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Thyroid cancer is one of the most common cancers, with a global increase in incidence rate for both genders. Ultrasound-guided fine-needle aspiration is the current gold standard to diagnose thyroid cancers, but the results are inaccurate, leading to repeated biopsies and unnecessary surgeries. To reduce the number of unnecessary biopsies, we explored the use of multiparametric photoacoustic (PA) analysis in combination with the American Thyroid Association (ATA) Guideline (ATAP). In this study, we performed in vivo multispectral PA imaging on thyroid nodules from 52 patients, comprising 23 papillary thyroid cancer (PTC) and 29 benign cases. From the multispectral PA data, we calculated hemoglobin oxygen saturation level in the nodule area, then classified the PTC and benign nodules with multiparametric analysis. Statistical analyses showed that this multiparametric analysis of multispectral PA responses could classify PTC nodules. Combining the photoacoustically indicated probability of PTC and the ATAP led to a new scoring method that achieved a sensitivity of 83% and a specificity of 93%. This study is the first multiparametric analysis of multispectral PA data of thyroid nodules with statistical significance. As a proof of concept, the results show that the proposed new ATAP scoring can help physicians examine thyroid nodules for fine-needle aspiration biopsy, thus reducing unnecessary biopsies. Significance: This report highlights a novel photoacoustic scoring method for risk stratification of thyroid nodules, where malignancy of the nodules can be diagnosed with 83% sensitivity and 93% specificity.

Graft-versus-host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT), mediated primarily by donor T cells that become activated and attack host tissues. Noninvasive strategies detecting T-cell activation would allow for early diagnosis and possibly more effective management of HCT recipients. PET imaging is a sensitive and clinically relevant modality ideal for GvHD diagnosis, and there is a strong rationale for the use of PET tracers that can monitor T-cell activation and expansion with high specificity. The TNF receptor superfamily member OX40 (CD134) is a cell surface marker that is highly specific for activated T cells, is upregulated during GvHD, and mediates disease pathogenesis. We recently reported the development of an antibody-based activated T-cell imaging agent targeting OX40. In the present study, we visualize the dynamics of OX40 expression in an MHC-mismatch mouse model of acute GvHD using OX40-immunoPET. This approach enabled visualization of T-cell activation at early stages of disease, prior to overt clinical symptoms with high sensitivity and specificity. This study highlights the potential utility of the OX40 PET imaging as a new strategy for GvHD diagnosis and therapy monitoring. Significance: OX40-immunoPET imaging is a promising noninvasive strategy for early detection of GvHD, capable of detecting signs of GvHD pathology even prior to the development of overt clinical symptoms.

Immunotherapy is innovating clinical cancer management. Nevertheless, only a small fraction of patient's benefit from current immunotherapies. To improve clinical management of cancer immunotherapy, it is critical to develop strategies for response monitoring and prediction. In this study, we describe inducible T-cell costimulator (ICOS) as a conserved mediator of immune response across multiple therapy strategies. ICOS expression was evaluated by flow cytometry, 89Zr-DFO-ICOS mAb PET/CT imaging was performed on Lewis lung cancer models treated with different immunotherapy strategies, and the change in tumor volume was used as a read-out for therapeutic response. ImmunoPET imaging of ICOS enabled sensitive and specific detection of activated T cells and early benchmarking of immune response. A STING (stimulator of interferon genes) agonist was identified as a promising therapeutic approach in this manner. The STING agonist generated significantly stronger immune responses as measured by ICOS ImmunoPET and delayed tumor growth compared with programmed death-1 checkpoint blockade. More importantly, ICOS ImmunoPET enabled early and robust prediction of therapeutic response across multiple treatment regimens. These data show that ICOS is an indicator of T-cell–mediated immune response and suggests ICOS ImmunoPET as a promising strategy for monitoring, comparing, and predicting immunotherapy success in cancer. Significance: ICOS ImmunoPET is a promising strategy to noninvasively predict and monitor immunotherapy response. See related commentary by Choyke, p. 2975

The cell's endogenous antioxidant system is vital to maintain redox homeostasis. Despite its central role in normal and pathophysiology, no noninvasive tools exist to measure this system in patients. The cystine/glutamate antiporter system xc− maintains the balance between intracellular reactive oxygen species and antioxidant production through the provision of cystine, a key precursor in glutathione biosynthesis. Here, we show that tumor cell retention of a system xc−-specific PET radiotracer, (S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), decreases in proportion to levels of oxidative stress following treatment with a range of redox-active compounds. The decrease in [18F]FSPG retention correlated with a depletion of intracellular cystine resulting from increased de novo glutathione biosynthesis, shown through [U-13C6, U-15N2]cystine isotopic tracing. In vivo, treatment with the chemotherapeutic doxorubicin decreased [18F]FSPG tumor uptake in a mouse model of ovarian cancer, coinciding with markers of oxidative stress but preceding tumor shrinkage and decreased glucose utilization. Having already been used in pilot clinical trials, [18F]FSPG PET could be rapidly translated to the clinic as an early redox indicator of tumor response to treatment. Significance: [18F]FSPG PET imaging provides a sensitive noninvasive measure of tumor redox status and provides an early marker of tumor response to therapy. See related commentary by Lee et al., p. 701

Thyroid cancer is one of the most common cancers, with a global increase in incidence rate for both genders. Ultrasound-guided fine-needle aspiration is the current gold standard to diagnose thyroid cancers, but the results are inaccurate, leading to repeated biopsies and unnecessary surgeries. To reduce the number of unnecessary biopsies, we explored the use of multiparametric photoacoustic (PA) analysis in combination with the American Thyroid Association (ATA) Guideline (ATAP). In this study, we performed

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[18F]-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. Significance: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells.

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.

Considerable advances in cancer-specific optical imaging have improved the precision of tumor resection. In comparison to traditional imaging modalities, this technology is unique in its ability to provide real-time feedback to the operating surgeon. Given the significant clinical implications of optical imaging, there is an urgent need to standardize surgical navigation tools and contrast agents to facilitate swift regulatory approval. Because fluorescence-enhanced surgery requires a combination of both device and drug, each may be developed in conjunction, or separately, which are important considerations in the approval process. This report is the result of a one-day meeting held on May 4, 2016 with officials from the National Cancer Institute, the FDA, members of the American Society of Image-Guided Surgery, and members of the World Molecular Imaging Society, which discussed consensus methods for FDA-directed human testing and approval of investigational optical imaging devices as well as contrast agents for surgical applications. The goal of this workshop was to discuss FDA approval requirements and the expectations for approval of these novel drugs and devices, packaged separately or in combination, within the context of optical surgical navigation. In addition, the workshop acted to provide clarity to the research community on data collection and trial design. Reported here are the specific discussion items and recommendations from this critical and timely meeting. Cancer Res; 77(9); 2197–206. ©2017 AACR.

BACKGROUND Glioblastoma (GBM) is the most lethal primary brain cancer (median survival: 15-17 months, 5-year survival: 5.6%). Standard interventions consist of aggressive surgical resection, radiotherapy, and chemotherapy; however, GBM is heterogeneous and present therapies are ineffective. Tumor treating fields (TTFields) is a form of alternating electric field therapy that has been shown to prolong survival in patients with newly-diagnosed GBM when combined with standard chemotherapy. The mechanism of TTFields' potentiation of standard chemotherapy against GBM is not well understood. We hypothesized that TTFields increases access of chemotherapy to cancer cells by disrupting the cell membrane. METHODS Human and murine GBM cells (GBM2, GBM39, U87-MG, KR158B) were isolated from primary gliomas. Cells were engineered to stably express firefly or renilla luciferase (fLuc or rLuc, respectively). Cells were either exposed to TTFields (50-500 kHz, 1-4 V/cm) or control conditions. Proliferation was assessed by bioluminescence imaging (BLI) and cell counting. Dextran-FITC binding and influx of 5-aminolevulinic acid (5-ALA) were also assessed. Scanning electron microscopy (SEM) studies were used to probe effects on cellular membranes. All experiments were performed in at least triplicate and 2-way ANOVA or univariate Mann-Whitney test was performed to compare the groups. RESULTS TTFields significantly inhibited growth of cells (p≤0.02, no TTFields vs. TTFields). BLI suggested alterations in membrane configuration when cancer cells were exposed to TTFields. This was validated with observations of greater fluorescence of membrane-associating Dextran-FITC to U87-MG cells that were subjected to TTFields (p< 0.01, no TTFields vs. TTFields). In GBM39 cells, the optimal effect by TTFields on enhancing Dextran-FITC binding occurred in the range of 100-300 kHz (p CONCLUSION The findings suggest a permeabilization of the GBM cell membrane upon exposure to TTFields. This may explain the previously observed synergy between TTFields and standard (e.g., temozolomide) and emerging (Withaferin A) therapies, whereby increased permeability on membranes confers greater accessibility to drugs. Such a strategy is thus a promising candidate for future clinical translation in glioblastoma. Citation Format: Edwin Chang, Chirag Patel, Caroline J. Young, Thomas Anthony Flores, Lydia-Marie Joubert, Yitian Zeng, Robert Sinclair, Sanjiv Gambhir. Combining the glioblastoma cell membrane-permeabilizing effect of tumor treating fields with chemotherapy [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 6258.

