Yingding Xu

Harnessing the Power of Radionuclides for Optical Imaging: Cerenkov Luminescence Imaging

Over the past several years, nuclear imaging modalities such as PET and SPECT have received much attention because they have been instrumental not only in preclinical cancer research but also in nuclear medicine. Yet nuclear imaging is limited by high instrumentation cost and subsequently low availability to basic researchers. Cerenkov radiation, a relativistic physical phenomenon that was discovered 70 years ago, has recently become an intriguing subject of study in molecular imaging because of its potential in augmenting nuclear imaging, particularly in preclinical small-animal studies. The intrinsic capability of radionuclides emitting luminescent light from decay is promising because of the possibility of bridging nuclear imaging with optical imaging—a modality that is much less expensive, is easier to use, and has higher throughput than its nuclear counterpart. Thus, with the maturation of this novel imaging technology using Cerenkov radiation, which is termed Cerenkov luminescence imaging, it is foreseeable that advances in both nuclear imaging and preclinical research involving radioisotopes will be significantly accelerated in the near future.

Tyrosinase as a multifunctional reporter gene for Photoacoustic/MRI/PET triple modality molecular imaging

Development of reporter genes for multimodality molecular imaging is highly important. In contrast to the conventional strategies which have focused on fusing several reporter genes together to serve as multimodal reporters, human tyrosinase (TYR) – the key enzyme in melanin production – was evaluated in this study as a stand-alone reporter gene for in vitro and in vivo photoacoustic imaging (PAI), magnetic resonance imaging (MRI) and positron emission tomography (PET). Human breast cancer cells MCF-7 transfected with a plasmid that encodes TYR (named as MCF-7-TYR) and non-transfected MCF-7 cells were used as positive and negative controls, respectively. Melanin targeted N-(2-(diethylamino)ethyl)-18F-5-fluoropicolinamide was used as a PET reporter probe. In vivo PAI/MRI/PET imaging studies showed that MCF-7-TYR tumors achieved significant higher signals and tumor-to-background contrasts than those of MCF-7 tumor. Our study demonstrates that TYR gene can be utilized as a multifunctional reporter gene for PAI/MRI/PET both in vitro and in vivo.

Proof-of-Concept Study of Monitoring Cancer Drug Therapy with Cerenkov Luminescence Imaging

Cerenkov luminescence imaging (CLI) has emerged as a less expensive, easier-to-use, and higher-throughput alternative to other nuclear imaging modalities such as PET. It is expected that CLI will find many applications in biomedical research such as cancer detection, probe development, drug screening, and therapy monitoring. In this study, we explored the possibility of using CLI to monitor drug efficacy by comparisons against PET. To assess the performance of both modalities in therapy monitoring, 2 murine tumor models (large cell lung cancer cell line H460 and prostate cancer cell line PC3) were given bevacizumab versus vehicle treatments. Two common radiotracers, 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) and 18F-FDG, were used to monitor bevacizumab treatment efficacy. Methods: One group of mice (n = 6) was implanted with H460 xenografts bilaterally in the shoulder region, divided into treatment and control groups (n = 3 each), injected with 18F-FLT, and imaged with PET immediately followed by CLI. The other group of mice (n = 6) was implanted with PC3 xenografts in the same locations, divided into treatment and control groups (n = 3 each), injected with 18F-FDG, and imaged by the same modalities. Bevacizumab treatment was performed by 2 injections of 20 mg/kg at days 0 and 2. Results: On 18F-FLT scans, both CLI and PET revealed significantly decreased signals from H460 xenografts in treated mice from pretreatment to day 3. Moderately increased to unchanged signals were observed in untreated mice. On 18F-FDG scans, both CLI and PET showed relatively unchanged signals from PC3 tumors in both treated and control groups. Quantifications of tumor signals of Cerenkov luminescence and PET images showed that the 2 modalities had excellent correlations (R2 > 0.88 across all study groups). Conclusion: CLI and PET exhibit excellent correlations across different tumor xenografts and radiotracers. This is the first study, to our knowledge, demonstrating the use of CLI for monitoring cancer treatment. The findings warrant further exploration and optimization of CLI as an alternative to PET in preclinical therapeutic monitoring and drug screening.

