Desiree Bos

SPECT- and Fluorescence Image–Guided Surgery Using a Dual-Labeled Carcinoembryonic Antigen–Targeting Antibody

Intraoperative visualization techniques promise to significantly improve the detection and resection of tumors. In this study, we used an anti–carcinoembryonic antigen (CEA) antibody (MN-14) tagged with both a radiolabel (111In) and a fluorophore (IRDye 800CW) for radionuclide detection and intraoperative fluorescence imaging, respectively. Methods: For this purpose, we prepared and characterized the dual-labeled antibody 111In-diethylenetriaminepentaacetic acid (DTPA)-MN-14-IRDye 800CW and performed 4 studies on mice with subcutaneous and intraperitoneal CEA–expressing tumors: a dose escalation study to determine the optimal MN-14 protein dose, a biodistribution study comparing dual-labeled MN-14 and radiolabeled MN-14, a study to determine the optimal time for SPECT and fluorescence imaging after injection of dual-labeled MN-14, and finally a SPECT and fluorescence image–guided surgery study using this dual-labeled antibody. Results: The optimal protein dose of dual-labeled MN-14 was 10 μg per mouse, yielding a tumor-to-blood ratio of 3.5 within 72 h. The biodistribution of 111In-DTPA-MN-14-IRDye 800CW in mice with subcutaneous LS174T tumors showed tumor uptake after 3 d (19.7% ± 17.0% injected dose/g) comparable to that of 111In-DTPA-MN-14 but higher accumulation in the liver. The optimal time for imaging after administration of the dual-labeled antibody was 2–3 d after injection. Finally, in mice with intraperitoneally growing LS174T tumor nodules that received 111In-DTPA-MN-14-IRDye 800CW, intraperitoneal tumor nodules could be localized with SPECT imaging after 3 d and subsequently resected using fluorescence image–guided surgery. Conclusion: Thus, we showed the feasibility for assessment and image-guided resection of CEA antigen–expressing tumors using dual-labeled MN-14. Both radionuclide detection and fluorescence imaging may provide useful information to improve localization of tumors and radical excision of tumor tissue. Because humanized MN-14 (labetuzumab) is available for clinical use, translation to a clinical setting is the next step.

Targeted Dual-Modality Imaging in Renal Cell Carcinoma: An Ex Vivo Kidney Perfusion Study

Purpose: Antibodies labeled with both a near-infrared fluorescent dye and a radionuclide can be used for tumor-targeted intraoperative dual-modality imaging. Girentuximab is a chimeric monoclonal antibody against carbonic anhydrase IX (CAIX), an antigen expressed in 95% of clear cell renal cell carcinoma (ccRCC). This study aimed to assess the feasibility of targeted dual-modality imaging with 111In-girentuximab-IRDye800CW using ex vivo perfusion of human tumorous kidneys. Experimental Design: Seven radical nephrectomy specimens from patients with ccRCC were perfused during 11 to 15 hours with dual-labeled girentuximab and subsequently rinsed during 2.5 to 4 hours with Ringers Lactate solution. Then, dual-modality imaging was performed on a 5- to 10-mm-thick lamella of the kidney. Fluorescence imaging was performed with a clinical fluorescence camera set-up as applied during image-guided surgery. The distribution of Indium-111 in the slice of tumor tissue was visualized by autoradiography. In two perfusions, an additional dual-labeled control antibody was added to demonstrate specific accumulation of dual-labeled girentuximab in CAIX-expressing tumor tissue. Results: Both radionuclide and fluorescence imaging clearly visualized uptake in tumor tissue and tumor-to-normal tissue borders, as confirmed (immuno)histochemically and by gamma counting. Maximum uptake of girentuximab in tumor tissue was 0.33% of the injected dose per gram (mean, 0.12 %ID/g; range, 0.01–0.33 %ID/g), whereas maximum uptake in the normal kidney tissue was 0.04 %ID/g (mean, 0.02 %ID/g; range, 0.00–0.04 %ID/g). Conclusions: Dual-labeled girentuximab accumulated specifically in ccRCC tissue, indicating the feasibility of dual-modality imaging to detect ccRCC. A clinical study to evaluate intraoperative dual-modality imaging in patients with ccRCC has been initiated. Clin Cancer Res; 22(18); 4634–42. ©2016 AACR.

