Israt Alam

All Publications

• Molecular Imaging of Acute Graft-Versus-Host Disease. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Bernardi, C., Garibotto, V., Mobashwera, B., Negrin, R. S., Alam, I. S., Simonetta, F. 2024

  Abstract

  Noninvasive molecular imaging of acute graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation has great potential to detect GvHD at the early stages, aid in grading of the disease, monitor treatment response, and guide therapeutic decisions. Although the specificity of currently available tracers appears insufficient for clinical GvHD diagnosis, recently, several preclinical studies have identified promising new imaging agents targeting one or more biologic processes involved in GvHD pathogenesis, ranging from T-cell activation to tissue damage. In this review, we summarize the different approaches reported to date for noninvasive detection of GvHD using molecular imaging with a specific focus on the use of PET. We discuss possible applications of molecular imaging for the detection of GvHD in the clinical setting, as well as some of the predictable challenges that are faced during clinical translation of these approaches.

  View details for DOI 10.2967/jnumed.123.266552

  View details for PubMedID 38360050

• PET Imaging of Innate Immune Activation Using 11C Radiotracers Targeting GPR84. JACS Au Kalita, M., Park, J. H., Kuo, R. C., Hayee, S., Marsango, S., Straniero, V., Alam, I. S., Rivera-Rodriguez, A., Pandrala, M., Carlson, M. L., Reyes, S. T., Jackson, I. M., Suigo, L., Luo, A., Nagy, S. C., Valoti, E., Milligan, G., Habte, F., Shen, B., James, M. L. 2023; 3 (12): 3297-3310

  Abstract

  Chronic innate immune activation is a key hallmark of many neurological diseases and is known to result in the upregulation of GPR84 in myeloid cells (macrophages, microglia, and monocytes). As such, GPR84 can potentially serve as a sensor of proinflammatory innate immune responses. To assess the utility of GPR84 as an imaging biomarker, we synthesized 11C-MGX-10S and 11C-MGX-11Svia carbon-11 alkylation for use as positron emission tomography (PET) tracers targeting this receptor. In vitro experiments demonstrated significantly higher binding of both radiotracers to hGPR84-HEK293 cells than that of parental control HEK293 cells. Co-incubation with the GPR84 antagonist GLPG1205 reduced the binding of both radiotracers by >90%, demonstrating their high specificity for GPR84 in vitro. In vivo assessment of each radiotracer via PET imaging of healthy mice illustrated the superior brain uptake and pharmacokinetics of 11C-MGX-10S compared to 11C-MGX-11S. Subsequent use of 11C-MGX-10S to image a well-established mouse model of systemic and neuro-inflammation revealed a high PET signal in affected tissues, including the brain, liver, lung, and spleen. In vivo specificity of 11C-MGX-10S for GPR84 was confirmed by the administration of GLPG1205 followed by radiotracer injection. When compared with 11C-DPA-713-an existing radiotracer used to image innate immune activation in clinical research studies-11C-MGX-10S has multiple advantages, including its higher binding signal in inflamed tissues in the CNS and periphery and low background signal in healthy saline-treated subjects. The pronounced uptake of 11C-MGX-10S during inflammation, its high specificity for GPR84, and suitable pharmacokinetics strongly support further investigation of 11C-MGX-10S for imaging GPR84-positive myeloid cells associated with innate immune activation in animal models of inflammatory diseases and human neuropathology.

  View details for DOI 10.1021/jacsau.3c00435

  View details for PubMedID 38155640

  View details for PubMedCentralID PMC10751761

• Application of Machine Learning Driven Computational Approaches for Novel CNS PET Tracer Development Jackson, I., Luo, A., Webb, E., Zhang, B., Guo, A., Nagy, S., Shao, X., Kuo, R., Carlson, M., Alam, I., Rodriguez, A., Winton, W., Stauff, J., Kalita, M., Scott, P., James, M. ELSEVIER SCIENCE INC. 2023: S40-S41

  View details for Web of Science ID 001128725600053

• Development and Initial Assessment of [18F]OP-801: a Novel Hydroxyl Dendrimer PET Tracer for Preclinical Imaging of Innate Immune Activation in the Whole Body and Brain. Molecular imaging and biology Carlson, M. L., Jackson, I. M., Azevedo, E. C., Reyes, S. T., Alam, I. S., Kellow, R., Castillo, J. B., Nagy, S. C., Sharma, R., Brewer, M., Cleland, J., Shen, B., James, M. L. 2023

  Abstract

  PURPOSE: Innate immune activation plays a critical role in the onset and progression of many diseases. While positron emission tomography (PET) imaging provides a non-invasive means to visualize and quantify such immune responses, most available tracers are not specific for innate immune cells. To address this need, we developed [18F]OP-801 by radiolabeling a novel hydroxyl dendrimer that is selectively taken up by reactive macrophages/microglia and evaluated its ability to detect innate immune activation in mice following lipopolysaccharide (LPS) challenge.PROCEDURES: OP-801 was radiolabeled in two steps: [18F]fluorination of a tosyl precursor to yield [18F]3-fluoropropylazide, followed by a copper-catalyzed click reaction. After purification and stability testing, [18F]OP-801 (150-250 muCi) was intravenously injected into female C57BL/6 mice 24 h after intraperitoneal administration of LPS (10 mg/kg, n=14) or saline (n=6). Upon completing dynamic PET/CT imaging, mice were perfused, and radioactivity was measured in tissues of interest via gamma counting or autoradiography.RESULTS: [18F]OP-801 was produced with >95% radiochemical purity, 12-52 muCi/mug specific activity, and 4.3±1.5% decay-corrected yield. Ex vivo metabolite analysis of plasma samples (n=4) demonstrated high stability in mice (97±3% intact tracer >120 min post-injection). PET/CT images of mice following LPS challenge revealed higher signal in organs known to be inflamed in this context, including the liver, lung, and spleen. Gamma counting confirmed PET findings, showing significantly elevated signal in the same tissues compared to saline-injected mice: the liver (p=0.009), lung (p=0.030), and spleen (p=0.004). Brain PET/CT images (summed 50-60 min) revealed linearly increasing [18F]OP-801 uptake in the whole brain that significantly correlated with murine sepsis score (r=0.85, p<0.0001). Specifically, tracer uptake was significantly higher in the brain stem, cortex, olfactory bulb, white matter, and ventricles of LPS-treated mice compared to saline-treated mice (p<0.05).CONCLUSION: [18F]OP-801 is a promising new PET tracer for sensitive and specific detection of activated macrophages and microglia that warrants further investigation.

  View details for DOI 10.1007/s11307-023-01850-5

  View details for PubMedID 37735280

• PET imaging of TREM1 identifies CNS-infiltrating myeloid cells in a mouse model of multiple sclerosis. Science translational medicine Chaney, A. M., Cropper, H. C., Jain, P., Wilson, E., Simonetta, F., Johnson, E. M., Alam, I. S., Patterson, I. T., Swarovski, M., Stevens, M. Y., Wang, Q., Azevedo, C., Nagy, S. C., Ramos Benitez, J., Deal, E. M., Vogel, H., Andreasson, K. I., James, M. L. 2023; 15 (702): eabm6267

  Abstract

  Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) that causes substantial morbidity and diminished quality of life. Evidence highlights the central role of myeloid lineage cells in the initiation and progression of MS. However, existing imaging strategies for detecting myeloid cells in the CNS cannot distinguish between beneficial and harmful immune responses. Thus, imaging strategies that specifically identify myeloid cells and their activation states are critical for MS disease staging and monitoring of therapeutic responses. We hypothesized that positron emission tomography (PET) imaging of triggering receptor expressed on myeloid cells 1 (TREM1) could be used to monitor deleterious innate immune responses and disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We first validated TREM1 as a specific marker of proinflammatory, CNS-infiltrating, peripheral myeloid cells in mice with EAE. We show that the 64Cu-radiolabeled TREM1 antibody-based PET tracer monitored active disease with 14- to 17-fold higher sensitivity than translocator protein 18 kDa (TSPO)-PET imaging, the established approach for detecting neuroinflammation in vivo. We illustrate the therapeutic potential of attenuating TREM1 signaling both genetically and pharmacologically in the EAE mice and show that TREM1-PET imaging detected responses to an FDA-approved MS therapy with siponimod (BAF312) in these animals. Last, we observed TREM1+ cells in clinical brain biopsy samples from two treatment-naïve patients with MS but not in healthy control brain tissue. Thus, TREM1-PET imaging has potential for aiding in the diagnosis of MS and monitoring of therapeutic responses to drug treatment.