The cell's endogenous antioxidant system is vital to maintenance of redox homeostasis. Despite its central role in normal and pathophysiology, no noninvasive tools exist to measure this system in patients. The cystine/glutamate antiporter system x

INTRODUCTION: Glioblastoma multiforme (GBM) is the most lethal primary brain cancer (median survival: 15-17 months, 5-year survival: 5%). Standard interventions consist of aggressive surgical resection, radiotherapy, and chemotherapy; however, GBM is heterogeneous and present therapies are often ineffective. Novel approaches need consideration. We investigated two such novel approaches: Withaferin A and Tumor Treating Fields (TTFields). Withaferin A is a steroidal lactone originating from extracts of the winter cherry plant (Withania somnifera). Extracts such as AshwaMAX can contain up to 4.3% (w/w) Withaferin A. Recently, the U.S. Food and Drug Administration approved another approach to treating glioblastoma: tumor treating fields (TTFields). TTFields involves the delivery of alternating electric fields to the tumor but mechanisms of action are not fully understood. METHODS: To investigate these two therapies, we used human glioblastoma cells (GBM2, GBM39, and U87-MG) isolated from primary tumors. Cells were modified to express firefly luciferase. Proliferation was monitored by: bioluminescent imaging, cell counting via hemocytometer, or cell titer blue viability assay. TTFields were imposed on cell cultures by the inovitroTM device from Novocure Ltd. RESULTS: Both AshwaMAX and Withaferin A inhibited growth of GBM cell lines (IC50 ~0.25 µM). In exploring Withaferin A and TTFields interventions in GBM cell culture, we discovered that the steroid worked synergistically with TTFields (p CONCLUSIONS: In summary, increased membrane permeability by TTFields helps to explain previous reports of additive vs. synergistic effects between TTFields and chemotherapies, including Withaferin A. These findings have implications for the design of combination therapies in glioblastoma and other cancers and may significantly alter standard of care strategies. Citation Format: Edwin Chang, Chirag Patel, Christoph Pohling, Caroline Young, Jonathan Song, Thomas A. Flores, Yitian Zeng, Lydia Marie Joubert, Hamed Arami, Arutselvan Natarajan, Robert Sinclair, Sanjiv S. Gambhir. Tumor treating fields increases membrane permeability in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 250.

Introduction: Pyruvate kinase M2 (PKM2) is an enzyme that catalyzes the final step in glycolysis, a key step in tumor metabolism and growth, making PKM2 an important marker of cancer glycolytic reprogramming. The 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) radiotracer has been developed to measure the expression of PKM2. Glioblastoma (GBM) is traditionally treated with surgical resection, temozolomide (TMZ) chemotherapy, and radiation therapy. Tumor treating fields (TTFields), the application of alternating electric fields (100-500 kHz, 1-4 V/cm) to tumors, is emerging as the fourth therapeutic modality in GBM. There is an important need to assess early on whether a patient’s GBM is responding to a given standard-of-care or experimental therapy. In this study we evaluated the ability of [18F]DASA-23 to detect changes in metabolism in response to standard-of-care (TMZ) and emerging (TTFields) therapies, in cell culture and an orthotopic murine model of human GBM. Methods: Human U87-MG GBM cells were subjected to either 200 kHz TTFields or the IC50 of TMZ for three or six days (n≥3/condition), followed by evaluation of 30-minute cellular uptake of [18F]DASA-23. In parallel experiments, immunofluorescence for PKM2 was performed to confirm the [18F]DASA-23 uptake results. Finally, U87-MG human GBM cells were orthotopically implanted in nude mice (N=5), and evaluated with 7T small animal magnetic resonance imaging (MRI) and [18F]DASA-23 microPET/CT before and one week post-initiation of vehicle or TMZ therapy. Results: There was a significant interaction between the treatment (vehicle, TMZ, or TTFields) and treatment duration (3 or 6 days) on PKM2 expression as measured by cellular uptake of [18F]DASA-23 (p=0.005, 2-way ANOVA). Immunofluorescence for PKM2 in TTFields-exposed and unexposed U87-MG cells revealed reduced cell count and less intense PKM2 staining due to TTFields. In the orthotopic mouse model, the tumor/normal brain (T/N) [18F]DASA-23 uptake ratio (unitless) at one week post-treatment was divided by the pre-treatment T/N ratio. The resulting ratio of ratios was significantly smaller in the TMZ cohort (1.0±0.4) compared to in the vehicle cohort (2.2±0.2), p=0.004. Conclusion: Together, these data suggest the potential for non-invasive assessment of GBM’s response to various therapies using [18F]DASA-23, a radiotracer that measures PKM2 expression. Clinical studies are currently being completed to evaluate the diagnostic ability of [18F]DASA-23 in patients with intracranial malignancies and future studies will evaluate the ability of [18F]DASA-23 to predict responders vs. non-responders to therapy in recurrent GBM. Citation Format: Chirag B. Patel, Corinne Beinat, Yuanyang Xie, Edwin Chang, Sanjiv S. Gambhir. Molecular imaging of pyruvate kinase M2 (PKM2) with [18F]DASA-23 detects temozolomide- and tumor treating fields (TTFields)-induced changes in glycolysis in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5271.

While there is an increasing role of ultrasound for breast cancer screening in patients with dense breast, conventional anatomical ultrasound lacks sensitivity and specificity for early breast cancer detection. In this study, we assessed the potential of ultrasound molecular imaging using clinically translatable vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted microbubbles (MBVEGFR2) to improve the diagnostic accuracy of ultrasound in earlier detection of breast cancer and ductal carcinoma in situ (DCIS) in a transgenic mouse model [FVB/N-Tg(MMTV-PyMT)634Mul]. In vivo binding specificity studies (n = 26 tumors) showed that ultrasound imaging signal was significantly higher (P < 0.001) using MBVEGFR2 than nontargeted microbubbles and imaging signal significantly decreased (P < 0.001) by blocking antibodies. Ultrasound molecular imaging signal significantly increased (P < 0.001) when breast tissue (n = 315 glands) progressed from normal [1.65 ± 0.17 arbitrary units (a.u.)] to hyperplasia (4.21 ± 1.16), DCIS (15.95 ± 1.31), and invasive cancer (78.1 ± 6.31) and highly correlated with ex vivo VEGFR2 expression [R2 = 0.84; 95% confidence interval (CI), 0.72–0.91; P < 0.001]. At an imaging signal threshold of 4.6 a.u., ultrasound molecular imaging differentiated benign from malignant entities with a sensitivity of 84% (95% CI, 78–88) and specificity of 89% (95% CI, 81–94). In a prospective screening trail (n = 63 glands), diagnostic performance of detecting DCIS and breast cancer was assessed and two independent readers correctly diagnosed malignant disease in more than 95% of cases and highly agreed between each other [intraclass correlation coefficient (ICC) = 0.98; 95% CI, 97–99]. These results suggest that VEGFR2-targeted ultrasound molecular imaging allows highly accurate detection of DCIS and breast cancer in transgenic mice and may be a promising approach for early breast cancer detection in women. Cancer Res; 73(6); 1689–98. ©2012 AACR.

MYC is a potential target for many cancers but is not amenable to existing pharmacologic approaches. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) by statins has shown potential efficacy against a number of cancers. Here, we show that inhibition of HMG-CoA reductase by atorvastatin (AT) blocks both MYC phosphorylation and activation, suppressing tumor initiation and growth in vivo in a transgenic model of MYC-induced hepatocellular carcinoma (HCC) as well as in human HCC-derived cell lines. To confirm specificity, we show that the antitumor effects of AT are blocked by cotreatment with the HMG-CoA reductase product mevalonate. Moreover, by using a novel molecular imaging sensor, we confirm that inhibition of HMG-CoA reductase blocks MYC phosphorylation in vivo. Importantly, the introduction of phosphorylation mutants of MYC at Ser62 or Thr58 into tumors blocks their sensitivity to inhibition of HMG-CoA reductase. Finally, we show that inhibition of HMG-CoA reductase suppresses MYC phosphorylation through Rac GTPase. Therefore, HMG-CoA reductase is a critical regulator of MYC phosphorylation, activation, and tumorigenic properties. The inhibition of HMG-CoA reductase may be a useful target for the treatment of MYC-associated HCC as well as other tumors. Cancer Res; 71(6); 2286–97. ©2011 AACR.

Adoptive immunotherapy is evolving to assume an increasing role in treating cancer. Most imaging studies in adoptive immunotherapy to date have focused primarily on locating tumor-specific T cells rather than understanding their effector functions. In this study, we report the development of a noninvasive imaging strategy to monitor T-cell activation in living subjects by linking a reporter gene to the Granzyme B promoter (pGB), whose transcriptional activity is known to increase during T-cell activation. Because pGB is relatively weak and does not lead to sufficient reporter gene expression for noninvasive imaging, we specifically employed 2 signal amplification strategies, namely the Two Step Transcription Amplification (TSTA) strategy and the cytomegalovirus enhancer (CMVe) strategy, to maximize firefly luciferase reporter gene expression. Although both amplification strategies were capable of increasing pGB activity in activated primary murine splenocytes, only the level of bioluminescence activity achieved with the CMVe strategy was adequate for noninvasive imaging in mice. Using T cells transduced with a reporter vector containing the hybrid pGB–CMVe promoter, we were able to optically image T-cell effector function longitudinally in response to tumor antigens in living mice. This methodology has the potential to accelerate the study of adoptive immunotherapy in preclinical cancer models. Cancer Res; 70(24); 10141–9. ©2010 AACR.

