case of nuclear fallout. Thirty-six male rats were i.v. injected with 0–4700 kBq 131 I, and killed at 24 h after injection (D thyroid = 0.0058–3.0 Gy). Total RNA was extracted from individual thyroid tissue samples and mRNA levels were determined using oligonucleotide microarray technique. Differentially expressed transcripts were determined using Nexus Expression 3.0. Hierarchical clustering was performed in the R statistical computing environment. Pathway analysis was performed using the Ingenuity Pathway Analysis tool and the Gene Ontol-ogy database. T4 and TSH plasma concentrations were measured using ELISA. Totally, 429 differentially regulated transcripts were identified. Downregulation of thyroid hormone biosynthesis associated genes (e.g. thyroglobulin, thyroid peroxidase, the sodium-iodine symporter) was identified in some groups, and an impact on thyroid function was supported by the pathway analysis. Recurring downregulation of Dbp and Slc47a2 was found. Dbp exhibited a pattern with monotonous reduction of downregulation with absorbed dose at 0.0058–0.22 Gy. T4 plasma levels were increased and decreased in rats whose thyroids were exposed to 0.057 and 0.22 Gy, respectively. Different amounts of injected 131 I gave distinct transcriptional responses in the rat thyroid. Transcriptional response related to thyroid function and changes in T4 plasma levels were found already at very low absorbed doses to thyroid.