Junling Li

Tumor Microenvironment–Dependent 18F-FDG, 18F-Fluorothymidine, and 18F-Misonidazole Uptake: A Pilot Study in Mouse Models of Human Non–Small Cell Lung Cancer

18F-FDG, 18F-fluorothymidine, and 18F-misonidazole PET scans have emerged as important clinical tools in the management of cancer; however, none of them have demonstrated conclusive superiority. The aim of this study was to compare the intratumoral accumulation of 18F-FDG, 18F-fluorothymidine, and 18F-misonidazole and relate this to specific components of the tumor microenvironment in mouse models of human non–small cell lung cancer (NSCLC). Methods: We used NSCLC A549 and HTB177 cells to generate subcutaneous and peritoneal xenografts in nude mice. Animals were coinjected with a PET radiotracer, pimonidazole (hypoxia marker), and bromodeoxyuridine (proliferation marker) intravenously 1 h before animal euthanasia. Tumor perfusion was assessed by Hoechst 33342 injection, given 1 min before sacrifice. The intratumoral distribution of PET radiotracers was visualized by digital autoradiography and related to microscopic visualization of proliferation, hypoxia, perfusion, stroma, and necrosis. Results: NSCLC xenografts had complex structures with intermingled regions of viable cancer cells, stroma, and necrosis. Cancer cells were either well oxygenated (staining negatively for pimonidazole) and highly proliferative (staining positively for bromodeoxyuridine) or hypoxic (pimonidazole-positive) and noncycling (little bromodeoxyuridine). Hypoxic cancer cells with a low proliferation rate had high18F-FDG and 18F-misonidazole uptake but low 18F-fluorothymidine accumulation. Well-oxygenated cancer cells with a high proliferation rate accumulated a high level of 18F-fluorothymidine but low 18F-FDG and18F-misonidazole. Tumor stroma and necrotic zones were always associated with low 18F-FDG, 18F-misonidazole, and 18F-fluorothymidine activity. Conclusion: In NSCLC A549 and HTB177 subcutaneously or intraperitoneally growing xenografts, 18F-fluorothymidine accumulates in well-oxygenated and proliferative cancer cells, whereas 18F-misonidazole and 18F-FDG accumulate mostly in poorly proliferative and hypoxic cancer cells. 18F-FDG and 18F-misonidazole display similar intratumoral distribution patterns, and both mutually exclude 18F-fluorothymidine.

Aptamer imaging with Cu-64 labeled AS1411: preliminary assessment in lung cancer.

INTRODUCTION AS1411 is a 26-base guanine-rich oligonucleotide aptamer shown binding to surface nucleolin, a protein over-expressed in multiple cancer cells, thus AS1411 labeled with a PET isotope can be explored as a potential diagnostic imaging agent. Our objective was to perform preliminary biological characterization of (64)Cu-labeled AS1411 in vitro and in vivo. METHODS Four chelators (DOTA, CB-TE2A, DOTA-Bn and NOTA-Bn) were selected to label AS1411 with Cu-64. 185kBq (5μCi) of each tracer was incubated in each well with H460 cells at 37°C for 1, 3, 6, 12, 24 and 48h, respectively (n=4). For microPET/CT imaging, 7.4MBq (200μCi) of AS1411 labeled with either (64)Cu-DOTA or (64)Cu-CB-TE2A was I.V. injected and multiple scans were obtained at 1, 3, 6 and 24h post injection. Afterward in vivo biodistribution studies were performed. RESULTS Percent uptake of (64)Cu-DOTA-AS1411 and (64)Cu-CB-TE2A-AS1411 was significantly higher than that of (64)Cu-DOTA-Bn-AS1411 and (64)Cu-NOTA-Bn-AS1411. About 90% of uptake for (64)Cu-DOTA-AS1411 and (64)Cu-CB-TE2A-AS1411 was internalized into cells within 3h and the internalization process was completed before 24h. Both tracers demonstrated reasonable in vivo stability and high binding affinity to the cells. MicroPET imaging with (64)Cu-CB-TE2A-AS1411 showed clear tumor uptake at both legs from 1 to 24h post injection, whereas both tumors were undetectable for up to 24h with (64)Cu-DOTA-AS1411. In addition, (64)Cu-CB-TE2A-AS1411 had faster in vivo pharmacokinetics than (64)Cu-DOTA-AS1411 with lower liver uptake and higher tumor to background contrast. CONCLUSION CB-TE2A is a preferred chelator with higher tumor-to-background ratio, lower liver uptake and faster clearance than DOTA. Aptamer imaging with (64)Cu-CB-TE2A-AS1411 may be feasible for detecting lung cancer, if an appropriate chelator can be identified and further validation can be performed with a known control oligonucleotide. It may also be used as a companion diagnostic imaging agent for AS1411 in the treatment of cancer.