Due to the high mortality of lung cancer, there is a critical need to develop diagnostic procedures enabling early detection of the disease while at a curable stage. Targeted molecular imaging builds on the positive attributes of positron emission tomography/computed tomography (PET/CT) to allow for a noninvasive detection and characterization of smaller lung nodules, thus increasing the chances of positive treatment outcome. In this study, we investigate the ability to characterize lung tumors that spontaneously arise in a transgenic mouse model. The tumors are first identified with small animal CT followed by characterization with the use of small animal PET with a novel 64Cu–1,4,7,10-tetra-azacylododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)–knottin peptide that targets integrins upregulated during angiogenesis on the tumor associated neovasculature. The imaging results obtained with the knottin peptide are compared with standard 18F-fluorodeoxyglucose (FDG) PET small animal imaging. Lung nodules as small as 3 mm in diameter were successfully identified in the transgenic mice by small animal CT, and both 64Cu-DOTA-knottin 2.5F and FDG were able to differentiate lung nodules from the surrounding tissues. Uptake and retention of the 64Cu-DOTA-knottin 2.5F tracer in the lung tumors combined with a low background in the thorax resulted in a statistically higher tumor to background (normal lung) ratio compared with FDG (6.01 ± 0.61 versus 4.36 ± 0.68; P < 0.05). Ex vivo biodistribution showed 64Cu-DOTA-knottin 2.5F to have a fast renal clearance combined with low nonspecific accumulation in the thorax. Collectively, these results show 64Cu-DOTA-knottin 2.5F to be a promising candidate for clinical translation for earlier detection and improved characterization of lung cancer. Cancer Res; 70(22); 9022–30. ©2010 AACR.

Clinical success of cancer immunotherapies has renewed interest in imaging the behavior of immune cells. Due to the spatiotemporally varying signatures of immune response, it has been difficult to monitor and predict patient outcomes using traditional clinical tests. ImmunoPET, defined herein as positron emission tomography utilizing radiolabeled antibodies, has the potential to enable noninvasive, sensitive and longitudinal interrogation of immune cell subset and state. Cell states including activation, anergy, and exhaustion may be more prognostic of disease outcome than the presence of tumor-infiltrating immune cells alone. In particular, T cell activation is thought to be critical to treatment success across many classes of cancer immunotherapy. In this work, we present the first radionuclide imaging of OX40, a novel and specific biomarker of activated antigen-specific T cells. Activation dependent and T cell restricted expression of OX40 was validated in vitro via flow cytometric analysis. Cell uptake studies with radiolabeled 64Cu-DOTA-AbOX40 demonstrated ~11 fold [p < .0001] higher uptake in dyna-bead activated T cells compared to resting. The tracer showed negligible nonspecific uptake in OX40 blocked or OX40-/- T cells and low background levels across a panel of 5 cancer cell lines tested. In vivo, ImmunoPET imaging revealed new insights into response following in situ tumor vaccination with CpG, an adjuvant immunotherapy currently in clinical trials. Balb-C mice bearing dual A20 lymphoma tumors were administered low dose CPG directly in the left tumor (n=7-10), while vehicle control mice received PBS (n=7-10). Early after vaccination, imaging revealed increased OX40 radiotracer uptake in the CPG treated tumor (TT) [~37%; p Citation Format: Aaron T. Mayer, Israt S. Alam, Idit Sagiv-Barfi, Kezheng Wang, Ophir Vermesh, Debra K. Czerwinski, Emily M. Johnson, Michelle L. James, Ronald Levy, Sanjiv S. Gambhir. Positron emission tomography imaging of activated T cells by targeting OX40 reveals spatiotemporal immune dynamics and predicts response to in situ tumor vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3031.

Glioblastoma (GBM) has a dismal prognosis due to therapy resistance of GBM initiating cells (GICs). GICs are preferentially found in the perivascular niche and novel therapeutic strategies are needed that target GICs and the vascular niche. The efficacy of current GBM therapies is hindered by their limited trans-endothelial permeability to the GIC perivascular niche and associated systemic toxicities. Here, we propose a new treatment option with tumor enzyme-activatable, combined therapeutic and diagnostic (theranostic) nanoparticles, which caused specific toxicity against GBM tumor cells and GICs. The theranostic cross-linked iron oxide nanoparticles (CLIO) were conjugated to a highly potent vascular disrupting agent (ICT) and secured with a matrix-metalloproteinase (MMP-14) cleavable protein cap. The purpose of this study was to evaluate tumor retention of CLIO-ICT through MR imaging and demonstrate its anti-GBM affects in vivo settings. Methods: GBM-bearing were treated i.v. with CLIO-ICT (80mg/kg of ICT), ICT (80mg/kg of ICT), CLIO (0.5 mmol Fe/kg) or PBS twice a week for 14 days. For combination treatment with temozolomide (33mg/kg), mice were treated with ICT and CLIO-ICT twice a week for 21 days. MRI studies of GBM-bearing mice were performed on a 7T MR scanner (Bruker-Agilent Technologies-General Electric Healthcare), using T2-weighted imaging sequences. BioLuminescent imaging was performed on an IVIS spectrum scanner (PerkinElmer). TNP delivery across the blood brain barrier (BBB) was confirmed by intra-vital microscopy (IVM) studies with a microscope (IV-100; Olympus, Tokyo, Japan). Flow cytometry and histopathology were performed to evaluate apoptosis in GICs upon CLIO-ICT treatment. Results are presented as mean ± SD unless otherwise presented. Tumor and organ relaxation rates and fluorescence signals were compared using one-way ANOVA. Kaplan-Meier survival curves were compared using the log-rank (Mantel-Cox) test. The level of significance was set at p < 0.05, as compared with the control group. Results: Treatment with CLIO-ICT disrupted tumor vasculature of MMP-14 expressing GBM, induced GIC apoptosis and significantly (p< 0.05) impaired tumor growth compared to PBS-treated animals. In addition, the iron core of CLIO-ICT enabled in vivo drug tracking with MR imaging. T2-weighted MR images before and after intravenous injection of CLIO-ICT in mice showed a signal drop or negative enhancement and significant (p< 0.05) tumor retention of CLIO-ICT in GBM tissue, which was confirmed with Prussian blue histopathological evaluations. Treatment with CLIO-ICT plus temozolomide achieved tumor remission and significantly (p< 0.05) increased survival of human GBM bearing mice compared to temozolomide alone. Conclusion: We present a new, clinically translatable therapeutic strategy with significant impact on survival. Further our approach of combining CLIO-ICT with TMZ offers refinements to current less optimal standards of care in order to achieve GBM remission. Citation Format: suchismita mohanty, Zixin Chen, Kai Li, Goreti Morais, Jessica Klockow, Ketan Yerneni, Laura Pisani, Frederick Chin, Siddhartha Mitra, Samuel Cheshier, Edwin Chang, Sanjiv Gambhir, Jianghong Rao, Paul M. Loadman, Robert A. Falconer, Heike E. Daldrup-Link. A novel theranostic strategy for MMP-14 expressing glioblastomas impacts survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-004.

There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity ( approximately 10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or (64)Cu-1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) to their N termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. NIR fluorescence and microPET imaging both showed that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 hour postinjection for two high-affinity (IC(50), approximately 20 nmol/L) (64)Cu-DOTA-conjugated knottin peptides was 4.47% +/- 1.21% and 4.56% +/- 0.64% injected dose/gram (%ID/g), compared with a low-affinity knottin peptide (IC(50), approximately 0.4 mumol/L; 1.48 +/- 0.53%ID/g) and c(RGDyK) (IC(50), approximately 1 mumol/L; 2.32 +/- 0.55%ID/g), a low-affinity cyclic pentapeptide under clinical development. Furthermore, (64)Cu-DOTA-conjugated knottin peptides generated lower levels of nonspecific liver uptake ( approximately 2%ID/g) compared with c(RGDyK) ( approximately 4%ID/g) 1 hour postinjection. MicroPET imaging results were confirmed by in vivo biodistribution studies. (64)Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers.

We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-CH3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 ± 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 ± 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 ± 4.0% ID/g). A minibody derived from a second anti-p185HER2 antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 ± 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 ± 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-CH2-CH3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 ± 2.4% ID/g) and reduced kidney uptake (13.1 ± 1.5% ID/g). Thus, by manipulating the size and format of anti-p185HER2 antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.

Background: Liquid biopsies have long promised to enable earlier cancer diagnosis and tailored therapy. However, circulating tumor cells (CTCs) are extremely rare (1-10 cells per mL blood), limiting their clinical utility. There are too few CTCs in a standard 5-10 mL blood sample for culture and drug susceptibility testing, or for comprehensively profiling a molecularly heterogeneous cancer and its metastases for drug resistance mutations. To achieve large-scale CTC enrichment, new strategies are needed that can rapidly and effectively interrogate large blood volumes. Aim: To design a flexible magnetic wire capable of high-throughput intravascular enrichment and retrieval of rare biomarkers, including CTCs, from the entire circulating blood volume to attain a much higher biomarker yield for earlier cancer detection and personalized treatment. Methods: We present a promising platform for in vivo enrichment of rare biomarkers, the Magnetic Wire for Intravascular Retrieval and Enrichment (MagWIRE): a flexible, self-contained magnetic wire consisting of a string of small magnets with alternating polarities, achieving high local field gradients along its entire length to capture magnetically labeled targets from a large surrounding volume. The platform is proposed to work as follows: Blood biomarkers are immunomagnetically labeled by injecting a patient with antibody-coated magnetic particles (MPs), similar to FDA-approved Feraheme®. The MagWIRE is then inserted through a standard IV catheter into a superficial vein in the arm or through an existing chemotherapy port to magnetically capture passing MP-bound biomarkers. Within an hour, ~5 liters has circulated through a 2-3-mm-diameter human vein, allowing most of the patient’s blood volume to be sampled. The magnets can then be displaced from the MagWIRE sheath to elute the bound targets into buffer for downstream analysis. We performed proof-of-concept demonstrations in: 1) a closed-loop blood circulation system consisting of a pump, tubing, and a blood reservoir, and 2) in vivo within a porcine ear vein model. To model CTC capture, we targeted H1650 lung cancer cells with 1-μm superparamagnetic iron oxide particles coated with antibodies against epithelial cell adhesion molecule (EpCAM), a commonly used CTC marker. We couple the MagWIRE with a unique approach for rapid ( Results: Considerable gains are achievable by sampling from large volumes, even at relatively low capture efficiencies. In our closed-loop setup, the MagWIRE demonstrated capture efficiencies in whole blood of 56.14+/-15.80% for pre-labeled cells and 10.17+/-5.41% for cells labeled in flow. In a porcine ear model, we captured cells with efficiencies up to 8%, corresponding to 80-fold enrichment when integrated over a 5-liter blood volume compared with a 5 mL blood draw. Citation Format: Ophir Vermesh, Amin Aalipour, Tianjia J. Ge, Yamil Saenz, Yue Guo, Seung-min Park, Yoshiaki Mitsutake, Michael Bachmann, Chin Chun Ooi, Kerstin Mueller, Hamed Arami, Alfredo Green, Shan X. Wang, Sanjiv S. Gambhir. An intravascular magnetic wire for high-throughput in vivo enrichment of rare circulating cancer biomarkers [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 3796. doi:10.1158/1538-7445.AM2017-3796

