Biodistribution of a Mitochondrial Metabolic Tracer, [18F]F-AraG, in Healthy Volunteers.

Purpose: [18F]F-AraG is a radiolabeled nucleoside analog that shows relative specificity for activated T cells. The aim of this study was to investigate the biodistribution of [18F]F-AraG in healthy volunteers and assess the preliminary safety and radiation dosimetry. Methods: Six healthy subjects (three female and three male) between the ages of 24 and 60 participated in the study. Each subject received a bolus venous injection of [18F]F-AraG (dose range: 244.2-329.3 MBq) prior to four consecutive PET/MR whole-body scans. Blood samples were collected at regular intervals and vital signs monitored before and after tracer administration. Regions of interest were delineated for multiple organs, and the area under the time-activity curves was calculated for each organ and used to derive time-integrated activity coefficient (TIAC). TIACs were input for absorbed dose and effective dose calculations using OLINDA. Results: PET/MR examination was well tolerated, and no adverse effects to the administration of [18F]F-AraG were noted by the study participants. The biodistribution was generally reflective of the expression and activity profiles of the enzymes involved in [18F]F-AraG's cellular accumulation, mitochondrial kinase dGK, and SAMHD1. The highest uptake was observed in the kidneys and liver, while the brain, lung, bone marrow, and muscle showed low tracer uptake. The estimated effective dose for [18F]F-AraG was 0.0162 mSv/MBq (0.0167 mSv/MBq for females and 0.0157 mSv/MBq for males). Conclusion: Biodistribution of [18F]F-AraG in healthy volunteers was consistent with its association with mitochondrial metabolism. PET/MR [18F]F-AraG imaging was well tolerated, with a radiation dosimetry profile similar to other commonly used [18F]-labeled tracers. [18F]F-AraG's connection with mitochondrial biogenesis and favorable biodistribution characteristics make it an attractive tracer with a variety of potential applications.

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging.

Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.

Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects.

Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and ß) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/ß)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/ß) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/ß)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90ß/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/ß)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.

Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe.

Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-ß-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.

Lymphoid-tissue-specific homing of bone-marrow-derived dendritic cells.

Because of their potent immunoregulatory capacity, dendritic cells (DCs) have been exploited as therapeutic tools to boost immune responses against tumors or pathogens, or dampen autoimmune or allergic responses. Murine bone marrow-derived DCs (BM-DCs) are the closest known equivalent of the blood monocyte-derived DCs that have been used for human therapy. Current imaging methods have proven unable to properly address the migration of injected DCs to small and deep tissues in mice and humans. This study presents the first extensive analysis of BM-DC homing to lymph nodes (and other selected tissues) after intravenous and intraperitoneal inoculation. After intravenous delivery, DCs accumulated in the spleen, and preferentially in the pancreatic and lung-draining lymph nodes. In contrast, DCs injected intraperitoneally were found predominantly in peritoneal lymph nodes (pancreatic in particular), and in omentum-associated lymphoid tissue. This uneven distribution of BM-DCs, independent of the mouse strain and also observed within pancreatic lymph nodes, resulted in the uneven induction of immune response in different lymphoid tissues. These data have important implications for the design of systemic cellular therapy with DCs, and in particular underlie a previously unsuspected potential for specific treatment of diseases such as autoimmune diabetes and pancreatic cancer.

Reproducibility study of [(18)F]FPP(RGD)2 uptake in murine models of human tumor xenografts.

