Legends for Supplemental Figures S1-S8.

Supplemental Figure S1. Effect of HDAC and Hsp90 inhibitors on Hsp90 (α/β)/p23 interactions.

Supplemental Figure S7. Inhibition of UKE-1 cell proliferation by the lead HDAC inhibitor 1A12 alone and in combination with the Doxorubicin and Hsp90 inhibitor PU-H71.

Supplemental Methods. Description of additional methods and procedures used in the study.

Supplemental Figure S2. 17-DMAG enhanced the effect of Tubacin on inhibition of Hsp90(α/β)/p23 interactions.

Supplemental Figure S8. Analyses of HDAC biomarkers in tumor xenografts in mice treated with 1A12.

Supplemental Table S1. Selectivity of lead HDAC inhibitors for inhibition of HDAC1-9 activities in vitro.

Supplemental Figures S1-4 and Tables 1-2. Supplemental Figure S1. Mechanism of 18F-C-SNAT activation, cyclization and intracellular trapping; Supplemental Figure S2. Flow cytometric analysis of cell death in EL-4 cell mixtures; Supplemental Figure S3. Radiotracer uptake and retention in carboplatin- and vehicle-treated PC9 and A549 non small cell lung cancer cells; Supplemental Figure S4. SPECT imaging of 99mTc-Annexin V uptake in vivo; Supplemental Table 1. Ex vivo biodistribution data for selected tissues in naive and drug-treated mice 90 min after injection; Supplemental Table 2. Radiotracer tumor-to-tissue ratios in naive and drug-treated EL-4 xenografts 90 min after injection.

CHO Cell Transfection Tracer Uptake and Efflux Assays for Adherent Cells Western Blot Immunofluorescence Staining of Lymphoid Organs Ex Vivo Donor T Cell Proliferation Analysis Automated preparation of [18F]FAraG for human imaging using the Neptis perform PET synthesizer Imaging Studies in Humans Statistics

Supplemental Video M2. 18FFDG uptake in drug-treated tumors

Supplementary Figure 1 shows [18F]F-AraG Structure and Schematic of AraG Uptake into T Cells Supplementary Figure 2 shows [18F]F-AraG and [3H]F-AraC Uptake and Retention Across Various Immune Cell Lines Supplementary Figure 3 shows AraG Competitive Inhibition of [18F]F-AraG Uptake Across Various Immune Cell Lines Supplementary Figure 4 shows Comparison of [18F]F-AraG and [3H]F-AraC Uptake and Retention Across Various Solid Tumor Cell Lines Supplementary Figure 5 shows Western Blot Analysis of dCK Expression in Wild-type and Mutant CCRF-CEM cells Supplementary Figure 6 shows dGK overexpression in CHO-K1 cells leads to increased [18F]F-AraG uptake and retention Supplementary Figure 7 shows [3H]F-AraC Accumulates in Cells via dCK Activity Supplementary Figure 8 shows Uptake of [18F]F-AraG in Activated Versus Resting Murine T Cells Supplementary Figure 9 shows Isolation and Activation of Primary Human T Cells Supplementary Figure 10 shows Uptake and Retention of [3H]AraG and [3H]F-AraC in Resting and Activated Human Primary T Cells Supplementary Figure 11 shows Evaluation of GFP+ Donor T cells in Spleens and Cervical Lymph Nodes (CLNs) at Day 3 and Day 10 of Acute GVHD Supplementary Figure 12 shows Evaluation of Proliferation of Donor T Cells in Cervical Lymph Nodes

Supplemental Figure S6. Specificity of 1A12 in inhibition of HDAC6.

Supplemental Figure S4. Up-regulation of HDAC biomarkers (p21waf1 and acetylated H3), Hsp70 and NRL-p23 fusion reporter in cells treated with lead HDAC inhibitors.

Supplemental Figure S5. Determination of the specificity of 1A12 in inhibition of HDACs.

Supplemental Figure S3. Syntheses and characterization of lead HDAC inhibitors.

It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade.

Abstract 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-β-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.

