Stuart Koelewijn

Dynamic and Static Small-Animal SPECT in Rats for Monitoring Renal Function After 177Lu-Labeled Tyr3-Octreotate Radionuclide Therapy

High kidney radiation doses during clinical peptide receptor radionuclide therapy (PRRT) with β-particle–emitting radiolabeled somatostatin analogs will lead to renal failure several months after treatment, urging the coinfusion of the cationic amino acids lysine and arginine to reduce the renal radiation dose. In rat PRRT studies, renal protection by the coadministration of lysine was confirmed by histologic examination of kidney specimens indicating nephrotoxicity. In the current study, we investigated dedicated small-animal SPECT/CT renal imaging in rats to monitor renal function in vivo during follow-up of PRRT, with and without lysine. Methods: The following 3 groups of rats were imaged using a multipinhole SPECT/CT camera: controls (group 1) and rats at more than 90 d after therapy with 460 MBq (15 μg) of 177Lu-DOTA-Tyr3-octreotate without (group 2) or with (group 3) a 400-mg/kg lysine coinjection as kidney protection (n ≥ 6 per group). At 90 and 140 d after therapy, static kidney scintigraphy was performed at 2 h after injection of 25 MBq of 99mTc-dimercaptosuccinic acid (99mTc-DMSA). In addition, dynamic dual-isotope renography was performed using 50 MBq of 111In-diethylenetriaminepentaacetic acid (111In-DTPA) and 50 MBq of 99mTc-mercaptoacetyltriglycine (99mTc-MAG3) at 100–120 d after therapy. Results: 111In-DTPA and 99mTc-MAG3 studies revealed a time–activity pattern comparable to those in patients, with a peak at 2–6 min followed by a decline of renal radioactivity. Reduced 111In-DTPA, 99mTc-MAG3, and 99mTc-DMSA uptake indicated renal damage in group 2, whereas group 3 showed only a decrease of 99mTc-MAG3 peak activity. These results indicating nephrotoxicity in group 2 and renal protection in group 3 correlated with levels of urinary protein and serum creatinine and urea and were confirmed by renal histology. Conclusion: Quantitative dynamic dual-isotope imaging using both 111In-DTPA and 99mTc-MAG3 and static 99mTc-DMSA imaging in rats is feasible using small-animal SPECT, enabling longitudinal monitoring of renal function. 99mTc-MAG3 renography, especially, appears to be a more sensitive marker of tubular function after PRRT than serum chemistry or 99mTc-DMSA scintigraphy.

mTOR Inhibitor RAD001 Promotes Metastasis in a Rat Model of Pancreatic Neuroendocrine Cancer

Inhibition of mTOR is commonly considered a valid target in cancer treatment, but this assertion does not address effects on the immune microenvironment that may be detrimental to cancer treatment. Here we show how administration of the mTOR inhibitor RAD001 (everolimus) results in the occurrence of distant metastasis in a rat model of pancreatic cancer. RAD001 was administered twice weekly for 4.5 weeks as a single treatment or combined with [(177)Lu-DOTA,Tyr3]octreotate ((177)Lu-DOTATATE), where the latter targets the somatostatin receptor-2. The hypothesized synergistic therapeutic effect of RAD001 combined with (177)Lu-DOTATATE was, however, not observed in our experiments. The combination was shown to be less effective than (177)Lu-DOTATATE alone. Unexpectedly, tumor metastasis was observed in 77% of the subjects treated with RAD001, either alone or as part of the combination treatment. This was a striking effect, because metastasis did not occur in control or (177)Lu-DOTATATE-treated animals, including those where the primary tumor was surgically removed. These findings may be important clinically among noncompliant patients or patients that discontinue RAD001 therapy because of adverse effects.

In Vitro and In Vivo Application of Radiolabeled Gastrin-Releasing Peptide Receptor Ligands in Breast Cancer

Breast cancer (BC) consists of multiple subtypes defined by various molecular characteristics, for instance, estrogen receptor (ER) expression. Methods for visualizing BC include mammography, MR imaging, ultrasound, and nuclear medicine–based methods such as 99mTc-sestamibi and 18F-FDG PET, unfortunately all lacking specificity. Peptide receptor scintigraphy and peptide receptor radionuclide therapy are successfully applied for imaging and therapy of somatostatin receptor–expressing neuroendocrine tumors using somatostatin receptor radioligands. On the basis of a similar rationale, radioligands targeting the gastrin-releasing peptide receptor (GRP-R) might offer a specific method for imaging and therapy of BC. The aim of this study was to explore the application of GRP-R radioligands for imaging and therapy of BC by introducing valid preclinical in vitro and in vivo models. Methods: GRP-R expression of 50 clinical BC specimens and the correlation with ER expression was studied by in vitro autoradiography with the GRP-R agonist 111In-AMBA. GRP-R expression was also analyzed in 9 BC cell lines applying 111In-AMBA internalization assays and quantitative reverse transcriptase polymerase chain reaction. In vitro cytotoxicity of 177Lu-AMBA was determined on the GRP-R–expressing BC cell line T47D. SPECT/CT imaging and biodistribution were studied in mice with subcutaneous and orthotopic ER-positive T47D and MCF7 xenografts after injection of the GRP-R antagonist 111In-JMV4168. Results: Most of the human BC specimens (96%) and BC cell lines (6/9) were found to express GRP-R. GRP-R tumor expression was positively (P = 0.026, χ2(4) = 12,911) correlated with ER expression in the human BC specimens. Treatment of T47D cells with 10−7 M/50 MBq of 177Lu-AMBA resulted in 80% reduction of cells in vitro. Furthermore, subcutaneous and orthotopic tumors from both BC cell lines were successfully visualized in vivo by SPECT/CT using 111In-JMV4168; T47D tumors exhibited a higher uptake than MCF7 xenografts. Conclusion: Targeting GRP-R–expressing BC tumors using GRP-R radioligands is promising for nuclear imaging and therapy, especially in ER-positive BC patients.

