Publications by authors named "Ronald Blasberg"

93 Publications

CTLA-4 blockade drives loss of T stability in glycolysis-low tumours.

Nature 2021 Feb 15. Epub 2021 Feb 15.

Ludwig Collaborative and Swim Across America Laboratory, MSK, New York, NY, USA.

Limiting the metabolic competition in the tumor microenvironment (TME) may increase the effectiveness of immunotherapy. Because of its critical role in glucose metabolism of activated T cells, CD28 signaling has been proposed as a T-cell metabolic biosensor. Conversely, CTLA-4 engagement has been shown to down-regulate T-cell glycolysis. Here, we investigated the impact of CTLA-4 blockade on the metabolic fitness of intra-tumor T cells in relationship to the tumor glycolytic capacity. We found that CTLA-4 blockade promotes immune cell infiltration and metabolic fitness especially in glycolysis-low tumors. Accordingly, anti-CTLA-4 achieved better therapeutic outcomes in mice bearing glycolysis-defective tumors. Intriguingly, tumor-specific CD8 T-cell responses correlated with phenotypic and functional destabilization of tumor-infiltrating regulatory T cells (Tregs) toward IFN-γ- and TNF-α-producing cells in glycolysis-defective tumors. By mimicking the highly and poorly glycolytic TME in vitro, we show that the effect of CTLA-4 blockade to promote Treg destabilization is dependent on Treg glycolysis and CD28 signaling. These findings indicate that decreasing tumor competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumor glycolysis. Moreover, these results reveal a new mechanism through which anti-CTLA-4 interferes with Treg function in the presence of glucose.
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http://dx.doi.org/10.1038/s41586-021-03326-4DOI Listing
February 2021

Lactate Dehydrogenase A Depletion Alters MyC-CaP Tumor Metabolism, Microenvironment, and CAR T Cell Therapy.

Mol Ther Oncolytics 2020 Sep 21;18:382-395. Epub 2020 Jul 21.

Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

To enhance human prostate-specific membrane antigen (hPSMA)-specific chimeric antigen receptor (CAR) T cell therapy in a hPSMA MyC-CaP tumor model, we studied and imaged the effect of lactate dehydrogenase A (LDH-A) depletion on the tumor microenvironment (TME) and tumor progression. Effective LDH-A short hairpin RNA (shRNA) knockdown (KD) was achieved in MyC-CaP:hPSMA Renilla luciferase (RLuc)-internal ribosome entry site (IRES)-GFP tumor cells, and changes in tumor cell metabolism and in the TME were monitored. LDH-A downregulation significantly inhibited cell proliferation and subcutaneous tumor growth compared to control cells and tumors. However, total tumor lactate concentration did not differ significantly between LDH-A knockdown and control tumors, reflecting the lower vascularity, blood flow, and clearance of lactate from LDH-A knockdown tumors. Comparing treatment responses of MyC-CaP tumors with LDH-A depletion and/or anti-hPSMA CAR T cells showed that the dominant effect on tumor growth was LDH-A depletion. With anti-hPSMA CAR T cell treatment, tumor growth was significantly slower when combined with tumor LDH-A depletion and compared to control tumor growth (p < 0.0001). The lack of a complete tumor response in our animal model can be explained in part by (1) the lower activity of human CAR T cells against hPSMA-expressing murine tumors in a murine host, and (2) a loss of hPSMA antigen from the tumor cell surface in progressive generations of tumor cells.
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http://dx.doi.org/10.1016/j.omto.2020.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452096PMC
September 2020

Imaging CXCR4 Expression with Iodinated and Brominated Cyclam Derivatives.

Mol Imaging Biol 2020 10;22(5):1184-1196

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Purpose: CXCR4 is one of several "chemokine" receptors expressed on malignant tumors (including GBM and PCNSL) and hematopoietic stem cells. Although Ga-pentixafor and Ga-NOTA-NFB have been shown to effectively image CXCR4 expression in myeloma and other systemic malignancies, imaging CXCR4 expression in brain tumors has been more limited due to the blood-brain barrier (BBB) and a considerable fraction of CXCR4 staining is intracellular.

Methods: We synthesized 6 iodinated and brominated cyclam derivatives with high affinity (low nM range) for CXCR4, since structure-based estimates of lipophilicity suggested rapid transfer across the BBB and tumor cell membranes.

Results: We tested 3 iodinated and 3 brominated cyclam derivatives in several CXCR4(+) and CXCR4(-) cell lines, with and without cold ligand blocking. To validate these novel radiolabeled cyclam derivatives for diagnostic CXCR4 imaging efficacy in brain tumors, we established appropriated murine models of intracranial GBM and PCNSL. Based on initial studies, I-HZ262 and Br-HZ270-1 were shown to be the most avidly accumulated radioligands. Br-HZ270-1 was selected for further study in the U87-CXCR4 and PCNSL #15 intracranial tumor models, because of its high uptake (9.5 ± 1.3 %ID/g, SD) and low non-specific uptake (1.6 ± 0.7 %ID/g, SD) in the s.c. U87-CXCR4 tumor models. However, imaging CXCR4 expression in intracranial U87-CXCR4 and PCNSL #15 tumors with Br-HZ270-1 was unsuccessful, following either i.v. or spinal-CSF injection.

Conclusions: Imaging CXCR4 expression with halogenated cyclam derivatives was successful in s.c. located tumors, but not in CNS located tumors. This was largely due to the following: (i) the hydrophilicity of the radiolabeled analogues-as reflected in the "measured" radiotracer distribution (LogD) in octanol/PBS-which stands in contrast to the structure-based estimate of LogP, which was the rationale for initiating the study and (ii) the presence of a modest BTB in intracranial U87-CXCR4 gliomas and an intact BBB/BTB in the intracranial PCNSL animal model.
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http://dx.doi.org/10.1007/s11307-020-01480-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497443PMC
October 2020

The AQARA Principle: Proposing Standard Requirements for Radionuclide-Based Images in Medical Journals.

J Nucl Med 2020 01;61(1):1-2

Memorial Sloan Kettering Cancer Center, New York, New York.

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http://dx.doi.org/10.2967/jnumed.119.240523DOI Listing
January 2020

F-Fluorocholine PET uptake correlates with pathologic evidence of recurrent tumor after stereotactic radiosurgery for brain metastases.

Eur J Nucl Med Mol Imaging 2020 06 21;47(6):1446-1457. Epub 2019 Dec 21.

Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA.