  View details for DOI 10.1126/scitranslmed.abm6267

  View details for PubMedID 37379371

• Clinical Radiosynthesis and Translation of [18F]OP-801: A Novel Radiotracer for Imaging Reactive Microglia and Macrophages. ACS chemical neuroscience Jackson, I. M., Carlson, M. L., Beinat, C., Malik, N., Kalita, M., Reyes, S., Azevedo, E. C., Nagy, S. C., Alam, I. S., Sharma, R., La Rosa, S. A., Moradi, F., Cleland, J., Shen, B., James, M. L. 2023

  Abstract

  Positron emission tomography (PET) is a powerful tool for studying neuroinflammatory diseases; however, current PET biomarkers of neuroinflammation possess significant limitations. We recently reported a promising dendrimer PET tracer ([18F]OP-801), which is selectively taken up by reactive microglia and macrophages. Here, we describe further important characterization of [18F]OP-801 in addition to optimization and validation of a two-step clinical radiosynthesis. [18F]OP-801 was found to be stable in human plasma for 90 min post incubation, and human dose estimates were calculated for 24 organs of interest; kidneys and urinary bladder wall without bladder voiding were identified as receiving the highest absorbed dose. Following optimization detailed herein, automated radiosynthesis and quality control (QC) analyses of [18F]OP-801 were performed in triplicate in suitable radiochemical yield (6.89 ± 2.23% decay corrected), specific activity (37.49 ± 15.49 GBq/mg), and radiochemical purity for clinical imaging. Importantly, imaging mice with tracer (prepared using optimized methods) 24 h following the intraperitoneal injection of liposaccharide resulted in the robust brain PET signal. Cumulatively, these data enable clinical translation of [18F]OP-801 for imaging reactive microglia and macrophages in humans. Data from three validation runs of the clinical manufacturing and QC were submitted to the Food and Drug Administration (FDA) as part of a Drug Master File (DMF). Subsequent FDA approval to proceed was obtained, and a phase 1/2 clinical trial (NCT05395624) for first-in-human imaging in healthy controls and patients with amyotrophic lateral sclerosis is underway.

  View details for DOI 10.1021/acschemneuro.3c00028

  View details for PubMedID 37310119

• Development and initial evaluation of a novel 11C-labeled PET tracer to image GPR84 expressing-myeloid cells during neuroinflammation Kalita, M., Park, J., Hayee, S., Marsango, S., Carlson, M., Reyes, S., Nagy, S., Straniero, V., Pandrala, M., Jackson, I., Alam, I., Valoti, E., Milligan, G., Shen, B., James, M. SOC NUCLEAR MEDICINE INC. 2023

  View details for Web of Science ID 001109210200159

• Highly Excretable Gold Supraclusters for Translatable In Vivo Raman Imaging of Tumors. ACS nano Yu, J. H., Jeong, M. S., Cruz, E. O., Alam, I. S., Tumbale, S. K., Zlitni, A., Lee, S. Y., Park, Y. I., Ferrara, K., Kwon, S., Gambhir, S. S., Rao, J. 2023

  Abstract

  Raman spectroscopy provides excellent specificity for in vivo preclinical imaging through a readout of fingerprint-like spectra. To achieve sufficient sensitivity for in vivo Raman imaging, metallic gold nanoparticles larger than 10 nm were employed to amplify Raman signals via surface-enhanced Raman scattering (SERS). However, the inability to excrete such large gold nanoparticles has restricted the translation of Raman imaging. Here we present Raman-active metallic gold supraclusters that are biodegradable and excretable as nanoclusters. Although the small size of the gold nanocluster building blocks compromises the electromagnetic field enhancement effect, the supraclusters exhibit bright and prominent Raman scattering comparable to that of large gold nanoparticle-based SERS nanotags due to high loading of NIR-resonant Raman dyes and much suppressed fluorescence background by metallic supraclusters. The bright Raman scattering of the supraclusters was pH-responsive, and we successfully performed in vivo Raman imaging of acidic tumors in mice. Furthermore, in contrast to large gold nanoparticles that remain in the liver and spleen over 4 months, the supraclusters dissociated into small nanoclusters, and 73% of the administered dose to mice was excreted during the same period. The highly excretable Raman supraclusters demonstrated here offer great potential for clinical applications of in vivo Raman imaging.

  View details for DOI 10.1021/acsnano.2c10378

  View details for PubMedID 36688431

• ICOS ImmunoPET Enables Visualization of Activated T Cells and Early Diagnosis of Murine Acute Gastrointestinal GvHD. Blood advances Xiao, Z., Alam, I. S., Simonetta, F., Chen, W., Scheller, L., Murty, S., Lohmeyer, J. K., Lopes Ramos, T. L., James, M. L., Negrin, R. S., Gambhir, S. S. 2022

  Abstract

  Allogeneic hematopoietic cell transplantation (HCT) is a well-established and potentially curative treatment for a broad range of hematological diseases, bone marrow failure states and genetic disorders. Acute graft-versus-host-disease (GvHD), mediated by donor T cells attacking host tissue, still represents a major cause of morbidity and mortality following allogeneic HCT. Current approaches to diagnosis of gastrointestinal acute GvHD rely on clinical and pathological criteria that manifest at late stages of disease. New strategies allowing for GvHD prediction and diagnosis, prior to symptom onset, are urgently needed. Noninvasive antibody-based PET (immunoPET) imaging of T cell activation post allogeneic HCT is a promising strategy towards this goal. In this work, we identified Inducible T-cell COStimulator (ICOS) as a potential immunoPET target for imaging activated T cells during GvHD. We demonstrate that the use of the 89Zr-DFO-ICOS monoclonal antibody (mAb) PET tracer, allows in vivo visualization of donor T cell activation in target tissues, namely the intestinal tract, in a murine model of acute GvHD. Importantly, we demonstrate that the 89Zr-DFO-ICOS mAb PET tracer does not affect GvHD pathogenesis or the graft-versus-tumor (GvT) effect of the transplant procedure. Our data identify ICOS immunoPET as a promising strategy for early GvHD diagnosis prior to the appearance of clinical symptoms.

  View details for DOI 10.1182/bloodadvances.2022007403

  View details for PubMedID 35790103

• Engineering genetically-encoded synthetic biomarkers for breath-based cancer detection Vermesh, O., D'Souza, A., Alam, I., Wardak, M., McLaughlin, T., El Rami, F., Sathirachinda, A., Bell, J., Pitteri, S., James, M., Hori, S., Gross, E., Gambhir, S. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509500130

• Multiparameter Longitudinal Imaging of Immune Cell Activity in Chimeric Antigen Receptor T Cell and Checkpoint Blockade Therapies. ACS central science Xie, J., El Rami, F., Zhou, K., Simonetta, F., Chen, Z., Zheng, X., Chen, M., Balakrishnan, P. B., Dai, S., Murty, S., Alam, I. S., Baker, J., Negrin, R. S., Gambhir, S. S., Rao, J. 2022; 8 (5): 590-602

  Abstract

  Longitudinal multimodal imaging presents unique opportunities for noninvasive surveillance and prediction of treatment response to cancer immunotherapy. In this work we first designed a novel granzyme B activated self-assembly small molecule, G-SNAT, for the assessment of cytotoxic T lymphocyte mediated cancer cell killing. G-SNAT was found to specifically detect the activity of granzyme B within the cytotoxic granules of activated T cells and engaged cancer cells in vitro. In lymphoma tumor-bearing mice, the retention of cyanine 5 labeled G-SNAT-Cy5 correlated to CAR T cell mediated granzyme B exocytosis and tumor eradication. In colorectal tumor-bearing transgenic mice with hematopoietic cells expressing firefly luciferase, longitudinal bioluminescence and fluorescence imaging revealed that after combination treatment of anti-PD-1 and anti-CTLA-4, the dynamics of immune cell trafficking, tumor infiltration, and cytotoxic activity predicted the therapeutic outcome before tumor shrinkage was evident. These results support further development of G-SNAT for imaging early immune response to checkpoint blockade and CAR T-cell therapy in patients and highlight the utility of multimodality imaging for improved mechanistic insights into cancer immunotherapy.