Investigation of Factors Determining the Enhanced Permeability and Retention Effect in Subcutaneous Xenografts

Liposomal chemotherapy offers several advantages over conventional therapies, including high intratumoral drug delivery, reduced side effects, prolonged circulation time, and the possibility to dose higher. The efficient delivery of liposomal chemotherapeutics relies, however, on the enhanced permeability and retention (EPR) effect, which refers to the ability of macromolecules to extravasate leaky tumor vessels and accumulate in the tumor tissue. Using a panel of human xenograft tumors, we evaluated the influence of the EPR effect on liposomal distribution in vivo by injection of pegylated liposomes radiolabeled with 111In. Liposomal accumulation in tumors and organs was followed over time by SPECT/CT imaging. We observed that fast-growing xenografts, which may be less representative of tumor development in patients, showed higher liposomal accumulation than slow-growing xenografts. Additionally, several other parameters known to influence the EPR effect were evaluated, such as blood and lymphatic vessel density, intratumoral hypoxia, and the presence of infiltrating macrophages. The investigation of various parameters showed a few correlations. Although hypoxia, proliferation, and macrophage presence were associated with tumor growth, no hard conclusions or predictions could be made regarding the EPR effect or liposomal uptake. However, liposomal uptake was significantly correlated with tumor growth, with fast-growing tumors showing a higher uptake, although no biological determinants could be elucidated to explain this correlation.

Multimodality imaging of somatostatin receptor-positive tumors with nuclear and bioluminescence imaging

Multimodal bioluminescence (BLI) and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging were investigated as means to monitor somatostatin receptor subtype 2 (SST2)-positive neuroendocrine tumors as both a subcutaneously implanted and a liver metastasis animal model in mice and rats. Ultimately, such a model will be of use for studying SST2-targeted peptide receptor radionuclide therapy (PRRT). CA20948 cells were transfected with a green fluorescent protein/luciferase plasmid construct. Cells were inoculated subcutaneously in the shoulder of nude mice: nontransfected cells in the left shoulder and transfected cells in the right shoulder. BLI, SPECT/CT imaging, biodistribution analysis, and ex vivo autoradiography of the tumors were performed. BLI and SPECT/CT imaging were also performed on an intrahepatic tumor model in the rat. Caliper volume measurement of transfected tumors could be correlated with BLI measurements (2 = .76). SPECT/CT imaging showed high levels of accumulation of 111In-DTPA-octreotide in control and transfected tumors, which was confirmed by biodistribution analysis and autoradiography. Subcapsular inoculation of transfected cells in rat liver resulted in an intrahepatic tumor, which could be visualized by both SPECT/CT and BLI. Transfection of CA20948 tumor cells did not alter the growth properties of the cell line or the expression of SST2. Transfected tumors could be clearly visualized by BLI and SPECT/CT imaging. The transfected SST2-positive tumor cell line could represent a novel preclinical model for tumor monitoring in studies that aim at further optimizing PRRT for neuroendocrine tumors.

Optimized time-resolved imaging of contrast kinetics (TRICKS) in dynamic contrast-enhanced MRI after peptide receptor radionuclide therapy in small animal tumor models

Anti-tumor efficacy of targeted peptide-receptor radionuclide therapy (PRRT) relies on several factors, including functional tumor vasculature. Little is known about the effect of PRRT on tumor vasculature. With dynamic contrast-enhanced (DCE-) MRI, functional vasculature is imaged and quantified using contrast agents. In small animals DCE-MRI is a challenging application. We optimized a clinical sequence for fast hemodynamic acquisitions, time-resolved imaging of contrast kinetics (TRICKS), to obtain DCE-MRI images at both high spatial and high temporal resolution in mice and rats. Using TRICKS, functional vasculature was measured prior to PRRT and longitudinally to investigate the effect of treatment on tumor vascular characteristics. Nude mice bearing H69 tumor xenografts and rats bearing syngeneic CA20948 tumors were used to study perfusion following PRRT administration with (177) lutetium octreotate. Both semi-quantitative and quantitative parameters were calculated. Treatment efficacy was measured by tumor-size reduction. Optimized TRICKS enabled MRI at 0.032 mm(3) voxel size with a temporal resolution of less than 5 s and large volume coverage, a substantial improvement over routine pre-clinical DCE-MRI studies. Tumor response to therapy was reflected in changes in tumor perfusion/permeability parameters. The H69 tumor model showed pronounced changes in DCE-derived parameters following PRRT. The rat CA20948 tumor model showed more heterogeneity in both treatment outcome and perfusion parameters. TRICKS enabled the acquisition of DCE-MRI at both high temporal resolution (Tres ) and spatial resolutions relevant for small animal tumor models. With the high Tres enabled by TRICKS, accurate pharmacokinetic data modeling was feasible. DCE-MRI parameters revealed changes over time and showed a clear relationship between tumor size and Ktrans .