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[18F]V-1008 localization observed in Ptf1a-Cre;LSL-KrasG12D/+;Tgfbr2+/- (KTC) pancreatic lesions

Purpose:Pancreatic cancer is among the most aggressive malignancies and is rarely discovered early. However, pancreatic “incidentalomas,” particularly cysts, are frequently identified in asymptomatic patients through anatomic imaging for unrelated causes. Accurate determination of the malignant potential of cystic lesions could lead to life-saving surgery or spare patients with indolent disease undue risk. Current risk assessment of pancreatic cysts requires invasive sampling, with attendant morbidity and sampling errors. Here, we sought to identify imaging biomarkers of high-risk pancreatic cancer precursor lesions.Experimental Design:Translocator protein (TSPO) expression, which is associated with cholesterol metabolism, was evaluated in premalignant and pancreatic cancer lesions from human and genetically engineered mouse (GEM) tissues. In vivo imaging was performed with [18F]V-1008, a TSPO-targeted PET agent, in two GEM models. For image-guided surgery (IGS), V-1520, a TSPO ligand for near-IR optical imaging based upon the V-1008 pharmacophore, was developed and evaluated.Results:TSPO was highly expressed in human and murine pancreatic cancer. Notably, TSPO expression was associated with high-grade, premalignant intraductal papillary mucinous neoplasms (IPMNs) and pancreatic intraepithelial neoplasia (PanIN) lesions. In GEM models, [18F]V-1008 exhibited robust uptake in early pancreatic cancer, detectable by PET. Furthermore, V-1520 localized to premalignant pancreatic lesions and advanced tumors enabling real-time IGS.Conclusions:We anticipate that combined TSPO PET/IGS represents a translational approach for precision pancreatic cancer care through discrimination of high-risk indeterminate lesions and actionable surgery.

Supplementary Information including Supplementary Tables S1, S2 and S3 and Legends for Supplementary Movies S1 and S2

Real-time image-guided surgery of Ptf1a-Cre;LSL-KrasG12D/+;Smad4fl/fl (KSC) mouse using V-1520

Altered metabolism is a hallmark of cancer. In addition to glucose, glutamine is an important nutrient for cellular growth and proliferation. Non-invasive imaging via positron emission tomography (PET) may help facilitate precision treatment of cancer through patient selection and monitoring of treatment response. L-[5-11C]-glutamine (11C-glutamine) is a PET tracer designed to study glutamine uptake and metabolism. The aim of this first-in-human study was to evaluate the radiologic safety and biodistribution of 11C-glutamine for oncologic PET imaging. Methods: Nine patients with confirmed metastatic colorectal cancer underwent PET/computed tomography (CT) imaging. Patients received 337.97 ± 44.08 MBq of 11C-glutamine. Dynamic PET acquisitions centered over the abdomen or thorax were initiated simultaneously with intravenous tracer administration. Following the dynamic acquisition, a whole-body PET/CT was acquired. Volume-of-interest analyses were carried out to obtain estimates of organ-based absorbed doses of radiation. Results:11C-glutamine was well-tolerated in all patients with no observed safety concerns. Organs with the highest radiation exposure included the bladder, pancreas, and liver. The estimated effective dose was 4.46E-03 ± 7.67E-04 mSv/MBq. Accumulation of 11C-glutamine was elevated and visualized in lung, brain, bone, and liver metastases, suggesting utility for cancer imaging. Conclusion: PET using 11C-glutamine appears safe for human use and allows non-invasive visualization of metastatic colon cancer lesions in multiple organs. Further studies are needed to elucidate its potential for other cancers and for monitoring response to treatment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2–4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME. Positron emission tomography measurements of nutrient uptake in cells of the tumour microenvironment reveal cell-intrinsic partitioning in which glucose uptake is higher in myeloid cells, whereas glutamine is preferentially acquired by cancer cells.