Purpose: Radiographic changes of brain metastases after stereotactic radiosurgery (SRS) can signify tumor recurrence and/or radiation necrosis (RN); however, standard imaging modalities cannot easily distinguish between these two entities. We investigated whether F-Fluorocholine uptake in surgical samples of the resected lesions correlates with pathologic evidence of recurrent tumor and PET imaging.

Methods: About 14 patients previously treated with SRS that developed radiographic changes were included. All patients underwent a preoperative 40-min dynamic PET/CT concurrent with 392 ± 11 MBq bolus injection of F-Fluorocholine. F-Fluorocholine pharmacokinetics were evaluated by standardized uptake value (SUV), graphical analysis (Patlak plot; K) and an irreversible two-compartment model (K, k, k, and K). 12 out of 14 patients were administered an additional 72 ± 14 MBq injection of F-Fluorocholine 95 ± 26 minutes prior to surgical resection. About 113 resected samples from 12 patients were blindly reviewed by a neuropathologist to assess the viable tumor and necrotic content, microvascular proliferation, reactive gliosis, and mono- and polymorphonuclear inflammatory infiltrates. Correlation between these metrics F-Fluorocholine SUV was investigated with a linear mixed model. Comparison of survival distributions of two groups of patients (population median split of PET SUV) was performed with the log-rank test.

Results: Exactly 10 out of 12 patients for which surgical samples were acquired exhibited pathologic recurrence. Strong correlation was observed between SUV as measured from a surgically removed sample with highest uptake and by PET (Pearson's r = 0.66). Patients with F-Fluorocholine PET SUV > 6 experienced poor survival. Surgical samples with viable tumor had higher F-fluorocholine uptake (SUV) than those without tumor (4.5 ± 3.7 and 2.6 ± 3.0; p = 0.01). F-fluorocholine count data from surgical samples is driven not only by the percentage viable tumor but also by the degree of inflammation and reactive gliosis (p ≤ 0.02; multivariate regression).

Conclusions: F-Fluorocholine accumulation is increased in viable tumor; however, inflammation and gliosis may also lead to elevated uptake. Higher F-Fluorocholine PET uptake portends worse prognosis. Kinetic analysis of dynamic F-Fluorocholine PET imaging supports the adequacy of the simpler static SUV metric.
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http://dx.doi.org/10.1007/s00259-019-04628-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596690PMC
June 2020

Molecular Imaging with Reporter Genes: Has Its Promise Been Delivered?

J Nucl Med 2019 12;60(12):1665-1681

Department of Neurology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York

The first reporter systems were developed in the early 1980s and were based on measuring the activity of an enzyme-as a surrogate measure of promoter-driven transcriptional activity-which is now known as a reporter gene system. The initial objective and application of reporter techniques was to analyze the activity of a specific promoter (namely, the expression of a gene that is under the regulation of the specific promoter that is linked to the reporter gene). This system allows visualization of specific promoter activity with great sensitivity. In general, there are 2 classes of reporter systems: constitutively expressed (always-on) reporter constructs used for cell tracking, and inducible reporter systems sensitive to endogenous signaling molecules and transcription factors that characterize specific tissues, tumors, or signaling pathways.This review traces the development of different reporter systems, using and proteins as well as reporter systems. The development and application of reporter systems is the focus of this review. The question at the end of the review is whether the "promise" of reporter gene imaging has been realized. What is required for moving forward with reporter systems, and what is required for successful translation to clinical applications?
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http://dx.doi.org/10.2967/jnumed.118.220004DOI Listing
December 2019

LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response.

PLoS One 2018 24;13(9):e0203965. Epub 2018 Sep 24.

Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.

Previous studies show that LDH-A knockdown reduces orthotopic 4T1 breast tumor lactate and delays tumor growth and the development of metastases in nude mice. Here, we report significant changes in the tumor microenvironment (TME) and a more robust anti-tumor response in immune competent BALB/c mice. 4T1 murine breast cancer cells were transfected with shRNA plasmids directed against LDH-A (KD) or a scrambled control plasmid (NC). Cells were also transduced with dual luciferase-based reporter systems to monitor HIF-1 activity and the development of metastases by bioluminescence imaging, using HRE-sensitive and constitutive promoters, respectively. The growth and metastatic profile of orthotopic 4T1 tumors developed from these cell lines were compared and a primary tumor resection model was studied to simulate the clinical management of breast cancer. Primary tumor growth, metastasis formation and TME phenotype were significantly different in LDH-A KD tumors compared with controls. In LDH-A KD cells, HIF-1 activity, hexokinase 1 and 2 expression and VEGF secretion were reduced. Differences in the TME included lower HIF-1α expression that correlated with lower vascularity and pimonidazole staining, higher infiltration of CD3+ and CD4+ T cells and less infiltration of TAMs. These changes resulted in a greater delay in metastases formation and 40% long-term survivors (>20 weeks) in the LDH-A KD cohort following surgical resection of the primary tumor. We show for the first time that LDH-depletion inhibits the formation of metastases and prolongs survival of mice through changes in tumor microenvironment that modulate the immune response. We attribute these effects to diminished HIF-1 activity, vascularization, necrosis formation and immune suppression in immune competent animals. Gene-expression analyses from four human breast cancer datasets are consistent with these results, and further demonstrate the link between glycolysis and immune suppression in breast cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203965PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153000PMC
March 2019

Impact of the Tumor Microenvironment on Tumor-Infiltrating Lymphocytes: Focus on Breast Cancer.

Breast Cancer (Auckl) 2017 25;11:1178223417731565. Epub 2017 Sep 25.

Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Immunotherapy is revolutionizing cancer care across disciplines. The original success of immune checkpoint blockade in melanoma has already been translated to Food and Drug Administration-approved therapies in a number of other cancers, and a large number of clinical trials are underway in many other disease types, including breast cancer. Here, we review the basic requirements for a successful antitumor immune response, with a focus on the metabolic and physical barriers encountered by lymphocytes entering breast tumors. We also review recent clinical trials of immunotherapy in breast cancer and provide a number of interesting questions that will need to be answered for successful breast cancer immunotherapy.
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http://dx.doi.org/10.1177/1178223417731565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617083PMC
September 2017

Biodistribution and Dosimetry of F-Meta-Fluorobenzylguanidine: A First-in-Human PET/CT Imaging Study of Patients with Neuroendocrine Malignancies.

J Nucl Med 2018 01 13;59(1):147-153. Epub 2017 Jul 13.

Pediatric Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.