PURPOSE: An (18)F-labeled PEGylated arginine-glycine-aspartic acid (RGD) dimer {[(18)F]FPP(RGD)(2)} has been used to image tumor α(v)ß(3) integrin levels in preclinical and clinical studies. Serial positron emission tomography (PET) studies may be useful for monitoring antiangiogenic therapy response or for drug screening; however, the reproducibility of serial scans has not been determined for this PET probe. The purpose of this study was to determine the reproducibility of the integrin α(v)ß(3)-targeted PET probe, [(18)F]FPP(RGD)(2,) using small animal PET. METHODS: Human HCT116 colon cancer xenografts were implanted into nude mice (n = 12) in the breast and scapular region and grown to mean diameters of 5-15 mm for approximately 2.5 weeks. A 3-min acquisition was performed on a small animal PET scanner approximately 1 h after administration of [(18)F]FPP(RGD)(2) (1.9-3.8 MBq, 50-100 µCi) via the tail vein. A second small animal PET scan was performed approximately 6 h later after reinjection of the probe to assess for reproducibility. Images were analyzed by drawing an ellipsoidal region of interest (ROI) around the tumor xenograft activity. Percentage injected dose per gram (%ID/g) values were calculated from the mean or maximum activity in the ROIs. Coefficients of variation and differences in %ID/g values between studies from the same day were calculated to determine the reproducibility. RESULTS: The coefficient of variation (mean±SD) for %ID(mean)/g and %ID(max)/g values between [(18)F]FPP(RGD)(2) small animal PET scans performed 6 h apart on the same day were 11.1 ± 7.6% and 10.4 ± 9.3%, respectively. The corresponding differences in %ID(mean)/g and %ID(max)/g values between scans were -0.025 ± 0.067 and -0.039 ± 0.426. Immunofluorescence studies revealed a direct relationship between extent of α(ν)ß(3) integrin expression in tumors and tumor vasculature with level of tracer uptake. Mouse body weight, injected dose, and fasting state did not contribute to the variability of the scans; however, consistent scanning parameters were necessary to ensure accurate studies, in particular, noting tumor volume, as well as making uniform: the time of imaging after injection and the ROI size. Reanalysis of ROI placement displayed variability for %ID(mean)/g of 6.6 ± 3.9% and 0.28 ± 0.12% for %ID(max)/g. CONCLUSION: [(18)F]FPP(RGD)(2) small animal PET mouse tumor xenograft studies are reproducible with relatively low variability.

Noninvasive detection of therapeutic cytolytic T cells with 18F-FHBG PET in a patient with glioma.

BACKGROUND: A 57-year-old man had been diagnosed with grade IV glioblastoma multiforme and was enrolled in a trial of adoptive cellular immunotherapy. The trial involved infusion of ex vivo expanded autologous cytolytic CD8+ T cells (CTLs), genetically engineered to express the interleukin 13 zetakine gene (which encodes a receptor protein that targets these T cells to tumor cells) and the herpes simplex virus 1 thymidine kinase (HSV1 tk) suicide gene, and PET imaging reporter gene. INVESTIGATIONS: MRI, whole-body and brain PET scan with (18)F-radiolabelled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) to detect CTLs that express HSV1 tk, and safety monitoring after injection of (18)F-FHBG. DIAGNOSIS: MRI detected grade III-IV glioblastoma multiforme plus two tumors recurrences that developed after resection of the initial tumor. MANAGEMENT: Surgical resection of primary glioblastoma tumor, enrollment in CTL therapy trial, reresection of glioma recurrences, infusion of approximately 1 x 10(9) CTLs into the site of tumor reresection, and (18)F-FHBG PET scan to detect infused CTLs.

Novel strategy for a cocktail 18F-fluoride and 18F-FDG PET/CT scan for evaluation of malignancy: results of the pilot-phase study.