Altered metabolism is a hallmark of cancer. In addition to glucose, glutamine is an important nutrient for cellular growth and proliferation. Noninvasive imaging via PET may help facilitate precision treatment of cancer through patient selection and monitoring of treatment response. l-[5-(11)C]-glutamine ((11)C-glutamine) is a PET tracer designed to study glutamine uptake and metabolism. The aim of this first-in-human study was to evaluate the radiologic safety and biodistribution of (11)C-glutamine for oncologic PET imaging. Methods: Nine patients with confirmed metastatic colorectal cancer underwent PET/CT imaging. Patients received 337.97 ± 44.08 MBq of (11)C-glutamine. Dynamic PET acquisitions that were centered over the abdomen or thorax were initiated simultaneously with intravenous tracer administration. After the dynamic acquisition, a whole-body PET/CT scan was acquired. Volume-of-interest analyses were performed to obtain estimates of organ-based absorbed doses of radiation. Results: (11)C-glutamine was well tolerated in all patients, with no observed safety concerns. The organs with the highest radiation exposure included the bladder, pancreas, and liver. The estimated effective dose was 4.46E−03 ± 7.67E−04 mSv/MBq. Accumulation of (11)C-glutamine was elevated and visualized in lung, brain, bone, and liver metastases, suggesting utility for cancer imaging. Conclusion: PET using (11)C-glutamine appears safe for human use and allows noninvasive visualization of metastatic colon cancer lesions in multiple organs. Further studies are needed to elucidate its potential for other cancers and for monitoring response to treatment.

The tumor microenvironment (TME) includes transformed cancer and infiltrating immune cells1,2. Cancer cells can consume large quantities of glucose through Warburg metabolism3,4 that can be visualized with positron emission tomography (PET). While infiltrating immune cells also rely on glucose, disruptions to metabolism can contribute to tumor immunological evasion5–9. How immune cell metabolism is programmed or restrained by competition with cancer cells for nutrients, remains uncertain. Here we used PET tracers to measure the accessibility of glucose and glutamine to cell subsets in the TME. Surprisingly, myeloid cells including macrophages were the greatest consumers of intra-tumoral glucose, followed by T cells and cancer cells. Cancer cells, in contrast, had the highest glutamine uptake. This distinct nutrient partitioning was programmed through selective mTORC1 signaling and glucose or glutamine-related gene expression. Inhibition of glutamine uptake enhanced glucose uptake across tumor resident cell types and shifted macrophage phenotype, demonstrating glucose is not limiting in the TME. Thus, cancer cells are not the only cells in tumors which exhibit high glucose uptake in vivo and instead preferentially utilize glutamine over other cell types. We observe that intrinsic cellular programs can play a major role in the use of some nutrients. Together, these data argue cell selective partitioning of glucose and glutamine can be exploited to develop therapies and imaging strategies to alter the metabolic programs of specific cell populations in the TME.

Cancer cells characteristically consume glucose through Warburg metabolism

The tumor microenvironment (TME) includes cancer and infiltrating immune cells. Tumors canonically consume glucose through Warburg metabolism, a process forming the basis of cancer imaging by positron emission tomography (PET). Activated immune cells also rely on glucose, and impaired immune cell metabolism in the TME contributes to tumor progression. It remains uncertain, however, if immune cell metabolism is dysregulated in the TME by cell intrinsic programs or by competition with cancer cells for limiting nutrients. Here we used PET tracers to measure access and uptake of glucose and glutamine by specific cell subsets in the TME. Surprisingly, myeloid cells had the greatest capacity to uptake glucose in vivo, followed by T cells and cancer cells across a range of cancer models. Cancer cells, in contrast, demonstrated high glutamine uptake. This distinct nutrient partitioning was cell intrinsically programmed through mTORC1 signaling and glucose and glutamine-related gene expression. Inhibiting glutamine uptake enhanced glucose uptake across tumor resident cell types, suggesting that glutamine metabolism suppresses glucose uptake without glucose being limiting in the TME. Thus, cell intrinsic programs dictate the preferential immune and cancer cell acquisition of glucose and glutamine. Cell selective partitioning of these nutrients may be exploited to develop therapies and imaging strategies to enhance or monitor the metabolism and activities of specific cell populations in the TME.