I-meta-iodobenzylguanidine (I-MIBG) imaging is currently a mainstay in the evaluation of many neuroendocrine tumors, especially neuroblastoma. I-MIBG imaging has several limitations that can be overcome by the use of a PET agent. F-meta-fluorobenzylguanidine (F-MFBG) is a PET analog of MIBG that may allow for single-day, high-resolution quantitative imaging. We conducted a first-in-human study of F-MFBG PET imaging to evaluate the safety, feasibility, pharmacokinetics, and dosimetry of F-MFBG in neuroendocrine tumors (NETs). Ten patients (5 with neuroblastoma and 5 with paraganglioma/pheochromocytoma) received 148-444 MBq (4-12mCi) of F-MFBG intravenously followed by serial whole-body imaging at 0.5-1, 1-2, and 3-4 after injection. Serial blood samples (a total of 6) were also obtained starting at 5 min after injection to as late as 4 h after injection; whole-body distribution and blood clearance data, lesion uptake, and normal-tissue uptake were determined, and radiation-absorbed doses to normal organs were calculated using OLINDA. No side effects were seen in any patient after F-MFBG injection. Tracer distribution showed prominent activity in the blood pool, liver, and salivary glands that decreased with time. Mild uptake was seen in the kidneys and spleen, which also decreased with time. Urinary excretion was prominent, with an average of 45% of the administered activity in the bladder by 1 h after injection; whole-body clearance was monoexponential, with a mean biologic half-life of 1.95 h, whereas blood clearance was biexponential, with a mean biologic half-life of 0.3 h (58%) for the rapid α phase and 6.1 h (42%) for the slower β phase. The urinary bladder received the highest radiation dose with a mean absorbed dose of 0.186 ± 0.195 mGy/MBq. The mean total-body dose was 0.011 ± 0.011 mGy/MBq, and the effective dose was 0.023 ± 0.012 mSv/MBq. Both skeletal and soft-tissue lesions were visualized with high contrast. The SUVmax (mean ± SD ) of lesions at 1-2 h after injection was 8.6 ± 9.6. Preliminary data show that F-MFBG imaging is safe and has favorable biodistribution and kinetics with good targeting of lesions. PET imaging with F-MFBG allows for same-day imaging of NETs. F-MFBG appears highly promising for imaging of patients with NETs, especially children with neuroblastoma.
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http://dx.doi.org/10.2967/jnumed.117.193169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750519PMC
January 2018

Enhancement of PSMA-Directed CAR Adoptive Immunotherapy by PD-1/PD-L1 Blockade.

Mol Ther Oncolytics 2017 Mar 14;4:41-54. Epub 2016 Dec 14.

Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Chimeric antigen receptor (CAR) T cell therapy in hematologic malignancies has shown remarkable responses, but the same level of success has not been observed in solid tumors. A new prostate cancer model (Myc-CaP:PSMA(+)) and a second-generation anti-hPSMA human CAR T cells expressing a Click Beetle Red luciferase reporter) were used to study hPSMA targeting and assess CAR T cell trafficking and persistence by bioluminescence imaging (BLI). We investigated the antitumor efficacy of human CAR T cells targeting human prostate-specific membrane antigen (hPSMA), in the presence and absence of the target antigen; first alone and then combined with a monoclonal antibody targeting the human programmed death receptor 1 (anti-hPD1 mAb). PDL-1 expression was detected in Myc-CaP murine prostate tumors growing in immune competent FVB/N and immune-deficient SCID mice. Endogenous CD3 T cells were restricted from the centers of Myc-CaP tumor nodules growing in FVB/N mice. Following anti-programmed cell death protein 1 (PD-1) treatment, the restriction of CD3 T cells was reversed, and a tumor-treatment response was observed. Adoptive hPSMA-CAR T cell immunotherapy was enhanced when combined with PD-1 blockade, but the treatment response was of comparatively short duration, suggesting other immune modulation mechanisms exist and restrict CAR T cell targeting, function, and persistence in hPSMA expressing Myc-CaP tumors. Interestingly, an "inverse pattern" of CAR T cell BLI intensity was observed in control and test tumors, which suggests CAR T cells undergo changes leading to a loss of signal and/or number following hPSMA-specific activation. The lower BLI signal intensity in the hPSMA test tumors (compared with controls) is due in part to a decrease in T cell mitochondrial function following T cell activation, which may limit the intensity of the ATP-dependent Luciferin-luciferase bioluminescence signal.
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http://dx.doi.org/10.1016/j.omto.2016.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363727PMC
March 2017

Inhibition of prostate cancer proliferation by Deferiprone.

NMR Biomed 2017 Jun 8;30(6). Epub 2017 Mar 8.

Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Cancer growth and proliferation rely on intracellular iron availability. We studied the effects of Deferiprone (DFP), a chelator of intracellular iron, on three prostate cancer cell lines: murine, metastatic TRAMP-C2; murine, non-metastatic Myc-CaP; and human, non-metastatic 22rv1. The effects of DFP were evaluated at different cellular levels: cell culture proliferation and migration; metabolism of live cells (time-course multi-nuclear magnetic resonance spectroscopy cell perfusion studies, with 1- C-glucose, and extracellular flux analysis); and expression (Western blot) and activity of mitochondrial aconitase, an iron-dependent enzyme. The 50% and 90% inhibitory concentrations (IC and IC , respectively) of DFP for the three cell lines after 48 h of incubation were within the ranges 51-67 μM and 81-186 μM, respectively. Exposure to 100 μM DFP led to: (i) significant inhibition of cell migration after different exposure times, ranging from 12 h (TRAMP-C2) to 48 h (22rv1), in agreement with the respective cell doubling times; (ii) significantly decreased glucose consumption and glucose-driven tricarboxylic acid cycle activity in metastatic TRAMP-C2 cells, during the first 10 h of exposure, and impaired cellular bioenergetics and membrane phospholipid turnover after 23 h of exposure, consistent with a cytostatic effect of DFP. At this time point, all cell lines studied showed: (iii) significant decreases in mitochondrial functional parameters associated with the oxygen consumption rate, and (iv) significantly lower mitochondrial aconitase expression and activity. Our results indicate the potential of DFP to inhibit prostate cancer proliferation at clinically relevant doses and plasma concentrations.
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http://dx.doi.org/10.1002/nbm.3712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505495PMC
June 2017

Tumor stroma interaction is mediated by monocarboxylate metabolism.

Exp Cell Res 2017 03 26;352(1):20-33. Epub 2017 Jan 26.