  View details for DOI 10.1021/acscentsci.2c00142

  View details for PubMedID 35647285

• Visualizing T cell responses: The T cell PET imaging toolbox. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Li, C., Han, C., Duan, S., Li, P., Alam, I., Xiao, Z. 2021

  Abstract

  T lymphocytes are key mediators of the adaptive immune response. Inappropriate or imbalanced T cell responses are underlying factors in cancer progression, allergy and other immune disorders. Monitoring the spatiotemporal dynamics of T cells and their functional status has the potential to provide unique biological insights in health and disease. Non-invasive positron emission tomography (PET) imaging represents an ideal whole-body modality for achieving this goal. With the appropriate PET imaging probes, T cell dynamics can be monitored in vivo, with high specificity and sensitivity. Herein, we provide a comprehensive overview of the applications of this state-of-the-art T cell PET imaging toolbox, and the potential it has to improve the clinical management of cancer immunotherapy and T cell- driven diseases. We also discuss future directions and prospects for clinical translation.

  View details for DOI 10.2967/jnumed.121.261976

  View details for PubMedID 34887338

• A Clinical PET Imaging Tracer ([18F]DASA-23) to Monitor Pyruvate Kinase M2 Induced Glycolytic Reprogramming in Glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research Beinat, C., Patel, C. B., Haywood, T., Murty, S., Naya, L., Castillo, J. B., Reyes, S. T., Phillips, M., Buccino, P., Shen, B., Park, J. H., Koran, M. E., Alam, I. S., James, M. L., Holley, D., Halbert, K., Gandhi, H., He, J. Q., Granucci, M., Johnson, E., Liu, D. D., Uchida, N., Sinha, R., Chu, P., Born, D. E., Warnock, G. I., Weissman, I., Hayden Gephart, M., Khalighi, M. M., Massoud, T. F., Iagaru, A., Davidzon, G., Thomas, R., Nagpal, S., Recht, L. D., Gambhir, S. S. 2021

  Abstract

  PURPOSE: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in healthy brain. We describe the development, validation, and translation of a novel positron emission tomography (PET) tracer to study PKM2 in GBM. We evaluated 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and GBM patients.EXPERIMENTAL DESIGN: [18F]DASA-23 was synthesized with a molar activity of 100.47 {plus minus} 29.58 GBq/mol and radiochemical purity >95%. We performed initial testing of [18F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next we produced [18F]DASA-23 under FDA oversight, and evaluated it in healthy volunteers, and a pilot cohort of glioma patients.RESULTS: In mouse imaging studies, [18F]DASA-23 clearly delineated the U87 GBM from surrounding healthy brain tissue and had a tumor-to-brain ratio (TBR) of 3.6 {plus minus} 0.5. In human volunteers, [18F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared. In GBM patients, [18F]DASA-23 successfully outlined tumors visible on contrast-enhanced magnetic resonance imaging (MRI). The uptake of [18F]DASA-23 was markedly elevated in GBMs compared to normal brain, and it identified a metabolic non-responder within 1-week of treatment initiation.CONCLUSIONS: We developed and translated [18F]DASA-23 as a new tracer that demonstrated the visualization of aberrantly expressed PKM2 for the first time in human subjects. These results warrant further clinical evaluation of [18F]DASA-23 to assess its utility for imaging therapy-induced normalization of aberrant cancer metabolism.

  View details for DOI 10.1158/1078-0432.CCR-21-0544

  View details for PubMedID 34475101

• Neurovascular, muscle, and skin changes on [18F]FDG PET/MRI in complex regional pain syndrome of the foot: A Prospective Clinical Study. Pain medicine (Malden, Mass.) Yoon, D., Xu, Y., Cipriano, P. W., Alam, I. S., Mari Aparici, C. A., Tawfik, V. L., Curtin, C. M., Carroll, I. R., Biswal, S. 2021

  Abstract

  The goal of this study is to demonstrate the feasibility of simultaneous [18F]-fluorodeoxyglucose (FDG) positron emission tomography (PET) and magnetic resonance imaging (MRI) for non-invasive visualization of muscular, neurovascular, and skin changes secondary to complex regional pain syndrome (CRPS).Seven adult patients with CRPS of the foot and seven healthy adult controls participated in our [18F]FDG PET/MRI study.All participants received whole-body PET/MRI scans one hour after the injection of 370MBq [18F]FDG. Resulting PET/MRI images were reviewed by two radiologists. Metabolic and anatomic abnormalities identified, were grouped into muscular, neurovascular, and skin lesions. The [18F]FDG uptake of each lesion was compared with that of corresponding areas in controls using a Mann-Whitney U-test.On PET images, muscular abnormalities were found in five patients, neurovascular abnormalities in four patients, and skin abnormalities in two patients. However, on MRI images, no muscular abnormalities were detected. Neurovascular abnormalities and skin abnormalities in the affected limb were identified on MRI in one and two patients, respectively. The difference in [18F]FDG uptake between the patients and the controls was significant in muscle (p = 0.018) and neurovascular bundle (p = 0.0005).The increased uptake of [18F]FDG in the symptomatic areas likely reflects the increased metabolism due to the inflammatory response causing pain. Therefore, our approach combining metabolic [18F]FDG PET and anatomic MR imaging may offer non-invasive monitoring of the distribution and progression of inflammatory changes associated with CRPS.

  View details for DOI 10.1093/pm/pnab315

  View details for PubMedID 34718774

• A miniaturized optoelectronic biosensor for real-time point-of-care total protein analysis. MethodsX Vermesh, O., Mahzabeen, F., Levi, J., Tan, M., Alam, I. S., Chan, C. T., Gambhir, S. S., Harris, J. S. 2021; 8: 101414

  Abstract

  A miniaturized optoelectronic sensor is demonstrated that measures total protein concentration in serum and urine with sensitivity and accuracy comparable to gold-standard methods. The sensor is comprised of a vertical cavity surface emitting laser (VCSEL), photodetector and other custom optical components and electronics that can be hybrid packaged into a portable, handheld form factor. In conjunction, a custom fluorescence assay has been developed based on the protein-induced fluorescence enhancement (PIFE) phenomenon, enabling real-time sensor response to changes in protein concentration. Methods are described for the following:Standard curves: Used to determine the sensitivity, dynamic range, and linearity of the VCSEL biosensor/PIFE assay system in buffer as well as in human blood and urine samples.Comparison of VCSEL biosensor performance with a benchtop fluorimetric microplate reader.Accuracy of the VCSEL biosensor/PIFE assay system: Evaluated by comparing sensor measurements with gold-standard clinical laboratory measurements of total protein in serum and urine samples from patients with diabetes.

  View details for DOI 10.1016/j.mex.2021.101414

  View details for PubMedID 34430309

• Minicircles for a two-step blood biomarker and PET imaging early cancer detection strategy. Journal of controlled release : official journal of the Controlled Release Society Robinson, E. R., Gowrishankar, G., D'Souza, A., Kheirolomoom, A., Haywood, T., Hori, S. S., Chuang, H. Y., Zeng, Y., Tumbale, S., Aalipour, A., Beinat, C., Alam, I. S., Sathirachinda, A., Kanada, M., Paulmurugan, R., Ferrara, K. W., Gambhir, S. S. 2021

  Abstract

  Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection agent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection agent. Secreted GLuc correlated with HSV-1 sr39TK expression with R2 = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.

