Scafoglio CR, Villegas B, Abdelhady G, Bailey ST, Liu J, Shirali AS, Wallace WD, Magyar CE, Grogan TR, Elashoff D, Walser T, Yanagawa J, Aberle DR, Barrio JR, Dubinett SM, Shackelford DB. Sodium-glucose transporter 2 is a diagnostic and therapeutic target for early-stage lung adenocarcinoma. Sci Transl Med. 2018;10. PMC6428683
Xu S, Catapang A, Doh H, Bayley NA, Lee JT, Braas D, Graeber TG, Herschman H. Hexokinase 2 is targetable for HK1 negative, HK2 positive tumors from a wide variety of tissues of origin. J Nucl Med 2018 Jun 7; doi:10.2967/jnumed.118.212365. [Epub ahead of print]. PMID: 29880505
Momcilovic N, Bailey ST, Lee JT, Fishbein MC, Braas D, Go J, Graeber TG, Parlati F, Demo S, Li R, Walser TC, Gricowski M, Shuman R, Ibarra J, Fridman D, Phelps ME, Badran K, St. John M, Bernthal NM, Federman N, Yanagawa J, Dubinett SM, Sadeghi S, Christofk HR, Shackelford DB. The GSK3 Signaling Axis Regulates Adaptive Glutamine Metabolism in Lung Squamous Cell Carcinoma. Cancer Cell. 2018 May 14;33(5):905–921. PMC6451645
Mai W, Gosa L, Daniels V, Ta L, Tsang J, Higgins B, Gilmore W, Bayley N, Harati M, Lee JT, Yong W, Kornblum H, Bensinger S, Mischel P, Rao N, Clark P, Cloughesy T, Letai A, Nathanson D. Cytoplasmic p53 couples oncogene-driven metabolism to apoptosis and is a therapeutic target in glioblastoma. Nat Med. 2017 Nov;23(11):1342-1351. PMCID: PMC5683421
Goodwin J, Neugent ML, Lee SY, Choe JH, Choi H, Jenkins DMR, Ruthenborg RJ, Robinson MW, Jeong JY, Wake M, Abe H, Takeda N, Endo H, Inoue M, Xuan Z, Yoo H, Chen M, Ahn JM, Minna JD, Helke KL, Singh PK, Shackelford DB, Kim JW. The distinct metabolic phenotype of lung squamous cell carcinoma defines selective vulnerability to glycolytic inhibition. Nat Commun. 2017 May 26;8:15503. PMC5458561
Momcilovic M*, Bailey ST*, Lee JT, Fishbein MC, Magyar C, Braas D, Graeber TG, Jackson NJ, Czernin J, Emberley E, Gross M, Jane J, Mackinnon A, Pan A, Rodriguez M, Works M, Zhang W, Parlati F, Demo S, Garon E, Krysan K, Walser TC, Dubinett SM, Sadeghi S, Christofk H, Shackelford DB. Targeted Inhibition of EGFR and Glutaminase Induces Metabolic Crisis in EGFR Mutant Lung Cancer. Cell Rep. 2017 Jan 17;18(3):601-610. PMCID: PMC5260616. *contributed equally to this work
Lee JK, Phillips JW, Smith BA, Park JW, Stoyanova T, McCaffrey EF, Baertsch R, Sokolov A, Meyerowitz JG, Mathis C, Cheng D, Stuart JM, Shokat KM, Gustafson WC, Huang J, Witte ON. N-Myc Drives Neuroendocrine Prostate Cancer Initiated from Human Prostate Epithelial Cells. Cancer Cell. 2016 Apr 11;29:536-547. PMC4829466
Sonn GA, Behesnilian AS, Jiang ZK, Zettlitz KA, Lepin EJ, Bentolila LA, Knowles SM, Lawrence D, Wu AM, Reiter RE. Fluorescent Image-Guided Surgery with an Anti-Prostate Stem Cell Antigen (PSCA) Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real Time. Clin Cancer Res. 2016 Mar 15;22:1403-12. PMC4794340
Kim W, Lea TM, Wei L, Austin WR, Bazzy J, Wang X, Uong NT, Abt ER, Poddar S, Capri JR, Van Valkenburgh JS, Steele D, Gipson RM, Slavik R, Cabebe AE, Taechariyakul T, Yaghoubi SS, Lee JT, Sadeghi S, Lavie A, Faull KF, Witte ON, Donahue TR, Phelps ME, Herschman HR, Herrmann K, Czernin J, Radu CG. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase. Proc Natl Acad Sci U S A. 2016 Mar 2; 113(15):4027-4032. PMCID: PMC4839461
Hong CS, Graham NA, Gu W, Espindola Camacho C, Mah V, Maresh EL, Alavi M, Bagryanova L, Krotee PA, Gardner BK, Behbahan IS, Horvath S, Chia D, Mellinghoff IK, Hurvitz SA, Dubinett SM, Critchlow SE, Kurdistani SK, Goodglick L, Braas D, Graeber TG, Christofk HR. MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4. Cell Rep. 2016 Feb 23;14:1590-1601. PMC4816454
Faltermeier CM, Drake JM, Clark PM, Smith BA, Zong Y, Volpe C, Mathis C, Morrissey C, Castor B, Huang J, Witte ON. Functional screen identifies kinases driving prostate cancer visceral and bone metastasis. Proc Natl Acad Sci U S A. 2016 Jan 12;113:E172-81. PMC4720329
Moughon DL, He H, Schokrpur S, Jiang ZK, Yaqoob M, David J, Lin C, Iruela-Arispe ML, Dorigo O, Wu L. Macrophage Blockade Using CSF1R Inhibitors Reverses the Vascular Leakage Underlying Malignant Ascites in Late-Stage Epithelial Ovarian Cancer. Cancer Res. 2015 Nov 15;75:4742-52. PMC4675660
Momcilovic M, McMickle R, Abt E, Seki A, Simko SA, Magyar C, Stout DB, Fishbein MC, Walser TC, Dubinett SM, Shackelford DB. Heightening Energetic Stress Selectively Targets LKB1-Deficient Non-Small Cell Lung Cancers. Cancer Res. 2015 Nov 15;75:4910-22. PMC4654699
Meng H, Wang M, Liu H, Liu X, Situ A, Wu B, Ji Z, Chang CH, Nel AE. Use of a lipid-coated mesoporous silica nanoparticle platform for synergistic gemcitabine and paclitaxel delivery to human pancreatic cancer in mice. ACS Nano. 2015;9:3540-57. PMC4415452.
Scafoglio C, Hirayama BA, Kepe V, Liu J, Ghezzi C, Satyamurthy N, Moatamed NA, Huang J, Koepsell H, Barrio JR, Wright EM. Functional expression of sodium-glucose transporters in cancer. Proc Natl Acad Sci U S A. 2015 Jul 28;112(30):E4111-9. doi: 10.1073/pnas.1511698112. Epub 2015 Jul 13.
Glucose is a major metabolic substrate required for cancer cell survival and growth. It is mainly imported into cells by facilitated glucose transporters (GLUTs). Here we demonstrate the importance of another glucose import system, the sodium-dependent glucose transporters (SGLTs), in pancreatic and prostate adenocarcinomas, and investigate their role in cancer cell survival. Three experimental approaches were used: (i) immunohistochemical mapping of SGLT1 and SGLT2 distribution in tumors; (ii) measurement of glucose uptake in fresh isolated tumors using an SGLT-specific radioactive glucose analog, α-methyl-4-deoxy-4-[(18)F]fluoro-D-glucopyranoside (Me4FDG), which is not transported by GLUTs; and (iii) measurement of in vivo SGLT activity in mouse models of pancreatic and prostate cancer using Me4FDG-PET imaging. We found that SGLT2 is functionally expressed in pancreatic and prostate adenocarcinomas, and provide evidence that SGLT2 inhibitors block glucose uptake and reduce tumor growth and survival in a xenograft model of pancreatic cancer. We suggest that Me4FDG-PET imaging may be used to diagnose and stage pancreatic and prostate cancers, and that SGLT2 inhibitors, currently in use for treating diabetes, may be useful for cancer therapy.
Lamkin DM, Sung HY, Yang GS, David JM, Ma JC, Cole SW, Sloan EK. α2-Adrenergic blockade mimics the enhancing effect of chronic stress on breast cancer progression. Psychoneuroendocrinology. 2015 Jan;51:262-70. doi: 10.1016/j.psyneuen.2014.10.004. Epub 2014 Oct 12.
Experimental studies in preclinical mouse models of breast cancer have shown that chronic restraint stress can enhance disease progression by increasing catecholamine levels and subsequent signaling of β-adrenergic receptors. Catecholamines also signal α-adrenergic receptors, and greater α-adrenergic signaling has been shown to promote breast cancer in vitro and in vivo. However, antagonism of α-adrenergic receptors can result in elevated catecholamine levels, which may increase β-adrenergic signaling, because pre-synaptic α2-adrenergic receptors mediate an autoinhibition of sympathetic transmission. Given these findings, we examined the effect of α-adrenergic blockade on breast cancer progression under non-stress and stress conditions (chronic restraint) in an orthotopic mouse model with MDA-MB-231HM cells. Chronic restraint increased primary tumor growth and metastasis to distant tissues as expected, and non-selective α-adrenergic blockade by phentolamine significantly inhibited those effects. However, under non-stress conditions, phentolamine increased primary tumor size and distant metastasis. Sympatho-neural gene expression for catecholamine biosynthesis enzymes was elevated by phentolamine under non-stress conditions, and the non-selective β-blocker propranolol inhibited the effect of phentolamine on breast cancer progression. Selective α2-adrenergic blockade by efaroxan also increased primary tumor size and distant metastasis under non-stress conditions, but selective α1-adrenergic blockade by prazosin did not. These results are consistent with the hypothesis that α2-adrenergic signaling can act through an autoreceptor mechanism to inhibit sympathetic catecholamine release and, thus, modulate established effects of β-adrenergic signaling on tumor progression-relevant biology.
Knowles SM, Tavaré R, Zettlitz KA, Rochefort MM, Salazar FB, Jiang ZK, Reiter RE, Wu AM. Applications of immunoPET: using 124I-anti-PSCA A11 minibody for imaging disease progression and response to therapy in mouse xenograft models of prostate cancer. Clin Cancer Res. 2014 Dec 15;20(24):6367-78. doi: 10.1158/1078-0432.CCR-14-1452. Epub 2014 Oct 17.
PURPOSE: Prostate stem cell antigen (PSCA) is highly expressed in local prostate cancers and prostate cancer bone metastases and its expression correlates with androgen receptor activation and a poor prognosis. In this study, we investigate the potential clinical applications of immunoPET with the anti-PSCA A11 minibody, an antibody fragment optimized for use as an imaging agent. We compare A11 minibody immunoPET to (18)F-Fluoride PET bone scans for detecting prostate cancer bone tumors and evaluate the ability of the A11 minibody to image tumor response to androgen deprivation.