Histone deacetylases (HDAC) that regulate gene expression are being explored as cancer therapeutic targets. In this study, we focused on HDAC6 based on its ability to inhibit cancerous Hsp90 chaperone activities by disrupting Hsp90/p23 interactions. To identify novel HDAC6 inhibitors, we used a dual-luciferase reporter system in cell culture and living mice by bioluminescence imaging (BLI). On the basis of existing knowledge, a library of hydrazone compounds was generated for screening by coupling cinnamic hydroxamates with aldehydes and ketones. Potency and selectivity were determined by in vitro HDAC profiling assays, with further evaluation to inhibit Hsp90(α/β)/p23 interactions by BLI. In this manner, we identified compound 1A12 as a dose-dependent inhibitor of Hsp90(α/β)/p23 interactions, UKE-1 myeloid cell proliferation, p21waf1 upregulation, and acetylated histone H3 levels. 1A12 was efficacious in tumor xenografts expressing Hsp90(α)/p23 reporters relative to carrier control–treated mice as determined by BLI. Small animal 18F-FDG PET/CT imaging on the same cohort showed that 1A12 also inhibited glucose metabolism relative to control subjects. Ex vivo analyses of tumor lysates showed that 1A12 administration upregulated acetylated-H3 by approximately 3.5-fold. Taken together, our results describe the discovery and initial preclinical validation of a novel selective HDAC inhibitor. Cancer Res; 74(24); 7475–86. ©2014 AACR.

Photoacoustic imaging (PAI) has the potential for real-time molecular imaging at high resolution and deep inside the tissue, using nonionizing radiation and not necessarily depending on exogenous imaging agents, making this technique very promising for a range of clinical applications. The fact that PAI systems can be made portable and compatible with existing imaging technologies favors clinical translation even more. The breadth of clinical applications in which photoacoustics could play a valuable role include: noninvasive imaging of the breast, sentinel lymph nodes, skin, thyroid, eye, prostate (transrectal), and ovaries (transvaginal); minimally invasive endoscopic imaging of gastrointestinal tract, bladder, and circulating tumor cells (in vivo flow cytometry); and intraoperative imaging for assessment of tumor margins and (lymph node) metastases. In this review, we describe the basics of PAI and its recent advances in biomedical research, followed by a discussion of strategies for clinical translation of the technique. Cancer Res; 74(4); 979–1004. ©2014 AACR.

Non-Hodgkin lymphoma (NHL) is a heterogeneous and highly disseminated disease, but the mechanisms of its growth and dissemination are not well understood. Using a mouse model of this disease, we used multimodal imaging, including intravital microscopy (IVM) combined with bioluminescence, as a powerful tool to better elucidate NHL progression. We injected enhanced green fluorescent protein and luciferase-expressing Eμ-Myc/Arf−/− (Cdkn2a−/−) mouse lymphoma cells (EL-Arf−/−) into C57BL/6NCrl mice intravenously. Long-term observation inside a peripheral lymph node was enabled by a novel lymph node internal window chamber technique that allows chronic, sequential lymph node imaging under in vivo physiologic conditions. Interestingly, during early stages of tumor progression we found that few if any lymphoma cells homed initially to the inguinal lymph node (ILN), despite clear evidence of lymphoma cells in the bone marrow and spleen. Unexpectedly, we detected a reproducible efflux of lymphoma cells from spleen and bone marrow, concomitant with a massive and synchronous influx of lymphoma cells into the ILN, several days after injection. We confirmed a coordinated efflux/influx of tumor cells by injecting EL-Arf−/− lymphoma cells directly into the spleen and observing a burst of lymphoma cells, validating that the burst originated in organs remote from the lymph nodes. Our findings argue that in NHL an efflux of tumor cells from one disease site to another, distant site in which they become established occurs in discrete bursts. Cancer Res; 72(23); 6111–8. ©2012 AACR.

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, d-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase (fl)] both in cell culture and in living mice revealed that mtfl possessed 6- to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes (jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 ± 0.1 versus 1.9 ± 0.1) × (106 p/s/cm2/sr)] but similar level of fluorine-18–labeled 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 ± 0.15 versus 1.37 ± 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk–expressing tumors compared with the fl-mrfp1-wttk–expressing tumors. Both tumors showed 4- to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18–labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques. [Cancer Res 2007;67(7):3085–93]

Background: Circulating tumor cells (CTCs), defined as epithelial cells shed from a primary tumor into the bloodstream, are valuable prognostic, and possibly diagnostic, biomarkers that contain actionable genetic information for cancer treatment. Unfortunately, the rarity of CTCs in comparison to other blood components necessitates high-throughput separation technologies for efficient enrichment and practical downstream analysis. Moreover, genetic data extraction from CTCs currently suffers from a dearth of reliable analytical methods capable of handling low cell numbers. Technological innovations are urgently required to developing platforms that can help to optimize cancer management. Aim: To measure gene expression profiles of individual CTCs for cancer management via an integrated nanoscale platform. Methods: We have developed a protocol to effectively enrich rare cells via a magnetic sifting technology, whose methodology is based on using magnetic nanoparticles to tag CTCs in conjunction with magnetic filtration to enable high-throughput enrichment with release capability. This magnetic sifter offers 1) high capture efficiency at fast flow rates due to extreme field gradients at the pore edges, 2) high throughput due to the density of pores (∼200 pores/mm2), 3) scalability via standard lithographic fabrication, and 4) harvesting of viable cells. For subsequent characterization, a robust nanowell-based assay was designed to circumvent experimental errors associated with ensemble measurements through detection of mRNA transcripts directly from single CTCs (using one-step RT-PCR). Using standard photolithography, 25,600 nanowells are positioned on top of polydimethyl-siloxane (PDMS) and designed for subsequent RT-PCR reaction from a single CTC. These massive single-cell arrays are able to isolate up to thousands of individual NSCLC cells to measure gene expression. Also, this device is easily interrogated by conventional fluorescence microscopy to detect a candidate panel of genes on CTCs that are relevant for cancer detection or therapy monitoring. Nanowell is innovative as a low-cost (PDMS-based), easily scalable (from currently 25k to more than 100k nanowells), adjunctive solution to existing diagnostic methods. Results and Discussion: To date, we have assayed 23 NSCLC patients using the MagSifter & Nanowell and detected CTCs with valid biomarkers (hTERT and cMET). Each 4-mL whole blood sample was processed within one working day using the current workflow. From these samples, individual CTCs were assessed for hTERT and cMet expression. Single CTCs displaying hTERT only, cMet only, and both were evident upon fluorescent imaging. Direct comparison with CTC enumeration confirms better sensitivity by Nanowell assay, since the Nanowell utilizes PCR amplification in fluorescence as a signal generator. We believe this is the first demonstration of ex vivo visualization of gene expression from individual lung cancer CTCs. Citation Format: Seung-min Park, Dawson J. Wong, Chin Chun Ooi, Viswam S. Nair, Ophir Vermesh, Sang Hun Lee, Susie Suh, Luke P. Lee, Shan X. Wang, Sanjiv S. Gambhir. Gene expression profiling of individual circulating tumor cells from non-small cell lung cancer (NSCLC) patients via integrated nanotechnologies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-280. doi:10.1158/1538-7445.AM2015-LB-280

Mitogen-activated protein kinases (MAPK) play important roles in malignancy. The ability to detect and quantitate MAPKs in live animal models of cancer will facilitate an understanding of disease progression. We have developed a gene expression-based imaging system that detects and quantifies MAPK activity in prostate cancer tumors implanted into severe combined immunodeficient mice. The imaging technology uses a modified version of two-step transcriptional amplification (TSTA). The tissue specificity of gene expression is imparted by an enhanced version of the prostate-specific antigen regulatory region that expresses GAL4-ELK1. GAL4-ELK1 confers MAPK specificity by activating a firefly luciferase (FLuc) reporter gene when the Ets-like transcription factor (ELK) 1 activation domain is phosphorylated by MAPK. FLuc activity in live animals was detected using the Xenogen In vivo Imaging System. We validated the TSTA-ELK1 system by analyzing its response to epidermal growth factor treatment in transfected tissue culture cells and in adenovirus (AdTSTA-ELK1)–injected prostate cancer xenograft tumors. We measured MAPK activity in two well-characterized xenograft models, CWR22 and LAPC9. Although no significant differences in MAPK levels were detected between androgen-dependent and androgen-independent xenografts, the CWR22 models display significantly higher levels of AdTSTA-ELK1 activity versus LAPC9. Western blots of tumor extracts showed that the elevated imaging signal in CWR22 xenografts correlated with elevated levels of phosphorylated extracellular signal-regulated kinase 1/2 but not p38 or c-Jun NH2-terminal kinase. We conclude that a gene expression-based optical imaging system can accurately detect and quantify MAPK activity in live animals. (Cancer Res 2006; 66(22): 10778-85)

The Ras-Raf-MAPK pathway is constitutively activated in the majority of melanomas because of a mutation in the BRAF gene. It has been hypothesized that activation of this pathway is crucial for the genesis and maintenance of melanoma and therefore represents an attractive clinical target for metastatic disease. We synthesized a previously characterized MAP kinase kinase inhibitor to test the effect that blocking the Ras-Raf-MAPK pathway would have on the establishment and maintenance of melanoma metastases. Oral administration of CI 1040 inhibited formation of pulmonary metastases and caused rapid regression of established pulmonary metastases in the mouse. Our findings indicate that Ras-Raf-MAPK activation provides crucial signals for the survival of melanoma cells at ectopic sites and that the pharmacological inhibition of this pathway is a promising target for melanoma therapy.