  View details for DOI 10.1016/j.jconrel.2021.05.026

  View details for PubMedID 34029631

• Nuclear Imaging of Endogenous Markers of Lymphocyte Response Nuclear Medicine and Immunology Alam, I. S., Shaffer, T. M., Gambhir, S. S. Springer. 2021: 15-59

  View details for DOI https://doi.org/10.1007/978-3-030-81261-4_2

• Real-time point-of-care total protein measurement with a miniaturized optoelectronic biosensor and fast fluorescence-based assay. Biosensors & bioelectronics Mahzabeen, F., Vermesh, O., Levi, J., Tan, M., Alam, I. S., Chan, C. T., Gambhir, S. S., Harris, J. S. 2020: 112823

  Abstract

  Measurement of total protein in urine is key to monitoring kidney health in diabetes. However, most total protein assays are performed using large, expensive laboratory chemistry analyzers that are not amenable to point-of-care analysis or home monitoring and cannot provide real-time readouts. We developed a miniaturized optoelectronic biosensor using a vertical cavity surface-emitting laser (VCSEL), coupled with a fast protein assay based on protein-induced fluorescence enhancement (PIFE), that can dynamically measure protein concentrations in protein-spiked buffer, serum, and urine in seconds with excellent sensitivity (urine LOD = 0.023 g/L, LOQ = 0.075 g/L) and over a broad range of physiologically relevant concentrations. Comparison with gold standard clinical assays and standard fluorimetry tools showed that the sensor can accurately and reliably quantitate total protein in clinical urine samples from patients with diabetes. Our VCSEL biosensor is amenable to integration with miniaturized electronics, which could afford a portable, low-cost, easy-to-use device for sensitive, accurate, and real-time total protein measurements from small biofluid volumes.

  View details for DOI 10.1016/j.bios.2020.112823

  View details for PubMedID 33715946

• Molecular Imaging of Chimeric Antigen Receptor T Cells by ICOS-ImmunoPET. Clinical cancer research : an official journal of the American Association for Cancer Research Simonetta, F., Alam, I. S., Lohmeyer, J. K., Sahaf, B., Good, Z., Chen, W., Xiao, Z., Hirai, T., Scheller, L., Engels, P., Vermesh, O., Robinson, E., Haywood, T., Sathirachinda, A., Baker, J., Malipatlolla, M. B., Schultz, L. M., Spiegel, J. Y., Lee, J. T., Miklos, D. B., Mackall, C. L., Gambhir, S. S., Negrin, R. 2020

  Abstract

  PURPOSE: Immunomonitoring of chimeric antigen receptor (CAR) T cells relies primarily on their quantification in the peripheral blood, which inadequately quantifies their biodistribution and activation status in the tissues. Non-invasive molecular imaging of CAR T cells by positron emission tomography (PET) is a promising approach with the ability to provide spatial, temporal and functional information. Reported strategies rely on the incorporation of reporter transgenes or ex vivo biolabeling, significantly limiting the application of CAR T cell molecular imaging. In the present study, we assessed the ability of antibody-based PET (immunoPET) to non-invasively visualize CAR T cells.EXPERIMENTAL DESIGN: After analyzing human CAR T cells in vitro and ex vivo from patient samples to identify candidate targets for immunoPET, we employed a syngeneic, orthotopic murine tumor model of lymphoma to assess the feasibility of in vivo tracking of CAR T cells by immunoPET using the 89Zr-DFO-anti-ICOS tracer we previously reported.RESULTS: Analysis of human CD19-CAR T cells during activation identified the Inducible T-cell COStimulator (ICOS) as a potential target for immunoPET. In a preclinical tumor model, 89Zr-DFO-ICOS mAb PET-CT imaging detected significantly higher signal in specific bone marrow-containing skeletal sites of CAR T cell treated mice compared with controls. Importantly, administration of ICOS-targeting antibodies at tracer doses did not interfere with CAR T cell persistence and function.CONCLUSIONS: This study highlights the potential of ICOS-immunoPET imaging for monitoring of CAR T cell therapy, a strategy readily applicable to both commercially available and investigational CAR T cells.

  View details for DOI 10.1158/1078-0432.CCR-20-2770

  View details for PubMedID 33087332

• PET reporter gene imaging and ganciclovir-mediated ablation of chimeric antigen receptor T-cells in solid tumors. Cancer research Murty, S., Labanieh, L., Murty, T., Gowrishankar, G., Haywood, T., Alam, I. S., Beinat, C., Robinson, E., Aalipour, A., Klysz, D. D., Cochran, J. R., Majzner, R. G., Mackall, C. L., Gambhir, S. S. 2020

  Abstract

  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. Additionally, 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 positron emission tomography (PET) reporter gene for imaging of T-cell trafficking in brain tumor patients. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir (GCV). Here we report the molecular engineering, imaging, and GCV-mediated destruction of B7H3 CAR T-cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for GCV. 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 GCV 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.

  View details for DOI 10.1158/0008-5472.CAN-19-3579

  View details for PubMedID 32958548

• Visualization of activated T cells by OX40-immunoPET as a strategy for diagnosis of acute Graft-versus-Host-Disease. Cancer research Alam, I. S., Simonetta, F., Scheller, L., Mayer, A. T., Murty, S., Vermesh, O., Nobashi, T. W., Lohmeyer, J. K., Hirai, T., Baker, J., Lau, K. H., Negrin, R., Gambhir, S. S. 2020

  Abstract

  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. Non-invasive strategies detecting T cell activation would allow for early diagnosis and possibly more effective management of HCT recipients. Positron emission tomography (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 tumor necrosis factor (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 a major histocompatibility complex (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.

  View details for DOI 10.1158/0008-5472.CAN-20-1149

  View details for PubMedID 32900772

• Clinical and radiological features of immune checkpoint inhibitor-related pneumonitis in lung cancer and non-lung cancers. The British journal of radiology Nobashi, T. W., Nishimoto, Y., Kawata, Y., Yutani, H., Nakamura, M., Tsuji, Y., Yoshida, A., Sugimoto, A., Yamamoto, T., Alam, I. S., Noma, S. 2020: 20200409

  Abstract

  OBJECTIVE: To investigate the clinical and radiological features of immune checkpoint inhibitor-related pneumonitis (ICI-P), a rare but serious pulmonary complication of cancer immunotherapy and to evaluate key differences between lung cancer (LC) and non-LC patients.METHODS: 247 patients (LC, n = 151) treated with ICI for malignancies were retrospectively screened in a single institute. The number of patients, history of other immune-related adverse events (irAE), the onset, serum KL-6 levels, and chest CT features (types of pneumonitis, symmetry, laterality, location) were recorded for the ICI-P population and compared for LC and non-LC groups.RESULTS: ICI-P was identified in 26 patients in total (LC, n = 19; non-LC, n = 7). The incidence of other irAE was significantly higher in ICI-P group (63%) compared with patients without ICI-P (34%) (p = 0.0056). An earlier onset of ICI-P was recorded in LC (78 days) compared to non-LC patients (186 days) (p = 0.0034). Serum KL-6 was significantly elevated only in the non-LC group when ICI-P was noticed (p = 0.029). Major CT findings of ICI-P, irrespective of primary disease, were organizing pneumonia pattern and ground glass opacities. LC patients commonly exhibited consolidation and traction bronchiectasis and were prone to asymmetrical shadows (p < 0.001). Non-LC patients were more likely to exhibit symmetrical infiltrations. A small fraction of both groups experienced relapse or moving patterns of ICI-P.CONCLUSION: ICI-P patients more often experienced other irAE prior to the development of ICI-P. The characteristics of ICI-P can differ in terms of the onset, KL-6 reliability, and chest CT findings between LC and non-LC patients.ADVANCES IN KNOWLEDGE: In ICI-P patients, a history of other irAE can be more frequently observed. Differences in disease onset and radiological patterns between LC and non-LC patients might be helpful to make a diagnosis of ICI-P; however, longitudinal observation of chest CT scans is advised to observe the pneumonitis activity irrespective of cancer types.