Validation of 2-18F-Fluorodeoxysorbitol as a Potential Radiopharmaceutical for Imaging Bacterial Infection in the Lung

2-18F-fluorodeoxysorbitol (18F-FDS) has been shown to be a promising agent with high selectivity and sensitivity in imaging bacterial infection. The objective of our study was to validate 18F-FDS as a potential radiopharmaceutical for imaging bacterial infection longitudinally in the lung. Methods: Albino C57 female mice were intratracheally inoculated with either live or dead Klebsiella pneumoniae to induce either lung infection or lung inflammation. One group of mice was imaged to monitor disease progression. PET/CT was performed on days 0, 1, 2, and 3 after inoculation using either 18F-FDS or 18F-FDG (n = 12 for each tracer). The other group was first screened by bioluminescent imaging (BLI) to select only mice with visible infection (region of interest > 108 ph/s) for PET/CT imaging with 18F-FDS (n = 12). For the inflammation group, 5 mice each were imaged with PET/CT using either 18F-FDS or 18F-FDG from days 1 to 4 after inoculation. Results: For studies of disease progression, BLI showed noticeable lung infection on day 2 after inoculation and significantly greater infection on day 3. Baseline imaging before inoculation showed no focal areas of lung consolidation on CT and low uptake in the lung for both PET radiotracers. On day 2, an area of lung consolidation was identified on CT, with a corresponding 2.5-fold increase over baseline for both PET radiotracers. On day 3, widespread areas of patchy lung consolidation were found on CT, with a drastic increase in uptake for both 18F-FDS and 18F-FDG (9.2 and 3.9). PET and BLI studies showed a marginal correlation between 18F-FDG uptake and colony-forming units (r = 0.63) but a much better correlation for 18F-FDS (r = 0.85). The uptake ratio of infected lung over inflamed lung was 8.5 and 1.7 for 18F-FDS and 18F-FDG on day 3. Conclusion: Uptake of both 18F-FDS and 18F-FDG in infected lung could be used to track the degree of bacterial infection measured by BLI, with a minimum detection limit of 107 bacteria. 18F-FDS, however, is more specific than 18F-FDG in differentiating K. pneumoniae lung infection from lung inflammation.

Characterization of 18F-dipicolylamine (DPA) derivatives in cells infected with influenza virus