Graft revascularization is essential for non-invasive monitoring of transplanted islets with radiolabeled exendin

Islet transplantation is a novel promising strategy to cure type 1 diabetes. However, the long-term outcome is still poor, because both function and survival of the transplant decline over-time. Non-invasive imaging methods have the potential to enable monitoring of islet survival after transplantation and the effects of immunosuppressive drugs on transplantation outcome. 111In-labeled exendin-3 is a promising tracer to visualize native and transplanted islets by SPECT (Single Photon Emission Computed Tomography). In the present study, we hypothesized that islet microvasculature plays an important role determining the uptake of exendin-3 in islets when monitoring transplant survival. We observed 111In-exendin-3 accumulation in the transplant as early as three days after transplantation and an increase in the uptake up to three weeks post-transplantation. Islet-revascularization correlated with the increase in 111In-exendin-3 uptake, whereas fully re-established islet vasculature coincided with a stabilized uptake of the radiotracer in the transplant. Here, we demonstrate the importance of islet vasculature for in vivo delivery of radiotracers to transplanted islets and we demonstrate that optimal and stable uptake of exendin four weeks after transplantation opens the possibility for long-term monitoring of islet survival by SPECT imaging.

Radionuclide and Fluorescence Imaging of Clear Cell Renal Cell Carcinoma Using Dual Labeled Anti-Carbonic Anhydrase IX Antibody G250

PURPOSE Tumor targeted optical imaging using antibodies labeled with near infrared fluorophores is a sensitive imaging modality that might be used during surgery to assure complete removal of malignant tissue. We evaluated the feasibility of dual modality imaging and image guided surgery with the dual labeled anti-carbonic anhydrase IX antibody preparation (111)In-DTPA-G250-IRDye800CW in mice with intraperitoneal clear cell renal cell carcinoma. MATERIALS AND METHODS BALB/c nu/nu mice with intraperitoneal SK-RC-52 lesions received 10 μg DTPA-G250-IRDye800CW labeled with 15 MBq (111)In or 10 μg of the dual labeled irrelevant control antibody NUH-82 (20 mice each). To evaluate when tumors could be detected, 4 mice per group were imaged weekly during 5 weeks with single photon emission computerized tomography/computerized tomography and the fluorescence imaging followed by ex vivo biodistribution studies. RESULTS As early as 1 week after tumor cell inoculation single photon emission computerized tomography and fluorescence images showed clear delineation of intraperitoneal clear cell renal cell carcinoma with good concordance between single photon emission computerized tomography/computerized tomography and fluorescence images. The high and specific accumulation of the dual labeled antibody conjugate in tumors was confirmed in the biodistribution studies. Maximum tumor uptake was observed 1 week after inoculation (mean ± SD 58.5% ± 18.7% vs 5.6% ± 2.3% injected dose per gm for DTPA-G250-IRDye800CW vs NUH-82, respectively). High tumor uptake was also observed at other time points. CONCLUSIONS This study demonstrates the feasibility of dual modality imaging with dual labeled antibody (111)In-DTPA-G250-IRDye800CW in a clear cell renal cell carcinoma model. Results indicate that preoperative and intraoperative detection of carbonic anhydrase IX expressing tumors, positive resection margins and metastasis might be feasible with this approach.

Detection of Micrometastases Using SPECT/Fluorescence Dual-Modality Imaging in a CEA-Expressing Tumor Model