  View details for DOI 10.1259/bjr.20200409

  View details for PubMedID 32783627

• Intravital imaging reveals synergistic effect of CAR T-cells and radiation therapy in a preclinical immunocompetent glioblastoma model Oncoimmunology Murty, S., Haile, S. T., Beinat, C., Aalipour, A., Alam, I. S., Murty, T., Shaffer, T. M., Patel, C. B., Graves, E. E., Mackall, C. L., Gambhir, S. S. 2020; 9 (1)

  View details for DOI 10.1080/2162402X.2020.1757360

• Evaluation of [18F]DASA-23 for non-invasive measurement of aberrantly expressed pyruvate kinase M2 in glioblastoma: preclinical and first in human studies Beinat, C., Patel, C., Haywood, T., Murty, S., Alam, I., Xie, Y., Gandhi, H., Holley, D., Gambhir, S. SOC NUCLEAR MEDICINE INC. 2019

  View details for Web of Science ID 000473116800052

• Development and Evaluation of an 18F-Radiolabeled Monocyclam Derivative for Imaging CXCR4 Expression. Molecular pharmaceutics Brickute, D., Braga, M., Kaliszczak, M. A., Barnes, C., Lau, D., Carroll, L., Stevens, E., Trousil, S., Alam, I. S., Nguyen, Q. D., Aboagye, E. O. 2019

  Abstract

  C-X-C chemokine receptor type 4 (CXCR4) is a protein that in humans is encoded by the CXCR4 gene and binds the ligand CXCL12 (also known as SDF-1). The CXCR4-CXCL12 interaction in cancer elicits biological activities that result in tumor progression and has accordingly been the subject of significant investigation for detection and treatment of disease. Peptidic antagonists have been labeled with a variety of radioisotopes for detection of CXCR4, but methodology utilizing small molecules have predominantly used radiometals. We report here the development of a 18F-radiolabeled cyclam-based small molecule radioprobe, [18F]MCFB, for imaging CXCR4 expression. The IC50 of [19F]MCFB for CXCR4 was similar to that of AMD3465 (111.3 and 89.9 nM, respectively). In vitro binding assays show that the tracer depicted differential CXCR4 expression, which was blocked in the presence of AMD3465, demonstrating specificity of [18F]MCFB. Positron emission tomography (PET) imaging studies showed distinct uptake of radioprobe in lymphoma and breast cancer xenografts. High liver and kidney uptake were seen with [18F]MCFB leading us to further examine the basis of its pharmacokinetics in relation to the tracer's cationic nature, thus, the role of organic cation transporters (OCTs). Substrate competition following the intravenous injection of metformin led to a marked decrease in urinary excretion of [18F]MCFB, with moderate changes observed in other organs, including the liver. Our results suggest involvement of OCTs in renal elimination of the tracer. In conclusion, the 18F radiolabeled monocyclam, [18F]MCFB, has potential to detect tumor CXCR4 in non-hepatic tissue.

  View details for DOI 10.1021/acs.molpharmaceut.9b00069

  View details for PubMedID 30883140

• The characterization of 18F-hGTS13 for molecular imaging of xC- transporter activity with positron emission tomography. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Beinat, C. n., Gowrishankar, G. n., Shen, B. n., Alam, I. S., Robinson, E. n., Haywood, T. n., Patel, C. B., Azevedo, E. C., Castillo, J. n., Ilovich, O. n., Koglin, N. n., Schmitt-Willich, H. n., Berndt, M. n., Mueller, A. n., Zerna, M. n., Srinivasan, A. n., Gambhir, S. S. 2019

  Abstract

  Purpose: The aim of this study was development of an improved positron emission tomography (PET) radiotracer for measuring xC- activity with increased tumor uptake and reduced uptake in inflammatory cells compared to (S)-4-(3-18F-Fluoropropyl)-L-glutamic acid (18F-FSPG). Experimental design: A racemic glutamate derivative, 18F-hGTS13 was evaluated in cell culture and animal tumor models. 18F-hGTS13 was separated into C5-epimers and the corresponding 18F-hGTS13-isomer1 and 18F-hGTS13-isomer2 evaluated in H460 tumor bearing rats. Preliminary studies investigate the cellular uptake of 18F-hGTS13-isomer2 in multiple immune cell populations and states. Results:18F-hGTS13 demonstrated excellent H460 tumor visualization with high tumor-to-background ratios, confirmed by ex vivo biodistribution studies. Tumor associated radioactivity of 18F-hGTS13 (7.5±0.9%ID/g, n = 3) was significantly higher than with 18F-FSPG (4.6±0.7%ID/g, n = 3, P = 0.01). 18F-hGTS13-isomer2 exhibited excellent H460 tumor visualization (6.3±1.1%ID/g, n-3), and significantly reduced uptake in multiple immune cell populations relative to 18F-FSPG. 18F-hGTS13-isomer2 exhibited increased liver uptake relative to 18F-FSPG (4.6±0.8%ID/g vs. 0.7±0.01%ID/g) limiting its application in hepatocellular carcinoma. Conclusion:18F-hGTS13-isomer2 is a new PET radiotracer for molecular imaging of xC- activity which may provide information regarding tumor oxidation states. 18F-hGTS13-isomer2 has potential for clinical translation for imaging cancers of the thorax due to the low background signal in healthy tissue.

  View details for DOI 10.2967/jnumed.119.225870

  View details for PubMedID 31171595

• Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence Front. Bioeng. Biotechnol Sônego, F., Bouccara, S., Pons, T., Alam, I. S., Shorte, S. L., Tournebize, R. 2019

  View details for DOI 10.3389/fbioe.2019.00073

• Positron emission tomography reporter gene strategy for use in the central nervous system PNAS Haywood, T., Beinat, C., Gowrishankar, G., Patel, C. B., Alam, I. S., Murty, S., Gambhir, S. S. 2019

  View details for DOI 10.1073/pnas.1901645116

• Tracking T Cell Activation By OX40 Immuno-PET: A Novel Strategy for Imaging of Graft Versus Host Disease Simonetta, F., Alam, I. S., Mayer, A. T., Murty, S., Vermesh, O., Hirai, T., Nobashi, T., Lau, K., Gambhir, S. S., Negrin, R. S. AMER SOC HEMATOLOGY. 2018

  View details for DOI 10.1182/blood-2018-99-116483

  View details for Web of Science ID 000454842804170

• An intravascular magnetic wire for the high-throughput retrieval of circulating tumour cells in vivo NATURE BIOMEDICAL ENGINEERING Vermesh, O., Aalipour, A., Ge, T., Saenz, Y., Guo, Y., Alam, I. S., Park, S., Adelson, C. N., Mitsutake, Y., Vilches-Moure, J., Godoy, E., Bachmann, M. H., Ooi, C., Lyons, J. K., Mueller, K., Arami, H., Green, A., Solomon, E., Wang, S. X., Gambhir, S. S. 2018; 2 (9): 696–705

  View details for DOI 10.1038/s41551-018-0257-3

  View details for Web of Science ID 000444282800014

• Positron emission tomography imaging of activated T cells by targeting OX40 reveals spatiotemporal immune dynamics and predicts response to in situ tumor vaccination Mayer, A. T., Alam, I. S., Sagiv-Barfi, I., Wang, K., Vermesh, O., Czerwinski, D. K., Johnson, E. M., James, M. L., Levy, R., Gambhir, S. S. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-3031

  View details for Web of Science ID 000468819500394

• Emerging Intraoperative Imaging Modalities to Improve Surgical Precision. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging Alam, I. S., Steinberg, I., Vermesh, O., van den Berg, N. S., Rosenthal, E. L., van Dam, G. M., Ntziachristos, V., Gambhir, S. S., Hernot, S., Rogalla, S. 2018

  Abstract

  Intraoperative imaging (IOI) is performed to guide delineation and localization of regions of surgical interest. While oncological surgical planning predominantly utilizes x-ray computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US), intraoperative guidance mainly remains on surgeon interpretation and pathology for confirmation. Over the past decades however, intraoperative guidance has evolved significantly with the emergence of several novel imaging technologies, including fluorescence-, Raman, photoacoustic-, and radio-guided approaches. These modalities have demonstrated the potential to further optimize precision in surgical resection and improve clinical outcomes for patients. Not only can these technologies enhance our understanding of the disease, they can also yield large imaging datasets intraoperatively that can be analyzed by deep learning approaches for more rapid and accurate pathological diagnosis. Unfortunately, many of these novel technologies are still under preclinical or early clinical evaluation. Organizations like the Intra-Operative Imaging Study Group of the European Society for Molecular Imaging (ESMI) support interdisciplinary interactions with the aim to improve technical capabilities in the field, an approach that can succeed only if scientists, engineers, and physicians work closely together with industry and regulatory bodies to resolve roadblocks to clinical translation. In this review, we provide an overview of a variety of novel IOI technologies, discuss their challenges, and present future perspectives on the enormous potential of IOI for oncological surgical navigation.