UNLABELLED: (18)F-FDG PET/CT is used for detecting cancer and monitoring cancer response to therapy. However, because of the variable rates of glucose metabolism, not all cancers are identified reliably. Sodium (18)F was previously used for bone imaging and can be used as a PET/CT skeletal tracer. The combined administration of (18)F and (18)F-FDG in a single PET/CT study for cancer detection has not been reported to date. METHODS: This is a prospective pilot study (November 2007-November 2008) of 14 patients with proven malignancy (6 sarcoma, 3 prostate cancer, 2 breast cancer, 1 colon cancer, 1 lung cancer, and 1 malignant paraganglioma) who underwent separate (18)F PET/CT and (18)F-FDG PET/CT and combined (18)F/(18)F-FDG PET/CT scans for the evaluation of malignancy (a total of 3 scans each). There were 11 men and 3 women (age range, 19-75 y; average, 50.4 y). RESULTS: Interpretation of the combined (18)F/(18)F-FDG PET/CT scans compared favorably with that of the (18)F-FDG PET/CT (no lesions missed) and the (18)F PET/CT scans (only 1 skull lesion seen on an (18)F PET/CT scan was missed on the corresponding combined scan). Through image processing, the combined (18)F/(18)F-FDG scan yielded results for bone radiotracer uptake comparable to those of the (18)F PET/CT scan performed separately. CONCLUSION: Our pilot-phase prospective trial demonstrates that the combined (18)F/(18)F-FDG administration followed by a single PET/CT scan is feasible for cancer detection. This combined method opens the possibility for improved patient care and reduction in health care costs.

A novel method for direct site-specific radiolabeling of peptides using [18F]FDG.

We have used the well-accepted and easily available 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) positron emission tomography (PET) tracer as a prosthetic group for synthesis of (18)F-labeled peptides. We herein report the synthesis of [(18)F]FDG-RGD ((18)F labeled linear RGD) and [(18)F]FDG-cyclo(RGD(D)YK) ((18)F labeled cyclic RGD) as examples of the use of [(18)F]FDG. We have successfully prepared [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) in 27.5% and 41% radiochemical yields (decay corrected) respectively. The receptor binding affinity study of FDG-cyclo(RGD(D)YK) for integrin alpha(v)beta(3), using alpha(v)beta(3) positive U87MG cells confirmed a competitive displacement with (125)I-echistatin as a radioligand. The IC(50) value for FDG-cyclo(RGD(D)YK) was determined to be 0.67 +/- 0.19 muM. High-contrast small animal PET images with relatively moderate tumor uptake were observed for [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) as PET probes in xenograft models expressing alpha(v)beta(3) integrin. In conclusion, we have successfully used [(18)F]FDG as a prosthetic group to prepare (18)F]FDG-RGD and [(18)F]FDG-cyclic[RGD(D)YK] based on a simple one-step radiosynthesis. The one-step radiosynthesis methodology consists of chemoselective oxime formation between an aminooxy-functionalized peptide and [(18)F]FDG. The results have implications for radiolabeling of other macromolecules and would lead to a very simple strategy for routine preclinical and clinical use.

Imaging of Activated T Cells as an Early Predictor of Immune Response to Anti-PD-1 Therapy.

Compelling evidence points to immune cell infiltration as a critical component of successful immunotherapy. However, there are currently no clinically available, noninvasive methods capable of evaluating immune contexture prior to or during immunotherapy. In this study, we evaluate a T-cell-specific PET agent, [18F]F-AraG, as an imaging biomarker predictive of response to checkpoint inhibitor therapy. We determined the specificity of the tracer for activated T cells in vitro and in a virally induced model of rhabdomyosarcoma. Of all immune cells tested, activated human CD8+ effector cells showed the highest accumulation of [18F]F-AraG. Isolation of lymphocytes from the rhabdomyosarcoma tumors showed that more than 80% of the intratumoral signal came from accumulation of [18F]F-AraG in immune cells, primarily CD8+ and CD4+. Longitudinal monitoring of MC38 tumor-bearing mice undergoing anti-PD-1 treatment revealed differences in signal between PD-1 and isotype antibody-treated mice early into treatment. The differences in [18F]F-AraG signal were also apparent between responders and nonresponders to anti-PD-1 therapy. Importantly, we found that the signal in the tumor-draining lymph nodes provides key information about response to anti-PD-1 therapy. Overall, [18F]F-AraG has potential to serve as a much needed immunomonitoring clinical tool for timely evaluation of immunotherapy. SIGNIFICANCE: These findings reveal differences in T-cell activation between responders and nonresponders early into anti-PD-1 treatment, which may impact many facets of immuno-oncology, including patient selection, management, and development of novel combinatorial approaches.