Department of Pharmacology and Graduate School of Biomedical Sciences, Rutgers Biomedical Health Sciences, Rutgers, The State University of New Jersey, 675 Hoes Lane West, Piscataway, NJ 08854, United States. Electronic address:

Human breast tumors contain significant amounts of stromal cells. There exists strong evidence that these stromal cells support cancer development and progression by altering various pathways (e.g. downregulation of tumor suppressor genes or autocrine signaling loops). Here, we suggest that stromal carcinoma-associated fibroblasts (CAFs), shown to be generated from bone marrow-derived mesenchymal stem cells, may (i) recycle tumor-derived lactate for their own energetic requirements, thereby sparing glucose for neighboring glycolytic tumor cells, and (ii) subsequently secrete surplus energetically and biosynthetically valuable metabolites of lactate oxidation, such as pyruvate, to support tumor growth. Lactate, taken up by stromal CAFs, is converted to pyruvate, which is then utilized by CAFs for energy needs as well as excreted and shared with tumor cells. We have interrogated lactate oxidation in CAFs to determine what metabolites may be secreted, and how they may affect the metabolism and growth of MDA-MB-231 breast cancer cells. We found that CAFs secrete pyruvate as a metabolite of lactate oxidation. Further, we show that pyruvate is converted to lactate to promote glycolysis in MDA-MB-231 cells and helps to control elevated ROS levels in these tumor cells. Finally, we found that inhibiting or interfering with ROS management, using the naturally occurring flavonoid phloretin (found in apple tree leaves), adds to the cytotoxicity of the conventional chemotherapeutic agent doxorubicin. Our work demonstrates that a lactate-pyruvate, reciprocally-supportive metabolic relationship may be operative within the tumor microenvironment (TME) to support tumor growth, and may be a useful drug target.
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http://dx.doi.org/10.1016/j.yexcr.2017.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476446PMC
March 2017

Synthesis and evaluation of an (18)F-labeled pyrimidine-pyridine amine for targeting CXCR4 receptors in gliomas.

Nucl Med Biol 2016 10 14;43(10):606-11. Epub 2016 May 14.

Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA; Department of Neurology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA; Program in Molecular Pharmacology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA; Weill Cornell Medical College, New York, NY, USA. Electronic address:

Introduction: Chemokine receptor-4 (CXCR4, fusin, CD184) is expressed on several tissues involved in immune regulation and is upregulated in many diseases including malignant gliomas. A radiolabeled small molecule that readily crosses the blood-brain barrier can aid in identifying CXCR4-expressing gliomas and monitoring CXCR4-targeted therapy. In the current work, we have synthesized and evaluated an [(18)F]-labeled small molecule based on a pyrimidine-pyridine amine for its ability to target CXCR4.

Experimental: The nonradioactive standards and the nitro precursor used in this study were prepared using established methods. An HPLC method was developed to separate the nitro-precursor from the nonradioactive standard and radioactive product. The nitro-precursor was radiolabeled with (18)F under inert, anhydrous conditions using the [(18)F]-kryptofix 2.2.2 complex to form the desired N-(4-(((6-[(18)F]fluoropyridin-2-yl)amino)methyl)benzyl)pyrimidin-2-amine ([(18)F]-3). The purified radiolabeled compound was used in serum stability, partition coefficient, cellular uptake, and in vivo cancer targeting studies.

Results: [(18)F]-3 was synthesized in 4-10% decay-corrected yield (to start of synthesis). [(18)F]-3 (tR ≈ 27 min) was separated from the precursor (tR ≈ 30 min) using a pentafluorophenyl column with an isocratic solvent system. [(18)F]-3 displayed acceptable serum stability over 2 h. The amount of [(18)F]-3 bound to the plasma proteins was determined to be > 97%. The partition coefficient (LogD7.4) is 1.4 ± 0.5. Competitive in vitro inhibition indicated 3 does not inhibit uptake of (67)Ga-pentixafor. Cell culture media incubation and ex vivo urine analysis indicate rapid metabolism of [(18)F]-3 into hydrophilic metabolites. Thus, in vitro uptake of [(18)F]-3 in CXCR4 overexpressing U87 cells (U87 CXCR4) and U87 WT indicated no specific binding. In vivo studies in mice bearing U87 CXCR4 and U87 WT tumors on the left and right shoulders were carried out using [(18)F]-3 and (68)Ga-pentixafor on consecutive days. The CXCR4 positive tumor was clearly visualized in the PET study using (68)Ga-pentixafor, but not with [(18)F]-3.

Conclusions: We have successfully synthesized both a radiolabeled analog to previously reported CXCR4-targeting molecules and a nitro precursor. Our in vitro and in vivo studies indicate that [(18)F]-3 is rapidly metabolized and, therefore, does not target CXCR4-expressing tumors. Optimization of the structure to improve the in vivo (and in vitro) stability, binding, and solubility could lead to an appropriate CXCR4-targeted radiodiagnositic molecule.
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http://dx.doi.org/10.1016/j.nucmedbio.2016.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363724PMC
October 2016

Metabolic plasticity of metastatic breast cancer cells: adaptation to changes in the microenvironment.

Neoplasia 2015 Aug;17(8):671-84

Department of Medical Physics, Memorial Sloan Kettering Cancer Center, 10065, New York, NY, USA. Electronic address:

Cancer cells adapt their metabolism during tumorigenesis. We studied two isogenic breast cancer cells lines (highly metastatic 4T1; nonmetastatic 67NR) to identify differences in their glucose and glutamine metabolism in response to metabolic and environmental stress. Dynamic magnetic resonance spectroscopy of (13)C-isotopomers showed that 4T1 cells have higher glycolytic and tricarboxylic acid (TCA) cycle flux than 67NR cells and readily switch between glycolysis and oxidative phosphorylation (OXPHOS) in response to different extracellular environments. OXPHOS activity increased with metastatic potential in isogenic cell lines derived from the same primary breast cancer: 4T1 > 4T07 and 168FARN (local micrometastasis only) > 67NR. We observed a restricted TCA cycle flux at the succinate dehydrogenase step in 67NR cells (but not in 4T1 cells), leading to succinate accumulation and hindering OXPHOS. In the four isogenic cell lines, environmental stresses modulated succinate dehydrogenase subunit A expression according to metastatic potential. Moreover, glucose-derived lactate production was more glutamine dependent in cell lines with higher metastatic potential. These studies show clear differences in TCA cycle metabolism between 4T1 and 67NR breast cancer cells. They indicate that metastases-forming 4T1 cells are more adept at adjusting their metabolism in response to environmental stress than isogenic, nonmetastatic 67NR cells. We suggest that the metabolic plasticity and adaptability are more important to the metastatic breast cancer phenotype than rapid cell proliferation alone, which could 1) provide a new biomarker for early detection of this phenotype, possibly at the time of diagnosis, and 2) lead to new treatment strategies of metastatic breast cancer by targeting mitochondrial metabolism.
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http://dx.doi.org/10.1016/j.neo.2015.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674487PMC
August 2015

Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes.