Improving response to epidermal growth factor receptor (EGFR)-targeted therapies in patients with advanced wild-type (WT) RAS colorectal cancer (CRC) remains an unmet need. In this preclinical work, we evaluated a new therapeutic combination aimed at enhancing efficacy by targeting cancer cell metabolism in concert with EGFR. We hypothesized that combined blockade of glutamine metabolism and EGFR represents a promising treatment approach by targeting both the "fuel" and "signaling" components that these tumors need to survive. To explore this hypothesis, we combined CB-839, an inhibitor of glutaminase 1 (GLS1), the mitochondrial enzyme responsible for catalyzing conversion of glutamine to glutamate, with cetuximab, an EGFR-targeted monoclonal antibody in preclinical models of CRC. 2D and 3D in vitro assays were executed following treatment with either single agent or combination therapy. The combination of cetuximab with CB-839 resulted in reduced cell viability and demonstrated synergism in several cell lines. In vivo efficacy experiments were performed in cell-line xenograft models propagated in athymic nude mice. Tumor volumes were measured followed by immunohistochemical (IHC) analysis of proliferation (Ki67), mechanistic target of rapamycin (mTOR) signaling (pS6), and multiple mechanisms of cell death to annotate molecular determinants of response. In vivo, a significant reduction in tumor growth and reduced Ki67 and pS6 IHC staining were observed with combination therapy, which was accompanied by increased apoptosis and/or necrosis. The combination showed efficacy in cetuximab-sensitive as well as resistant models. In conclusion, this therapeutic combination represents a promising new precision medicine approach for patients with refractory metastatic WT RAS CRC.

Background: Gln metabolism plays a critical role in cancer. In CRC, the epidermal growth factor receptor (EGFR) and Gln cooperate to provide signal transduction and fuel for mitogen activated protein kinase-dependent cell growth. Our preclinical data illustrate that Gln abrogates EGFR inhibition and blockade of Gln metabolism restores EGFR sensitivity, forming the basis of a phase I/II clinical trial exploring dual EGFR and Gln metabolism in mCRC (NCT03263429). As a biomarker correlate, we are performing dual tracer PET imaging of Gln flux (11C-Gln) and Gln to glutamate conversion (18F-FSPG). We hypothesize that tumors dependent on Gln will exhibit high 11C-Gln uptake at baseline, while inhibition of Gln metabolism will reduce 18F-FSPG uptake. Notably, this is the first-in-human study with 11C-Gln. Methods: Adult patients (pts) with previously treated anti-EGFR, RAS WT, mCRC are enrolling and receive panitumumab (6 mg/kg Day 1 and 15) and CB-839 (800 mg twice daily) in 28-day treatment cycles. 11C-Gln and 18F-FSPG scans are performed at baseline and Cycle 1 Day 28. Lesion to blood pool ratio (LBR) is calculated for each target lesion and compared with change in tumor size as measured by RECIST 1.1 after 2 cycles of therapy. Results: The study is ongoing. To date, we have collected pre- and post-therapy PET data on 4 pts; individual lesion data for 2 pts are presented in the table. A mixed response with 11C-Gln uptake was observed for Pt #1, which could reflect different underlying lesion pathology; varying degree of lesion size change was also observed in this patient. The greatest decrease in 18F-FSPG uptake corresponded to the lesion with the smallest growth. For Pt #2, 11C-Gln uptake at baseline was highest in the lesion with the largest decrease in tumor size at Day 56; 18F-FSPG uptake was greatly reduced in all lesions, which corresponded to decreases in tumor size. Best overall response was progressive disease and partial response for Pt #1 and #2, respectively. Gln PET as a function of lesion response11C-Gln18F-FSPG% Change in lesion size at Day 56Baseline LBRDay 28 LBR% ChangeBaseline LBRDay 28 LBR% ChangePatient#1L lung nodule+44%2.171.93-11.1%2.001.06-47.0%Posterior hepatic lobe+6%5.404.27-20.9%6.422.84-55.8%Segment IVA liver+107%5.114.88-4.5%5.583.90-30.1%Adrenal mass+152%3.504.4727.7%2.862.84-0.7%Patient#2R infrahilar pulmonary metastasis-15%2.471.74-29.6%3.131.72-45.0%L pulmonary metastasis-15%0.750.784.0%0.960.82-14.6%Hepatic Metastasis #1-100%8.976.81-24.1%7.003.50-50.0% Conclusions: Quantitative biomarkers that predict response early in the course of therapy are a means to achieve the promise of precision medicine. Our preliminary clinical imaging data suggest that the employed investigational PET molecular imaging tracers have potential for prioritizing patients for combined EGFR/Gln targeted therapy and to predict response early in the treatment course. Additional PET data for all pts and summary statistics will be presented. Citation Format: Jennifer Gray Whisenant, Gary Smith, Allison Cohen, Kristen K. Ciombor, Dana Cardin, Cathy Eng, Laura Goff, Satya Das, Tiffany Hickman, Anna Fisher, Adria Payne, Robert Coffey, G. Dan Ayers, Jordan D. Berlin, H. Charles Manning. Molecular imaging of glutamine (Gln) metabolism in RAS wildtype (WT) metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4260.