  View details for PubMedID 29916118

• [F-18] FSPG-PET reveals increased cystine/glutamate antiporter (xc-) activity in a mouse model of multiple sclerosis JOURNAL OF NEUROINFLAMMATION Hoehne, A., James, M. L., Alam, I. S., Ronald, J. A., Schneider, B., D'Souza, A., Witney, T. H., Andrews, L. E., Cropper, H. C., Behera, D., Gowrishankar, G., Ding, Z., Wyss-Coray, T., Chin, F. T., Biswal, S., Gambhir, S. S. 2018; 15

  View details for DOI 10.1186/s12974-018-1080-1

  View details for Web of Science ID 000425975200001

• Eradication of spontaneous malignancy by local immunotherapy. Science translational medicine Sagiv-Barfi, I., Czerwinski, D. K., Levy, S., Alam, I. S., Mayer, A. T., Gambhir, S. S., Levy, R. 2018; 10 (426)

  Abstract

  It has recently become apparent that the immune system can cure cancer. In some of these strategies, the antigen targets are preidentified and therapies are custom-made against these targets. In others, antibodies are used to remove the brakes of the immune system, allowing preexisting T cells to attack cancer cells. We have used another noncustomized approach called in situ vaccination. Immunoenhancing agents are injected locally into one site of tumor, thereby triggering a T cell immune response locally that then attacks cancer throughout the body. We have used a screening strategy in which the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents, and the resulting immune response is detected by the regression of the distant, untreated tumor. Using this assay, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG)-a Toll-like receptor 9 (TLR9) ligand-and anti-OX40 antibody provided the most impressive results. TLRs are components of the innate immune system that recognize molecular patterns on pathogens. Low doses of CpG injected into a tumor induce the expression of OX40 on CD4+ T cells in the microenvironment in mouse or human tumors. An agonistic anti-OX40 antibody can then trigger a T cell immune response, which is specific to the antigens of the injected tumor. Remarkably, this combination of a TLR ligand and an anti-OX40 antibody can cure multiple types of cancer and prevent spontaneous genetically driven cancers.

  View details for DOI 10.1126/scitranslmed.aan4488

  View details for PubMedID 29386357

  View details for PubMedCentralID PMC5997264

• Imaging activated T cells predicts response to cancer vaccines. The Journal of clinical investigation Alam, I. S., Mayer, A. T., Sagiv-Barfi, I. n., Wang, K. n., Vermesh, O. n., Czerwinski, D. K., Johnson, E. M., James, M. L., Levy, R. n., Gambhir, S. S. 2018

  Abstract

  In situ cancer vaccines are under active clinical investigation, given their reported ability to eradicate both local and disseminated malignancies. Intratumoral vaccine administration is thought to activate a T cell-mediated immune response, which begins in the treated tumor and cascades systemically. In this study, we describe a PET tracer (64Cu-DOTA-AbOX40) that enabled noninvasive and longitudinal imaging of OX40, a cell-surface marker of T cell activation. We report the spatiotemporal dynamics of T cell activation following in situ vaccination with CpG oligodeoxynucleotide in a dual tumor-bearing mouse model. We demonstrate that OX40 imaging was able to predict tumor responses on day 9 after treatment on the basis of tumor tracer uptake on day 2, with greater accuracy than both anatomical and blood-based measurements. These studies provide key insights into global T cell activation following local CpG treatment and indicate that 64Cu-DOTA-AbOX40 is a promising candidate for monitoring clinical cancer immunotherapy strategies.

  View details for PubMedID 29596062

• A Novel Positron Emission Tomography Reporter Gene/Reporter Probe for the Central Nervous System SOC NUCLEAR MEDICINE INC Haywood, T., Beinat, C., Gowrishankar, G., Patel, C., Alam, I. S., Gambhir, S. S. 2018
• Intraoperative Molecular Imaging in Lung Cancer: The State of the Art and the Future. Molecular therapy : the journal of the American Society of Gene Therapy Rogalla, S. n., Joosten, S. C., Alam, I. S., Gambhir, S. S., Vermesh, O. n. 2018; 26 (2): 338–41

  View details for PubMedID 29398484

• The Utility of [18F]DASA-23 for Molecular Imaging of Prostate Cancer with Positron Emission Tomography. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging Beinat, C. n., Haywood, T. n., Chen, Y. S., Patel, C. B., Alam, I. S., Murty, S. n., Gambhir, S. S. 2018

  Abstract

  There is a strong, unmet need for superior positron emission tomography (PET) imaging agents that are able to measure biochemical processes specific to prostate cancer. Pyruvate kinase M2 (PKM2) catalyzes the concluding step in glycolysis and is a key regulator of tumor growth and metabolism. Elevation of PKM2 expression was detected in Gleason 8-10 tumors compared to Gleason 6-7 carcinomas, indicating that PKM2 may potentially be a marker of aggressive prostate cancer. We have recently reported the development of a PKM2-specific radiopharmaceutical [18F]DASA-23 and herein describe its evaluation in cell culture and preclinical models of prostate cancer.The cellular uptake of [18F]DASA-23 was evaluated in a panel of prostate cancer cell lines and compared to that of [18F]FDG. The specificity of [18F]DASA-23 to measure PKM2 levels in cell culture was additionally confirmed through the use of PKM2-specific siRNA. PET imaging studies were then completed utilizing subcutaneous prostate cancer xenografts using either PC3 or DU145 cells in mice.[18F]DASA-23 uptake values over 60-min incubation period in PC3, LnCAP, and DU145 respectively were 23.4 ± 4.5, 18.0 ± 2.1, and 53.1 ± 4.6 % tracer/mg protein. Transient reduction in PKM2 protein expression with siRNA resulted in a 50.1 % reduction in radiotracer uptake in DU145 cells. Small animal PET imaging revealed 0.86 ± 0.13 and 1.6 ± 0.2 % ID/g at 30 min post injection of radioactivity in DU145 and PC3 subcutaneous tumor bearing mice respectively.Herein, we evaluated a F-18-labeled PKM2-specific radiotracer, [18F]DASA-23, for the molecular imaging of prostate cancer with PET. [18F]DASA-23 revealed rapid and extensive uptake levels in cellular uptake studies of prostate cancer cells; however, there was only modest tumor uptake when evaluated in mouse subcutaneous tumor models.

  View details for PubMedID 29736561

• A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant. Cancer research Ronald, J. A., Kim, B., Gowrishankar, G., Namavari, M., Alam, I. S., D'Souza, A., Nishikii, H., Chuang, H., Ilovich, O., Lin, C., Reeves, R., Shuhendler, A., Hoehne, A., Chan, C. T., Baker, J., Yaghoubi, S. S., VanBrocklin, H. F., Hawkins, R., Franc, B. L., Jivan, S., Slater, J. B., Verdin, E. F., Gao, K. T., Benjamin, J., Negrin, R., Gambhir, S. S. 2017; 77 (11): 2893-2902

  Abstract

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

  View details for DOI 10.1158/0008-5472.CAN-16-2953

  View details for PubMedID 28572504

• Rapid Imaging of Tumor Cell Death In Vivo Using the C2A Domain of Synaptotagmin-I JOURNAL OF NUCLEAR MEDICINE Neves, A. A., Xie, B., Fawcett, S., Alam, I. S., Witney, T. H., de Backer, M. M., Summers, J., Hughes, W., McGuire, S., Soloviev, D., Miller, J., Howat, W. J., Hu, D., Rodrigues, T. B., Lewis, D. Y., Brindle, K. M. 2017; 58 (6): 881–87

  Abstract

  Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of synaptotagmin-I. Methods: The capability of near-infrared fluorophore-labeled and 99mTc- and 111In-labeled derivatives of C2Am for imaging tumor cell death, using planar near-infrared fluorescence imaging and SPECT, respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft. Results: The fluorophore-labeled C2Am derivative showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumor cell death (2%-5%). There was a significant correlation (R > 0.9, P < 0.05) between fluorescently labeled C2Am binding and histologic markers of cell death, including cleaved caspase-3, whereas there was no such correlation with a site-directed mutant of C2Am (iC2Am) that does not bind phosphatidylserine. 99mTc-C2Am and 111In-C2Am also showed favorable biodistribution profiles, with predominantly renal clearance and low nonspecific retention in the liver and spleen at 24 h after probe administration. 99mTc-C2Am and 111In-C2Am generated tumor-to-muscle ratios in drug-treated tumors of 4.3× and 2.2×, respectively, at 2 h and 7.3× and 4.1×, respectively, at 24 h after administration. Conclusion: Given the favorable biodistribution profile of 99mTc- and 111In-labeled C2Am, and their ability to produce rapid and cell death-specific image contrast, these agents have potential for clinical translation.