A metastatic renal cell carcinoma (RCC) tumor model xenograft that expresses the targetable, membrane-bound tumor-associated antigen carbonic anhydrase type 9 (CA IX) is described. The xenograft, established from a high-grade type-2 chromophil RCC (cRCC), has been serially transplanted in immune compromised mice, in which it grows orthotopically under the renal capsule, doubling its size every 9 weeks and sending metastases to the lung and liver at approximately 20 weeks. Tumors were capable of being imaged using a micro-PET (micro-positron emission tomograph) with an 18-fluorodeoxyglucose (18-FDG) tracer. Subsequent xenograft generations have conserved immunohistochemical and ultrastructural properties typical for malignant renal epithelium-derived neoplasia (vimentin+, CK-19+, CA IX+ with hypoxia-inducible factor (HIF)-1 alpha constitutive expression) and have demonstrated extensive proliferation, lack of apoptosis, severe genetic alterations, and molecular expression alterations; transforming growth factor beta 1 (TGF-beta 1), hepatocyte growth factor (HGF), proto-oncogene (c-met), matrix metalloproteinase (MMP)-1, and vascular endothelial growth factor (VEGF) C and D were overexpressed, whereas human epidermal growth factor receptor (HER)-2, MMP-2 and MMP-9, VEGF-R3, p53, and p27 were severely down-regulated, suggesting a proangiogenic environment, local invasiveness, and facilitated lymphatic metastasis. Altogether, LABAZ1 provides a relevant and flexible model to study the biology of cRCC, the role of CA IX in RCC tumorigenesis, progression, and metastasis, and a platform for testing new targeted therapeutic strategies.

The early androgen-dependent (AD) phase of prostate cancer is dependent on the androgen receptor (AR). However, it is unclear whether AR is fully functional in recurrent prostate cancer after androgen withdrawal. To address this issue we interrogated AR signaling in AD and recurrent prostate cancer xenografts using molecular imaging, chromatin immunoprecipitation, and immunohistochemistry. In the imaging experiments, an adenovirus bearing a two-step transcriptional activation cassette, which amplifies AR-dependent firefly luciferase reporter gene activity, was injected into tumors implanted into severe combined immunodeficiency mice. A charge-coupled device optical imaging system detected the initial loss and then resumption of AR transcriptional activity in D-luciferin-injected mice as tumors transitioned from AD to recurrent growth. The results of chromatin immunoprecipitation and immunohistochemical localization experiments correlated with the Ad two-step transcriptional activation imaging signal. AR localized to the nucleus and bound to the endogenous prostate-specific antigen enhancer in AD tumors but exited the nucleus and dissociated from the enhancer upon castration. However, AR reentered the nucleus and rebound the prostate-specific antigen enhancer as the cancer transitioned into the recurrent phase. Surprisingly, RNA polymerase II and the general factor TFIIB remained bound to the gene throughout the transition. Our data support the concept that AR is fully functional in recurrent cancer and suggest a model by which a poised but largely inactive transcription complex facilitates reactivation by AR at castrate levels of ligand.

Noninvasive imaging of reporter gene expression using various imaging modalitiesis playing an increasingly important role in defining molecular events in the field of cancer biology, cell biology, and gene therapy. In this study, a novel reporter vector was constructed encoding a fusion protein comprised of a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) (tk), a positron emission tomography (PET) reporter gene, and renilla luciferase (rl), a bioluminescence optical reporter gene joined by a 20 amino acid long spacer sequence. We validated the activity of the two enzymes encoded by the fusion protein (tk(20)rl) in cell culture. Then, tumors stably expressing the tk(20)rl fusion gene were imaged both by microPET and optically using a cooled charge coupled device camera in xenograft-bearing living mice. Using a single fusion reporter (PET/optical) gene should accelerate the validation of reporter gene approaches developed in cell culture for translation into preclinical and clinical models.

Introduction: The detection of proteins or other biomarkers secreted or released (shed) from tumor cells into blood may facilitate early cancer screening, particularly for lethal cancers such as ovarian carcinoma, which typically remains asymptomatic and undetected until advanced stages. But the utility of blood-based assays is confounded because it is unknown whether cancer biomarker levels correlate with viable tumor burden, and it is unclear how highly shed a biomarker must be to enable detection of early (< 1 cm diameter) tumors. No clinical or animal data is available to correlate blood biomarker levels with cancer state, before and after disease diagnosis, for multiple time points in the same subject, without intervention or treatment. Aim: To determine whether cancer blood biomarkers may be used as measures of tumor viability in an early-stage ovarian cancer mouse model. Methods: We genetically engineered human ovarian cancer cell line 2008 to stably coexpress a secretory reporter (secreted alkaline phosphatase, SEAP) not produced endogenously in any living animal, and a bifusion optical imaging reporter (firefly luciferase-eGFP, FL-eGFP) to enable in vivo bioluminescence imaging of tumor viability. We orthotopically implanted as few as 3×103 2008-SEAP-FL-eGFP cells into the ovary of 6-week-old female Nu/Nu mice, and monitored subsequent tumor growth for up to 28 days using serial 25-μl plasma SEAP sampling to assess biomarker shedding, in vivo bioluminescence imaging to quantify tumor viability, and in vivo ultrasound imaging to assess tumor volume. Plasma SEAP concentration was measured using a chemiluminescence assay (Clontech). Mice were sacrificed when tumor volume reached 1-cm diameter (or by Day 28) and tumors were excised for histological analysis. Results and Discussion: This is the first study to show that cancer plasma biomarker measurements may be used as surrogates of tumor viability during the early tumor growth stages in a living animal model. Orthotopic implantation of at least 3×104 transfected cells in the ovary resulted in tumor growth (confirmed by ultrasound imaging) that was detectable by plasma SEAP levels and FL imaging within 4 hours and up to 28 days. Clinical translation of this preclinical work was aided by the use of mathematical modeling, which yielded novel estimates of biomarker shedding rates never before measured in vivo (2×10-5 ng/day/cell) and were consistent with predicted rates from cell culture. Plasma SEAP levels correlated well with increasing FL average radiance during the first 15 days post-implantation (R2=0.86), indicating that a cancer-specific biomarker, when shed highly, may reflect early tumor viability. This integrative mouse model and mathematical modeling approach may be used to further elucidate the biology of cancer biomarker shedding in vivo, thereby helping to define guidelines for selecting better biomarkers for early cancer detection. Citation Format: Sharon S. Hori, Amelie M. Lutz, Ramasamy Paulmurugan, Sanjiv S. Gambhir. Correlation of plasma biomarker levels with early-stage tumor viability in an orthotopic ovarian cancer mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 873. doi:10.1158/1538-7445.AM2014-873

Introduction: There is an urgent clinical need to develop noninvasive biomarkers that early detect tumor responses to anti-angiogenic therapy. Treatment response in ovarian cancer patients is assessed with transvaginal ultrasound imaging of tumor size and CA125 screening. We hypothesized that combining photoacoustic (PA) imaging to non-invasively visualize tumor vascular architecture, with serum diagnostics using endothelial biomarkers, would yield a sensitive and readily translatable approach for monitoring response to anti-angiogenic therapy. Methods: Ovarian tumors were established in nude mice by orthotopic injection of OV2008 cells at day 0. At day 10 mice were randomized into groups of at least n = 10. Baseline PA imaging and submandibular blood draw were performed, then repeated 24h after dosing with a peptibody that prevents the interaction of secreted angiopoietin 1 (Ang1; agonist) and 2 (Ang2; antagonist) with the receptor tyrosine kinase Tie2, or vehicle, on days 13, 16 and 19. Mice were then sacrificed and tumors excised for histology. Results: PA imaging uses endogenous hemoglobin light absorption to generate contrast. At 797nm, we found a 1.5-fold increase in hemoglobin-weighted signal in vehicle mice by day 19, while treated mice remained close to the baseline (n=9; p=0.008). The oxyhemoglobin-weighted PA signal (837nm/797nm) was elevated by 20% in treated mice but decreased 15% in vehicle mice (n=9; p Conclusions: We have shown for the first time that PA imaging and serum angiopoietin levels measure complementary parameters that indicate response to angiopoietin blockade in ovarian cancer. PA imaging noninvasively detected drug-induced vessel regression and normalization, giving a functional readout of response. This modality can be readily combined with transvaginal ultrasound. The drug action was mediated by normalization of mAng1 levels, indicated by serum measures and IF. Importantly, this work supports the combined use of in vitro and in vivo diagnostics to monitor response to anti-angiogenic therapy in clinical trials. Citation Format: Sarah E. Bohndiek, Laura Sasportas, Steven Machtaler, Jesse V. Jokerst, Sharon Hori, Sanjiv S. Gambhir. Molecular photoacoustic imaging and serum diagnostics rapidly detect response to angiopoietin 1 and 2 blockade in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2047. doi:10.1158/1538-7445.AM2014-2047