PURPOSE: There is an urgent need for the development of novel positron emission tomography (PET) tracers for glioma imaging. In this study, we developed a novel PET probe ([(18)F]VUIIS1018A) by targeting translocator protein (TSPO), an imaging biomarker for glioma. The purpose of this preclinical study was to evaluate this novel TSPO probe for glioma imaging. PROCEDURES: In this study, we synthesized [(19)F]VUIIS1018A and the precursor for radiosynthesis of [(18)F]VUIIS1018A. TSPO binding affinity was confirmed using a radioligand competitive binding assay in C6 glioma cell lysate. Further, dynamic imaging studies were performed in rats using a microPET system. These studies include displacement and blocking studies for ligand reversibility and specificity evaluation, and compartment modeling of PET data for pharmacokinetic parameter measurement using metabolite-corrected arterial input functions and PMOD. RESULTS: Compared to previously reported TSPO tracers including [(18)F]VUIIS1008 and [(18) F]DPA-714, the novel tracer [(18)F]VUIIS1018A demonstrated higher binding affinity and BP(ND). Pretreatment with the cold analog [(19)F]VUIIS1018A could partially block tumor accumulation of this novel tracer. Further, compartment modeling of this novel tracer also exhibited a greater tumor-to-background ratio, a higher tumor binding potential and a lower brain binding potential when compared with other TSPO probes, such as [(18)F]DPA-714 and [(18)F]VUIIS1008. CONCLUSIONS: These studies illustrate that [(18)F]VUIIS1018A can serve as a promising TSPO PET tracer for glioma imaging and potentially imaging of other solid tumors.

The tumor microenvironment is composed of multiple cell types, including malignant cancer cells and tumor-infiltrating leukocytes (TIL). A possible mechanism of immunosuppression in the tumor microenvironment (TME) is cancer cells outcompeting anti-cancer TIL for nutrients such as glucose. 18F-deoxyglucose (FDG) positron emission tomography (PET) imaging is a staple of diagnosing and monitoring many types of cancer and is based on the Warburg model in which cancer cells within the tumor utilize glucose for growth and proliferation. Here, we use magnetic bead sorting to fractionate FDG-avid murine tumors and measure tumor cell-specific glucose uptake. We find that CD45+ immune cells are more FDG-avid than CD45− cancer cells. We further fractionate immune cell subsets into CD4/8+ T cells and CD11b+ myeloid cells to demonstrate that TIL T cells take up more glucose than CD45− cancer cells and resting splenic T cells. Strikingly, CD11b+ myeloid cells are the most FDG-avid cells in the TME. In MC38 colorectal cancer tumors, we show that CD11b+ F4/80 hi macrophages have high glucose uptake, and by extracellular flux analysis demonstrate higher metabolic activity than tumor T cells and CD45− cancer cells. Intriguingly, while glucose uptake is low in CD45− cancer cells, 18F-glutamine uptake is higher in cancer cells than immune cells. Our results illustrate a novel approach to measuring nutrient uptake in the TME and suggest that TIL are not starved of nutrients in the TME. Future work will determine the effects of immunotherapy and metabolism-targeted therapeutics on cell-specific glucose and glutamine uptake in the TME, as well as determine the immune contribution to cancer PET scans in the context of immunotherapy response.