EXPERIMENTAL DESIGN: Osteoblastic, PSCA-expressing, LAPC-9 intratibial xenografts were imaged with serial (124)I-anti-PSCA A11 minibody immunoPET and (18)F-Fluoride bone scans. Mice bearing LAPC-9 subcutaneous xenografts were treated with either vehicle or MDV-3100 and imaged with A11 minibody immunoPET/CT scans pre- and posttreatment. Ex vivo flow cytometry measured the change in PSCA expression in response to androgen deprivation.
RESULTS: A11 minibody demonstrated improved sensitivity and specificity over (18)F-Fluoride bone scans for detecting LAPC-9 intratibial xenografts at all time points. LAPC-9 subcutaneous xenografts showed downregulation of PSCA when treated with MDV-3100 which A11 minibody immunoPET was able to detect in vivo.
CONCLUSIONS: A11 minibody immunoPET has the potential to improve the sensitivity and specificity of clinical prostate cancer metastasis detection over bone scans, which are the current clinical standard-of-care. A11 minibody immunoPET additionally has the potential to image the activity of the androgen signaling axis in vivo which may help evaluate the clinical response to androgen deprivation and the development of castration resistance.
Nathanson DA, Armijo AL, Tom M, Li Z, Dimitrova E, Austin WR, Nomme J, Campbell DO, Ta L, Le TM, Lee JT, Darvish R, Gordin A, Wei L, Liao HI, Wilks M, Martin C, Sadeghi S, Murphy JM, Boulos N, Phelps ME, Faull KF, Herschman HR, Jung ME, Czernin J, Lavie A, Radu CG. Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication. J Exp Med. 2014 Mar 10;211(3):473-86. doi: 10.1084/jem.20131738. Epub 2014 Feb 24.
Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.http://www.ncbi.nlm.nih.gov/pubmed/24567448
Shackelford DB, Abt E, Gerken L, Vasquez DS, Seki A, Leblanc M, Wei L, Fishbein MC, Czernin J, Mischel PS, Shaw RJ. LKB1 inactivation dictates therapeutic response of non-small cell lung cancer to the metabolism drug phenformin. Cancer Cell. 2013 Feb 11;23(2):143-58. doi: 10.1016/j.ccr.2012.12.008. Epub 2013 Jan 24.
The LKB1 (also called STK11) tumor suppressor is mutationally inactivated in ∼20% of non-small cell lung cancers (NSCLC). LKB1 is the major upstream kinase activating the energy-sensing kinase AMPK, making LKB1-deficient cells unable to appropriately sense metabolic stress. We tested the therapeutic potential of metabolic drugs in NSCLC and identified phenformin, a mitochondrial inhibitor and analog of the diabetes therapeutic metformin, as selectively inducing apoptosis in LKB1-deficient NSCLC cells. Therapeutic trials in Kras-dependent mouse models of NSCLC revealed that tumors with Kras and Lkb1 mutations, but not those with Kras and p53 mutations, showed selective response to phenformin as a single agent, resulting in prolonged survival. This study suggests phenformin as a cancer metabolism-based therapeutic to selectively target LKB1-deficient tumors.
Braas D, Ahler E, Tam B, Nathanson D, Riedinger M, Benz MR, Smith KB, Eilber FC, Witte ON, Tap WD, Wu H, Christofk HR. Metabolomics strategy reveals subpopulation of liposarcomas sensitive to gemcitabine treatment. Cancer Discov. 2012 Dec;2(12):1109-17. doi: 10.1158/2159-8290.CD-12-0197.
Unlike many cancers that exhibit glycolytic metabolism, high-grade liposarcomas often exhibit low 2[18F]fluoro-2-deoxy-D-glucose uptake by positron emission tomography (PET), despite rapid tumor growth. Here, we used liquid chromatography tandem mass spectrometry to identify carbon sources taken up by liposarcoma cell lines derived from xenograft tumors in patients. Interestingly, we found that liposarcoma cell lines consume nucleosides from culture media, suggesting nucleoside salvage pathway activity. The nucleoside salvage pathway is dependent on deoxycytidine kinase (dCK) and can be imaged in vivo by PET with 1-(2'-deoxy-2'-[18F]fluoroarabinofuranosyl) cytosine (FAC). We found that liposarcoma cell lines and xenograft tumors exhibit dCK activity and dCK-dependent FAC uptake in vitro and in vivo. In addition, liposarcoma cell lines and xenograft tumors are sensitive to treatment with the nucleoside analogue prodrug gemcitabine, and gemcitabine sensitivity is dependent on dCK expression. Elevated dCK activity is evident in 7 of 68 clinical liposarcoma samples analyzed. These data suggest that a subpopulation of liposarcoma patients have tumors with nucleoside salvage pathway activity that can be identified noninvasively using [18F]-FAC-PET and targeted using gemcitabine.
SIGNIFICANCE: Patients with high-grade liposarcoma have poor prognoses and often fail to respond to chemotherapy. This report identifies elevated nucleoside salvage activity in a subset of liposarcomas that are identifiable using noninvasive PET imaging with FAC and that are sensitive to gemcitabine. Thus, we suggest a new treatment paradigm for liposarcoma patients that uses [18F]-FAC-PET in the clinic to delineate gemcitabine responders from nonresponders.
Lee JT, Campbell DO, Satyamurthy N, Czernin J, Radu CG. Stratification of nucleoside analog chemotherapy using 1-(2′-deoxy-2′-18F-fluoro-beta-D-arabinofuranosyl)cytosine and 1-(2′-deoxy-2′-18F-fluoro-beta-L-arabinofuranosyl)-5-methylcytosine PET. J Nucl Med. 2012;53:275–280.
The ability to measure tumor determinants of response to nucleoside analog (NA) chemotherapy agents such as gemcitabine and related compounds could significantly affect the management of several types of cancer. Previously we showed that the accumulation in tumors of the new PET tracer 1-(2'-deoxy-2'-(18)F-fluoro-β-d-arabinofuranosyl)cytosine ((18)F-FAC) is predictive of responses to gemcitabine. (18)F-FAC retention in cells requires deoxycytidine kinase (dCK), a rate-limiting enzyme in the deoxyribonucleoside salvage metabolism and in gemcitabine conversion from an inactive prodrug to a cytotoxic compound. The objectives of the current study were to determine whether (18)F-FAC tumor uptake is also influenced by cytidine deaminase (CDA), a determinant of resistance to gemcitabine; to develop a new PET assay using (18)F-FAC and the related probe 1-(2'-deoxy-2'-(18)F-fluoro-β-l-arabinofuranosyl)-5-methylcytosine (l-(18)F-FMAC) to profile tumor lesions for both dCK and CDA enzymatic activities; and to determine whether this PET assay can identify the most effective NA chemotherapy against tumors with differential expression of dCK and CDA.
METHODS: Isogenic murine leukemic cell lines with defined dCK and CDA activities were generated by retroviral transduction. A cell viability assay was used to determine the sensitivity of the isogenic cell lines to the dCK-dependent NA prodrugs gemcitabine and clofarabine. In vitro enzymatic and cell-based tracer uptake assays and in vivo PET with (18)F-FAC and l-(18)F-FMAC were used to predict tumor responses to gemcitabine and clofarabine.
RESULTS: dCK and CDA activities measured by kinase and tracer uptake assays correlated with the sensitivity of isogenic cell lines to gemcitabine and clofarabine. Coexpression of CDA decreased the sensitivity of dCK-positive cells to gemcitabine treatment in vitro by 15-fold but did not affect responses to clofarabine. Coexpression of CDA decreased (18)F-FAC but not l-(18)F-FMAC, phosphorylation, and uptake by dCK-positive cells. (18)F-FAC and l-(18)F-FMAC PET estimates of the enzymatic activities of dCK and CDA in tumor implants in mice were predictive of responses to gemcitabine and clofarabine treatment in vivo.
CONCLUSION: These findings support the utility of PET-based phenotyping of tumor nucleoside metabolism for guiding the selection of NA prodrugs.
Ishikawa TO, Herschman HR. Conditional bicistronic Cre reporter line expressing both firefly luciferase and β-galactosidase. Mol Imaging Biol. 2011 Apr;13(2):284-92. doi: 10.1007/s11307-010-0333-x.
PURPOSE: The Cre-loxP system has become an important strategy for conditional gene deletion and conditional gene expression in genetically engineered mice. To evaluate Cre recombinase expression, we generated reporter mice that permit both noninvasive imaging in living animals and either ex vivo histochemical/immunohistochemical tissue transgene expression analysis or quantitative enzyme analysis in the same animal.
PROCEDURES: Transgenic reporter mice were generated in which a loxP-flanked enhanced green fluorescent protein (EGFP) reporter gene and STOP sequence are placed after the nearly ubiquitously expressed CAG promoter, but before a bicistronic transcriptional unit containing luciferase and β-galactosidase reporter gene coding sequences.
RESULTS: After global deletion of the floxed STOP sequence by germ line Cre deletion, the reporter mouse expresses luciferase and β-galactosidase in all tissues examined. Tissue-specific expression of both reporter genes occurs in reporter mouse strains expressing Cre in skin (K14 keratin Cre), heart (myosin light chair Cre), or colon (Villin Cre).
CONCLUSION: The luc-gal(Tg) reporter mouse allows noninvasive imaging of target Cre activation both in living animals and in tissues and cells following necropsy, using loss of EGFP expression, gain of luciferase expression, and gain of β-galactosidase expression as alternatives within the same animal for qualitative analysis of Cre expression.