Blood biomarkers can revolutionize early cancer detection by earlier detection of circulating tumor biomarkers. We have shown that cell membrane permeabilization by low frequency ultrasound can release biomarkers from tumor cells. We use this non-invasive method to increase the detectable blood biomarker level from orthotopic liver tumors in mice, allowing for tumor characterization. The ability to focus ultrasound enables localization of the biomarker-release site. This novel strategy could lead to the earlier characterization of unknown incidental lesions leading to earlier detection and localization of cancer. METHODS: The colon cancer cell line LS174T transfected with the firefly luciferase2 reporter gene was used to produce liver tumors in nude mice. This cell line produces the biomarkers carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA-19-9), which were detected using an enzyme-linked immunosorbant assay. The liver tumors were produced by either injecting 3 million cells in matrigel or transplantation of a piece of previously established subcutaneous tumor into the liver. The tumor growth was followed using bioluminescence and size was measured using ultrasound. The liver was exposed to low frequency (1MHz) ultrasound for 6 minutes, serum was collected and biomarkers measured. The increase in serum biomarker was determined by the difference in pre and post-ultrasound-treatment levels. Control samples were from tumor-bearing mice not ultrasound treated. Cells in culture were treated with the low frequency and biomarker release studied with an increase in time (0, 10, 30 min). RESULTS: Both the methods used to produce the liver tumors showed an 80% rate of tumor establishment within a week of implantation. The other 20% showed tumor growth at a slower rate. The transplantation of the previously established subcutaneous tumor piece showed a better control of the tumor size compared to the cells implanted. The tumors showed an exponential growth in three weeks (week-3 mean volume 0.815 cm3). The ultrasound treated tumors showed a significant increase in blood biomarkers released (CEA: p CONCLUSIONS: Increase in biomarkers released from cells in culture and liver tumors was observed with ultrasound-treatment. This allows for characterization of tumors depending on the amplification of tumor specific blood biomarkers. This orthotopic liver model can be used to study tumor characterization as well as has implications in diagnostic monitoring of lesions and therapy. This novel method has a clear pathway for application of MR-guided focused ultrasound in characterization of incidental tumors, merging the fields of imaging and in vitro diagnostics. Citation Format: Aloma L. D'Souza, Xinrui Yan, Sanjiv S. Gambhir. Novel method of liver tumor detection and characterization using ultrasound-induced biomarker release. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4287. doi:10.1158/1538-7445.AM2014-4287

Purpose: MicroRNAs (miRNAs) are being investigated as stable, non-invasive, circulating biomarkers for detecting human disease, including cancer. We sought to determine whether select, circulating miRNAs can define a “footprint” for circulating tumor cells (CTCs) in the blood of lung cancer patients. Methods: We analyzed plasma from patients with non-small cell lung cancer (NSCLC) for nine candidate miRNAs using Taqman™ qRT-PCR primers in duplicate and normalized by spiked-in synthetic cel-miR-39. We then associated miRNA expression levels with CTCs using a morphometric assay that is independent of Epithelial Cell Adhesion Molecule (EpCAM) and designed for the ultra-sensitive detection of individual CTCs and tumor cell clusters. We chose candidate plasma miRNA markers based on criteria including low or absent expression in other blood components, epithelial-specific expression, and/or significance in the literature. This included miRs -21, -135b, -141, -200a/b/c, -205, -210 and -429. We analyzed Pearson and Spearman rank correlations and associations by group using ANOVA with Bonferonni adjustments for multiple comparisons. Lastly, we integrated patients' clinical and tumors' imaging data for modeling using multiple logistic regression to assess how miRNAs associated with CTCs. Results: We evaluated 68 patients with predominantly stage I disease (75%) and adenocarcinoma histology (69%). Median CTC/mL was 4.2 (Interquartile Range [IQR] 0-19), 49% (33/68) of patients had more than 5 CTCs/mL and 51% (35/68) of these patients also had tumor clusters present. MiRNAs were only weakly correlated with absolute CTC/mL levels in the circulation but at a threshold of 5 CTCs/mL there was a strong relationship with miR-141 (p-value = 0.0004, adj p-value = 0.004). Several miRNAs were associated with the presence of tumor clusters, including miR-205 (p-value = 0.007; adj p-value = 0.06) and miR-429 (p-value = 0.006, adj p-value = 0.05). A logistic model integrating clinical, imaging and miRNA features selected several miRNAs preferentially to predict the presence of tumor clusters (miR-21, miR-200b, miR-205, and miR-429). Conclusion: Circulating oncomiRs may indirectly associate with CTCs and tumor clusters in patients with NSCLC. Our “footprint” is concordant with one previous investigation in metastatic breast cancer showing common results for miR-141 and the miR-200-429 cluster. Further investigations examining miRNAs expressed directly in CTCs would be useful to determine the origin of circulating cancer-associated miRNAs. Citation Format: Viswam S. Nair, Maria Giraldez, Madelyn Luttgen, Khun Visith Keu, Minal Vasanawala, George Horng, Mehran Jamali, Anand Kolatkar, Ware Kuschner, Peter Kuhn, Sanjiv Sam Gambhir, Muneesh Tewari. MicroRNA footprints of circulating tumor cells in patients with non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4006. doi:10.1158/1538-7445.AM2014-4006

Introduction: Reliable, non-invasive diagnostic blood tests to assess high-risk pulmonary nodules are needed to avoid unnecessary invasive procedures and the risk of missing lung cancer. Methods: We used a prospective, observational, case-control study design of consecutively enrolled patients undergoing imaging for a lung nodule or diagnosed malignancy to investigate whether a non-EpCAM based circulating tumor cell (CTC) platform could accurately diagnose stage I non-small cell lung cancer (NSCLC). Phlebotomy was performed according to standardized protocols at all three participating medical centers and the number of CTCs was analyzed in a single blinded fashion on a continuous scale per mL of blood. For determining test characteristics, cases consisted of patients with stage I NSCLC, and controls consisted of advanced NSCLC, malignant non-NSCLC lung nodules, and benign lung nodules. Differences by disease group were compared formally by ANOVA or Chi-square analysis, and test performance for identifying stage I NSCLC compared to benign disease was assessed by sensitivity, specificity and C statistics. Results: Seventy-three of 79 samples were adequate for analysis after processing. The median age was 69 years (interquartile range [IQR] 65-76), 73% were male, 70% were Caucasian, 79% had a smoking history, and 45% had a history of cancer. Diagnoses consisted of 56 NSCLC patients, of which two-thirds were adenocarcinoma and 45 were stage I, 8 malignant non-NSCLC cases, and 9 benign nodules. There were no differences in basic clinical characteristics across diagnosis type other than plasma white blood cell count. Average nodule diameter was 2.2 cm (IQR 1.6-2.8) and this did not vary significantly by diagnosis. Median CTC/ml was 3.3, 3.0, 0.60 and 0.70 for Stage I NSCLC, Stage II-IV NSCLC, other non-NSCLCs, and benign nodules respectively. At a threshold of 1.0 CTC/mL, 2.0 CTC/mL and 5.0 CTC/mL respectively 63%, 54% and 44% of stage I NSCLCs had a detectable CTC burden. For these three thresholds sensitivity (89%, 89%, 100%) and specificity (64%, 53%, 44%) was calculated for discriminating stage I NSCLC from benign disease. The accuracy (C-statistic) for identifying stage I disease only relative to benign disease only improved from 0.84 to 0.91 when adding CTC/mL data to age, gender, smoking and cancer history, nodule size and location, and SUVmax. Discussion: CTCs may be useful for identifying stage I NSCLC from benign lung nodules. Addition of these data to non-invasive clinical and imaging parameters we currently use to risk stratify patients could improve clinical diagnosis for this group. Citation Format: Madelyn S. Luttgen, Khun Visith Keu, Viswam S. Nair, George Horng, Minal Vasanawala, Anand Kolatkar, Anders Carlsson, Mohsen Sabouri, Billy W. Loo, Joseph B. Shrager, Andrei Iagaru, Ware Kuschner, Peter Kuhn, Sanjiv S. Gambhir. Developing a non-invasive, diagnostic test for stage I non-small cell lung cancer using circulating tumor cells. [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 3485. doi:10.1158/1538-7445.AM2013-3485

BACKGROUND: Circulating tumor cells (CTCs) represent disease derived cells identified in the blood of cancer patients with prognostic implications. 18F-2-fluoro-2-deoxyglucose (FDG) PET tumor uptake in non-small cell lung cancer (NSCLC) may be associated with poor prognosis despite therapy since the tracer accumulates in increasing amounts in more aggressive cancers. We therefore set out to investigate whether CTCs correlate with FDG uptake. METHODS: For this multi-center, prospective, observational study patients were consented and underwent phlebotomy into a cell preservative blood collection tube. Samples were shipped at ambient temperature and processed within 36 hours. CTC detection was defined by immunofluorescence (CK, DAPI, CD45) followed by automated morphometric analysis that was validated manually. Patients who underwent surgery or had medical treatment prior to imaging were excluded and only those with biopsy proven NSCLC who had PET images acquired within 4 weeks of CTC sample procurement were eligible. Semi-quantitative values (maximum standardized uptake value [SUVmax]) were extracted from images using a region of interest drawn over the tumor by the interpreting physician. Correlations were calculated by either a Pearson or Spearman-rank test depending on the distribution of the underlying variable. RESULTS: Twenty-six patients with treatment naïve NSCLC underwent phlebotomy for CTCs and had FDG PET imaging done at four medical centers during the pilot phase of this study. Five patients were excluded for further analysis due to pending results or more than 4 weeks from PET imaging to CTC draw. The average age for this cohort was 67 (± 11) years, 13 of 21 patients were male (62%) and 71% were stage I or II NSCLC. Mean tumor size was 3.0 cm (range 1.5-7.5 cm), median SUVmax was 7.4 (IQR 4.4-16.1) and two-thirds of patients had adenocarcinoma. At least one CTC per ml was detected in 81% of patients (17/21), and the median CTC count was 27 per ml (IQR 2.4-59.0) across all samples and histologic types. CTCs appeared to correlate more with tumor size (r=0.42) than with glucose metabolism as measured by SUVmax (r=0.22). CONCLUSION: We have established a multi-center workflow to expeditiously study tumor glucose metabolism using imaging and in-vitro diagnostics. Preliminary data show good sensitivity for a combined immunofluorescent and cytometric CTC detection approach in early-stage disease. CTC count may not correlate well with tumor glucose metabolism in NSCLC but further study is required. 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 2369. doi:1538-7445.AM2012-2369