J Nucl Med 2015 Jul 29;56(7):1055-60. Epub 2015 May 29.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York Sloan Kettering Institute Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, New York

Unlabelled: Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging.

Methods: Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodium-iodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 10(5) to 3 × 10(6) reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (background) was measured.

Results: The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-(18)F-fluorobenzylguanidine ((18)F-MFBG) reporter system could detect less than 1 × 10(5) T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/(124)I-iodide reporter system could detect approximately 1 × 10(6) T cells; (124)I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2'-(18)F-fluoro-5-ethyl-1-β-d-arabinofuranosyluracil ((18)F-FEAU) and hsvTK/(18)F-FEAU reporter systems detected approximately 3 × 10(5) T cells, respectively. (18)F-FEAU was a more efficient probe (higher uptake, lower background) than (124)I-1-(2-deoxy-2-fluoro-1-d-arabinofuranosyl)-5-iodouracil for both hdCKDM and hsvTK.

Conclusion: A comparison of different reporter gene-reporter probe systems for imaging of T cell number was performed, and the hNET/(18)F-MFBG PET reporter system was found to be the most sensitive and capable of detecting approximately 35-40 × 10(3) T cells at the site of T cell injection in the animal model.
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http://dx.doi.org/10.2967/jnumed.115.159855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511596PMC
July 2015

Surface-enhanced resonance Raman scattering nanostars for high-precision cancer imaging.

Sci Transl Med 2015 Jan;7(271):271ra7

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Brain Tumor Center, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Center for Molecular Imaging and Nanotechnology (CMINT), Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Department of Radiology, Weill Cornell Medical College, New York, NY 10065, USA.

The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread, and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the true extent of tumors is desired clinically and surgically. We show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal locoregional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed SERRS nanostars. The SERRS nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In genetically engineered mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, and in one human sarcoma xenograft model, SERRS nanostars enabled accurate detection of macroscopic malignant lesions, as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 fM limit of detection) of SERRS nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS nanostars a promising imaging agent for more precise cancer imaging and resection.
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http://dx.doi.org/10.1126/scitranslmed.3010633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414254PMC
January 2015

Understanding the pharmacological properties of a metabolic PET tracer in prostate cancer.

Proc Natl Acad Sci U S A 2014 May 1;111(20):7254-9. Epub 2014 May 1.

Program in Molecular Pharmacology and Chemistry, andDepartments of Radiology,

Generally, solid tumors (>400 mm(3)) are inherently acidic, with more aggressive growth producing greater acidity. If the acidity could be targeted as a biomarker, it would provide a means to gauge the pace of tumor growth and degree of invasiveness, as well as providing a basis for predicting responses to pH-dependent chemotherapies. We have developed a (64)Cu pH (low) insertion peptide (pHLIP) for targeting, imaging, and quantifying acidic tumors by PET, and our findings reveal utility in assessing prostate tumors. The new pHLIP version limits indiscriminate healthy tissue binding, and we demonstrate its targeting of extracellular acidification in three different prostate cancer models, each with different vascularization and acid-extruding protein carbonic anhydrase IX (CAIX) expression. We then describe the tumor distribution of this radiotracer ex vivo, in association with blood perfusion and known biomarkers of acidity, such as hypoxia, lactate dehydrogenase A, and CAIX. We find that the probe reveals metabolic variations between and within tumors, and discriminates between necrotic and living tumor areas.
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http://dx.doi.org/10.1073/pnas.1405240111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034234PMC
May 2014

Imaging the norepinephrine transporter in neuroblastoma: a comparison of [18F]-MFBG and 123I-MIBG.

Clin Cancer Res 2014 Apr 26;20(8):2182-91. Epub 2014 Feb 26.

Authors' Affiliations: Departments of Radiology, Pediatrics, Neurology, Medical Physics, Epidemiology and Biostatistics, and Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center (MSKCC), New York, New York.

Purpose: The norepinephrine transporter (NET) is a critical regulator of catecholamine uptake in normal physiology and is expressed in neuroendocrine tumors like neuroblastoma. Although the norepinephrine analog, meta-iodobenzylguanidine (MIBG), is an established substrate for NET, (123)I/(131)I-MIBG has several clinical limitations for diagnostic imaging. In the current studies, we evaluated meta-[(18)F]-fluorobenzylguanidine ([(18)F]-MFBG) and compared it with (123)I-MIBG for imaging NET-expressing neuroblastomas.

Experimental Design: NET expression levels in neuroblastoma cell lines were determined by Western blot and (123)I-MIBG uptake assays. Five neuroblastoma cell lines and two xenografts (SK-N-BE(2)C and LAN1) expressing different levels of NET were used for comparative in vitro and in vivo uptake studies.

Results: The uptake of [(18)F]-MFBG in cells was specific and proportional to the expression level of NET. Although [(18)F]-MFBG had a 3-fold lower affinity for NET and an approximately 2-fold lower cell uptake in vitro compared with that of (123)I-MIBG, the in vivo imaging and tissue radioactivity concentration measurements demonstrated higher [(18)F]-MFBG xenograft uptake and tumor-to-normal organ ratios at 1 and 4 hours after injection. A comparison of 4 hours [(18)F]-MFBG PET (positron emission tomography) imaging with 24 hours (123)I-MIBG SPECT (single-photon emission computed tomography) imaging showed an approximately 3-fold higher tumor uptake of [(18)F]-MFBG, but slightly lower tumor-to-background ratios in mice.

Conclusions: [(18)F]-MFBG is a promising radiopharmaceutical for specifically imaging NET-expressing neuroblastomas, with fast pharmacokinetics and whole-body clearance. [(18)F]-MFBG PET imaging shows higher sensitivity, better detection of small lesions with low NET expression, allows same day scintigraphy with a shorter image acquisition time, and has the potential for lower patient radiation exposure compared with (131)I/(123)I-MIBG.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-1153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072734PMC
April 2014

Synthesis and evaluation of 18F-labeled benzylguanidine analogs for targeting the human norepinephrine transporter.

Eur J Nucl Med Mol Imaging 2014 Feb 31;41(2):322-32. Epub 2013 Oct 31.

Department of Radiology, Memorial Sloan-Kettering Cancer Center (MSKCC), New York, NY, 10065, USA.