  View details for DOI 10.2967/jnumed.116.183004

  View details for Web of Science ID 000402572500010

  View details for PubMedID 28209913

• F]DASA-23 for Imaging Tumor Glycolysis Through Noninvasive Measurement of Pyruvate Kinase M2. Molecular imaging and biology Beinat, C., Alam, I. S., James, M. L., Srinivasan, A., Gambhir, S. S. 2017

  Abstract

  A hallmark of cancer is metabolic reprogramming, which is exploited by cancer cells to ensure rapid growth and survival. Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key step in tumor metabolism and growth. Recently, we reported the radiosynthesis of the first positron emission tomography tracer for visualizing PKM2 in vivo-i.e., [(11)C]DASA-23. Due to the highly promising imaging results obtained with [(11)C]DASA-23 in rodent model glioblastoma, we set out to generate an F-18-labeled version of this tracer, with the end goal of clinical translation in mind. Herein, we report the radiosynthesis of 1-((2-fluoro-6-[(18)F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([(18)F]DASA-23) and our initial investigation of its binding properties in cancer cells.We synthesized [(18)F]DASA-23 via fluorination of 1-((2-fluoro-6-nitrophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine (10) with K[(18)F]F/K2.2.2 in N,N-dimethylformamide at 110 °C for 20 min. Subsequently, we evaluated uptake of [(18)F]DASA-23 in HeLa cervical adenocarcinoma cells and in vitro stability in human and mouse serum.We successfully prepared [(18)F]DASA-23 in 2.61 ± 1.54 % radiochemical yield (n = 10, non-decay corrected at end of synthesis) with a specific activity of 2.59 ± 0.44 Ci/μmol. Preliminary cell uptake experiments revealed high uptake in HeLa cells, which was effectively blocked by pretreating cells with the structurally distinct PKM2 activator, TEPP-46. [(18)F]DASA-23 remained intact in human and mouse serum up to 120 min.Herein, we have identified a F-18-labeled PKM2 specific radiotracer which shows potential for in vivo imaging. The promising cell uptake results reported herein warrant the further evaluation of [(18)F]DASA-23 for its ability to detect and monitor cancer noninvasively.

  View details for DOI 10.1007/s11307-017-1068-8

  View details for PubMedID 28236227

• Microwave gallium-68 radiochemistry for kinetically stable bis(thiosemicarbazone) complexes: structural investigations and cellular uptake under hypoxia DALTON TRANSACTIONS Alam, I. S., Arrowsmith, R. L., Cortezon-Tamarit, F., Twyman, F., Kociok-Koehn, G., Botchway, S. W., Dilworth, J. R., Carroll, L., Aboagye, E. O., Pascu, S. I. 2016; 45 (1): 144-155

  View details for DOI 10.1039/c5dt02537k

  View details for Web of Science ID 000366429000017

• Radiopharmaceuticals as probes to characterize tumour tissue EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Alam, I. S., Arshad, M. A., Quang-De Nguyen, Q. D., Aboagye, E. O. 2015; 42 (4): 537-561

  View details for DOI 10.1007/s00259-014-2984-3

  View details for Web of Science ID 000350685700002

• Acetyl-CoA Synthetase 2 Promotes Acetate Utilization and Maintains Cancer Cell Growth under Metabolic Stress CANCER CELL Schug, Z. T., Peck, B., Jones, D. T., Zhang, Q., Grosskurth, S., Alam, I. S., Goodwin, L. M., Smethurst, E., Mason, S., Blyth, K., McGarry, L., James, D., Shanks, E., Kalna, G., Saunders, R. E., Jiang, M., Howell, M., Lassailly, F., Thin, M. Z., Spencer-Dene, B., Stamp, G., van den Broek, N. J., Mackay, G., Bulusu, V., Kamphorst, J. J., Tardito, S., Strachan, D., Harris, A. L., Aboagye, E. O., Critchlow, S. E., Wakelam, M. J., Schulze, A., Gottlieb, E. 2015; 27 (1): 57-71

  Abstract

  A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.

  View details for DOI 10.1016/j.ccell.2014.12.002

  View details for Web of Science ID 000347906900010

  View details for PubMedID 25584894

  View details for PubMedCentralID PMC4297291

• Preclinical Evaluation of 3-F-18-Fluoro-2,2-Dimethylpropionic Acid as an Imaging Agent for Tumor Detection JOURNAL OF NUCLEAR MEDICINE Witney, T. H., Pisaneschi, F., Alam, I. S., Trousil, S., Kaliszczak, M., Twyman, F., Brickute, D., Nguyen, Q., Schug, Z., Gottlieb, E., Aboagye, E. O. 2014; 55 (9): 1506-1512

  Abstract

  Deregulated cellular metabolism is a hallmark of many cancers. In addition to increased glycolytic flux, exploited for cancer imaging with (18)F-FDG, tumor cells display aberrant lipid metabolism. Pivalic acid is a short-chain, branched carboxylic acid used to increase oral bioavailability of prodrugs. After prodrug hydrolysis, pivalic acid undergoes intracellular metabolism via the fatty acid oxidation pathway. We have designed a new probe, 3-(18)F-fluoro-2,2-dimethylpropionic acid ((18)F-FPIA), for the imaging of aberrant lipid metabolism and cancer detection.Cell intrinsic uptake of (18)F-FPIA was measured in murine EMT6 breast adenocarcinoma cells. In vivo dynamic imaging, time course biodistribution, and radiotracer stability testing were performed. (18)F-FPIA tumor retention was further compared in vivo to (18)F-FDG uptake in several xenograft models and inflammatory tissue.(18)F-FPIA rapidly accumulated in EMT6 breast cancer cells, with retention of intracellular radioactivity predicted to occur via a putative (18)F-FPIA carnitine-ester. The radiotracer was metabolically stable to degradation in mice. In vivo imaging of implanted EMT6 murine and BT474 human breast adenocarcinoma cells by (18)F-FPIA PET showed rapid and extensive tumor localization, reaching 9.1% ± 0.5% and 7.6% ± 1.2% injected dose/g, respectively, at 60 min after injection. Substantial uptake in the cortex of the kidney was seen, with clearance primarily via urinary excretion. Regarding diagnostic utility, uptake of (18)F-FPIA was comparable to that of (18)F-FDG in EMT6 tumors but superior in the DU145 human prostate cancer model (54% higher uptake; P = 0.002). Furthermore, compared with (18)F-FDG, (18)F-FPIA had lower normal-brain uptake resulting in a superior tumor-to-brain ratio (2.5 vs. 1.3 in subcutaneously implanted U87 human glioma tumors; P = 0.001), predicting higher contrast for brain cancer imaging. Both radiotracers showed increased localization in inflammatory tissue.(18)F-FPIA shows promise as an imaging agent for cancer detection and warrants further investigation.