OBJECTIVE Bis(Zn-dipicolylamine (Zn-DPA)) coordination complexes represent a new class of synthetic small molecules that can target anionic phosphatidylserine (PS) in the apoptotic cells with high affinity and specificity. In this study, we labeled Zn-DPA and Cy7-Zn-DPA with different (18)F-prosthetic groups and characterized their uptake in A549 cells infected with influenza A virus from the 2009 pandemic (H1N1pdm). METHODS DPA was labeled with N-succinimidyl 4-(18)F-fluorobenzoate ((18)F-SFB), 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP), 2-(18)F-Fluoroethyl toslyate ((18)F-FET), and (18)F-aluminum (Al(18)F), respectively. Cy7-DPA was labeled with (18)F-SFB and (18)F-NFP only. The tracers were reconstituted with zinc nitrate before use. Apoptosis in A549 cells was induced by infection with the H1N1pdm virus for 48 h. Three μCi of each tracer was added to each well and incubated at 37 °C. The effect of different prosthetic groups, different MOI, and incubation time on percent cellular uptake was studied. Cell internalization and efflux was evaluated within 2h of incubation. The competitive binding assay was performed with increasing concentration (10(-12)-10(-5)M) of Zn-DPA or Cy7-Zn-DPA prior to the addition of either (18)F-FB-Zn-DPA or (18)F-FB-Cy7-Zn-DPA into each well. IC50 values for the two Zn-DPA analogues were estimated by GraphPad Prism 6.0. RESULTS Among all the four prosthetic groups, the (18)F-SFB method provided the highest conjugation yield for DPA and the highest uptake ratio between the infection cells and the control when both Zn-DPA and Cy7-Zn-DPA were present in the complex. The uptake ratio was similar for (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA. Uptake of (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA was proportional to the degree of apoptosis with a plateau at MOI 3. Uptake of (18)F-FB-Cy7-Zn-DPA also increased over incubation time and reached a plateau at 1h, whereas uptake of (18)F-FB-Zn-DPA did not show any significant change over time. Cell internalization studies showed that more than 70% of (18)F-FB-Zn-DPA remained on the cell surface over a time course of 2 hr in the cell media, but over 90% of (18)F-FB-Cy7-Zn-DPA was internalized within 15 min of incubation. IC50 values were estimated to be 1.5±0.3 nM and 26.2±5.1 nM for Zn-DPA and Cy7-Zn-DPA, respectively. CONCLUSIONS (18)F-SFB was the optimal labeling method for Zn-DPA and Cy7-Zn-DPA with respect to radiochemistry and provided complexes with high target-to-background ratios. (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA appeared to have a completely different internalization mechanism, while Zn-DPA showed higher binding affinity than Cy7-Zn-DPA. Based on these favorable characteristics, (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA should be further evaluated as potential imaging agents for viral infection.

Assessment of radiopharmaceutical retention for vascular access ports using positron emission tomography imaging

Abstract Purpose The purpose of this study was to resolve the issue of whether various generations of CR Bard peripheral vascular access ports and catheters are prone to retain PET radiopharmaceuticals. The study evaluates the residual radioactivity remaining following injection for two PET radiopharmaceuticals currently used extensively in the clinic, FDG and Na18F. Methods FDG was purchased from a local cyclotron facility and Na18F was prepared in‐house. Three generations of currently marketed vascular access ports were tested. A total of five (n = 5) of each model was tested. Radiopharmaceutical of 2–3 mCi of each was injected into each port and flushed with 10, 30, 60, and 120 ml of saline. MicroPET scans were performed after each flush to detect the residual radioactivity on each port. A dose calibrator was used to detect the retention of radioactivity after each flush. Results Radioactivity retention for all vascular port models measured by microPET imaging was similar for both FDG and Na18F, with less than 1% residual activity following a 10 ml saline flush. Based on the microPET images, all the subsequent flushes of 30, 60, and 120 ml were also considered. Dose calibrator activity measurements validated microPET measurements as negligible for all the ports, even with the first 10 ml flush. Conclusions MicroPET imaging was more sensitive than the dose calibrator in determining the radioactivity retention of the vascular access ports from CR Bard. These ports may be used for the injection of FDG and Na18F to track glucose metabolism and bone uptake with PET imaging. It is recommended to apply at least a 10 ml flush after radiopharmaceutical administration, to reduce residual activity to baseline levels.