Purpose: Both (131)I- and (123)I-labeled meta-iodobenzylguanidine (MIBG) have been widely used in the clinic for targeted imaging of the norepinephrine transporter (NET). The human NET (hNET) gene has been imaged successfully with (124)I-MIBG positron emission tomography (PET) at time points of >24 h post-injection (p.i.). (18)F-labeled MIBG analogs may be ideal to image hNET expression at time points of <8 h p.i. We developed improved methods for the synthesis of known MIBG analogs, [(18)F]MFBG and [(18)F]PFBG and evaluated them in hNET reporter gene-transduced C6 rat glioma cells and xenografts.

Methods: [(18)F]MFBG and [(18)F]PFBG were synthesized manually using a three-step synthetic scheme. Wild-type and hNET reporter gene-transduced C6 rat glioma cells and xenografts were used to comparatively evaluate the (18)F-labeled analogs with [(123)I]/[(124)I]MIBG.

Results: The fluorination efficacy on benzonitrile was predominantly determined by the position of the trimethylammonium group. The para-isomer afforded higher yields (75 ± 7%) than meta-isomer (21 ± 5%). The reaction of [(18)F]fluorobenzylamine with 1H-pyrazole-1-carboximidamide was more efficient than with 2-methyl-2-thiopseudourea. The overall radiochemical yields (decay-corrected) were 11 ± 2% (n = 12) for [(18)F]MFBG and 41 ± 12% (n = 5) for [(18)F]PFBG, respectively. The specific uptakes of [(18)F]MFBG and [(18)F]PFBG were similar in C6-hNET cells, but 4-fold less than that of [(123)I]/[(124)I]MIBG. However, in vivo [(18)F]MFBG accumulation in C6-hNET tumors was 1.6-fold higher than that of [(18)F]PFBG at 1 h p.i., whereas their uptakes were similar at 4 h. Despite [(18)F]MFBG having a 2.8-fold lower affinity to hNET and approximately 4-fold lower cell uptake in vitro compared to [(123)I]/[(124)I]MIBG, PET imaging demonstrated that [(18)F]MFBG was able to visualize C6-hNET xenografts better than [(124)I]MIBG. Biodistribution studies showed [(18)F]MFBG and (123)I-MIBG had a similar tumor accumulation, which was lower than that of no-carrier-added [(124)I]MIBG, but [(18)F]MFBG showed a significantly more rapid body clearance and lower uptake in most non-targeting organs.

Conclusion: [(18)F]MFBG and [(18)F]PFBG were synthesized in reasonable radiochemical yields under milder conditions. [(18)F]MFBG is a better PET ligand to image hNET expression in vivo at 1-4 h p.i. than both [(18)F]PFBG and [(123)I]/[(124)I]MIBG.
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http://dx.doi.org/10.1007/s00259-013-2558-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947152PMC
February 2014

Targeting the Hsp90-associated viral oncoproteome in gammaherpesvirus-associated malignancies.

Blood 2013 Oct 13;122(16):2837-47. Epub 2013 Aug 13.

Department of Pathology and Laboratory Medicine, and.

PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV(+) primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.
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http://dx.doi.org/10.1182/blood-2013-01-479972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798998PMC
October 2013

Relationships between LDH-A, lactate, and metastases in 4T1 breast tumors.

Clin Cancer Res 2013 Sep 5;19(18):5158-69. Epub 2013 Jul 5.

Authors' Affiliations: Departments of Medical Physics, Neurology, Radiology, and Medicine, Bioinformatics Core, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center; and Department of Physiology and Biophysics, Weill Cornell Graduate School of Medical Sciences, New York, New York.

Purpose: To investigate the relationship between lactate dehydrogenase A (LDH-A) expression, lactate concentration, cell metabolism, and metastases in murine 4T1 breast tumors.

Experimental Design: Inhibition of LDH-A expression and protein levels were achieved in a metastatic breast cancer cell line (4T1) using short hairpin RNA (shRNA) technology. The relationship between tumor LDH-A protein levels and lactate concentration (measured by magnetic resonance spectroscopic imaging, MRSI) and metastases was assessed.

Results: LDH-A knockdown cells (KD9) showed a significant reduction in LDH-A protein and LDH activity, less acid production, decreased transwell migration and invasion, lower proliferation, reduced glucose consumption and glycolysis, and increase in oxygen consumption, reactive oxygen species (ROS), and cellular ATP levels, compared with control (NC) cells cultured in 25 mmol/L glucose. In vivo studies showed lower lactate levels in KD9, KD5, and KD317 tumors than in NC or 4T1 wild-type tumors (P < 0.01), and a linear relationship between tumor LDH-A protein expression and lactate concentration. Metastases were delayed and primary tumor growth rate decreased.

Conclusions: We show for the first time that LDH-A knockdown inhibited the formation of metastases, and was accompanied by in vivo changes in tumor cell metabolism. Lactate MRSI can be used as a surrogate to monitor targeted inhibition of LDH-A in a preclinical setting and provides a noninvasive imaging strategy to monitor LDH-A-targeted therapy. This imaging strategy can be translated to the clinic to identify and monitor patients who are at high risk of developing metastatic disease.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-3300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877680PMC
September 2013

Molecular imaging of expression of vascular endothelial growth factor a (VEGF a) in femoral bone grafts transplanted into living mice.

Cell Transplant 2014 12;23(7):901-12. Epub 2013 Apr 12.

Research Division, Hospital for Special Surgery, New York, NY, USA.

The biology of cells transplanted with bone grafts is incompletely understood. Focusing on the early angiogenic response postgrafting, we report a mouse femur graft model in which grafts were derived from mice transgenic for a firefly luciferase (FLuc) bioluminescence reporter gene driven by a promoter for the angiogenic signaling molecule vascular endothelial growth factor (VEGF). Upon transplantation into wild-type (wt) mice, in vivo bioluminescence imaging (BLI) permitted longitudinal visualization and measurements of VEGF promoter activity in the transplanted graft cells and demonstrated a lag period of 7 days posttransplantation prior to robust induction of the promoter. To determine cellular mediators of VEGF induction in graft bone, primary graft-derived osteoblastic cells (GDOsts) were characterized. In vitro BLI on GDOsts showed hypoxia-induced VEGF expression and that this induction depended on PI3K signaling and, to a lesser degree, on the MEK pathway. This transcriptional regulation correlated with VEGF protein production and was validated in GDOsts seeded on demineralized bone matrix (DBM), a bone graft substitute material. Together, combined imaging of VEGF expression in living animals and in live cells provided clues about the regulation of VEGF in cells post-bone grafting. These data are particularly significant toward the development of future smart bone graft substitutes.
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http://dx.doi.org/10.3727/096368912X667015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477423PMC
January 2015

Noninvasive molecular imaging using reporter genes.