Lung cancer is the leading cause of cancer deaths in the United States. Novel lung cancer targeted therapeutic and molecular imaging agents are needed to improve outcomes and enable personalized care. Since these agents typically cannot cross the plasma membrane while carrying cytotoxic payload or imaging contrast, discovery of cell-surface targets is a necessary initial step. Herein, we report the discovery and characterization of lung cancer cell-surface markers for use in development of targeted agents. To identify putative cell-surface markers, existing microarray gene expression data from patient specimens were analyzed to select markers with differential expression in lung cancer compared to normal lung. Greater than 200 putative cell-surface markers were identified as being overexpressed in lung cancers. Ten cell-surface markers (CA9, CA12, CXorf61, DSG3, FAT2, GPR87, KISS1R, LYPD3, SLC7A11 and TMPRSS4) were selected based on differential mRNA expression in lung tumors vs. non-neoplastic lung samples and other normal tissues, and other considerations involving known biology and targeting moieties. Protein expression was confirmed by immunohistochemistry (IHC) staining and scoring of patient tumor and normal tissue samples. As further validation, marker expression was determined in lung cancer cell lines using microarray data and Kaplan-Meier survival analyses were performed for each of the markers using patient clinical data. High expression for six of the markers (CA9, CA12, CXorf61, GPR87, LYPD3, and SLC7A11) was significantly associated with worse survival. These markers should be useful for the development of novel targeted imaging probes or therapeutics for use in personalized care of lung cancer patients.

The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein–protein or viral–host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles.

574 Background: Epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) are approved in RAS WT mCRC; however, patients (pts) will develop resistance to these agents. Alterations in glutamine (Gln) metabolism play a critical role in cancer cell growth. In cancers such as CRC, EGFR and Gln cooperate to provide signals and fuel for mitogen activated protein kinase-dependent cell growth. Our in vitro data show that Gln abrogates EGFR inhibition, and blockade of Gln transport restores sensitivity. We also observed a greater antitumor response in vivo with EGFR mAb plus CB-839, an inhibitor of a rate-limiting enzyme of Gln metabolism, than either agent alone. We designed a phase I/II study (NCT03263429) to evaluate CB-839 + panitumumab + irinotecan in anti-EGFR refractory RAS WT mCRC. Methods: Dose escalation used a Bayesian continual reassessment method targeting a 25% toxicity probability. CB-839 (600 mg or 800 mg twice daily [BID]) were evaluated with panitumumab (6 mg/kg) and irinotecan (180 mg/m2). Irinotecan was included in phase I to establish a future phase II dose of the triplet. Prior EGFR mAb treatment (tx) was not required for phase I. Dose-limiting toxicity (DLT) was any tx-related non-hematologic ≥Gr 3 toxicity (except fatigue, rash, or elevated liver enzymes) or ≥Gr 4 hematologic toxicity during the first 28 days. Results: Nine pts have been enrolled; 2 were not evaluable for DLT and replaced. Zero DLTs were observed at dose level 1 (n = 3) or 2 (n = 4); 2 more pts are needed to confirm the maximum tolerated dose (MTD). Most frequent toxicities were anemia and hypomagnesemia (88%) and elevated alkaline phosphatase, nausea, and rash (75%), most ≤Gr 2. One of 7 evaluable pts (14%) has an ongoing partial response, and 5 pts had stable disease (SD; 71%). Three pts have been on tx > 6 months, and 3 pts with prior EGFR mAb tx achieved SD. Conclusions: Triplet combination was tolerable at full doses of each drug, and preliminary antitumor activity was observed in a majority of pts. Phase II will begin after phase I completion and will evaluate efficacy of CB-839 (800 mg BID) and panitumumab (6 mg/kg). Imaging studies using investigational PET tracers to evaluate Gln metabolism as a function of tumor response are planned. Clinical trial information: NCT03263429.

Irradiation of solutions of tetraphenylporphyrin (H2TPP) in chloroform causes decomposition of the chloroform at UV wavelengths higher than those that decompose chloroform directly. The catalytic cycle involves photooxidation of the porphyrin followed by thermal reduction. Photocatalysis continues after H2TPP has been completely protonated by the HCl produced during decomposition, and the rate of HCl production accelerates as a second pathway, in which CCl3OOH oxidizes the porphyrin, becomes important.