Tissue-targeted therapy of autoimmune diabetes using dendritic cells transduced to express IL-4 in NOD mice.

A deficit in IL-4 production has been previously reported in both diabetic human patients and non-obese diabetic (NOD) mice. In addition, re-introducing IL-4 into NOD mice systemically, or as a transgene, led to a beneficial outcome in most studies. Here, we show that prediabetic, 12-week old female NOD mice have a deficit in IL-4 expression in the pancreatic lymph nodes (PLN) compared to age-matched diabetes-resistant NOD.B10 mice. By bioluminescence imaging, we demonstrated that the PLN was preferentially targeted by bone marrow-derived dendritic cells (DCs) following intravenous (IV) administration. Following IV injection of DCs transduced to express IL-4 (DC/IL-4) into 12-week old NOD mice, it was possible to significantly delay or prevent the onset of hyperglycemia. We then focused on the PLN to monitor, by microarray analysis, changes in gene expression induced by DC/IL-4 and observed a rapid normalization of the expression of many genes, that were otherwise under-expressed compared to NOD.B10 PLN. The protective effect of DC/IL-4 required both MHC and IL-4 expression by the DCs. Thus, adoptive cellular therapy, using DCs modified to express IL-4, offers an effective, tissue-targeted cellular therapy to prevent diabetes in NOD mice at an advanced stage of pre-diabetes, and may offer a safe approach to consider for treatment of high risk human pre-diabetic patients.

Preclinical efficacy of the c-Met inhibitor CE-355621 in a U87 MG mouse xenograft model evaluated by 18F-FDG small-animal PET.

UNLABELLED: The purpose of this study was to evaluate the efficacy of CE-355621, a novel antibody against c-Met, in a subcutaneous U87 MG xenograft mouse model using (18)F-FDG small-animal PET. METHODS: CE-355621 or control vehicle was administered intraperitoneally into nude mice (drug-treated group, n = 12; control group, n = 14) with U87 MG subcutaneous tumor xenografts. Drug efficacy was evaluated over 2 wk using (18)F-FDG small-animal PET and compared with tumor volume growth curves. RESULTS: The maximum %ID/g (percentage injected dose per gram of tissue) of (18)F-FDG accumulation in mice treated with CE-355621 remained essentially unchanged over 2 wk, whereas the %ID/g of the control tumors increased 66% compared with the baseline. Significant inhibition of (18)F-FDG accumulation was seen 3 d after drug treatment, which was earlier than the inhibition of tumor volume growth seen at 7 d after drug treatment. CONCLUSION: CE-355621 is an efficacious novel antineoplastic chemotherapeutic agent that inhibits (18)F-FDG accumulation earlier than tumor volume changes in a mouse xenograft model. These results support the use of (18)F-FDG PET to assess early tumor response for CE-355621.

Applications of lentiviral vectors in noninvasive molecular imaging.

Noninvasive imaging of molecular-genetic and cellular processes is an effective way to determine the location(s), magnitude, and time variation of action of gene products used for many therapeutic strategies. Lentiviral vectors provide effective means for the delivery, integration, and expression of transgenes in cultured mammalian cells as well as in vivo. Therefore, the combination of lentiviral vector-mediated therapeutic and imaging-targeted reporter gene delivery to various target organs holds promise for the future treatment of diseases. In this chapter, we provide protocols for developing lentiviral vectors that can be utilized for noninvasive monitoring/imaging of reporter gene expression. We have described the procedures to perform cellular assays and animal imaging based on positron emission tomography (PET), optical bioluminescence, and fluorescence reporter genes. The protocols described here are standardized for mouse models, which can also be adapted for other small animal models (e.g., rats).

Multimodality imaging of T-cell hybridoma trafficking in collagen-induced arthritic mice: image-based estimation of the number of cells accumulating in mouse paws.