Pancreatic cancer is a lethal disease largely due to presence of advanced and inoperable disease at the time of diagnosis. Evidence from current screening programs for high-risk individuals suggests that aggressive detection at operable stages could dramatically improve prognosis; however, current radiological imaging screen methods are not standardized and rely on solid mass recognition from anatomical/morphological imaging strategies. Incorporation of a molecular imaging strategy can provide a highly reliable and cancer-specific diagnosis, as well as, improved delineation of tumor boundaries for therapeutic and surgical guidance. Molecular ultrasound imaging is accomplished by conjugating targeting molecules (peptides/antibodies) to the surface of gas-filled microbubble contrast agents (1-4 μm diameter), and these contrast agents bind to markers overexpressed on disease-associated endothelium. In this study, we investigated the ability to detect small pancreatic cancers with contrast microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) and small animal transabdominal ultrasound (US) imaging in a transgenic mouse model of pancreatic cancer (Pdx1-Cre; KRasV12G; Ink4a-/-; PC mice). Small tumors ranging from 0.8 mm to 3.0 mm in diameter (mean: 1.7 ± 0.6 mm) could be detected with high-resolution B-mode US imaging, and showed 7.8-fold higher (P < 0.001) VEGFR2-targeted molecular US signal (mean signal intensity: 6.5 ± 8.4 arbitrary units; n=32 tumors) in PC mice compared with normal pancreas in wildtype mice (mean signal intensity: 0.8 ± 0.6 arbitrary units; n=14 mice). Small pancreatic tumors showed 3-fold higher VEGFR2-targeted US signal (mean signal intensity: 10.5 ± 9.4 arbitrary units) compared with US signal measured with non-targeted microbubbles (mean signal intensity: 3.6 ± 1.8 arbitrary units), indicating high specificity for VEGFR2. Ex vivo histological (H&E) analysis and immunostaining of VEGFR2 confirmed millimeter-sized tumors in the pancreas and VEGFR2 expression in PC mice (VEGFR2-positive tumors); in contrast, pancreas tissue from normal, wildtype mice was VEGFR2-negative. Taken together, these studies indicate that VEGFR2-targeted US imaging can detect small pancreatic cancers that are not normally recognized with conventional anatomical imaging, and may be highly useful for establishing a standardized pancreatic cancer screening/monitoring method for high-risk patients. Supported by the Canary Foundation, and NIH/NCI grants R21 CA139279 and Fellowship R25 CA11868. 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 LB-509. doi:1538-7445.AM2012-LB-509

A novel Raman imaging device that is capable of rotationally scanning hollow structures has been developed to be used in conjunction with tumor targeting Surface enhanced Raman scattering (SERS) nanoparticles. SERS nanoparticles can act as excellent contrast agents in that they offer ultra high sensitivity, and multiplexing capabilities due to the unique spectral signatures associated with them. One key advantage of multiplexing includes the ability to utilize several targeted SERS probes to target and localize a multitude of unique biomarkers associated with a specific disease, and effectively increasing cancer specificity. The device can potentially be used to distinguish a variety of tissues of interest in the colon such as: flat lesions, varying types of adenomas and carcinomas. There are also applications beyond the colon, such as the esophagus, stomach, oral cavity and bladder. In addition, multiplexing can allow for real-time functional imaging. A 45-degree scanning mirror, actuated with micro-motor, is used to perform circumferential scans of the lumen of the colon at a rate of 1 revolution per second and a spatial resolution or approximately 1.5 mm. The device is able to be used with currently available endoscopes by inserting it into the working channel of an endoscope. The use of many (36) multi-mode fibers is used to increase the flexibility of the fiber bundle so that it can be inserted and used in a fully articulating endoscope while still being able to collect a sufficient amount scattered Raman light. When in the circumferential scanning mode, American National Standards Institute (ANSI) regulations will allow us to increase the laser illumination power to about 580 mW at a scan rate of 60rpm, resulting in a detection limit of approximately 1 pM of SERS nanoparticles. One of the key features of our Raman device is that it has been designed for efficient use over a wide range of working distances. This is necessary to accommodate for imperfect centering of the device within the colon, as well as variable working distances due to folds and bends within the colon. These techniques could allow endoscopists to distinguish between normal and cancerous tissues instantly, as well as to identify flat lesions that are easily missed during conventional screening endoscopy. After a suspicious region has been identified, the rotating mirror can be held at a certain angle, and used to help guide biopsies or resection of tissues in real-time. 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 LB-512. doi:1538-7445.AM2012-LB-512

Visualizing adaptations of tumor metabolism by employing positron emission tomography (PET) with a specific imaging probe has the potential to better characterize tumors with higher sensitivity and specificity. The glutamate-cystine exchanger (system xC-, a SLC7A11/SLC3A2 heterodimer) is an important transporter in tumor cells and a promising new target for imaging. This exchanger transports the amino acids L-cystine and L-glutamate, thus providing a rate-limiting precursor for glutathione biosynthesis. This contributes to redox maintenance, which is especially important in tumor cells. Recently, it was reported that a splice variant of the stem cell marker CD44 stabilizes the xCT subunit of system xC- at the cell membrane and promotes its proper function. FSPG (BAY 94-9392) is an 18F-labeled glutamate derivative that provides specific visualization of system xC- activity with PET/CT. Through this multicenter pilot trial, FSPG has successfully been translated into cancer patients and shows favorable pharmacokinetics and an absence of adverse events. A favorable biodistribution pattern previously seen in mice was also confirmed in these patients with different tumor types. A high lesion detection rate (80-100%) was found in lung cancer (n=10), hepatocellular cancer (n=5), head and neck cancer (n=5), and colorectal cancer (n=5) patients who also had positive FDG-PET/CT scans. Importantly, very low background uptake in healthy brain parenchyma enabled the detection of high-grade brain cancers as well as small brain metastases and skull base lesions (n=4) not well seen with FDG-PET. Primary breast tumors (n=4) and a low-grade brain tumor, however, showed minimal uptake of FSPG. Immunohistochemical (IHC) analyses on these subjects’ pathology samples showed significant correlation between FSPG uptake and protein expression of both the xCT subunit and CD44. In breast tumor samples specifically, IHC showed that absence of CD44 results in low signal from FSPG-PET even if xCT is present, indicating the importance of CD44 co-expression for system xC- function. Future clinical studies will further explore the potential of this compound in additional tumor types and expand upon the initial findings in a larger number of subjects. Here we present the first clinical examples demonstrating the ability of the tracer to measure system xC- activity in patients. FSPG-PET/CT may provide improved characterization and sensitivity of tumor detection in patients and further improve the understanding of the role of system xC- in cancer biology. It could be a valuable tool for improved tumor classification in patients, linking uptake of the tracer with malignancy and cancer stem cell properties of tumors. Furthermore, the ability to noninvasively probe this important pathway in humans may allow for more rational treatment planning, patient stratification and better development of new drugs. 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 LB-227. doi:1538-7445.AM2012-LB-227

Earlier cancer diagnosis and monitoring of therapy could be improved by the earlier detection of circulating biomarkers. Ultrasound at low frequencies is known to permeabilize cell membranes and has been used for delivery of molecules into the cell. We hypothesize and prove that this bioeffect of ultrasound also causes the release of both protein and nucleic acid biomarkers from cells in culture and mice. The ability to focus ultrasonic waves allows for the localization of the potential biomarker source in vivo. This novel strategy could lead to the earlier identification, characterization and localization of incidental lesions, cancer and other disease. Methods: The colon cancer cell line LS174T that produces protein biomarkers (carcinoembryonic antigen-CEA and cancer antigen 19-9 – CA19-9) and microRNAs (miRNA – miR -16 and miR-141) was exposed to low frequency ultrasound (1 MHz) in culture using varying intensities (0, 0.1, 0.3. 0.5, 0.7, 1.0 W/cm2) and time (0, 10, 30 min). Subcutaneous tumors of LS174T in mice were also exposed to ultrasound (2 W/cm2; 6 min), directly over the tumors or at a non-tumor bearing site. Samples were collected pre and post-ultrasound treatment and compared for changes in biomarker levels. Protein biomarkers were detected using an enzyme-linked immunosorbant assay and miRNAs were detected using quantitative reverse-transcription polymerase chain reaction. Cell death was studied using Trypan blue staining. Results: LS174T cells treated with 1 MHz ultrasound in culture (n=4) at a low intensity of 0.3 W/cm2 released both CEA and CA19-9 with an increase in time (0, 10, 30 min; p1000-fold (1.0 W/cm2) for miR-16 and 100-fold (0.1 W/cm2) to 500-fold (1 W/cm2) for miR-141, within 30 min of ultrasound treatment. Cell death was less than 5% across all conditions. Subcutaneous tumors (n=10) showed an increase release of protein biomarkers when treated with ultrasound at 2 W/cm2 (CEA p Conclusions: Increase in protein and miRNA biomarkers were observed when ultrasound was directly applied to cells. We have developed a simple non-invasive method to amplify and spatially localize the biomarker signal from tumors. This method has implications in diagnosis and monitoring of therapy and has a clear pathway into clinical applications uniting the fields of imaging and in vitro diagnostics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2011-4881