Fluorescence molecular imaging can be employed for the development of novel cancer targeting agents. Herein, we investigated the pharmacokinetics (PK), biodistribution (BD) and cellular uptake of Dmt-Tic-Cy5, a delta-opioid receptor (δOR) antagonist-fluorescent dye conjugate, as a tumor-targeting molecular imaging agent. δOR expression is observed normally in the CNS, and pathologically in some tumors, including lung liver and breast cancers. In vitro, in vivo and ex vivo experiments were conducted to image and quantify the fluorescence signal associated with Dmt-Tic-Cy5 over time using in vitro and intravital fluorescence microscopy and small animal fluorescence imaging of tumor-bearing mice. We observed specific retention of Dmt-Tic-Cy5 in tumors with maximum uptake in δOR-expressing positive tumors at 3 h and observable persistence for >96 h; clearance from δOR non-expressing negative tumors by 6 h; and systemic clearance from normal organs by 24 h. Live-cell and intravital fluorescence microscopy demonstrated that Dmt-Tic-Cy5 had sustained cell-surface binding lasting at least 24 h with gradual internalization over the initial 6 h following administration. Dmt-Tic-Cy5 is a δOR-targeted agent that exhibits long lasting and specific signal in δOR-expressing tumors, is rapidly cleared from systemic circulation and is not retained in non-δOR expressing tissues. Hence, Dmt-Tic-Cy5 has potential as a fluorescent tumor imaging agent.

Lung cancer is the leading cause of cancer deaths worldwide. Many lung cancer patients are diagnosed with advanced disease. These patients have a low 5-year survival rate and limited treatment options. Novel therapies, which target biomarkers that are overexpressed in lung cancer but have basal expression in benign tissue, are desperately needed. The goal of this work was to develop a targeted agent for immune checkpoint therapy of lung cancer. Recently, immune checkpoint inhibitors have been approved for use in lung cancer and many more are being tested in clinical trials. The current immune checkpoint inhibitor agents are not tumor-targeted. Targeting the immune checkpoint inhibitor to tumor cell-surface markers should concentrate the conjugate in the tumor microenvironment and enhance the immune response in the tumor while reducing the systemic dosages needed, resulting in lower systemic toxicity. The delta opioid receptor (DOR) is expressed in some lung cancers, but is not expressed or is expressed only at basal levels in normal tissues outside the brain. We have previously synthesized fluorescently-labeled DOR-targeted imaging agents based on a synthetic peptide antagonist (DORL). These targeted fluorescent agents have high affinity and selectivity for DOR, and exhibit good pharmacokinetic (PK) and biodistribution (BD) profiles, i.e. specific tumor uptake with rapid systemic clearance and no uptake in tissues of concern, e.g. brain. We are now developing lung cancer-specific immunotherapy agents that target the DOR by conjugating DORL to immunomodulatory molecules. In the current work, we synthesized a fluorescently-labeled DOR targeting ligand and conjugated it to an anti-PD1 antibody (DORL-PD1). We synthesized immunoconjugates with several targeting ligand-to-antibody ratios (TARs). We engineered murine lung cancer cells to constitutively express the DOR. By lanthanide time-resolved fluorescence (LTRF) competitive binding assays, we have shown that the agents have high avidity for the DOR in vitro with higher TARs resulting in higher binding avidity. We characterized the uptake of DORL-PD1 in vitro using live-cell fluorescence microscopy. Using syngeneic engraftment tumor models in immunocompetent mice, we performed longitudinal fluorescence imaging studies to determine the agent circulation time (PK), tumor selectivity and tissue distribution (BD). Immune checkpoint efficacy studies were performed using the DOR negative mouse tumor models. In conclusion, we have synthesized fluorescent DOR-targeted immune checkpoint therapy agents, DORL-PD1; demonstrated avidity and selectivity for the DOR in vitro and in vivo; and immune checkpoint therapy efficacy in vivo. Future studies will evaluate the efficacy of DORL-PD1 in immune competent mice bearing DOR positive tumors. These agents could be useful for increasing the efficacy and reducing systemic toxicity of immune-checkpoint therapy of lung cancer. Citation Format: Allison S. Cohen, Michael L. Doligalski, Hong Zheng, Narges K. Tafreshi, Veronica Estrella, Nella Delva, Jonathan Nguyen, Amer Beg, Mark L. McLaughlin, David L. Morse. Targeting immune checkpoint therapy to the lung tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1709. doi:10.1158/1538-7445.AM2017-1709