Freise AC, Zettlitz KA, Salazar FB, Tavaré R, Tsai WK, Hadjioannou A, Rozengurt N, Braun J, Wu AM, Freise AC, Salazar FB, Chatziioannou AF. Immuno-PET in Inflammatory Bowel Disease: Imaging CD4-Positive T Cells in a Murine Model of Colitis. J Nucl Med. 2018 Jun 1;59:980-985. PMC6004558
Zettlitz KA, Tavare R, Knowles SM, Steward KK, Timmerman JM, Wu AM, Tavaré R. ImmunoPET of Malignant and Normal B Cells with 89Zr- and 124I-Labeled Obinutuzumab Antibody Fragments Reveals Differential CD20 Internalization In Vivo. Clin Cancer Res. 2017 Dec 1;23:7242-7252. PMC5880625
Lu J, Liu X, Liao YP, Salazar F, Sun B, Jiang W, Chang CH, Jiang J, Wang X, Wu AM, Meng H, Nel AE. Nano-enabled pancreas cancer immunotherapy using immunogenic cell death and reversing immunosuppression. Nat Commun. 2017 Nov 27;8:1811. PMC5703845
Antonios JP, Soto H, Everson RG, Moughon D, Wang AC, Orpilla J, Radu CG, Ellingson BM, Lee JT, Cloughesy T, Phelps ME, Czernin J, Liau LM, Prins RM. Detection of immune responses after immunotherapy in glioblastoma using PET and MRI. Proc Natl Acad Sci U S A. 2017 Sep 19;114(38):10220-10225. PMCID: PMC5617282
Freise AC, Zettlitz KA, Salazar FB, Lu X, Tavaré R, Wu AM. ImmunoPET Imaging of Murine CD4+T Cells Using Anti-CD4 Cys-Diabody: Effects of Protein Dose on T Cell Function and Imaging. Mol Imaging Biol. 2017 Aug 1;19:599-609. PMC5524218
Tavaré R, Escuin-Ordinas H, Mok S, McCracken MN, Zettlitz KA, Salazar FB, Witte ON, Ribas A, Wu AM. An Effective Immuno-PET Imaging Method to Monitor CD8-Dependent Responses to Immunotherapy. Cancer Res. 2016 Jan 1;76:73-82. PMC4703530
McCracken MN, Vatakis DN, Dixit D, McLaughlin J, Zack JA, Witte ON. Noninvasive detection of tumor-infiltrating T cells by PET reporter imaging. J Clin Invest. 2015 May 1;125:1815-26. PMC4463193
Escamilla J, Schokrpur S, Liu C, Priceman SJ, Moughon D, Jiang Z, Pouliot F, Magyar C, Sung JL, Xu J, Deng G, West BL, Bollag G, Fradet Y, Lacombe L, Jung ME, Huang J, Wu L. CSF1 receptor targeting in prostate cancer reverses macrophage-mediated resistance to androgen blockade therapy. Cancer Res. 2015 Feb 15;75:950-62. PMC4359956
Tavaré R, McCracken MN, Zettlitz KA, Knowles SM, Salazar FB, Olafsen T, Witte ON, Wu AM. Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo. Proc Natl Acad Sci U S A. 2014 Jan 21;111(3):1108-13. doi: 10.1073/pnas.1316922111. Epub 2014 Jan 3.
The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS220.127.116.11) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8(+) T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8(+) T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for (64)Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both (64)Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.
McCracken MN, Gschweng EH, Nair-Gill E, McLaughlin J, Cooper AR, Riedinger M, Cheng D, Nosala C, Kohn DB, Witte ON. Long-term in vivo monitoring of mouse and human hematopoietic stem cell engraftment with a human positron emission tomography reporter gene. Proc Natl Acad Sci U S A. 2013 Jan 29;110(5):1857-62. doi: 10.1073/pnas.1221840110. Epub 2013 Jan 14.
Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-β-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.
Koya RC, Mok S, Otte N, Blacketor KJ, Comin-Anduix B, Tumeh PC, Minasyan A, Graham NA, Graeber TG, Chodon T, Ribas A. BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy. Cancer Res. 2012 Aug 15;72(16):3928-37. doi: 10.1158/0008-5472.CAN-11-2837. Epub 2012 Jun 12.
Combining immunotherapy with targeted therapy blocking oncogenic BRAFV600 may result in improved treatments for advanced melanoma. In this study, we developed a BRAFV600E-driven murine model of melanoma, SM1, which is syngeneic to fully immunocompetent mice. SM1 cells exposed to the BRAF inhibitor vemurafenib (PLX4032) showed partial in vitro and in vivo sensitivity resulting from the inhibition of MAPK pathway signaling. Combined treatment of vemurafenib plus adoptive cell transfer therapy with lymphocytes genetically modified with a T-cell receptor (TCR) recognizing chicken ovalbumin (OVA) expressed by SM1-OVA tumors or pmel-1 TCR transgenic lymphocytes recognizing gp100 endogenously expressed by SM1 resulted in superior antitumor responses compared with either therapy alone. T-cell analysis showed that vemurafenib did not significantly alter the expansion, distribution, or tumor accumulation of the adoptively transferred cells. However, vemurafenib paradoxically increased mitogen-activated protein kinase (MAPK) signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells. Taken together, our findings, derived from 2 independent models combining BRAF-targeted therapy with immunotherapy, support the testing of this therapeutic combination in patients with BRAFV600 mutant metastatic melanoma.http://www.ncbi.nlm.nih.gov/pubmed/22693252
Lisiero DN, Soto H, Liau LM, Prins RM. Enhanced sensitivity to IL-2 signaling regulates the clinical responsiveness of IL-12-primed CD8(+) T cells in a melanoma model. J Immunol. 2011 May 1;186(9):5068-77. doi: 10.4049/jimmunol.1003317. Epub 2011 Mar 23.
The optimal expansion, trafficking, and function of adoptively transferred CD8(+) T cells are parameters that currently limit the effectiveness of antitumor immunity to established tumors. In this study, we addressed the mechanisms by which priming of self tumor-associated Ag-specific CD8(+) T cells influenced antitumor functionality in the presence of the inflammatory cytokine IL-12. In vitro priming of mouse tumor-specific CD8(+) T cells in the presence of IL-12 induced a diverse and rapid antitumor effector activity while still promoting the generation of memory cells. Importantly, IL-12-primed effector T cells dramatically reduced the growth of well-established s.c. tumors and significantly increased survival to highly immune resistant, established intracranial tumors. Control of tumor growth by CD8(+) T cells was dependent on IL-12-mediated upregulation of the high-affinity IL-2R (CD25) and a subsequent increase in the sensitivity to IL-2 stimulation. Finally, IL-12-primed human PBMCs generated tumor-specific T cells both phenotypically and functionally similar to IL-12-primed mouse tumor-specific T cells. These results highlight the ability of IL-12 to obviate the strict requirement for administering high levels of IL-2 during adoptive cell transfer-mediated antitumor responses. Furthermore, acquisition of a potent effector phenotype independent of cytokine support suggests that IL-12 could be added to adoptive cell transfer clinical strategies in cancer patients.
Radu CG, Shu CJ, Nair-Gill E, Shelly SM, Barrio JR, Satyamurthy N, Phelps ME, Witte ON. Molecular imaging of lymphoid organs and immune activation by positron emission tomography with a new [18F]-labeled 2'-deoxycytidine analog. Nat Med. 2008 Jul;14(7):783-8. doi: 10.1038/nm1724. Epub 2008 Jun 8.
Monitoring immune function with molecular imaging could have a considerable impact on the diagnosis and treatment evaluation of immunological disorders and therapeutic immune responses. Positron emission tomography (PET) is a molecular imaging modality with applications in cancer and other diseases. PET studies of immune function have been limited by a lack of specialized probes. We identified [(18)F]FAC (1-(2'-deoxy-2'-[(18)F]fluoroarabinofuranosyl) cytosine) by differential screening as a new PET probe for the deoxyribonucleotide salvage pathway. [(18)F]FAC enabled visualization of lymphoid organs and was sensitive to localized immune activation in a mouse model of antitumor immunity. [(18)F]FAC microPET also detected early changes in lymphoid mass in systemic autoimmunity and allowed evaluation of immunosuppressive therapy. These data support the use of [(18)F]FAC PET for immune monitoring and suggest a wide range of clinical applications in immune disorders and in certain types of cancer.
Hu-Lieskovan S, Mok S, Homet Moreno B, Tsoi J, Robert L, Goedert L, Pinheiro EM, Koya RC, Graeber TG, Comin-Anduix B, Ribas A. Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAFV600E melanoma. Sci Transl Med. 2015 Mar 18;7(279):279ra41. doi: 10.1126/scitranslmed.aaa4691.
Combining immunotherapy and BRAF targeted therapy may result in improved antitumor activity with the high response rates of targeted therapy and the durability of responses with immunotherapy. However, the first clinical trial testing the combination of the BRAF inhibitor vemurafenib and the CTLA4 antibody ipilimumab was terminated early because of substantial liver toxicities. MEK [MAPK (mitogen-activated protein kinase) kinase] inhibitors can potentiate the MAPK inhibition in BRAF mutant cells while potentially alleviating the unwanted paradoxical MAPK activation in BRAF wild-type cells that lead to side effects when using BRAF inhibitors alone. However, there is the concern of MEK inhibitors being detrimental to T cell functionality. Using a mouse model of syngeneic BRAF(V600E)-driven melanoma, SM1, we tested whether addition of the MEK inhibitor trametinib would enhance the antitumor activity of combined immunotherapy with the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression, increased T cell infiltration into tumors, and improved in vivo cytotoxicity. Single-agent dabrafenib increased tumor-associated macrophages and T regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapy resulted in increased melanosomal antigen and major histocompatibility complex (MHC) expression and global immune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen-specific ACT, we tested the combination of dabrafenib, trametinib, and anti-PD1 therapy in SM1 tumors, and observed superior antitumor effect. Our findings support the testing of triple combination therapy of BRAF and MEK inhibitors with immunotherapy in patients with BRAF(V600E) mutant metastatic melanoma.
Gschweng EH, McCracken MN, Kaufman ML, Ho M, Hollis RP, Wang X, Saini N, Koya RC, Chodon T, Ribas A, Witte ON, Kohn DB. HSV-sr39TK positron emission tomography and suicide gene elimination of human hematopoietic stem cells and their progeny in humanized mice. Cancer Res. 2014 Sep 15;74(18):5173-83. doi: 10.1158/0008-5472.CAN-14-0376. Epub 2014 Jul 18.
Engineering immunity against cancer by the adoptive transfer of hematopoietic stem cells (HSC) modified to express antigen-specific T-cell receptors (TCR) or chimeric antigen receptors generates a continual supply of effector T cells, potentially providing superior anticancer efficacy compared with the infusion of terminally differentiated T cells. Here, we demonstrate the in vivo generation of functional effector T cells from CD34-enriched human peripheral blood stem cells modified with a lentiviral vector designed for clinical use encoding a TCR recognizing the cancer/testes antigen NY-ESO-1, coexpressing the PET/suicide gene sr39TK. Ex vivo analysis of T cells showed antigen- and HLA-restricted effector function against melanoma. Robust engraftment of gene-modified human cells was demonstrated with PET reporter imaging in hematopoietic niches such as femurs, humeri, vertebrae, and the thymus. Safety was demonstrated by the in vivo ablation of PET signal, NY-ESO-1-TCR-bearing cells, and integrated lentiviral vector genomes upon treatment with ganciclovir, but not with vehicle control. Our study provides support for the efficacy and safety of gene-modified HSCs as a therapeutic modality for engineered cancer immunotherapy.http://www.ncbi.nlm.nih.gov/pubmed/25038231
Escuin-Ordinas H, Elliott MW, Atefi M, Lee M, Ng C, Wei L, Comin-Anduix B, Montecino-Rodriguez E, Avramis E, Radu C, Sharp LL, Ribas A. PET imaging to non-invasively study immune activation leading to antitumor responses with a 4-1BB agonistic antibody. J Immunother Cancer. 2013 Aug 27;1:14. doi: 10.1186/2051-1426-1-14. eCollection 2013.