6″-18F-fluoromaltotriose is a PET tracer that can potentially be used to image and localize most bacterial infections, much like 18F-FDG has been used to image and localize most cancers. However, unlike 18F-FDG, 6″-18F-fluoromaltotriose is not taken up by inflammatory lesions and appears to be specific to bacterial infections by targeting the maltodextrin transporter that is expressed in gram-positive and gram-negative strains of bacteria. Methods: 6″-18F-fluoromaltotriose was synthesized with high radiochemical purity and evaluated in several clinically relevant bacterial strains in cultures and in living mice. Results: 6″-18F-fluoromaltotriose was taken up in both gram-positive and gram-negative bacterial strains. 6″-18F-fluoromaltotriose was also able to detect Pseudomonas aeruginosa in a clinically relevant mouse model of wound infection. The utility of 6″-18F-fluoromaltotriose to help monitor antibiotic therapies was also evaluated in rats. Conclusion: 6″-18F-fluoromaltotriose is a promising new tracer that has significant diagnostic utility, with the potential to change the clinical management of patients with infectious diseases of bacterial origin.

Abstract Purpose: An early readout of tumor response to therapy through measurement of drug or radiation-induced cell death may provide important prognostic indications and improved patient management. It has been shown that the uptake of 18F-C-SNAT can be used to detect early response to therapy in tumors by positron emission tomography (PET) via a mechanism of caspase-3–triggered nanoaggregation. Experimental Design: Here, we compared the preclinical utility of 18F-C-SNAT for the detection of drug-induced cell death to clinically evaluated radiotracers, 18F-FDG, 99mTc-Annexin V, and 18F-ML-10 in tumor cells in culture, and in tumor-bearing mice in vivo. Results: In drug-treated lymphoma cells, 18F-FDG, 99mTc-Annexin V, and 18F-C-SNAT cell-associated radioactivity correlated well to levels of cell death (R2 > 0.8; P < 0.001), with no correlation measured for 18F-ML-10 (R2 = 0.05; P > 0.05). A similar pattern of response was observed in two human NSCLC cell lines following carboplatin treatment. EL-4 tumor uptake of 99mTc-Annexin V and 18F-C-SNAT were increased 1.4- and 2.1-fold, respectively, in drug-treated versus naïve control animals (P < 0.05), although 99mTc-Annexin V binding did not correlate to ex vivo TUNEL staining of tissue sections. A differential response was not observed with either 18F-FDG or 18F-ML-10. Conclusions: We have demonstrated here that 18F-C-SNAT can sensitively detect drug-induced cell death in murine lymphoma and human NSCLC. Despite favorable image contrast obtained with 18F-C-SNAT, the development of next-generation derivatives, using the same novel and promising uptake mechanism, but displaying improved biodistribution profiles, are warranted for maximum clinical utility. Clin Cancer Res; 21(17); 3896–905. ©2015 AACR.

Immune checkpoint signaling through the programmed death 1 (PD-1) axis to its ligand (PD-L1) significantly dampens anti-tumor immune responses. Cancer patients treated with checkpoint inhibitors that block this suppressive signaling have exhibited objective response rates of 20-40% for advanced solid tumors, lymphomas, and malignant melanomas. This represents a tremendous advance in cancer treatment. Unfortunately, all patients do not respond to immune checkpoint blockade. Recent findings suggest that patients with tumor infiltrating lymphocytes (TILs) expressing PD-1 may be most likely to respond to αPD-1/PD-L1 checkpoint inhibitors. There is a compelling need for diagnostic and prognostic imaging tools to assess the PD-1 status of TILs in vivo. Here we have developed a novel immunoPET tracer to image PD-1 expressing TILs in a transgenic mouse model bearing melanoma. A (64)Cu labeled anti-mouse antibody (IgG) PD-1 immuno positron emission tomography (PET) tracer was developed to detect PD-1 expressing murine TILs. Quality control of the tracer showed >95% purity by HPLC and >70% immunoreactivity in an in vitro cell binding assay. ImmunoPET scans were performed over 1-48 h on Foxp3+.LuciDTR4 mice bearing B16-F10 melanoma tumors. Mice receiving anti-PD-1 tracer (200 ± 10 μCi/10-12 μg/200 μL) revealed high tracer uptake in lymphoid organs and tumors. BLI images of FoxP3(+) CD4(+) Tregs known to express PD-1 confirmed lymphocyte infiltration of tumors at the time of PET imaging. Biodistribution measurements performed at 48 h revealed a high (11×) tumor to muscle uptake ratio of the PET tracer (p < 0.05). PD-1 tumors exhibited 7.4 ± 0.7%ID/g tracer uptake and showed a 2× fold signal decrease when binding was blocked by unlabeled antibody. To the best of our knowledge this data is the first report to image PD-1 expression in living subjects with PET. This radiotracer has the potential to assess the prognostic value of PD-1 in preclinical models of immunotherapy and may ultimately aid in predicting response to therapies targeting immune checkpoints.