J Nucl Med 2013 Feb 14;54(2):167-72. Epub 2013 Jan 14.

Department of Radiology, Division of Molecular Imaging, Medical University of Vienna, Vienna, Austria.

Noninvasive reporter gene imaging is a component of molecular imaging. Reporter imaging can provide noninvasive assessments of endogenous biologic processes in living subjects and can be performed using different imaging modalities. This review will focus on radionuclide-based reporter gene imaging as developed and applied in preclinical and clinical studies. Examples of different reporter systems are presented, with a focus on human reporter systems. Selected applications are discussed, including adoptive cell therapies, gene and oncoviral therapies, oncogenesis, signal pathway monitoring, and imaging drug treatment. Molecular imaging, and noninvasive reporter gene imaging in particular, are making important contributions to our understanding of disease development, progression, and treatment in our current era of molecular medicine and individualized patient care.
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http://dx.doi.org/10.2967/jnumed.111.099788DOI Listing
February 2013

Integrin αvβ3-targeted IRDye 800CW near-infrared imaging of glioblastoma.

Clin Cancer Res 2012 Oct 22;18(20):5731-40. Epub 2012 Aug 22.

Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Purpose: Integrin α(v)β(3) plays an important role in tumor angiogenesis, growth, and metastasis. We have tested a targeted probe to visualize integrin receptor expression in glioblastomas using near-infrared fluorescent (NIRF) imaging.

Experimental Design: A transgenic glioblastoma mouse model (RCAS-PDGF-driven/tv-a glioblastoma, which mimics the infiltrative growth pattern of human glioblastomas) and two human orthotopic glioblastoma models (U-87 MG with high integrin β(3) expression and TS543 with low integrin β(3) expression) were studied. An integrin-targeting NIRF probe, IRDye 800CW-cyclic-RGD peptide (IRDye 800CW-RGD), was tested by in vivo and ex vivo NIRF imaging.

Results: We show that the IRDye 800CW-RGD peptide: (i) specifically binds to integrin receptors; (ii) is selectively localized to glioblastoma tissue with overexpressed integrin receptors and is retained over prolonged periods of time; (iii) is associated with minimal autofluorescence and photobleaching because of imaging at 800 nm; (iv) provides delineation of tumor tissue with high precision because of a high tumor-to-normal brain fluorescence ratio (79.7 ± 6.9, 31.2 ± 2.8, and 16.3 ± 1.3) in the U-87 MG, RCAS-PDGF, and TS543 models, respectively; P < 0.01); and (v) enables fluorescence-guided glioblastoma resection. Importantly, small foci of residual fluorescence were observed after resection was completed using white light imaging alone, and these fluorescent foci were shown to represent residual tumor tissue by histology.

Conclusions: NIRF imaging with the IRDye 800CW-RGD probe provides a simple, rapid, low-cost, nonradioactive, and highly translatable approach for improved intraoperative glioblastoma visualization and resection. It also has the potential to serve as an imaging platform for noninvasive cancer detection and drug efficacy evaluation studies.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-0374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733255PMC
October 2012

Monitoring the induction of heat shock factor 1/heat shock protein 70 expression following 17-allylamino-demethoxygeldanamycin treatment by positron emission tomography and optical reporter gene imaging.

Mol Imaging 2012 Feb;11(1):67-76

Department of Neurology, Memorial Hospital,Sloan-KetteringInstitute, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

The cell response to proteotoxic cell stresses is mediated primarily through activation of heat shock factor 1 (HSF1). This transcription factor plays a major role in the regulation of the heat shock proteins (HSPs), including HSP70. We demonstrate that an [124I]iodide-pQHNIG70 positron emission tomography (PET) reporter system that includes an inducible HSP70 promoter can be used to image and monitor the activation of the HSF1/HSP70 transcription factor in response to drug treatment (17-allylamino-demethoxygeldanamycin [17-AAG]). We developed a dual imaging reporter (pQHNIG70) for noninvasive imaging of the heat shock response in cell culture and living animals previously and now study HSF1/HSP70 reporter activation in both cell culture and tumor-bearing animals following exposure to 17-AAG. 17-AAG (10-1,000 nM) induced reporter expression; a 23-fold increase was observed by 60 hours. Good correspondence between reporter expression and HSP70 protein levels were observed. MicroPET imaging based on [124I]iodide accumulation in pQHNIG70-transduced RG2 xenografts showed a significant 6.2-fold reporter response to 17-AAG, with a corresponding increase in tumor HSP70 and in tumor human sodium iodide symporter and green fluorescent protein reporter proteins. The HSF1 reporter system can be used to screen anticancer drugs for induction of cytotoxic stress and HSF1 activation both in vitro and in vivo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400108PMC
February 2012

Imaging: strategies, controversies, and opportunities.

Clin Cancer Res 2012 Feb;18(3):631-7

Department of Neurology, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

At a Clinical and Translational Cancer Research Think Tank meeting sponsored by the American Association for Cancer Research in 2010, one of the breakout groups focused on new technologies and imaging. The discussions emphasized new opportunities in translational imaging and its role in the future, rather than established techniques that are currently in clinical practice. New imaging methods under development are changing the approach of imaging science from a focus on the anatomic description of disease to a focus on the molecular basis of disease. Broadly referred to as molecular imaging, these new strategies directly embrace the incorporation of cell and molecular biology concepts and techniques into image generation and can involve the introduction of genes into cells with the explicit intent to image the end products of gene expression with external imaging devices. These new methods hold the promise of providing clinicians with (i) robust linkages between cell and animal models and clinical trials, (ii) in vivo biomarkers that can be measured repeatedly and sequentially over time to observe dynamic disease processes and responses to treatment, and (iii) tools for preselection and patient population enrichment in phase II and III trials to improve outcomes and better direct treatment. These strategies provide real-time pharmacodynamic parameters and can be powerful tools to monitor therapeutic effects in a spatially and tissue-specific manner, which may reduce cost during drug development, because pharmacodynamic studies in animals can inform clinical trials and accelerate the translation process. The Imaging Response Assessment Team (IRAT) program serves as an example of how imaging techniques can be incorporated into clinical trials. IRATs work to advance the role of imaging in assessment of response to therapy and to increase the application of quantitative anatomic, functional, and molecular imaging endpoints in clinical trials, and imaging strategies that will lead to individualized patient care.
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http://dx.doi.org/10.1158/1078-0432.CCR-11-2020DOI Listing
February 2012

MYCN and MYC regulate tumor proliferation and tumorigenesis directly through BMI1 in human neuroblastomas.