BACKGROUND: Molecular imaging with positron emission tomography (PET) may allow the non-invasive study of the pharmacodynamic effects of agonistic monoclonal antibodies (mAb) to 4-1BB (CD137). 4-1BB is a member of the tumor necrosis factor family expressed on activated T cells and other immune cells, and activating 4-1BB antibodies are being tested for the treatment of patients with advanced cancers.
METHODS: We studied the antitumor activity of 4-1BB mAb therapy using [(18) F]-labeled fluoro-2-deoxy-2-D-glucose ([(18) F]FDG) microPET scanning in a mouse model of colon cancer. Results of microPET imaging were correlated with morphological changes in tumors, draining lymph nodes as well as cell subset uptake of the metabolic PET tracer in vitro.
RESULTS: The administration of 4-1BB mAb to Balb/c mice induced reproducible CT26 tumor regressions and improved survival; complete tumor shrinkage was achieved in the majority of mice. There was markedly increased [(18) F]FDG signal at the tumor site and draining lymph nodes. In a metabolic probe in vitro uptake assay, there was an 8-fold increase in uptake of [(3)H]DDG in leukocytes extracted from tumors and draining lymph nodes of mice treated with 4-1BB mAb compared to untreated mice, supporting the in vivo PET data.
CONCLUSION: Increased uptake of [(18) F]FDG by PET scans visualizes 4-1BB agonistic antibody-induced antitumor immune responses and can be used as a pharmacodynamic readout to guide the development of this class of antibodies in the clinic.
Shu CJ, Radu CG, Shelly SM, Vo DD, Prins R, Ribas A, Phelps ME, Witte ON. Quantitative PET reporter gene imaging of CD8+ T cells specific for a melanoma-expressed self-antigen. Int Immunol. 2009 Feb;21(2):155-65. doi: 10.1093/intimm/dxn133. Epub 2008 Dec 23.
Adoptive transfer (AT) T-cell therapy provides significant clinical benefits in patients with advanced melanoma. However, approaches to non-invasively visualize the persistence of transferred T cells are lacking. We examined whether positron emission tomography (PET) can monitor the distribution of self-antigen-specific T cells engineered to express an herpes simplex virus 1 thymidine kinase (sr39tk) PET reporter gene. Micro-PET imaging using the sr39tk-specific substrate 9-[4-[(18)F]fluoro-3-(hydroxymethyl)-butyl]guanine ([(18)F]FHBG) enabled the detection of transplanted T cells in secondary lymphoid organs of recipient mice over a 3-week period. Tumor responses could be predicted as early as 3 days following AT when a >25-fold increase of micro-PET signal in the spleen and 2-fold increase in lymph nodes (LNs) were observed in mice receiving combined immunotherapy versus control mice. The lower limit of detection was approximately 7 x 10(5) T cells in the spleen and 1 x 10(4) T cells in LNs. Quantification of transplanted T cells in the tumor was hampered by the sr39tk-independent trapping of [(18)F]FHBG within the tumor architecture. These data support the feasibility of using PET to visualize the expansion, homing and persistence of transferred T cells. PET may have significant clinical utility by providing the means to quantify anti-tumor T cells throughout the body and provide early correlates for treatment efficacy.
Prins RM, Shu CJ, Radu CG, Vo DD, Khan-Farooqi H, Soto H, Yang MY, Lin MS, Shelly S, Witte ON, Ribas A, Liau LM. Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain. Cancer Immunol Immunother. 2008 Sep;57(9):1279-89. doi: 10.1007/s00262-008-0461-1. Epub 2008 Feb 6.
It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.c.) tumors underwent total body irradiation (TBI) to induce transient lymphopenia, followed by the adoptive transfer of gp100(25-33)-specific CD8+ T cells (Pmel-1). Pmel-1 cells were transduced to express the bioluminescent imaging (BLI) gene luciferase. Following adoptive transfer, recipient mice were vaccinated with hgp100(25-33) peptide-pulsed dendritic cells (hgp100(25-33)/DC) and systemic interleukin 2 (IL-2). This treatment regimen resulted in significant reduction in tumor size and extended survival. Imaging of T cell trafficking demonstrated early accumulation of transduced T cells in lymph nodes draining the hgp100(25-33)/DC vaccination sites, the spleen and the cervical lymph nodes draining the CNS tumor. Subsequently, transduced T cells accumulated in the bone marrow and brain tumor. BLI could also detect significant differences in the expansion of gp100-specific CD8+ T cells in the treatment group compared with mice that did not receive either DC vaccination or IL-2. These differences in BLI correlated with the differences seen both in survival and tumor infiltrating lymphocytes (TIL). These studies demonstrate that peripheral tolerance to endogenous TAA can be overcome to treat tumors in the brain and suggest a novel trafficking paradigm for the homing of tumor-specific T cells that target CNS tumors.
Tavare R, McCracken MN, Zettlitz KA, Salazar FB, Olafsen T, Witte ON, Wu AM. ImmunoPET of murine T cell reconstitution post-adoptive stem cell transplant using anti-CD4 and anti-CD8 cys-diabodies. J Nucl Med. 2015 May 7. pii: jnumed.114.153338.
The proliferation and trafficking of T lymphocytes in immune responses are crucial events in determining inflammatory responses. To study whole body T lymphocyte dynamics non-invasively in vivo, we have generated anti-CD4 and -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 and 2.43, respectively, for 89Zr-immunoPET detection of helper and cytotoxic T cell populations.
METHODS: Anti-CD4 and -CD8 cys-diabodies were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography, and characterized for T cell binding. The cys-diabodies were site-specifically conjugated to maleimide-desferrioxamine for 89Zr radiolabeling and subsequent microPET/CT acquisition and ex vivo biodistribution in both wild type mice and a model of hematopoietic stem cell (HSC) transplantation.
RESULTS: ImmunoPET and biodistribution studies demonstrate targeting and visualization of CD4 and CD8 T cell populations in vivo in the spleen and lymph nodes of wild type mice, with specificity confirmed through in vivo blocking and depletion studies. Subsequently, a murine model of HSC transplantation demonstrated successful in vivo detection of T cell repopulation at 2, 4, and 8 weeks post-HSC transplant using the 89Zr-radiolabeled anti-CD4 and -CD8 cDbs.
CONCLUSION: These newly developed anti-CD4 and -CD8 immunoPET reagents represent a powerful resource to monitor T cell expansion, localization and novel engraftment protocols. Future potential applications of T cell targeted immunoPET include monitoring immune cell subsets in response to immunotherapy, autoimmunity, and lymphoproliferative disorders, contributing overall to preclinical immune cell monitoring.
Yu AS, Hirayama BA, Timbol G, Liu J, Diez-Sampedro A, Kepe V, Satyamurthy N, Huang SC, Wright EM, Barrio JR. Regional distribution of SGLT activity in rat brain in vivo. Am J Physiol Cell Physiol. 2013 Feb 1;304(3):C240-7. doi: 10.1152/ajpcell.00317.2012. Epub 2012 Nov 14.
Na(+)-glucose cotransporter (SGLT) mRNAs have been detected in many organs of the body, but, apart from kidney and intestine, transporter expression, localization, and functional activity, as well as physiological significance, remain elusive. Using a SGLT-specific molecular imaging probe, α-methyl-4-deoxy-4-[(18)F]fluoro-D-glucopyranoside (Me-4-FDG) with ex vivo autoradiography and immunohistochemistry, we mapped in vivo the regional distribution of functional SGLTs in rat brain. Since Me-4-FDG is not a substrate for GLUT1 at the blood-brain barrier (BBB), in vivo delivery of the probe into the brain was achieved after opening of the BBB by an established procedure, osmotic shock. Ex vivo autoradiography showed that Me-4-FDG accumulated in regions of the cerebellum, hippocampus, frontal cortex, caudate nucleus, putamen, amygdala, parietal cortex, and paraventricular nucleus of the hypothalamus. Little or no Me-4-FDG accumulated in the brain stem. The regional accumulation of Me-4-FDG overlapped the distribution of SGLT1 protein detected by immunohistochemistry. In summary, after the BBB is opened, the specific substrate for SGLTs, Me-4-FDG, enters the brain and accumulates in selected regions shown to express SGLT1 protein. This localization and the sensitivity of these neurons to anoxia prompt the speculation that SGLTs may play an essential role in glucose utilization under stress such as ischemia. The expression of SGLTs in the brain raises questions about the potential effects of SGLT inhibitors under development for the treatment of diabetes.
Teng E, Kepe V, Frautschy SA, Liu J, Satyamurthy N, Yang F, Chen PP, Cole GB, Jones MR, Huang SC, Flood DG, Trusko SP, Small GW, Cole GM, Barrio JR. [F-18]FDDNP microPET imaging correlates with brain Aβ burden in a transgenic rat model of Alzheimer disease: effects of aging, in vivo blockade, and anti-Aβ antibody treatment. Neurobiol Dis. 2011 Sep;43(3):565-75. doi: 10.1016/j.nbd.2011.05.003. Epub 2011 May 13.
In vivo detection of Alzheimer's disease (AD) neuropathology in living patients using positron emission tomography (PET) in conjunction with high affinity molecular imaging probes for β-amyloid (Aβ) and tau has the potential to assist with early diagnosis, evaluation of disease progression, and assessment of therapeutic interventions. Animal models of AD are valuable for exploring the in vivo binding of these probes, particularly their selectivity for specific neuropathologies, but prior PET experiments in transgenic mice have yielded conflicting results. In this work, we utilized microPET imaging in a transgenic rat model of brain Aβ deposition to assess [F-18]FDDNP binding profiles in relation to age-associated accumulation of neuropathology. Cross-sectional and longitudinal imaging demonstrated that [F-18]FDDNP binding in the hippocampus and frontal cortex progressively increases from 9 to 18months of age and parallels age-associated Aβ accumulation. Specificity of in vivo [F-18]FDDNP binding was assessed by naproxen pretreatment, which reversibly blocked [F-18]FDDNP binding to Aβ aggregrates. Both [F-18]FDDNP microPET imaging and neuropathological analyses revealed decreased Aβ burden after intracranial anti-Aβ antibody administration. The combination of this non-invasive imaging method and robust animal model of brain Aβ accumulation allows for future longitudinal in vivo assessments of potential therapeutics for AD that target Aβ production, aggregation, and/or clearance. These results corroborate previous analyses of [F-18]FDDNP PET imaging in clinical populations.