Intraoperative dual-modality imaging can help the surgeon distinguish tumor from normal tissue. This technique may prove particularly valuable if small tumors need to be removed that are difficult to detect with the naked eye. The humanized anticarcinoembryonic antigen (anti-CEA) monoclonal antibody, labetuzumab, can be used as a tumor-targeting agent in colorectal cancer, since CEA is overexpressed in approximately 95% of colorectal cancer. Dual-labeled labetuzumab, labeled with both a near-infrared fluorescent dye (IRDye800CW) and a radioactive label (111In), can be used as a tracer for dual-modality imaging. This study aimed to assess whether dual-modality imaging using 111In-diethylenetriaminepentaacetic acid (DTPA)–labetuzumab-IRDye800CW can detect pulmonary micrometastases in a mouse model. Methods: Pulmonary GW-39 human colonic carcinoma microcolonies were induced in athymic BALB/c mice by intravenous injection of 100 μL of a GW-39 cell suspension. After 1, 2, 3, and 4 wk of tumor growth, dual-modality imaging was performed 3 d after intravenous injection of 111In-DTPA-labetuzumab-IRDye800CW (10 μg, 25 MBq). Small-animal SPECT images and optical images were acquired, and image-guided surgery was performed. Finally, the biodistribution of the dual-labeled tracer was determined. Formalin-fixed sections of the lungs were analyzed using fluorescence imaging, autoradiography, and immunohistochemistry. Results: Submillimeter pulmonary tumor colonies could be visualized with both small-animal SPECT and fluorescence imaging from the first week of tumor growth, before they became visible to the naked eye. Furthermore, dual-modality imaging could be used to guide resection of tumors. Mean uptake (percentage injected dose per gram) of the dual-labeled tracer in tumors was 17.2 ± 5.4 and 16.5 ± 4.4 at weeks 3 and 4, respectively. Immunohistochemical analysis of the tumorous lungs showed that the distribution of the radioactive and fluorescent signal colocalized with CEA-expressing tumors. Conclusion: Dual-modality imaging after injection of 111In-labetuzumab-IRDye800CW can be used to detect submillimeter CEA-expressing pulmonary tumors before they become visible to the naked eye, supporting the added value of this technique in the resection of small tumors.

Optimization of Dual-Labeled Antibodies for Targeted Intraoperative Imaging of Tumors

For intraoperative imaging, antibodies labeled with both a radionuclide and a fluorophore may be used to tag the tumor lesion with a radiolabel and a fluorescent signal at high tumor to background ratios. However, labeling antibodies with fluorescent moieties may affect the in vivo behavior of the antibody depending on the dye to antibody substitution ratio. To investigate the optimal substitution ratio for use in dual-modality image-guided surgery, we conjugated three different antibodies, MN-14 (anti-CEACAM5), girentuximab (anti-CAIX), and cetuximab (anti-EGFR), with both diethylene triamine pentaacetic acid (DTPA, for labeling with 111In) and IRdye 800CW at dye to antibody ratios of 0, 1, 1.5, 2, and 3 and assessed in vivo behavior. Biodistribution studies showed that at high dye to antibody ratios, liver uptake of the dual-labeled antibodies increased, whereas tumor uptake decreased. Conversely, very low ratios may not be optimal either because in that case, only a few antibody molecules will be dual-labeled (i.e., contain both a DTPA and an IRDye 800CW moiety), which may complicate interpretation of dual-modality data. The present study shows that, provided that the chelator to antibody ratio is high enough, a dye to antibody ratio in the range of 1 to 1.5 is optimal for antibody-targeted dual-modality imaging applications. However, the optimal configuration is antibody dependent and should be determined for each dual-labeled antibody individually.

Optimizing lutetium 177-anti-carbonic anhydrase IX radioimmunotherapy in an intraperitoneal clear cell renal cell carcinoma xenograft model

A new approach in the treatment of clear cell renal carcinoma (ccRCC) is radioimmunotherapy (RIT) using anti-carbonic anhydrase IX (CAIX) antibody G250. To investigate the potential of RIT with lutetium 177 (177Lu)-labeled G250, we conducted a protein dose escalation study and subsequently an RIT study in mice with intraperitoneally growing ccRCC lesions. Mice with intraperitoneal xenografts were injected with 1, 3, 10, 30, or 100 μg of G250 labeled with 10 MBq indium 111 (111In) to determine the optimal protein dose. The optimal protein dose determined with imaging and biodistribution studies was used in a subsequent RIT experiment in three groups of 10 mice with intraperitoneal SK-RC-52 tumors. One group received 13 MBq 177LU-DOTA-G250, a control group received 13 MBq nonspecific 177LU-MOPC21, and the second control group was not treated and received 20 MBq 111In-DOTA-G250. The optimal G250 protein dose to target ccRCC in this model was 10 μg G250. Treatment with 13 MBq 177LU-DOTA-G250 was well tolerated and resulted in significantly prolonged median survival (139 days) compared to controls (49-53 days, p = .015), indicating that RIT has potential in this metastatic ccRCC model.