Evaluation of Four Affibody-Based Near-Infrared Fluorescent Probes for Optical Imaging of Epidermal Growth Factor Receptor Positive Tumors

UNLABELLED The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity for EGFR, ZEGFR:1907, has been reported. We are interested in translating Affibody molecules to potential clinical optical imaging of EGFR positive cancers. In this study, four anti-EGFR Affibody based near-infrared (NIR) fluorescent probes were thus prepared, and their in vivo performance was evaluated in the mice bearing EGFR positive subcutaneous A431 tumors. METHODS The Affibody analogue, Ac-Cys-ZEGFR:1907, was synthesized using solid-phase peptide synthesis method. The purified small protein was then site-specifically conjugated with four NIR fluorescent dyes, Cy5.5-monomaleimide, Alex-Fluor-680-maleimide, SRfluor680-maleimide, or IRDye-800CW-maleimide, to produce four optical probes-Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, SR680-ZEGFR:1907, and 800CW-ZEGFR:1907. The EGFR binding property and specificity of the four NIR fluorescent Affibody probes were studied by fluorescence microscopy using high EGFR expressing A431 cells and low expressing MCF7 cells. The binding affinities of the probes (KD) to EGFR were further determined by flow cytometry. In vivo optical imaging of the four probes was performed in the mice bearing subcutaneous A431 tumors. RESULTS The four NIR optical probes were prepared in high purity. In vitro cell imaging studies demonstrated that all of them could specifically bind to EGFR positive A431 cells while showing minimum uptake in low EGFR expressing MCF7 cells. Flow cytometry showed that Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 possessed high binding affinity in low nanomolar range (43.6 ± 8.4 and 28.3 ± 4.9, respectively). In vivo optical imaging of the four probes revealed that they all showed fast tumor targeting ability and good tumor-to-normal tissue contrast as early as 0.5 h postinjection (p.i.). The tumor-to-normal tissue ratio reached a peak at 2 to 4 h p.i. by regional of interest (ROI) analysis of images. Ex vivo studies further demonstrated that the four probes had high tumor uptakes. Particularly, Cy5.5-ZEGFR:1907 and Alex680-ZEGFR:1907 displayed higher tumor-to-normal tissue ratios than those of the other two probes. CONCLUSION This work demonstrates that Affibody proteins can be modified with different NIR fluorescent dyes and used for imaging of EGFR expressing tumors. Different NIR fluorescent dyes have variable impact on the in vitro binding and in vivo performance of the resulting Affibody based probes. Therefore, selection of an appropriate NIRF label is important for optical probe development. The probes developed are promising for further tumor imaging applications and clinical translation. Particularly, Alex680-ZEGFR:1907 and Cy5.5-ZEGFR:1907 are excellent candidates as EGFR-targeted probes for optical imaging.

Cerenkov Luminescence Imaging (CLI) for cancer therapy monitoring.

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues. Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI). CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one (18)F decay was estimated to produce an average of 3 photons in water. Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET. Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Lymphoid Hyperplasia of the Appendix: A Potential Pitfall in the Sonographic Diagnosis of Appendicitis

OBJECTIVE The objective of this study was to test the hypothesis that thickening of the lamina propria, a finding produced by lymphoid hyperplasia, is significantly associated with false-positive sonographic diagnoses of appendicitis in 6- to 8-mm noncompressible appendixes. MATERIALS AND METHODS Sonograms of 119 consecutive patients with suspected appendicitis and 6- to 8-mm noncompressible appendixes were retrospectively blindly evaluated for thickening of the lamina propria (short axis thickness ≥ 1 mm). The reference standard for appendicitis was pathologic analysis of resected specimens. Results were compared with the two-tailed Fisher exact test. RESULTS Thirty-one patients (26.1%) had a thickened lamina propria and 88 (73.9%) did not. Of the 27 pediatric patients with a thickened lamina propria, five (18.5%) had true-positive and 22 (81.5%) had false-positive sonograms for appendicitis; among the 55 pediatric patients without a thickened lamina propria, 27 (49.1%) had true-positive and 28 (50.9%) had false-positive sonograms for appendicitis (p = 0.009). Similar differences in adult patients were not statistically significant. All five pediatric patients with appendicitis and thickened lamina propria also showed two or more findings of periappendiceal fluid, hyperechoic periappendiceal fat, or mural hyperemia on color Doppler examination, compared with two of 22 similar pediatric patients without appendicitis (p < 0.001). CONCLUSION Lymphoid hyperplasia may result in a noncompressible appendix 6-8 mm in diameter and may be misdiagnosed as appendicitis in pediatric patients. True-positive diagnoses of appendicitis can be accurately identified by the presence of at least two additional findings from the group of periappendiceal fluid, hyperechoic periappendiceal fat, and mural hyperemia. Identifying the characteristic sonographic appearance of lymphoid hyperplasia may help prevent false-positive misdiagnoses of appendicitis.

Color Doppler Imaging of the Appendix Criteria to Improve Specificity for Appendicitis in the Borderline-Size Appendix

To test the hypothesis that continuous intramural vascular signal measuring at least 3 mm on color Doppler imaging is highly specific for appendicitis in patients with diagnostically borderline‐size appendices.

Sonographic Differentiation of Complicated From Uncomplicated Appendicitis: Implications for Antibiotics-First Therapy

To evaluate sonographic findings as indicators of complicated versus uncomplicated appendicitis in the setting of known appendicitis, a necessary distinction in deciding whether to proceed with antibiotic therapy or with appendectomy.