Hsu JJ, Lu J, Umar S, Lee JT, Kulkarni RP, Ding Y, Chang CC, Hsiai T, Hokugo A, Gkouveris I, Tetradis S, Nishimura I, Demer LL, Tintut Y. Effects of bone anabolic therapy on progression of calcific aortic vasculopathy in Apoe-/- mice. Am J Physiol Heart Circ Physiol. 2018 Feb 16. PMC6032086https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032086/
Pillai ICL, Li S, Romay M, Lam L, Lu Y, Huang J, Dillard N, Zemanova M, Rubbi L, Wang Y, Lee JT, Xia M, Liang O, Xie YH, Pellegrini M, Lusis AJ and Deb A. Cardiac Fibroblasts Adopt Osteogenic Fates and Can Be Targeted to Attenuate Pathological Heart Calcification. Cell Stem Cell. 2017 Feb; 20(2):1–15. PMCID: PMC5291784https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291784
Xu S, Zhou T, Doh HM, Trinh R, Catapang A, Lee JT, Braas D, Bayley NA, Yamada RE, Vasuthasawat A, Sasine JP, Timmerman JM, Larson SM, Kim Y, MacLeod AR, Morrison SL, Herschman HR. An HK2 antisense oligonucleotide induces synthetic lethality in HK1-HK2+ multiple myeloma. Cancer Res. 2019 March 18. doi:10.1158/0008-5472.CAN-18-2799. PMID: 30885978https://www.ncbi.nlm.nih.gov/pubmed/30885978
Xu S, Catapang A, Braas D, Stiles L, Doh HM, Lee JT, Graeber TG, Damoiseaux R, Shirihai O, Herschman HR. A precision therapeutic strategy for hexokinase 1-null, hexokinase 2-positive cancers. Cancer Metab. 2018 Jun 28;6:7. doi: 10.1186/s40170-018-0181-8. eCollection 2018. PMC6022704https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022704
Liu X, Lin P, Perrett I, Lin J, Liao YP, Chang CH, Jiang J, Wu N, Donahue T, Wainberg Z, Nel AE, Meng H. Tumor-penetrating peptide enhances transcytosis of silicasome-based chemotherapy for pancreatic cancer. J Clin Invest. 2017 May 1;127:2007-2018. PMC5409788https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409788
Liu X, Situ A, Kang Y, Villabroza KR, Liao Y, Chang CH, Donahue T, Nel AE, Meng H. Irinotecan Delivery by Lipid-Coated Mesoporous Silica Nanoparticles Shows Improved Efficacy and Safety over Liposomes for Pancreatic Cancer. ACS Nano. 2016 Feb 23;10:2702-15. PMC4851343https://www.ncbi.nlm.nih.gov/pubmed/26835979
Murphy JM, Armijo AL, Nomme J, Lee CH, Smith QA, Li Z, Campbell DO, Liao HI, Nathanson DA, Austin WR, Lee JT, Darvish R, Wei L, Wang J, Su Y, Damoiseaux R, Sadeghi S, Phelps ME, Herschman HR, Czernin J, Alexandrova AN, Jung ME, Lavie A, Radu CG. Development of new deoxycytidine kinase inhibitors and noninvasive in vivo evaluation using positron emission tomography. J Med Chem. 2013 Sep 12;56(17):6696-708. doi: 10.1021/jm400457y. Epub 2013 Aug 15.
Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest, and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using (18)F-L-1-(2'-deoxy-2'-FluoroArabinofuranosyl) Cytosine ((18)F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC50 = ∼1-12 nM) using the (18)F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.http://www.ncbi.nlm.nih.gov/pubmed/23947754
Lee JH, Chen KJ, Noh SH, Garcia MA, Wang H, Lin WY, Jeong H, Kong BJ, Stout DB, Cheon J, Tseng HR. On-demand drug release system for in vivo cancer treatment through self-assembled magnetic nanoparticles. Angew Chem Int Ed Engl. 2013 Apr 15;52(16):4384-8. doi: 10.1002/anie.201207721. Epub 2013 Mar 20.
On-demand drug release: Magnetothermally responsive drug-encapsulated supramolecular nanoparticles for on-demand drug release in vivo have been developed. The remote application of an alternative magnetic field heats the magnetic particles that effectively trigger the release of the drug. An acute drug concentration can be delivered to the tumor in vivo, resulting in an improved therapeutic outcome.
Gu Z, Taschereau R, Vu N, Prout DL, Silverman RW, Lee JT, Chatziioannou AF. Performance evaluation of G8, a high sensitivity benchtop preclinical PET/CT tomograph. J Nucl Med. 2018 Jun 14. doi: 10.2967/jnumed.118.208827. [Epub ahead of print]. PMC6354226.https://www.ncbi.nlm.nih.gov/pubmed/29903933
Sergeev ME, Lazari M, Morgia F, Collins J, Javed MR, Sergeeva O, Jones J, Phelps ME, Lee JT, Keng PY, van Dam RM. Performing radiosynthesis in microvolumes to maximize molar activity of tracers for positron emission tomography. Comm Chemistry. 2018 Mar 22; 1(10). [Epub ahead of print].https://www.nature.com/articles/s42004-018-0009-z
Collins J, Waldmann CM, Drake C, Slavik R, Ha NS, Sergeev M, Lazari M, Shen B, Chin FT, Moore M, Sadeghi S, Phelps ME, Murphy JM, van Dam RM. Production of diverse PET probes with limited resources: 2418F-labeled compounds prepared with a single radiosynthesizer. Proc Natl Acad Sci U S A. 2017 Oct 24;114:11309-11314. PMC5664529https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664529
Chao P, Collins J, Argus J, Tseng W-Y, Lee JT, van Dam RM. Automatic concentration and reformulation of PET tracers via microfluidic membrane distillation. Lab on a Chip. 2017 May 16;17(10):1802-1816. PMCID: PMC5497730.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497730
Dooraghi AA, Carroll L, Collins J, van Dam RM, Chatziioannou AF. ARAS: an automated radioactivity aliquoting system for dispensing solutions containing positron-emitting radioisotopes. EJNMMI Res. 2016 Dec 1;6:22. PMC4783308https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783308
Gu Z, Prout DL, Silverman RW, Herman H, Dooraghi A, Chatziioannou AF. A DOI Detector With Crystal Scatter Identification Capability for High Sensitivity and High Spatial Resolution PET Imaging. IEEE Trans Nucl Sci. 2015;62:740-747. PMC4608445https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608445
Shin YS, Kim J, Johnson D, Dooraghi AA, Mai WX, Ta L, Chatziioannou AF, Phelps ME, Nathanson DA, Heath JR. Quantitative assessments of glycolysis from single cells. Technology (Singap World Sci). 2015 Jun 1;3:172-178. PMC4728151.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728151
Wang H, Stout DB, Chatziioannou AF. A deformable atlas of the laboratory mouse. Mol Imaging Biol. 2015 Feb;17(1):18-28. doi: 10.1007/s11307-014-0767-7.
PURPOSE: This paper presents a deformable mouse atlas of the laboratory mouse anatomy. This atlas is fully articulated and can be positioned into arbitrary body poses. The atlas can also adapt body weight by changing body length and fat amount.
PROCEDURES: A training set of 103 micro-CT images was used to construct the atlas. A cage-based deformation method was applied to realize the articulated pose change. The weight-related body deformation was learned from the training set using a linear regression method. A conditional Gaussian model and thin-plate spline mapping were used to deform the internal organs following the changes of pose and weight.
RESULTS: The atlas was deformed into different body poses and weights, and the deformation results were more realistic compared to the results achieved with other mouse atlases. The organ wights of this atlas matched well with the measurements of real mouse organ weights. This atlas can also be converted into voxelized images with labeled organs, pseudo CT images and tetrahedral mesh for phantom studies.
CONCLUSIONS: With the unique ability of articulated pose and weight changes, the deformable laboratory mouse atlas can become a valuable tool for preclinical image analysis.http://www.ncbi.nlm.nih.gov/pubmed/25049072
Herman H, Flores G, Quinn K, Eddings M, Olma S, Moore MD, Ding H, Bobinski KP, Wang M, Williams D, Wiliams D, Shen CK, Phelps ME, Michael van Dam R. Plug-and-play modules for flexible radiosynthesis. Appl Radiat Isot. 2013 Aug;78:113-24. doi: 10.1016/j.apradiso.2013.04.023. Epub 2013 Apr 25.
We present a plug-and-play radiosynthesis platform and accompanying computer software based on modular subunits that can easily and flexibly be configured to implement a diverse range of radiosynthesis protocols. Modules were developed that perform: (i) reagent storage and delivery, (ii) evaporations and sealed reactions, and (iii) cartridge-based purifications. The reaction module incorporates a simple robotic mechanism that removes tubing from the vessel and replaces it with a stopper prior to sealed reactions, enabling the system to withstand high pressures and thus provide tremendous flexibility in choice of solvents and temperatures. Any number of modules can rapidly be connected together using only a few fluidic connections to implement a particular synthesis, and the resulting system is controlled in a semi-automated fashion by a single software interface. Radiosyntheses of 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG), 1-[(18)F]fluoro-4-nitrobenzene ([(18)F]FNB), and 2'-deoxy-2'-[(18)F]fluoro-1-β-d-arabinofuranosyl cytosine (d-[(18)F]FAC) were performed to validate the system and demonstrate its versatility.
Lazari M, Quinn KM, Claggett SB, Collins J, Shah GJ, Herman HE, Maraglia B, Phelps ME, Moore MD, van Dam RM. ELIXYS - a fully automated, three-reactor high-pressure radiosynthesizer for development and routine production of diverse PET tracers. EJNMMI Res. 2013 Jul 12;3(1):52. doi: 10.1186/2191-219X-3-52.
BACKGROUND: Automated radiosynthesizers are vital for routine production of positron-emission tomography tracers to minimize radiation exposure to operators and to ensure reproducible synthesis yields. The recent trend in the synthesizer industry towards the use of disposable kits aims to simplify setup and operation for the user, but often introduces several limitations related to temperature and chemical compatibility, thus requiring reoptimization of protocols developed on non-cassette-based systems. Radiochemists would benefit from a single hybrid system that provides tremendous flexibility for development and optimization of reaction conditions while also providing a pathway to simple, cassette-based production of diverse tracers.