  View details for DOI 10.2967/jnumed.114.140343

  View details for Web of Science ID 000341286900019

  View details for PubMedID 25012458

• Radiolabeled RGD Tracer Kinetics Annotates Differential alpha(v)beta(3) Integrin Expression Linked to Cell Intrinsic and Vessel Expression MOLECULAR IMAGING AND BIOLOGY Alam, I. S., Witney, T. H., Tomasi, G., Carroll, L., Twyman, F. J., Quang-De Nguyen, Q. D., Aboagye, E. O. 2014; 16 (4): 558-566

  View details for DOI 10.1007/s11307-013-0710-3

  View details for Web of Science ID 000339377900014

• A Novel Radiotracer to Image Glycogen Metabolism in Tumors by Positron Emission Tomography CANCER RESEARCH Witney, T. H., Carroll, L., Alam, I. S., Chandrashekran, A., Quang-De Nguyen, Q. D., Sala, R., Harris, R., DeBerardinis, R. J., Agarwal, R., Aboagye, E. O. 2014; 74 (5): 1319-1328

  Abstract

  The high rate of glucose uptake to fuel the bioenergetic and anabolic demands of proliferating cancer cells is well recognized and is exploited with (18)F-2-fluoro-2-deoxy-d-glucose positron emission tomography ((18)F-FDG-PET) to image tumors clinically. In contrast, enhanced glucose storage as glycogen (glycogenesis) in cancer is less well understood and the availability of a noninvasive method to image glycogen in vivo could provide important biologic insights. Here, we demonstrate that (18)F-N-(methyl-(2-fluoroethyl)-1H-[1,2,3]triazole-4-yl)glucosamine ((18)F-NFTG) annotates glycogenesis in cancer cells and tumors in vivo, measured by PET. Specificity of glycogen labeling was demonstrated by isolating (18)F-NFTG-associated glycogen and with stable knockdown of glycogen synthase 1, which inhibited (18)F-NFTG uptake, whereas oncogene (Rab25) activation-associated glycogen synthesis led to increased uptake. We further show that the rate of glycogenesis is cell-cycle regulated, enhanced during the nonproliferative state of cancer cells. We demonstrate that glycogen levels, (18)F-NFTG, but not (18)F-FDG uptake, increase proportionally with cell density and G1-G0 arrest, with potential application in the assessment of activation of oncogenic pathways related to glycogenesis and the detection of posttreatment tumor quiescence. Cancer Res; 74(5); 1319-28. ©2014 AACR.

  View details for DOI 10.1158/0008-5472.CAN-13-2768

  View details for Web of Science ID 000332475900006

  View details for PubMedID 24590807

• RGD-targeted MnO nanoparticles as T-1 contrast agents for cancer imaging - the effect of PEG length in vivo JOURNAL OF MATERIALS CHEMISTRY B Gallo, J., Alam, I. S., Lavdas, I., Wylezinska-Arridge, M., Aboagye, E. O., Long, N. J. 2014; 2 (7): 868-876

  View details for DOI 10.1039/c3tb21422b

  View details for Web of Science ID 000330118500012

• PET imaging with multimodal upconversion nanoparticles DALTON TRANSACTIONS Gallo, J., Alam, I. S., Jin, J., Gu, Y., Aboagye, E. O., Wong, W., Long, N. J. 2014; 43 (14): 5535-5545

  Abstract

  A series of new upconversion nanoparticles have been functionalised with tumour-targeting molecules and metal chelates, prepared following standard peptidic and thiol chemistry. The targeting strategy has been delivered via the αvβ3 integrin, which is a heterodimeric cell surface receptor that is up-regulated in a variety of cancers, such as melanoma and breast cancer. The well-known DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) motif allows coordination to the radionuclide (68)Ga. Radiolabelling experiments were optimised under relatively mild conditions, and are rare amongst nanoparticulate materials. In vivo application of these probes in mouse tumour models revealed their potential as specific cancer contrast agents for PET imaging.

  View details for DOI 10.1039/c3dt53095g

  View details for Web of Science ID 000332929200033

  View details for PubMedID 24535647

• Evaluation of Deuterated F-18- and C-11-Labeled Choline Analogs for Cancer Detection by Positron Emission Tomography CLINICAL CANCER RESEARCH Witney, T. H., Alam, I. S., Turton, D. R., Smith, G., Carroll, L., Brickute, D., Twyman, F. J., Quang-De Nguyen, Q. D., Tomasi, G., Awais, R. O., Aboagye, E. O. 2012; 18 (4): 1063-1072

  Abstract

  (11)C-Choline-positron emission tomography (PET) has been exploited to detect the aberrant choline metabolism in tumors. Radiolabeled choline uptake within the imaging time is primarily a function of transport, phosphorylation, and oxidation. Rapid choline oxidation, however, complicates interpretation of PET data. In this study, we investigated the biologic basis of the oxidation of deuterated choline analogs and assessed their specificity in human tumor xenografts.(11)C-Choline, (11)C-methyl-[1,2-(2)H(4)]-choline ((11)C-D4-choline), and (18)F-D4-choline were synthesized to permit comparison. Biodistribution, metabolism, small-animal PET studies, and kinetic analysis of tracer uptake were carried out in human colon HCT116 xenograft-bearing mice.Oxidation of choline analogs to betaine was highest with (11)C-choline, with reduced oxidation observed with (11)C-D4-choline and substantially reduced with (18)F-D4-choline, suggesting that both fluorination and deuteration were important for tracer metabolism. Although all tracers were converted intracellularly to labeled phosphocholine (specific signal), the higher rate constants for intracellular retention (K(i) and k(3)) of (11)C-choline and (11)C-D4-choline, compared with (18)F-D4-choline, were explained by the rapid conversion of the nonfluorinated tracers to betaine within HCT116 tumors. Imaging studies showed that the uptake of (18)F-D4-choline in three tumors with similar radiotracer delivery (K(1)) and choline kinase α expression-HCT116, A375, and PC3-M-were the same, suggesting that (18)F-D4-choline has utility for cancer detection irrespective of histologic type.We have shown here that both deuteration and fluorination combine to provide protection against choline oxidation in vivo. (18)F-D4-choline showed the highest selectivity for phosphorylation and warrants clinical evaluation.

  View details for DOI 10.1158/1078-0432.CCR-11-2462

  View details for Web of Science ID 000300628100016

  View details for PubMedID 22235095

• Imaging sialylated tumor cell glycans in vivo FASEB JOURNAL Neves, A. A., Stoeckmann, H., Harmston, R. R., Pryor, H. J., Alam, I. S., Ireland-Zecchini, H., Lewis, D. Y., Lyons, S. K., Leeper, F. J., Brindle, K. M. 2011; 25 (8): 2528-2537

  Abstract

  Cell surface glycans are involved in numerous physiological processes that involve cell-cell interactions and migration, including lymphocyte trafficking and cancer metastasis. We have used a bioorthogonal metabolic labeling strategy to detect cell surface glycans and demonstrate, for the first time, fluorescence and radionuclide imaging of sialylated glycans in a murine tumor model in vivo. Peracetylated azido-labeled N-acetyl-mannosamine, injected intraperitoneally, was used as the metabolic precursor for the biosynthesis of 5-azidoneuraminic, or azidosialic acid. Azidosialic acid-labeled cell surface glycans were then reacted, by Staudinger ligation, with a biotinylated phosphine injected intraperitoneally, and the biotin was detected by subsequent intravenous injection of a fluorescent or radiolabeled avidin derivative. At 24 h after administration of NeutrAvidin, labeled with either a far-red fluorophore or (111)In, there was a significant azido-labeled N-acetyl-mannosamine-dependent increase in tumor-to-tissue contrast, which was detected using optical imaging or single-photon-emission computed tomography (SPECT), respectively. The technique has the potential to translate to the clinic, where, given the prognostic relevance of altered sialic acid expression in cancer, it could be used to monitor disease progression.

  View details for DOI 10.1096/fj.10-178590

  View details for Web of Science ID 000293337800004

  View details for PubMedID 21493886

• Comparison of the C2A Domain of Synaptotagmin-I and Annexin-V As Probes for Detecting Cell Death BIOCONJUGATE CHEMISTRY Alam, I. S., Neves, A. A., Witney, T. H., Boren, J., Brindle, K. M. 2010; 21 (5): 884-891

  Abstract

  The induction of apoptosis is frequently accompanied by the exposure of phosphatidylserine (PS) on the cell surface, which has been detected using radionuclide and fluorescently labeled derivatives of the PS-binding protein, Annexin V. The fluorescently labeled protein has been used extensively in vitro as a diagnostic reagent for detecting cell death, and radionuclide-labeled derivatives have undergone clinical trials for detecting tumor cell death in vivo following treatment. We show here that the C2A domain of Synaptotagmin-I, which had been fluorescently labeled at a single cysteine residue introduced by site-directed mutagenesis, detected the same levels of cell death as a similarly labeled Annexin-V derivative, in drug-treated murine lymphoma and human breast cancer cell lines in vitro. However, the C2A derivative showed significantly less binding to viable cells and, as a consequence, up to 4-fold more specific binding to apoptotic and necrotic cells when compared with Annexin-V. C2A offers a potential route for the development of a new generation of more specific imaging probes for the detection of tumor cell death in the clinic.

  View details for DOI 10.1021/bc9004415

  View details for Web of Science ID 000277683300013

  View details for PubMedID 20402461