In Vivo Imaging of Pancreatic Islet Grafts in Diabetes Treatment

Transplantation of pancreatic islets has potential to offer life-long blood glucose management in type I diabetes and severe type II diabetes without the need of exogenous insulin administration. However, islet cell therapy suffers from autoimmune and allogeneic rejection as well as non-immune related factors. Non-invasive techniques to monitor and evaluate the fate of cell implants in vivo are essential to understand the underlying causes of graft failure, and hence to improve the precision and efficacy of islet therapy. This review describes how imaging technology has been employed to interrogate the distribution, number or volume, viability, and function of islet implants in vivo. To date, fluorescence imaging, PET, SPECT, BLI, MRI, MPI, and ultrasonography are the many imaging modalities being developed to fulfill this endeavor. We outline here the advantages, limitations, and clinical utility of each particular imaging approach.


INTRODUCTION
Moment-to-moment regulation of blood glucose in type I diabetes (T1D) and severe type II diabetes (T2D) patients may be achieved by transplantation of pancreatic islets without the need for exogeneous insulin administration (1-3). Engrafted islets have been shown to stabilize blood glucose control, reduce the occurrence of hypoglycemia and lead to insulin independence albeit for only a short period of time. Indeed, phase 3 clinical studies on allogeneic islet transplantation in 48 T1D patients have been conducted at eight centers in North America. At 1 and 2 years post-treatment, 88 and 71% of patients successfully met the primary end point (HbA1c <7.0%), respectively (4). Islet therapy however suffers from autoimmune and allogeneic rejection as well as non-immune related factors, such as inadequate neovascularization of islet grafts. It has been a major challenge to elucidate the fate of islets after engraftment (5)(6)(7). At present, clinical trials rely on blood glucose levels and metabolic tests before and after transplantation to gauge the therapeutic performance (8,9). These values do not accurately reflect the mass of successfully engrafted islets, and the survival and insulin secretion of islets in vivo. Many of these tests detect failed engraftment long after the majority of islet grafts have died and thus, the window to apply interventional therapy to save the implants has passed.
Recent work strives to develop imaging technology for non-invasive and clinically relevant interrogation of the distribution, number or volume, viability, and function of islet implants in vivo. The main challenges are the small size of islets (50-300 µm) and the lack of inherent contrast between islet grafts and the surrounding host tissue. Such limitations demand a high detection sensitivity of labeled islets. Histology, bioluminescence (BLI), and fluorescence microscopy imaging (FMI) have been widely utilized to assess transplanted islets in animal models but all three methods are not suitable for patient studies due to its invasiveness or lack of light penetration in deep tissues. PET, SPECT, magnetic resonance imaging (MRI), magnetic particle imaging (MPI), and ultrasonography are currently being developed for this specific purpose and, unlike BLI or FMI, have shown promising potential for clinical translation.

LABELING OF ISLETS
For in vivo imaging, islets or cells are typically labeled prior to transplantation with the exception of SPECT and PET radiolabeling. For BLI, islets or beta-like cells, such as INS-1E cells, can be transfected or transduced with luciferase gene commonly derived from firefly (10)(11)(12)(13)(14)(15)(16)(17). Baculovirus has been used to mediate radiolabeling by 125 I for SPECT imaging (18). However, genetic modification of cells raises serious concerns about adverse cell differentiation as well as their immunogenicity, leading to a long path of FDA approval if any. SPECT and PET tracers are designed to target beta cell-specific receptors and therefore label islet grafts in situ (19)(20)(21)(22). For FMI, MRI, and MPI, islets are labeled ex vivo prior to transplantation by direct probe incubation. Here, nanoparticles can serve as a versatile platform for creating probes that can be detected by more than one imaging modality (10,15,23). The limiting factor in islet labeling is the total amount of probe that an islet can carry while maintaining preservation of islet viability and function as well as in vivo detection sensitivity. Henceforth, the development of optimum islet labeling probes and protocols remains an actively pursued field.