METHODS: We have designed, built, and tested an automated three-reactor radiosynthesizer (ELIXYS) to provide a flexible radiosynthesis platform suitable for both tracer development and routine production. The synthesizer is capable of performing high-pressure and high-temperature reactions by eliminating permanent tubing and valve connections to the reaction vessel. Each of the three movable reactors can seal against different locations on disposable cassettes to carry out different functions such as sealed reactions, evaporations, and reagent addition. A reagent and gas handling robot moves sealed reagent vials from storage locations in the cassette to addition positions and also dynamically provides vacuum and inert gas to ports on the cassette. The software integrates these automated features into chemistry unit operations (e.g., React, Evaporate, Add) to intuitively create synthesis protocols. 2-Deoxy-2-[18F]fluoro-5-methyl-β-l-arabinofuranosyluracil (l-[18F]FMAU) and 2-deoxy-2-[18F]fluoro-β-d-arabinofuranosylcytosine (d-[18F]FAC) were synthesized to validate the system.
RESULTS: l-[18F]FMAU and d-[18F]FAC were successfully synthesized in 165 and 170 min, respectively, with decay-corrected radiochemical yields of 46% ± 1% (n = 6) and 31% ± 5% (n = 6), respectively. The yield, repeatability, and synthesis time are comparable to, or better than, other reports. d-[18F]FAC produced by ELIXYS and another manually operated apparatus exhibited similar biodistribution in wild-type mice.
CONCLUSION: The ELIXYS automated radiosynthesizer is capable of performing radiosyntheses requiring demanding conditions: up to three reaction vessels, high temperatures, high pressures, and sensitive reagents. Such flexibility facilitates tracer development and the ability to synthesize multiple tracers on the same system without customization or replumbing. The disposable cassette approach simplifies the transition from development to production.http://www.ncbi.nlm.nih.gov/pubmed/23849185
Gu Z, Taschereau R, Vu NT, Wang H, Prout DL, Silverman RW, Bai B, Stout DB, Phelps ME, Chatziioannou AF. NEMA NU-4 performance evaluation of PETbox4, a high sensitivity dedicated PET preclinical tomograph. Phys Med Biol. 2013 Jun 7;58(11):3791-814. doi: 10.1088/0031-9155/58/11/3791. Epub 2013 May 10.
PETbox4 is a new, fully tomographic bench top PET scanner dedicated to high sensitivity and high resolution imaging of mice. This manuscript characterizes the performance of the prototype system using the National Electrical Manufacturers Association NU 4-2008 standards, including studies of sensitivity, spatial resolution, energy resolution, scatter fraction, count-rate performance and image quality. The PETbox4 performance is also compared with the performance of PETbox, a previous generation limited angle tomography system. PETbox4 consists of four opposing flat-panel type detectors arranged in a box-like geometry. Each panel is made by a 24 × 50 pixelated array of 1.82 × 1.82 × 7 mm bismuth germanate scintillation crystals with a crystal pitch of 1.90 mm. Each of these scintillation arrays is coupled to two Hamamatsu H8500 photomultiplier tubes via a glass light guide. Volumetric images for a 45 × 45 × 95 mm field of view (FOV) are reconstructed with a maximum likelihood expectation maximization algorithm incorporating a system model based on a parameterized detector response. With an energy window of 150-650 keV, the peak absolute sensitivity is approximately 18% at the center of FOV. The measured crystal energy resolution ranges from 13.5% to 48.3% full width at half maximum (FWHM), with a mean of 18.0%. The intrinsic detector spatial resolution is 1.5 mm FWHM in both transverse and axial directions. The reconstructed image spatial resolution for different locations in the FOV ranges from 1.32 to 1.93 mm, with an average of 1.46 mm. The peak noise equivalent count rate for the mouse-sized phantom is 35 kcps for a total activity of 1.5 MBq (40 µCi) and the scatter fraction is 28%. The standard deviation in the uniform region of the image quality phantom is 5.7%. The recovery coefficients range from 0.10 to 0.93. In comparison to the first generation two panel PETbox system, PETbox4 achieves substantial improvements on sensitivity and spatial resolution. The overall performance demonstrates that the PETbox4 scanner is suitable for producing high quality images for molecular imaging based biomedical research.
Keng PY, Chen S, Ding H, Sadeghi S, Shah GJ, Dooraghi A, Phelps ME, Satyamurthy N, Chatziioannou AF, Kim CJ, van Dam RM. Micro-chemical synthesis of molecular probes on an electronic microfluidic device. Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):690-5. doi: 10.1073/pnas.1117566109. Epub 2011 Dec 30.
We have developed an all-electronic digital microfluidic device for microscale chemical synthesis in organic solvents, operated by electrowetting-on-dielectric (EWOD). As an example of the principles, we demonstrate the multistep synthesis of [(18)F]FDG, the most common radiotracer for positron emission tomography (PET), with high and reliable radio-fluorination efficiency of [(18)F]FTAG (88 ± 7%, n = 11) and quantitative hydrolysis to [(18)F]FDG (> 95%, n = 11). We furthermore show that batches of purified [(18)F]FDG can successfully be used for PET imaging in mice and that they pass typical quality control requirements for human use (including radiochemical purity, residual solvents, Kryptofix, chemical purity, and pH). We report statistical repeatability of the radiosynthesis rather than best-case results, demonstrating the robustness of the EWOD microfluidic platform. Exhibiting high compatibility with organic solvents and the ability to carry out sophisticated actuation and sensing of reaction droplets, EWOD is a unique platform for performing diverse microscale chemical syntheses in small volumes, including multistep processes with intermediate solvent-exchange steps.
Lu Y, Machado HB, Bao Q, Stout D, Herschman H, Chatziioannou AF. In vivo mouse bioluminescence tomography with radionuclide-based imaging validation. Mol Imaging Biol. 2011 Feb;13(1):53-8. doi: 10.1007/s11307-010-0332-y.
INTRODUCTION: Bioluminescence imaging, especially planar bioluminescence imaging, has been extensively applied in in vivo preclinical biological research. Bioluminescence tomography (BLT) has the potential to provide more accurate imaging information due to its 3D reconstruction compared with its planar counterpart.
METHODS: In this work, we introduce a positron emission tomography (PET) radionuclide imaging-based strategy to validate the BLT results. X-ray computed tomography, PET, spectrally resolved bioluminescence imaging, and surgical excision were performed on a tumor xenograft mouse model expressing a bioluminescent reporter gene.
SULTS: With different spectrally resolved measured data, the BLT reconstructions were acquired based on the third-order simplified spherical harmonics (SP3) approximation and the diffusion approximation (DA). The corresponding tomographic images were obtained for validation of bioluminescence source reconstruction.
CONCLUSION: Our results show the strength of PET imaging compared with other validation methods for BLT and improved source localization accuracy based on the SP(3) approximation compared with the diffusion approximation
Cho JS, Taschereau R, Olma S, Liu K, Chen YC, Shen CK, van Dam RM, Chatziioannou AF. Cerenkov radiation imaging as a method for quantitative measurements of beta particles in a microfluidic chip. Phys Med Biol. 2009 Nov 21;54(22):6757-71. doi: 10.1088/0031-9155/54/22/001. Epub 2009 Oct 21.
It has been observed that microfluidic chips used for synthesizing (18)F-labeled compounds demonstrate visible light emission without nearby scintillators or fluorescent materials. The origin of the light was investigated and found to be consistent with the emission characteristics from Cerenkov radiation. Since (18)F decays through the emission of high-energy positrons, the energy threshold for beta particles, i.e. electrons or positrons, to generate Cerenkov radiation was calculated for water and polydimethylsiloxane (PDMS), the most commonly used polymer-based material for microfluidic chips. Beta particles emitted from (18)F have a continuous energy spectrum, with a maximum energy that exceeds this energy threshold for both water and PDMS. In addition, the spectral characteristics of the emitted light from (18)F in distilled water were also measured, yielding a broad distribution from 300 nm to 700 nm, with higher intensity at shorter wavelengths. A photograph of the (18)F solution showed a bluish-white light emitted from the solution, further suggesting Cerenkov radiation. In this study, the feasibility of using this Cerenkov light emission as a method for quantitative measurements of the radioactivity within the microfluidic chip in situ was evaluated. A detector previously developed for imaging microfluidic platforms was used. The detector consisted of a charge-coupled device (CCD) optically coupled to a lens. The system spatial resolution, minimum detectable activity and dynamic range were evaluated. In addition, the calibration of a Cerenkov signal versus activity concentration in the microfluidic chip was determined. This novel method of Cerenkov radiation measurements will provide researchers with a simple yet robust quantitative imaging tool for microfluidic applications utilizing beta particles.
Zoller S, Park H, Olafsen T, Zamilpa C, Burke ZD, Blumstein G, Sheppard WL, Hamad CD, Hori KR, Tseng JC, Czupryna J, McMannus C, Lee JT, Bispo M, Pastrana FR, Raineri EJM, Miller LS, Dijl JM, Francis KP, Bernthal NM. Multimodal Imaging Guides Surgical Management in a Preclinical Spinal Implant Infection Model. JCI Insight. 2019 Feb 7;4(3):e124813. doi: 10.1172/jci.insight.124813. PMC6413782https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413782
Salas JR, Chen BY, Wong A, Duarte S, Angarita SAK, Lipshutz GS, Witte ON, Clark PM. Noninvasive Imaging of Drug-Induced Liver Injury with 18F-DFA PET. J Nucl Med. 2018 Aug;59:1308-1315. PMC6071498https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071498
Lisova K, Sergeev M, Evans-Axelsson S, Stuparu AD, Beykan S, Collins J, Jones J, Lassmann M, Herrmann K, Perrin D, Lee JT, Slavik R, van Dam M. Microfluidic radiosynthesis, preclinical imaging and dosimetry study of [18F]AMBF3-TATE: a potential PET tracer for clinical imaging of somatostatin receptors. Nucl Med Biol. 2018 Apr 20;61:36-44. PMCID: PMC6015542https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015542
Gonzalez C, Sanchez A, Collins J, Lisova K, Lee JT, van Dam RM, Barbieri MA, Ramanchandran C, Wnuk SF. The 4-N-Acyl and 4-N-Alkyl Gemcitabine Analogues with Silicon-Fluoride-Acceptor: Application to 18F-Radiolabeling. Eur J Med Chem. 2018 Mar 25;148:314-324. [Epub ahead of print]. PMCID: PMC5841594https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841594
Radiochemistry on electrodes: synthesis of an 18F-labelled and in vivo-stable COX-2 inhibitorhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413030
Lee JT, Zhang H, Moroz MA, Likar Y, Shenker L, Sumzin N, Lobo J, Zurita J, Collins J, van Dam RM, Ponomarev V. Comparative Analysis of Human Nucleoside Kinase-Based Reporter Systems for PET Imaging. Mol Imaging Biol. 2017 Feb;19(1):100-108. PMCID: PMC5345744.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345744
Lee JT*, Boschi S*, Beykan S, Slavik R, Wei L, Spick C, Eberlein U, Buck AK, Lodi F, Cicoria G, Czernin J, Lassmann M, Fanti S, Herrmann K. Synthesis and preclinical evaluation of an Al18F radiofluorinated GLU-UREA-LYS(AHX)-HBED-CC PSMA ligand. Eur J Nucl Med Mol Imaging. 2016 Nov; 43(12):2122-2130. PMC5050145. *contributed equally to this work.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050145
Hou S, Choi JS, Garcia MA, Xing Y, Chen KJ, Chen YM, Jiang ZK, Ro T, Wu L, Stout DB, Tomlinson JS, Wang H, Chen K, Tseng HR, Lin WY. Pretargeted Positron Emission Tomography Imaging That Employs Supramolecular Nanoparticles with in Vivo Bioorthogonal Chemistry. ACS Nano. 2016 Jan 26;10:1417-24. PMC4893318https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893318
Mosessian S, Duarte-Vogel SM, Stout DB, Roos KP, Lawson GW, Jordan MC, Ogden A, Matter C, Sadeghi S, Mills GQ, Schelbert HR, Shu CG, Czernin J, Couto M, Phelps ME. INDs for PET molecular imaging probes-approach by an academic institution. Mol Imaging Biol. 2014 Aug;16(4):441-8. doi: 10.1007/s11307-014-0735-2.