Appropriate targeting of therapeutic cells is essential in adoptive cellular gene therapy (ACGT). Imaging cell trafficking in animal models and patients will guide development of ACGT protocols. Collagen type II (C-II)-specific T cell hybridomas are transduced with a lentivirus carrying a triple fusion reporter gene (TFR) construct consisting of a fluorescent reporter gene (RG), a bioluminescent RG (hRluc), and a positron emission tomography (PET) RG. Collagen-induced arthritic (CIA) mice are scanned with a bioluminescence imaging camera before and after implantation of various known cell quantities in their paws. Linear regression analysis yields equations relating two parameters of image signal intensity in mice paws to the quantity of hRluc expressing cells in the paws. Afterward, trafficking of intravenously injected cells is studied by quantitative analysis of bioluminescence images. Comparison of the average cell numbers does not demonstrate consistently higher accumulation of T-cell hybridomas in the paws with higher inflammation scores, and injecting more cells does not cause increased accumulation. MicroPET images illustrate above background signal in the inflamed paws and chest areas of CIA mice. The procedures described in this study can be used to derive equations for cells expressing other bioluminescent RGs and in other animal models.

Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma.

High-grade gliomas are aggressive cancers that often become rapidly fatal. Immunotherapy using CD8+ cytotoxic T lymphocytes (CTLs), engineered to express both herpes simplex virus type 1 thymidine kinase (HSV1-TK) and interleukin-13 (IL-13) zetakine chimeric antigen receptor (CAR), is a treatment strategy with considerable potential. To optimize this and related immunotherapies, it would be helpful to monitor CTL viability and trafficking to glioma cells. We show that noninvasive positron emission tomography (PET) imaging with 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG) can track HSV1-tk reporter gene expression present in CAR-engineered CTLs. [18F]FHBG imaging was safe and enabled the longitudinal imaging of T cells stably transfected with a PET reporter gene in patients. Further optimization of this imaging approach for monitoring in vivo cell trafficking should greatly benefit various cell-based therapies for cancer.

A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant.

A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-ß-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR.

Preclinical safety evaluation of 18F-FHBG: a PET reporter probe for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk's expression.

UNLABELLED: 9-(4-(18)F-Fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG) is a sensitive and specific PET reporter probe for imaging the PET reporter genes, herpes simplex 1 thymidine kinase (HSV1-tk) and its mutant HSV1-sr39tk. (18)F-FHBG has suitable pharmacokinetics and dosimetry for clinical applications and imaging of HSV1-TK has been demonstrated in the livers of hepatocellular cancer patients. METHODS: Male and female Sprague-Dawley rats and New Zealand White rabbits were divided into equal groups receiving either 14 microg/kg cold FHBG or carrier solution, for a 14-d acute toxicity assessment. We monitored body weight, food and water consumption, body temperature, cardiovascular electrical and functional indices, respiratory performance and oxygen saturation, comprehensive blood chemistry, complete blood count (CBC), and urinalysis. We conducted daily cage-side examinations for the detection of any clinical abnormalities. Tissues of the animals that were euthanized and necropsied on day 14 were prepared for histopathologic examination. RESULTS: No significant differences in cardiovascular and respiratory parameters, food consumption, body weight, urine components, or clinical signs attributable to test article toxicity were observed between the treatment and control groups. Any differences noted in the blood chemistry and CBC parameters were deemed to be incidental findings unrelated to the administration of the FHBG. CONCLUSION: Acute toxicity evaluation of FHBG at 100 times the expected human dose does not indicate harm to organ function or tissues. The Food and Drug Administration has approved FHBG as an Investigational New Drug.

Molecular Imaging in Genetic Medicine.

The field of biomedical imaging has made significant advances in recent times. This includes extremely high-resolution anatomic imaging and functional imaging of physiologic and pathologic processes as well as novel modalities in optical imaging to evaluate molecular features within the cellular environment. The latter has made it possible to image phenotypic markers of various genotypes that are implicated in human development, behavior, and disease. This article discusses the role of molecular imaging in genetic and precision medicine.