In Vitro PET Imaging of a Miniature Ventricular Assist Device

Interactions between the life-sustaining ventricular assist devices and diagnostic therapies must be carefully considered to decrease the risk of inaccurate diagnostic imaging or pump failure. Methods: The MVAD® pump, currently under investigational use, was tested for interaction with radiotracers in an in vitro flow-loop study. The radiotracers 18F-sodium fluoride and 18F-FDG were injected into a closed loop to determine the feasibility of direct imaging of the MVAD® pump in a PET scanner. Results: No real-time changes were observed in pump operation, and there were no statistical differences in pump parameters (power consumption, speed, and estimated flow rate) between the baseline and circulation conditions. In addition, no effect was observed on any external components, including the permissive-action-link controller and the batteries powering the device. Imaging of the internal pump components was possible, with obscuration observed only in the portion of the pump where the spinning impeller is located. Retention of radiotracer in the pump components after circulation was minimal (<1%). Conclusion: PET imaging is an attractive diagnostic tool for patients with a ventricular assist device and may have additional utility outside its current use, detection of infection.

Abstract 2439: Molecular imaging of spatial and temporal heterogeneity of tumor micro-environment in mouse models of non-small cell lung cancer macroscopic xenografts and micro-metastases

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 18F-fluoro-2-deoxyglucose (18F-FDG, imaging glucose metabolism), 18F-fluorothymidine (18F-FLT, cell proliferation) and 18F-misonidazole (18F-FMISO, hypoxia) PET scans have emerged as important clinical tools for management of cancer. The objective of this study was to spatially and temporally visualize tumor microenvironment of tumor hypoxia, proliferation and glucose metabolism using 18F-FDG, 18F-FLT and 18F-FMISO digital autoradiography and microPET comparing with histological findings. Methods: We used human non-small cell lung cancer (NSCLC) A549 and HTB177 cells to generate subcutaneous and peritoneal metastases in nude mice. Animals were coinjected with the mixture of one PET radiotracer, pimonidazole (hypoxia marker) and bromodeoxyuridine (proliferation marker) intravenously 1 hour before animal euthanasia. The intratumoral distributions of radiotracers were visualized by digital autoradiography (DAR) and related to microscopic visualization of cellular proliferation, tumor hypoxia, stroma and necrosis. Serial microPET scans (day 1to day5) were also performed in the same animals to investigate change in glucose metabolism (using 18F-FDG), proliferation (18F-FLT) and hypoxia (18F-FMISO). Results: NSCLC microenvironment was complex and highly heterogeneous: xenografts had complex structures with intermingled regions of well oxygenated (negative pimonidazole) and highly proliferative (positive bromodeoxyuridine) cancer cells, hypoxic (positive pimonidazole) and low proliferation (little bromodeoxyuridine) cancer cells stroma and necrosis. Hypoxic cancer cells had high18F-FDG and 18F-FMISO but low 18F-FLT accumulation, indicating increased glucose metabolism is not a common feature of cancer cells but only hypoxic ones. Well oxygenated cancer cells with high proliferation rate accumulated high level of 18F-FLT, but low 18F-FDG and18F-FMISO. Stroma and necrotic zones always associated with low activity in all radiotracers we tested. MicroPET scans revealed apparent change in intratumor distribution of 18F-FLT, 18F-FDG as well as 18F-FMISO in as short as ∼48 hrs interval, indicating temporal heterogeneity of tumor microenvironment in term of proliferation, glucose metabolism and hypoxia. Conclusions: Both macroscopic xenografts and micrometastases of NSCLC have spatial heterogeneity of tumor microenvironment. Temporal change of tumor microenvironment did occur in a very short of interval of natural growth process in NSCLC. We have investigated to spatial and temporal behavior of heterogeneity of tumor microenvironment which is important for better understanding cancer biology and cancer management, if our findings in mice models are clinically applicable. Acknowledgement: This study was supported by Kentucky Lung Cancer Research Program Award to Dr. Xiao-Feng Li. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2439. doi:1538-7445.AM2012-2439