We have developed an efficient, streamlined, cost-effective approach to obtain Investigational New Drug (IND) approvals from the Food and Drug Administration (FDA) for positron emission tomography (PET) imaging probes (while the FDA uses the terminology PET drugs, we are using "PET imaging probes," "PET probes," or "probes" as the descriptive terms). The required application and supporting data for the INDs were collected in a collaborative effort involving appropriate scientific disciplines. This path to INDs was successfully used to translate three [(18) F]fluoro-arabinofuranosylcytosine (FAC) analog PET probes to phase 1 clinical trials. In doing this, a mechanism has been established to fulfill the FDA regulatory requirements for translating promising PET imaging probes from preclinical research into human clinical trials in an efficient and cost-effective manner.
Clark PM, Flores G, Evdokimov NM, Clark MN, Chai T, Nair-Gill E, O'Mahony F, Beaven SW, Faull KF, Phelps ME, Jung ME, Witte ON. Positron emission tomography probe demonstrates a striking concentration of ribose salvage in the liver. Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):E2866-74. doi: 10.1073/pnas.1410326111. Epub 2014 Jun 30.
PET is a powerful technique for quantifying and visualizing biochemical pathways in vivo. Here, we develop and validate a novel PET probe, [(18)F]-2-deoxy-2-fluoroarabinose ([(18)F]DFA), for in vivo imaging of ribose salvage. DFA mimics ribose in vivo and accumulates in cells following phosphorylation by ribokinase and further metabolism by transketolase. We use [(18)F]DFA to show that ribose preferentially accumulates in the liver, suggesting a striking tissue specificity for ribose metabolism. We demonstrate that solute carrier family 2, member 2 (also known as GLUT2), a glucose transporter expressed in the liver, is one ribose transporter, but we do not know if others exist. [(18)F]DFA accumulation is attenuated in several mouse models of metabolic syndrome, suggesting an association between ribose salvage and glucose and lipid metabolism. These results describe a tool for studying ribose salvage and suggest that plasma ribose is preferentially metabolized in the liver.http://www.ncbi.nlm.nih.gov/pubmed/24982199
Gil JS, Machado HB, Campbell DO, Clark M, Shu C, Witte ON, Herschman HR. Application of a rapid, simple, and accurate adenovirus-based method to compare PET reporter gene/PET reporter probe systems. Mol Imaging Biol. 2013 Jun;15(3):273-81. doi: 10.1007/s11307-012-0596-5.
PURPOSE: This study aims to use a simple, quantitative method to compare the HSV1sr39TK/(18) F-FHBG PET reporter gene/PET reporter probe (PRG/PRP) system with PRGs derived from human nucleoside kinases.
PROCEDURES: The same adenovirus vector is used to express alternative PRGs. Equal numbers of vectors are injected intravenously into mice. After PRP imaging, quantitative hepatic PET signals are normalized for transduction by measuring hepatic viral genomes.
RESULTS: The same adenovirus vector was used to express equivalent amounts of HSV1sr39TK, mutant human thymidine kinase 2 (TK2-DM), and mutant human deoxycytidine kinase (dCK-A100VTM) in mouse liver. HSV1sr39TK expression was measured with (18) F-FHBG, TK2-DM and dCK-A100VTM with (18) F-L-FMAU. TK2-DM/(18) F-L-FMAU and HSV1sr39TK/(18) F-FHBG had equivalent sensitivities; dCK-A100VTM/(18) F-L-FMAU was twice as sensitive as HSV1sr39TK/(18) F-FHBG.
CONCLUSIONS: The human PRG/PRP sensitivities are comparable and/or better than HSV1sr39TK/(18) F-FHBG. However, for clinical use, identification of the best PRP substrate for each enzyme, characterization of probe distribution, and consequences of overexpressing nucleoside kinases must be evaluated.http://www.ncbi.nlm.nih.gov/pubmed/23054556
Campbell DO, Campbell SS, Su Y, Lee JT, Auerbach MS, Herschman H, Satyamurthy N, Czernin J, Lavie A, Shu CG. Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe. J Biol Chem. 2012 Jan 2;287(1):446-54. doi: 10.1074/jbc.M111.314666. Epub 2011 Nov 9.
Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-β-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.
Shu CJ, Campbell DO, Lee JT, Tran AQ, Wengrod JC, Witte ON, Phelps ME, Satyamurthy N, Czernin J, Shu CG. Novel PET probes specific for deoxycytidine kinase. J Nucl Med. 2010 Jul;51(7):1092-8. doi: 10.2967/jnumed.109.073361. Epub 2010 Jun 16.
Deoxycytidine kinase (dCK) is a rate-limiting enzyme in the deoxyribonucleoside salvage pathway and a critical determinant of therapeutic activity for several nucleoside analog prodrugs. We have previously reported the development of 1-(2'-deoxy-2'-(18)F-fluoro-beta-D-arabinofuranosyl)cytosine ((18)F-FAC), a new probe for PET of dCK activity in immune disorders and certain cancers. The objective of the current study was to develop PET probes with improved metabolic stability and specificity for dCK. Toward this goal, several candidate PET probes were synthesized and evaluated in vitro and in vivo.
METHODS: High-pressure liquid chromatography was used to analyze the metabolic stability of (18)F-FAC and several newly synthesized analogs with the natural D-enantiomeric sugar configuration or the corresponding unnatural L-configuration. In vitro kinase and uptake assays were used to determine the affinity of the (18)F-FAC L-nucleoside analogs for dCK. The biodistribution of selected L-analogs in mice was determined by small-animal PET/CT.
RESULTS: Candidate PET probes were selected using the following criteria: low susceptibility to deamination, high affinity for purified recombinant dCK, high uptake in dCK-expressing cell lines, and biodistribution in mice reflective of the tissue-expression pattern of dCK. Among the 10 newly developed candidate probes, 1-(2'-deoxy-2'-(18)F-fluoro-beta-L-arabinofuranosyl)cytosine (L-(18)F-FAC) and 1-(2'-deoxy-2'-(18)F-fluoro-beta-L-arabinofuranosyl)-5-methylcytosine (L-(18)F-FMAC) most closely matched the selection criteria. The selection of L-(18)F-FAC and L-(18)F-FMAC was validated by showing that these two PET probes could be used to image animal models of leukemia and autoimmunity.
CONCLUSION: Promising in vitro and in vivo data warrant biodistribution and dosimetry studies of L-(18)F-FAC and L-(18)F-FMAC in humans.http://www.ncbi.nlm.nih.gov/pubmed/20554721
Yong J, Rasooly J, Dang H, Lu Y, Middleton B, Zhang Z, Hon L, Namavari M, Stout DB, Atkinson MA, Tian J, Gambhir SS, Kaufman DL. Multimodality imaging of β-cells in mouse models of type 1 and 2 diabetes. Diabetes. 2011 May;60(5):1383-92. doi: 10.2337/db10-0907. Epub 2011 Mar 25.
OBJECTIVE: β-Cells that express an imaging reporter have provided powerful tools for studying β-cell development, islet transplantation, and β-cell autoimmunity. To further expedite diabetes research, we generated transgenic C57BL/6 "MIP-TF" mice that have a mouse insulin promoter (MIP) driving the expression of a trifusion (TF) protein of three imaging reporters (luciferase/enhanced green fluorescent protein/HSV1-sr39 thymidine kinase) in their β-cells. This should enable the noninvasive imaging of β-cells by charge-coupled device (CCD) and micro-positron emission tomography (PET), as well as the identification of β-cells at the cellular level by fluorescent microscopy.
RESEARCH DESIGN AND METHODS: MIP-TF mouse β-cells were multimodality imaged in models of type 1 and type 2 diabetes.
RESULTS: MIP-TF mouse β-cells were readily identified in pancreatic tissue sections using fluorescent microscopy. We show that MIP-TF β-cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed a progressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing proinsulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse β-cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, providing a new experimental model of type 1 diabetes. Mice fed a high-fat diet to model early type 2 diabetes displayed a progressive increase in their pancreatic bioluminescent signals, which were positively correlated with area under the curve-intraperitoneal glucose tolerance test (AUC-IPGTT).
CONCLUSIONS: MIP-TF mice provide a new tool for monitoring β-cells from the single cell level to noninvasive assessments of β-cells in models of type 1 diabetes and type 2 diabetes.
Wilks MQ, Knowles SM, Wu AM, Huang SC. Improved modeling of in vivo kinetics of slowly diffusing radiotracers for tumor imaging. J Nucl Med. 2014 Sep;55(9):1539-44. doi: 10.2967/jnumed.114.140038. Epub 2014 Jul 3.
Large-molecule tracers, such as labeled antibodies, have shown success in immuno-PET for imaging of specific cell surface biomarkers. However, previous work has shown that localization of such tracers shows high levels of heterogeneity in target tissues, due to both the slow diffusion and the high affinity of these compounds. In this work, we investigate the effects of subvoxel spatial heterogeneity on measured time-activity curves in PET imaging and the effects of ignoring diffusion limitation on parameter estimates from kinetic modeling.
METHODS: Partial differential equations (PDE) were built to model a radially symmetric reaction-diffusion equation describing the activity of immuno-PET tracers. The effects of slower diffusion on measured time-activity curves and parameter estimates were measured in silico, and a modified Levenberg-Marquardt algorithm with Bayesian priors was developed to accurately estimate parameters from diffusion-limited data. This algorithm was applied to immuno-PET data of mice implanted with prostate stem cell antigen-overexpressing tumors and injected with (124)I-labeled A11 anti-prostate stem cell antigen minibody.