Cell-surface glycans are attractive targets for molecule imaging due to their reflection of cellular processes associated with development and disease progression. In this paper, we describe the design, synthesis, and biological application of a new phosphine probe for real-time imaging of cell-surface glycans using bioluminescence. To accomplish this goal, we took advantage of the bioorthogonal chemical reporter technique. This strategy uses a two-step labeling procedure in which an unnatural sugar analogue containing a functional handle is (1) incorporated into sugar-bearing proteins via the cell’s own biosynthetic machinery and then (2) detected with an exogenously added probe. We designed phosphine−luciferin reagent 1 to activate bioluminescence in response to Staudinger ligation with azide-labeled glycans. We chose to use a phosphine probe because, despite their slow reaction kinetics, they remain the best-performing reagents for tagging azidosugars in mice. Given the sensitivity and negligible background provided by bioluminescence imaging (BLI), we reasoned that 1 might be able to overcome some of the limitations encountered with fluorescent phosphine probes. In this work, we synthesized the first phosphine−luciferin probe for use in real-time BLI and demonstrated that azide-labeled cell-surface glycans can be imaged with 1 using concentrations as low as single digit nanomolar and times as little as 5 min, a feat that cannot be matched by any previous fluorescent phosphine probes. Even though we have only demonstrated its use in visualizing glycans, it can be envisioned that this probe could also be used for bioluminescence imaging of any azide-containing biomolecule, such as proteins and lipids, since azides have been previously incorporated into these molecules. The phosphine−luciferin probe is therefore poised for many applications in real-time imaging in cells and whole animals. These studies are currently in progress in our laboratory.

In vivo fluorescence lifetime imaging allows for the isolation of probe-specific fluorescence lifetime from surrounding tissue auto-fluorescence. Fluorescence lifetime is not affected by agent concentration or depth, or image acquisition parameters. However, fluorescence lifetime can be affected by the in vivo tissue microenvironment, e.g. pH or hypoxia. In this work, fluorescence lifetime is used to characterize the presence and fate of a high-affinity peptidyl delta opioid receptor (δOR) ligand conjugated to the near-infrared fluorescent IRDye800CW (Li-Cor). HCT116 colorectal cancer cells engineered to over-express the δOR and parental non-expressing HCT116 cells were bilaterally xenografted subcutaneously into the flanks of athymic nude mice. At a series of time-points following tail vein injection of the ligand, in vivo fluorescence images images were acquired using the Optix MX3 scanner (Advanced Research Technologies). For 24 h, the ligand fluoresence lifetime was significantly different (p In vitro studies demonstrated that the fluoresecence lifetime of this ligand is not altered within the biological pH range. However, the tumor microenvironment is complex and multiple factors may be influencing the observed differences. Future plans include studies of 3D cell cultures using varied microenvironmental conditions and studies on δOR endogenous cell lines. Citation Format: Yoli JeuneSmith, Allison S. Cohen, Narges K. Tafreshi, Josef Vagner, Robert Gillies, David Morse. In vivo fluorescence lifetime imaging differentiates the bound versus unbound status of a cell surface receptor targeted ligand. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5519. doi:10.1158/1538-7445.AM2013-5519

Detection and quantification of fatty acid fluxes in animal model systems following physiological, pathological, or pharmacological challenges is key to our understanding of complex metabolic networks as these macronutrients also activate transcription factors and modulate signaling cascades including insulin-sensitivity. To enable non-invasive, real-time, spatiotemporal quantitative imaging of fatty acid fluxes in animals, we created a bioactivatable molecular imaging probe based on long-chain fatty acids conjugated to a reporter molecule (luciferin). We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used in animal systems as a valuable tool to localize and quantitate in real-time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. This imaging approach should further our understanding of basic metabolic processes and pathological alterations in multiple disease models.