BIOLUMINESCENCE IMAGING
Upon administration of the substrate D-luciferin to the islet recipients, the luciferase gene of labeled islets emits light. This photon emission is subsequently detected with a BLI instrument. BLI signals are registered as "hot spots", therefore avoiding any confusion with background artifacts (Figures 1A, B). BLI is commonly overlaid on a regular photograph of the animal to provide anatomical context. This modality offers non-invasive quantitative imaging as the number of labeled islets linearly corresponds to photon emission, i.e., the intensity of the BLI signal (12). Since only live labeled islets produce photons, BLI can be used to monitor the survival of islet grafts, and to study the causes and timeline of islet death as well as graft distribution in a serial and quantitative manner.
However, deep tissues attenuate or scatter emitted photons measured by BLI (24) and compromise the readout. It is highly unlikely that the FDA will approve D-luciferin injection in patients in addition to issues related to genetic manipulation of cells and the expression of a xenogeneic protein (firefly luciferase). A typical D-luciferin injection dose for small rodents is no less than 150 µg/g body weight. Thus, the use of BLI has been confined to the study of cells and tissues in small rodents. BLI has been used to monitor the effects of 3D stem cell spheroids (13), poly(ethylene glycol)-encapsulants (14), mesenchymal stem cell-enriched scaffolds (16) or heparinreleasing silk fibroin scaffolds (17) on islet survival in vivo in rodents. The overall tolerance of islets for labeling them with perfluoro-15-crown-5-ether (PCE) emulsions for 19 F MRI (10) or poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with PCE and indocyanine green dye for 19 F MRI and nearinfrared (NIR) fluorescence imaging (15) has also been evaluated with BLI. Lastly, using TLR4-deficient mice and BLI, Gao et al. (11) showed that TLR4 activation mediated graft failure following intraportal islet transplantation.

FLUORESCENCE MICROSCOPIC IMAGING
The limited depth of light penetration has largely limited FMI to examination of cells and tissues ex vivo or superficially. However, some recent developments have focused on in vivo imaging using FMI. Several potential clinical graft sites for islets have been explored, including subcutaneous chambers or intramuscular transplantation. As long as islets are transplanted superficially, FMI can be used to visualize the grafts. Nilsson et al. (25) implanted 20-30 human islets in the anterior chamber of the mouse eye, serving as an optical window for longitudinal monitoring with a high resolution 2-photon microscope. This study utilized transgenic NOD. (Cg)-Gt(ROSA)26Sortm4-Rag2 −/− mice whose cells and tissues expressed a membrane-targeted tomato fluorescent protein to facilitate identification of the recipient and the donor tissues. Although the eyes have been considered to be an immunoprivileged site (due to the bloodocular barrier), the clinical relevancy for this form of islet transplantation is a subject of debate. In a different study, iron oxide and human serum albumin-bound indocyanine green were encapsulated in clinically applicable PLGA nanoparticles. Three hundred syngeneic islets labeled with these nanoparticles and engrafted in the kidney capsule of C57/Bl6 mice could be well identified by NIR fluorescence microscopy (23)

SPECT AND PET IMAGING
SPECT and PET imaging offer high detection sensitivity, deep tissue penetration and are well suited for clinical translation. Both modalities require the islets to be labeled with radionuclides, such as 125 In, 99m Tc, 68 Ga, or 18 F. Hence, a drawback of SPECT and PET is radiation exposure, which should be avoided by pregnant or highrisk patients. In addition, the need for a cyclotron and/or radiochemical laboratory with trained personnel carries a high price tag. Due to the lack of anatomical information of SPECT and PET scans, both need to be co-registered with CT or MRI to provide tissue background anatomy. PET and SPECT signals appear as "hot spots" which can be used to quantify the number of radiolabeled islets. For clinical use, the sensitivity and spatial resolution of PET are approximately 2-3 orders of magnitude better than SPECT (26), but for small animal imaging, the resolution of SPECT is about 5-fold better than PET. However, SPECT radiotracers and equipment are less expensive than PET. SPECT radiotracers typically have a longer decay time than those of PET, thus allowing a longer observation period. On the other hand, a faster decay time may be desired to limit longer-term radioactive exposure.
For SPECT imaging, islets have been transduced with baculovirus vectors expressing the sodium iodide symporter for an enhanced uptake of sodium tracer agents (18). One to 8 h post-IV injection of 18.5 MBq of the radiotracer sodium 125 I and 24 h after islet transplantation, SPECT/CT was able to visualize 2000 rat islet equivalents (IEQ) implanted in the axillary cavity of NOD-SCID mice (Figures 2A-C). Because of its low gamma energy, 125 In is only suitable for small animal imaging, while 123 In is the appropriate radionuclide for human imaging. Targeting receptors that are naturally present in islets is preferred instead of viral infection or genetic manipulation of islets. Demine et al. (29) developed a 99m Tc-labeled camelid antibody which bound specifically to alpha and beta islet cells by virtue of their dipeptidyl peptidase 6 expression. A high number of human islets (a cluster of 1,000 or 3,000 IEQ) subcutaneously engrafted in SCID mice could be detected by SPECT 1 h after probe administration (32-39 MBq per mouse) for 4 weeks posttransplantation. Willekens et al. (19,20) used 123 I-labeled iodobenzamide (58 MBq) to target D2 receptors expressed by beta cells for serial SPECT, and quantified syngeneic 1,000, 2,000 or 3,000 islet graft volume in the calf muscle of WAG/Rij rats for 10 weeks. The SPECT signal correlated linearly with the insulinpositive graft volume, as confirmed by histology.
Studies to enable imaging lower numbers of islets are underway. Kroon et al. (21,22) used 111 In-exendin-3 to target glucagonlike peptide-1 receptors, specifically expressed on beta In a clinical trial, human islets were labeled with 20 MBq/ml of 18 F-fluorodeoxyglucose for 60 min at 37°C. PET imaging of five T1DM patients receiving an intraportal infusion of 69,000 to 153,000 labeled, allogeneic islets revealed a 25% loss of islets during delivery (27). A heterogenous distribution of islet grafts with a marked concentration in small multifocal areas in the liver could be seen ( Figure 2D). However, this technique was only applicable for 1 to 2 h post-implantation due to the radiotracer half-life of 110 min and short retention within the islets. In a different clinical study (33), eight patients with intraportal islet allografts (2-5 implant sessions of 210,000-800,000 IEQ each) underwent two PET sessions 8 months apart after i.v. administration of the serotonin precursor [ 11 C]5hydroxytryptophan (2-5 MBq/kg). However, the signals from liver uptake overwhelmed the signals from islet grafts although a few hotspots were seen in a few of the patients. In one patient, a change in hotspot uptake predicted graft function loss. With in situ labeling of entities that bind probes after i.v. injection, obfuscation of graft signal can occur by probe accumulation in other organs involved with metabolic clearance, such as the bladder, kidneys, and lungs, where potential toxic side effects need to be considered.