Preventing Radiobleaching of Cyanine Fluorophores Enhances Stability of Nuclear/NIRF Multimodality Imaging Agents

Despite the large interest in nuclear/optical multimodality imaging, the effect of radiation on the fluorescence of fluorophores remains largely unexplored. Herein, we report on the radiobleaching of cyanine fluorophores and describe conditions to provide robust radioprotection under practical (pre)clinical settings. We determined the radiosensitivity of several cyanine fluorescent compounds, including IRDye 800CW (800CW) and a dual modality imaging tetrapeptide containing DOTA as chelator and Dylight 800 as fluorophore, exposed to increasing activities of 111In, 68Ga, or 213Bi (γ, EC/β, and α emitter, respectively). An activity and type of radiation-dependent radiation-induced loss of fluorescence, radiobleaching, of 800CW was observed upon incubation with escalating activities of 111In, 68Ga, or 213Bi. 68Ga showed the largest radiolytic effect, followed by 111In and 213Bi. The addition of oxygen radical scavengers including ethanol, gentisic acid, and ascorbic acid (AA), provided a concentration dependent radioprotective effect. These results supported the hypothesis of a free radical-mediated radiobleaching mechanism. AA provided the most robust radioprotection over a wide range of concentrations and preserved fluorescence at much higher radioactivity levels. Overall, both near-infrared fluorescent compounds displayed similar sensitivity, except for 213Bi-irradiated solutions, where the dual modality construct exhibited enhanced radiolysis, presumably due to direct radiation damage from α particles. Concurrently, AA was not able to preserve fluorescence of the dual-modality molecule labeled with 213Bi. Our findings have important consequences for several research areas including ROS sensing, radiation-mediated drug release (uncaging), fluorescent dosimetry, and in the preparation of dual-modality radiopharmaceuticals.

Noninvasive Imaging of Islet Transplants with 111In-Exendin-3 SPECT/CT

Islet transplantation is a promising treatment for type 1 diabetic patients. However, there is acute as well as chronic loss of islets after transplantation. A noninvasive imaging method that could monitor islet mass might help to improve transplantation outcomes. In this study, islets were visualized after transplantation in a rat model with a dedicated small-animal SPECT scanner by targeting the glucagonlike peptide-1 receptor (GLP-1R), specifically expressed on β-cells, with 111In-labeled exendin-3. Methods: Targeting of 111In-exendin-3 to GLP-1R was tested in vitro on isolated islets of WAG/Rij rats. For in vivo evaluation, 400 or 800 islets were transplanted into the calf muscle of WAG/Rij rats (6–8 wk old). Four weeks after transplantation, SPECT/CT images were acquired 1 h after injection of 111In-labeled exendin-3. After SPECT acquisition, the muscles containing the transplant were analyzed immunohistochemically and autoradiographically. Results: The binding assay, performed on isolated islets, showed a linear correlation between the number of islets and 111In-exendin-3 accumulation (Pearson r = 0.98). In vivo, a 1.70 ± 0.44-fold difference in tracer uptake between 400 and 800 transplanted islets was observed. Ex vivo analysis of the islet transplant showed colocalization of tracer accumulation on autoradiography, with insulin-positive cells and GLP-1R expression on immunohistochemistry. Conclusion: 111In-exendin-3 accumulates specifically in the β-cells after islet transplantation and is a promising tracer for noninvasive monitoring of the islet mass.

Non-invasive in vivo determination of viable islet graft volume by 111In-exendin-3

Pancreatic islet transplantation is a promising therapy for patients with type 1 diabetes. However, the duration of long-term graft survival is limited due to inflammatory as well as non-inflammatory processes and routine clinical tests are not suitable to monitor islet survival. 111In-exendin-SPECT (single photon emission computed tomography) is a promising method to non-invasively image islets after transplantation and has the potential to help improve the clinical outcome. Whether 111In-exendin-SPECT allows detecting small differences in beta-cell mass (BCM) and measuring the actual volume of islets that were successfully engrafted has yet to be demonstrated. Here, we evaluated the performance of 111In-exendin-SPECT using an intramuscular islet transplantation model in C3H mice. In vivo imaging of animals transplanted with 50, 100, 200, 400 and 800 islets revealed an excellent linear correlation between SPECT quantification of 111In-exendin uptake and insulin-positive area of islet transplants, demonstrating that 111In-exendin-SPECT specifically and accurately measures BCM. The high sensitivity of the method allowed measuring small differences in graft volumes, including grafts that contained less than 50 islets. The presented method is reliable, convenient and holds great potential for non-invasive monitoring of BCM after islet transplantation in humans.