FASEB J 2011 Dec 19;25(12):4138-49. Epub 2011 Aug 19.

Department of Neurology, Memorial Sloan Kettering Cancer Center, 1275 York Ave., New York, NY 10065, USA.

The BMI1 gene is overexpressed in ≈ 90% of human neuroblastomas. However, little is known about the regulation of BMI1 expression. Using microarray and immunohistochemical analysis, we show that BMI1 expression correlated with MYCN levels in MYCN-amplified human neuroblastomas, and with MYC levels in the MYCN-nonamplified group. We further demonstrated that BMI1 is a direct target gene of MYCN/MYC in 3 neuroblastoma cell lines: BE (2)-C, LAN1, and SH-SY5Y. Overexpression of MYCN or MYC transactivated the BMI1 promoter and up-regulated BMI1 gene expression. shRNA-mediated knockdown of MYCN or MYC decreased BMI1 gene expression. Chromatin immunoprecipitation and point-mutation assays revealed that both MYCN and MYC bind to the E-box within the BMI1 promoter. Overexpression of BMI1, MYCN, and MYC independently increased both cell proliferation and tumor growth. Conversely, specific inhibition of BMI1, MYCN, and MYC decreased tumor cell proliferation and tumor growth. Interestingly, BMI1 suppression in MYCN/MYC-overexpressing cells resulted in significantly greater inhibition compared to that in mock-transduced and parental cells. Our results indicate that MYCN and MYC regulate BMI1 gene expression at the transcriptional level and that dysregulation of the BMI1 gene mediated by MYCN or MYC overexpression, confers increased cell proliferation during neuroblastoma genesis and tumor progression.
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http://dx.doi.org/10.1096/fj.11-185033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3236625PMC
December 2011

Metabolic imaging: a link between lactate dehydrogenase A, lactate, and tumor phenotype.

Clin Cancer Res 2011 Oct 15;17(19):6250-6261. Epub 2011 Aug 15.

Department of Medical Physics, Memorial Sloan Kettering Cancer Center, 415 E68 Street, New York, NY 10065, USA.

Purpose: We compared the metabolic profiles and the association between LDH-A expression and lactate production in two isogenic murine breast cancer cell lines and tumors (67NR and 4T1). These cell lines were derived from a single mammary tumor and have different growth and metabolic phenotypes.

Experimental Design: LDH-A expression, lactate concentration, glucose utilization, and oxygen consumption were measured in cells, and the potential relationship between tumor lactate levels [measured by magnetic resonance spectroscopic imaging (MRSI)] and tumor glucose utilization [measured by [(18)F]2-deoxy-2-fluoro-D-glucose positron emission tomography ([(18)F]FDG-PET)] was assessed in orthotopic breast tumors derived from these cell lines.

Results: We show a substantial difference in LDH-A expression between 67NR and 4T1 cells under normoxia and hypoxia. We also show that small orthotopic 4T1 tumors generate 10-fold more lactate than corresponding 67NR tumors. The high lactate levels in small primary 4T1 tumors are associated with intense pimonidazole staining (a hypoxia indicator). Less-intense hypoxia staining was observed in the larger 67NR tumors and is consistent with the gradual increase and plateau of lactate concentration in enlarging 67NR tumors.

Conclusions: Lactate-MRSI has a greater dynamic range than [(18)F]FDG-PET and may be a more sensitive measure with which to evaluate the aggressive and metastatic potential of primary breast tumors.
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http://dx.doi.org/10.1158/1078-0432.CCR-11-0397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4217119PMC
October 2011

The synthesis and evaluation of N1-(4-(2-[18F]-fluoroethyl)phenyl)-N8-hydroxyoctanediamide ([18F]-FESAHA), a PET radiotracer designed for the delineation of histone deacetylase expression in cancer.

Nucl Med Biol 2011 Jul 3;38(5):683-96. Epub 2011 Mar 3.

Radiochemistry Service, Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

Introduction: Given the significant utility of suberoylanilide hydroxamic acid (SAHA) in chemotherapeutic protocols, a PET tracer that mimics the histone deacetylase (HDAC) inhibition of SAHA could be a valuable tool in the diagnosis, treatment planning and treatment monitoring of cancer. Here, we describe the synthesis, characterization and evaluation of N(1)-(4-(2-[(18)F]-fluoroethyl)phenyl)-N(8)-hydroxyoctanediamide ([(18)F]-FESAHA), a PET tracer designed for the delineation of HDAC expression in cancer.

Methods: FESAHA was synthesized and biologically characterized in vivo and in vitro. [(18)F]-FESAHA was then synthesized in high radiochemical purity, and the logP and serum stability of the radiotracer were determined. In vitro cellular uptake experiments and acute biodistribution and small-animal PET studies were performed with [(18)F]-FESAHA in mice bearing LNCaP xenografts.

Results: [(18)F]-FESAHA was synthesized in high radiochemical purity via an innovative one-pot procedure. Enzymatic inhibition assays illustrated that FESAHA is a potent HDAC inhibitor, with IC(50) values from 3 nM to 1.7 μM against the 11 HDAC subtypes. Cell proliferation experiments revealed that the cytostatic properties of FESAHA very closely resemble those of SAHA in both LNCaP cells and PC-3 cells. Acute biodistribution and PET imaging experiments revealed tumor uptake of [(18)F]-FESAHA and substantially higher values in the small intestine, kidneys, liver and bone.

Conclusion: The significant non-tumor background uptake of [(18)F]-FESAHA presents a substantial obstacle to the use of the radiotracer as an HDAC expression imaging agent. The study at hand, however, does present a number of lessons critical to both the synthesis of hydroxamic acid containing PET radiotracers and imaging agents aimed at delineating HDAC expression.
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http://dx.doi.org/10.1016/j.nucmedbio.2010.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145497PMC
July 2011

ATP-binding cassette transporters modulate both coelenterazine- and D-luciferin-based bioluminescence imaging.

Mol Imaging 2011 Jun;10(3):215-26

Department of Neurology and Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Bioluminescence imaging (BLI) of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI readout intensity from intact living cells. To investigate the effect of ATP-binding cassette (ABC) transporters on BLI readout, we generated click beetle (cLuc), firefly (fLuc), Renilla (rLuc), and Gaussia (gLuc) luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2). In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type-independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052835PMC
June 2011