ULTRASOUND IMAGING
Ultrasonography is a fast, safe, and easy to operate imaging modality that involves no radiation. The device is portable, relatively inexpensive and available in most smaller clinics. This modality however suffers from a small window for imaging, an operator-dependent outcome and low detection sensitivity. Sakata et al. (28) employed high-frequency ultrasonography (HF-US) to monitor to monitor 200, 500, or 1,000 islets transplanted under the renal capsule of BALB/c mice ( Figure  2E). Signals from syngeneic islets persisted while those from xenogeneic Sprague Dawley rat islets vanished by day 28, suggesting the ability of HF-US to report on islet survival. The islet volume calculated by HF-US correlated with the number as well as the metabolic function of islet grafts. In this study, islets were imaged without the use of labeling, avoiding potential adverse effects of labels. However, HF-US could only detect islet clusters, and not individual islets. In the clinic, ultrasound imaging revealed 230,000 autografted islets in one pancreatectomy patient as hyperechoic regions in the liver (34).

MAGNETIC RESONANCE IMAGING
MRI has the distinct advantage of being able to provide wholebody, detailed anatomical images of the subject with excellent contrast between soft tissues. Implanted labeled islets can be visualized along with the recipient anatomy using the same imaging system, avoiding any potential discrepancies that may be encountered in co-registration of images from two different systems as can be the case in SPECT/CT, PET/CT, or PET/MRI. The most common MRI technique involves the manipulation of water protons which are abundantly present in living tissue. 1 H MRI contrast agents are categorized as T1-weighted or T2weighted agents. T1-weighted agents enhance MRI signal and appear as bright signals or hyperintensities. In contrast, T2weighted agents decrease MRI signal and appear as hypointensities.
Gadolinium (Gd)-based molecules are the most commonly used T1-weighted contrast agents. Gd is conjugated to biocompatible molecules/chelates to improve biocompatibility, circulation life and cell uptake. One thousand human and 500 murine BALB/c islets labeled with 50 mM Gd-HP-DO3A could be imaged after engraftment under the kidney capsule of mice as hyperintense entities (35). However, the risk of nephrogenic systemic fibrosis associated with dissociated free Gd has dampened the overall enthusiasm in using Gd-based contrast agents for clinical cell tracking (36).
SPIO-labeled islets transplanted in the intraportal vein or kidney capsule of rodents were easily visualized as hypointensities from a few days up to 6 weeks post-surgery (23,37,39,40,43,44). Hwang et al. (23) engrafted 300 syngeneic islets labeled with 250-500 µg/ml PLGA-coated SPIOs under the renal capsules of chemically induced diabetic C57Bl/6 mice which were imaged at 4.7T up to 4 weeks post-transplantation. In another study, islets were labeled with 800 mg/ml ferumoxytol, a clinically used ultrasmall (U)SPIO formulation, for 48 h ex vivo (37). Labeled syngeneic C57Bl/6 murine islet grafts could be visualized at 7T at 1 and 2 weeks after implantation under the kidney capsule or in the liver, with 300-600 IEQ given per mouse. Meanwhile, Ribeiro et al. created cationic phospholipid-coated SPIOs, also called magnetoliposomes (39,40). MRI performed at 9.4T was able to visualize 200 labeled Sprague Dawley rat islets (containing~35 ng of iron/islet) xenografted under the kidney capsule of C57Bl/6 mice for 4 days after engraftment. On the other hand, 50-1,000 labeled syngeneic islets could be detected much longer, up to 6 weeks post-transplantation in the same graft sites in Lewis rats.
When SPIOs are released from dying islets, the nanoparticles may linger inside the host tissue. As there are no means to differentiate T2-weighted signals originating from live vs. dead implants vs. free SPIOs, the accuracy of this modality to elucidate the survival of islet grafts is questionable. Furthermore, in vivo quantification of islet transplants from MRI signals is hampered by several factors (45,46). It is impossible to discern between single islets and multiple islet clusters when the hypointense contrast becomes so high that no signal is left. Other confounding sources of negative contrast from intestinal air pockets and hemorrhage/endogenous iron deposits also generate T2-weighted effects. Furthermore, depending on the location of the graft, peristaltic movements may introduce motion artifacts.