Early cancer detection may eventually be enabled by a quick, highly sensitive assay to monitor abnormal cancer biomarker levels in blood, followed by a localized imaging scan to visualize tumor burden. To improve screening of high-risk patients prior to imaging, blood assay sensitivity/specificity is being increased and biomarker secretion processes are being studied, but it is not clear which strategies will provide better detection of smaller tumors. To address this, we developed a linear 1-compartment model with 9 parameters to describe plasma biomarker kinetics in relation to the growth of cancer, beginning with a single tumor cell. In this model, plasma biomarker levels are dependent on the number of biomarker-secreting cells (cancerous and healthy), the biomarker secretion rate, the percent of biomarker entering plasma from the interstitium, and the elimination rate of plasma biomarker. To exemplify a realistic case, we based parameter values on ovarian cancer and CA125 secretion because CA125 secretion rates and assay sensitivities are readily available in published literature. Gompertzian tumor growth was modeled using published human ovarian carcinoma growth rates. The model predicted that tumor burden must grow to a volume of (20.4 mm)3 to reach plasma biomarker levels that are greater than in 99.9% of healthy patients; this may require >10 years after the birth of the initial tumor cell. These predictions are consistent with published cancer progression timelines recently uncovered from sequencing of metastatic genome data. Furthermore, by perturbing parameter values over log-orders of magnitude, we identified alternative strategies that could allow detection of Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5079. doi:10.1158/1538-7445.AM2011-5079

BAY 87–2243 is a novel small molecule inhibitor of hypoxia inducible factor-1α (HIF-1α) activity. Preclinical studies with BAY 87–2243 in a number of murine tumor models showed moderate to high tumor growth inhibition. The present study describes a non-invasive positron emission tomographic (PET) imaging method that monitors early therapeutic efficacy of this drug. To identify optimal tracer(s) for monitoring BAY 87–2243 anti-tumor activity, female nude mice were implanted on both scapulae with either 3.5×106 human non-small cell lung cancer (H460) or 2×106 human prostate cancer cells (PC3). Both are growth-inhibited by BAY 87–2243. Xenografts were grown to 200 mm3 over 10 days. Four PET tracers were assessed for uptake into tumor xenografts: 18F-FDG, 18F-FPP(RGD)2, 18F-FLT and 18F-FAZA. Tracers were injected via the tail vein. After pre-scan, BAY 87–2243 was orally administered, daily at 9mg/kg to 7 days. Tracer uptake was examined 1 and 3 day(s) after initial administration. Each treatment group for each PET tracer tested consisted of 6 tumors in 3 animals. At each imaging time point, RNA from BAY 87–2243-treated and vehicle-treated H460 tumor xenografts (3 additional animals each) was isolated. HIF target genes were quantified by Real-Time polymerase chain (RT-PCR) reaction. For both 18F-FDG and 18F-FPP(RGD)2, uptake was not significantly altered. However, 18F-FAZA tumor uptake (%ID mean/g) declined by 55–70% (1.21 ± 0.10 %ID/g to 0.35 ± 0.1 %ID/g, n=6, vehicle vs. treatment) in both H460 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-147. doi:10.1158/1538-7445.AM2011-LB-147

MYC protein regulates a variety of human genes and is deregulated in most of human cancers. The deregulation of MYC is at multiple levels, from gene transcription to mRNA translation and protein activity. Activation of Myc protein is phosphorylation-mediated and has been found to be essential for development of many cancers. Here, we developed a bioluminescent sensor, using protein assisted split luciferase complementation system, to noninvasively detect the phosphorylation-mediated activation of the cytoplasmic Myc (c-Myc). In this sensor system, the c-Myc phosphorylation motif and the phosphorylation recognition domain of GSK3beta were fused with the two split luciferase fragments respectively. Phosphorylation induced c-Myc interaction with GSK3beta resulted in complementation of the two split luciferases and reconstitution of luciferase activity. Signal of the optimal sensor was specific for the c-Myc phosphorylation. It detected as low as 2-fold higher levels of c-Myc phosphorylation in cancer cells versus normal cells. It also detected the temporal regulation pattern of c-Myc phosphorylation upon serum stimulation in normal cells. MAP kinase inhibitor, PD98059 and U0126, and Atorvastatin (AT), induced dose dependent signal reduction in tumor cells, which was correlated with the decrease of endogenous c-Myc phosphorylation. In mouse tumor xenograft models, the sensor detected the inhibitory effects of AT well before there was a detectable difference in tumor size (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5223.

The central nervous system (CNS) is emerging as the first site of metastatic disease in women with breast cancer. Women with hormone unresponsive tumors and/or HER-2 overexpressing tumors are disproportionately affected. Breast cancer brain metastasis represents a unique therapeutic challenge because of the limited access to the CNS of many anticancer drugs. Radioiodide (131I) therapy may provide a therapeutic alternative since over 70% of invasive breast cancers, and some brain metastases express the sodium-iodide symporter (NIS). The rationale for this approach is based on the success of 131I radioablative therapy for thyroid cancers. Objectives: To evaluate CNS permeability, kinetics of radioiodine uptake and retention in our NIS expressing breast cancer brain metastasis (BCBM) mouse model. Methods: A brain-seeking clone of the hormone independent ((ER-/PR-/HER2-) human breast cancer cell line MDA-MB-231 (231BR) was transduced with a lentiviral vector carrying a bicistronic cassette with NIS and the firefly luciferase (Fluc) genes separated by an internal ribosomal entry site (IRES). Single cell clones were selected and characterized for iodide uptake and bioluminescence. 1 Million NIS-Fluc-MDAMB231 cells were implanted subcutaneously in the mammary fat pad (mfp) of nude mice (NCr nude) and tumor growth was monitored by serial in vivo bioluminescent imaging and external tumor volume measurements. NIS-Fluc mfp tumor xenografts were then explanted, 1×1 mm pieces excised and inserted stereotactically into the basal ganglia of the mouse. Bioluminescence imaging was used to follow intracranial tumor growth over time. Kinetics of 123I uptake and biodistribution in mfp xenografts mice was evaluated by ex vivo radioactive count and biodistribution of 124I was evaluated in vivo by positron emission tomography (PET) imaging on BCBM mice. Results: Brain tumors 124I uptake was evident on the PET 1 hour post injection of the tracer, with values ranging from 1.60 to 3.25 % injected dose/gram of tissue (%ID/g) whereas thyroid uptake was between 6.99 and 11.19 %ID/g. At 20 hours post injection, the brain tumors showed a sustained 124I retention while uptake in most other organs (except thyroid and stomach) was close to background levels; brain tumor to normal brain tissue mean uptake ratio was ranging from to 217.12 to 541.50 (versus 2.02 to 2.68 at 1 hour post injection). The kinetics of 124I uptake was in agreement with the data obtained from the ex vivo counts in mfp xenografts. Conclusions: We have characterized the kinetics of iodide uptake and retention in a model of breast cancer brain metastases. We showed that NIS expressing BCBM are able to concentrate and retain 124I for prolonged period of time. These results together with dosimetric calculations will allow us to adjust the dosage and timing for 131I therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 477.

Abstract #2011 Background: An increasing number of women develop brain metastases (BM) after breast cancer (BC) treatment. A large proportion of these are estrogen/progesterone receptor-negative (ER-/PR-) and/or Her-2/neu overexpressing tumors. 131I radioablative therapy may provide a therapeutic alternative to treat metastases at this anatomic sanctuary since over 70% of invasive breast cancers, including a majority of ER- tumors and some brain metastases (unpublished data) express the sodium-iodide symporter (NIS). This approach relies on the success of radioiodide as a targeted treatment for thyroid cancers. To test this concept, we developed a BC BM model using tumor cells engineered to express NIS. Methods: MDAMB231 and SKBr3 cell lines were transduced with a lentiviral vector carrying a bicistronic cassette with NIS and the firefly luciferase (Fluc) genes separated by an internal ribosomal entry site. Single cell clones were selected and characterized for iodide uptake and bioluminescence. NIS-Fluc-MDAMB231 or NIS-Fluc SKBr3 cells (2.5 x 106 cells) were implanted subcutaneously (sc) in the mammary fat pad (mfp) of nude mice (NCr nude; 5-6 weeks old; n=5). NIS-Fluc mfp tumor xenografts were then explanted, 1x1 mm pieces excised and inserted stereotactically into the basal ganglia of the animal. All tumor development was monitored by serial in vivo bioluminescent imaging. Once established, brain tumors were excised, dissociated, established in tissue culture and re-implanted sc in the mfp of a new set of mice. Successive passages in the mfp then in the brain were performed in an attempt to increase tumor take. A second strategy tested with MDAMB231 cells consisted of direct implantation of cells into the basal ganglia. NIS expression was evaluated on tissue sections with a polyclonal antibody raised against the C-terminus of the human NIS. Results: All mice survived and were healthy in appearance. Intracranial implantation of mfp xenografts was highly successful with 66% take in both MDAMB231 (after two passages) and SKBr3 (after first passage). Bioluminescent imaging revealed sustained growth of tumors for more than 4 weeks. Microscopically, the explanted brain tumors had a cellular appearance without stromal cell or lymphocytic infiltration and were congruent with the histology of mfp xenografts. However, the tumor cell population was heterogeneous as NIS expression was present with plasma membrane staining in about 50% of SKBr3 and 15% of MDAMB231 cells. Direct cell implantations failed as no discernible bioluminescence was noted over a period of 3 weeks and no visible tumor at necropsy. Conclusions: A BCBM model has been developed by implanting intracranially mfp xenografts obtained with ER-/PR- +/- Her-2/neu overexpressing cells. Using this model, it will be possible to evaluate the effects of 131I on NIS-expressing BCBM. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2011.