Indeed, clinical trials of SPIO-labeled islets infused into the liver via the intraportal vein have been performed with varying outcomes. The first demonstration on a safe use of SPIO-labeled islets in patients was reported in 2008 by Toso et al. (45). Here, islet allogeneic implants could be detected at 1.5T as hypointensities scattered in the liver parenchyma in three out of four T1D patients at 5 days, 6 weeks, and 6 months after transplantation ( Figure 3A). Each patient received two deliveries of 50,000-375,000 IEQ per session, labeled with 280 µg iron/ml Resovist ® . This study was followed by Saudek et al. (46) using Resovist ® -labeled islets in eight T1DM patients. Each patient underwent one or two engraftments of 73,00-687,000 total IEQ (140 µg iron/ml), which was followed at 3T for 24 weeks postdelivery. About a 60% MRI signal decrease was observed within 1-week post engraftment, suggesting early rejection or erroneous transplantation, but hypointense signals persisted up to 24 weeks. 19 F MRI directly detects tracers carrying 19 F nuclei. Fluorine labeling of islets did not appear to affect their viability and function (47,49). Unlike T1-weighted or T2-weighted MRI, 19 F labeled-islets are visible as "hot spots" (50), very much alike PET and SPECT tracer imaging. Due to the scant amount of 19 F atoms naturally present in the body, 19 F MRI practically has no background signal interference. The images are overlaid on anatomical images first acquired by 1 H MRI. 19 F MRI enables non-invasive quantification of islet implants since the signal intensity correlates linearly to the number of labeled islets. Barnett et al. (47) was the first who labeled human islets with perfluoropolyether (PFPE) emulsions for 19

MULTIMODAL IMAGING
As each imaging tool possesses its own advantages as well as limitations, researchers have created multi-modal probes in order to combine different imaging modalities within a single labeling platform. Liang

MICROENCAPSULATION FOR IMAGING AND IMMUNOPROTECTION OF ISLETS
A different approach for imaging islet grafts is to add imaging agents during immunoprotective microencapsulation of islets (54)(55)(56). In this design, the microcapsules are labeled instead of islets, thus avoiding direct manipulation of cells. A high concentration of imaging agents can be achieved here, higher than what can be obtained with labeling islets themselves, significantly augmenting detection sensitivity to the level of single capsule/islet detection.   4D-G). Furthermore, due to their intrinsic radiopacity, microcapsules synthesized using Ba 2+ ion cross-linker could be visualized with micro-CT without the need of further labeling (64). Upon the rupture of 19 F MRI-visible microcapsules or fluorocapsules, the 19 F MRI signal disappeared due to the release of fluorine into host tissues in a T1D model of NOD/ Shiltj mice (53). This new approach may be used to noninvasively investigate the loss of immunoprotection imparted by disintegrating microcapsules and the subsequent rejection and death of islet grafts as was confirmed by BLI in this study.

FUTURE OUTLOOK
Long-term labeling stability, toxicity of labels and detection sensitivity are issues that need to be addressed for successful translations of islet imaging techniques to the clinics. At present, the majority of imaging modalities only detect the localization and distribution of islet grafts not long after transplantation. Better methods to elucidate islet viability, function as well as the number or volume of islets throughout the treatment period will provide a more complete information on the fate of islets after delivery. Despite recent progress in the field of in vivo islet imaging, much work remains to be done to further improve the precision and efficacy of image-guided islet therapy.

AUTHOR CONTRIBUTIONS
DA performed a literature search and wrote the manuscript. JB edited the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
The authors are supported by NIH R01 DK106972.