RESULTS: Slow diffusion of tracers in linear binding models resulted in heterogeneous localization in silico but no measurable differences in time-activity curves. For more realistic saturable binding models, measured time-activity curves were strongly dependent on diffusion rates of the tracers. Fitting diffusion-limited data with regular compartmental models led to parameter estimate bias in an excess of 1,000% of true values, while the new model and fitting protocol could accurately measure kinetics in silico. In vivo imaging data were also fit well by the new PDE model, with estimates of the dissociation constant (Kd) and receptor density close to in vitro measurements and with order of magnitude differences from a regular compartmental model ignoring tracer diffusion limitation.
CONCLUSION: Heterogeneous localization of large, high-affinity compounds can lead to large differences in measured time-activity curves in immuno-PET imaging, and ignoring diffusion limitations can lead to large errors in kinetic parameter estimates. Modeling of these systems with PDE models with Bayesian priors is necessary for quantitative in vivo measurements of kinetics of slow-diffusion tracers.
Wong KP, Zhang X, Huang SC. Improved derivation of input function in dynamic mouse [18F]FDG PET using bladder radioactivity kinetics. Mol Imaging Biol. 2013 Aug;15(4):486-96. doi: 10.1007/s11307-013-0610-6.
PURPOSE: Accurate determination of the plasma input function (IF) is essential for absolute quantification of physiological parameters in positron emission tomography (PET). However, it requires an invasive and tedious procedure of arterial blood sampling that is challenging in mice because of the limited blood volume. In this study, a hybrid modeling approach is proposed to estimate the plasma IF of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) in mice using accumulated radioactivity in urinary bladder together with a single late-time blood sample measurement.
METHODS: Dynamic PET scans were performed on nine isoflurane-anesthetized male C57BL/6 mice after a bolus injection of [18F]FDG at the lateral caudal vein. During a 60- or 90-min scan, serial blood samples were taken from the femoral artery. Image data were reconstructed using filtered backprojection with computed tomography-based attenuation correction. Total accumulated radioactivity in the urinary bladder at late times was fitted to a renal compartmental model with the last blood sample and a one-exponential function that described the [18F]FDG clearance in blood. Multiple late-time blood sample estimates were calculated by the blood [18F]FDG clearance equation. A sum of four-exponentials was assumed for the plasma IF that served as a forcing function to all tissues. The estimated plasma IF was obtained by simultaneously fitting the [18F]FDG model to the time-activity curves (TACs) of liver and muscle and the forcing function to early (0-1 min) left-ventricle data (corrected for delay, dispersion, partial-volume effects, and erythrocyte uptake) and the late-time blood estimates. Using only the blood sample collected at the end of the study to estimate the IF and the use of liver TAC as an alternative IF were also investigated.
RESULTS: The area under the plasma IFs calculated for all studies using the hybrid approach was not significantly different from that using all blood samples. [18F]FDG uptake constants in brain, myocardium, skeletal muscle, and liver computed by the Patlak analysis using estimated and measured plasma IFs were in excellent agreement (slope∼1; R2>0.983). The IF estimated using only the last blood sample drawn at the end of the study and the use of liver TAC as the plasma IF provided less reliable results.
CONCLUSIONS: The estimated plasma IFs obtained with the hybrid method agreed well with those derived from arterial blood sampling. Importantly, the proposed method obviates the need of arterial catheterization, making it possible to perform repeated dynamic [18F]FDG PET studies on the same animal. Liver TAC is unsuitable as an input function for absolute quantification of [18F]FDG PET data.
Sha W, Ye H, Iwamoto KS, Wong KP, Wilks MQ, Stout D, McBride W, Huang SC. Factors affecting tumor (18) F-FDG uptake in longitudinal mouse PET studies. EJNMMI Res. 2013 Jul 10;3:51. doi: 10.1186/2191-219X-3-51. eCollection 2013.
BACKGROUND: Many biological factors of 2-[(18) F]fluoro-2-deoxy-d-glucose ((18) F-FDG) in blood can affect (18) F-FDG uptake in tumors. In this study, longitudinal (18) F-FDG positron emission tomography (PET) studies were performed on tumor-bearing mice to investigate the effect of blood glucose level and tumor size on (18) F-FDG uptake in tumors.
METHODS: Six- to eight-week-old severe combined immunodeficiency mice were implanted with glioblastoma U87 (n = 8) or adenocarcinoma MDA-MB-231 (MDA) (n = 11) in the shoulder. When the tumor diameter was approximately 2.5 mm, a 60-min dynamic (18) F-FDG PET scan was performed weekly until the tumor diameter reached 10 mm. Regions of interests were defined in major organs and tumor. A plasma curve was derived based on a modeling method that utilizes the early heart time-activity curve and a late-time blood sample. The (18) F-FDG uptake constant K i was calculated using Patlak analysis on the tumors without an apparent necrotic center shown in the PET images. For each tumor type, the measured K i was corrected for partial volume (PV), and multivariate regression analysis was performed to examine the effects of blood glucose level ([Glc]) and tumor growth. Corrected Akaike's information criterion was used to determine the best model.
RESULTS: The regression model that best fit the PV-corrected K i for U87 data was K i /RC = (1/[Glc]) × (0.27 ± 0.027) mL/min/mL (where [Glc] is in mmol/L), and for MDA, it was K i /RC = (0.04 ± 0.005) mL/min/mL, where K i /RC denotes the PV-corrected K i using an individual recovery coefficient (RC). The results indicated that (18) F-FDG K i /RC for U87 was inversely related to [Glc], while [Glc] had no effect on (18) F-FDG K i /RC of MDA. After the effects of PV and [Glc] were accounted for, the data did not support any increase of (18) F-FDG K i as the tumor (of either type) grew larger in size.
CONCLUSIONS: The effect of [Glc] on the tumor (18) F-FDG K i was tumor-dependent. PV- and [Glc]-corrected (18) F-FDG K i did not show significant increase as the tumor of either type grew in size.
Kreissl MC, Stout DB, Wong KP, Wu HM, Caglayan E, Ladno W, Zhang X, Prior JO, Reiners C, Huang SC, Schelbert HR. Influence of dietary state and insulin on myocardial, skeletal muscle and brain [F]-fluorodeoxyglucose kinetics in mice. EJNMMI Res. 2011 Jul 6;1:8. doi: 10.1186/2191-219X-1-8.
BACKGROUND: We evaluated the effect of insulin stimulation and dietary changes on myocardial, skeletal muscle and brain [(18)F]-fluorodeoxyglucose (FDG) kinetics and uptake in vivo in intact mice.
METHODS: Mice were anesthetized with isoflurane and imaged under different conditions: non-fasted (n = 7; "controls"), non-fasted with insulin (2 IU/kg body weight) injected subcutaneously immediately prior to FDG (n = 6), fasted (n = 5), and fasted with insulin injection (n = 5). A 60-min small-animal PET with serial blood sampling and kinetic modeling was performed.
RESULTS: We found comparable FDG standardized uptake values (SUVs) in myocardium in the non-fasted controls and non-fasted-insulin injected group (SUV 45-60 min, 9.58 ± 1.62 vs. 9.98 ± 2.44; p = 0.74), a lower myocardial SUV was noted in the fasted group (3.48 ± 1.73; p < 0.001). In contrast, the FDG uptake rate constant (K(i)) for myocardium increased significantly by 47% in non-fasted mice by insulin (13.4 ± 3.9 ml/min/100 g vs. 19.8 ± 3.3 ml/min/100 g; p = 0.030); in fasted mice, a lower myocardial K(i) as compared to controls was observed (3.3 ± 1.9 ml/min/100 g; p < 0.001). Skeletal muscle SUVs and K(i) values were increased by insulin independent of dietary state, whereas in the brain, those parameters were not influenced by fasting or administration of insulin. Fasting led to a reduction in glucose metabolic rate in the myocardium (19.41 ± 5.39 vs. 3.26 ± 1.97 mg/min/100 g; p < 0.001), the skeletal muscle (1.06 ± 0.34 vs. 0.34 ± 0.08 mg/min/100 g; p = 0.001) but not the brain (3.21 ± 0.53 vs. 2.85 ±0.25 mg/min/100 g; p = 0.19).
CONCLUSIONS: Changes in organ SUVs, uptake rate constants and metabolic rates induced by fasting and insulin administration as observed in intact mice by small-animal PET imaging are consistent with those observed in isolated heart/muscle preparations and, more importantly, in vivo studies in larger animals and in humans. When assessing the effect of insulin on the myocardial glucose metabolism of non-fasted mice, it is not sufficient to just calculate the SUV - dynamic imaging with kinetic modeling is necessary.
Wong KP, Sha W, Zhang X, Huang SC. Effects of administration route, dietary condition, and blood glucose level on kinetics and uptake of 18F-FDG in mice. J Nucl Med. 2011 May;52(5):800-7. doi: 10.2967/jnumed.110.085092. Epub 2011 Apr 15.
The effects of dietary condition and blood glucose level on the kinetics and uptake of (18)F-FDG in mice were systematically investigated using intraperitoneal and tail-vein injection.
METHODS: Dynamic PET was performed for 60 min on 23 isoflurane-anesthetized male C57BL/6 mice after intravenous (n = 11) or intraperitoneal (n = 12) injection of (18)F-FDG. Five and 6 mice in the intravenous and intraperitoneal groups, respectively, were kept fasting overnight (18 ± 2 h), and the others were fed ad libitum. Serial blood samples were collected from the femoral artery to measure (18)F-FDG and glucose concentrations. Image data were reconstructed using filtered backprojection with CT-based attenuation correction. The standardized uptake value (SUV) was estimated from the 45- to 60-min image. The metabolic rate of glucose (MRGlu) and (18)F-FDG uptake constant (K(i)) were derived by Patlak graphical analysis.
RESULTS: In the brain, SUV and K(i) were significantly higher in fasting mice with intraperitoneal injection, but MRGlu did not differ significantly under different dietary states and administration routes. Cerebral K(i) was inversely related to elevated blood glucose levels, irrespective of administration route or dietary state. In myocardium, SUV, K(i), and MRGlu were significantly lower in fasting than in nonfasting mice for both routes of injection. Myocardial SUV and K(i) were strongly dependent on the dietary state, and K(i) did not correlate with the blood glucose level. Similar results were obtained for skeletal muscle, although the differences were not as pronounced.
CONCLUSION: Intraperitoneal injection is a valid alternative route, providing pharmacokinetic data equivalent to data from tail-vein injection for small-animal (18)F-FDG PET. Cerebral K(i) varies inversely with blood glucose level, but the measured cerebral MRGlu does not correlate with blood glucose level or dietary condition. Conversely, the K(i) values of the myocardium and skeletal muscle are strongly dependent on dietary condition but not on blood glucose level. In tissue in which (18)F-FDG uptake declines with increasing blood glucose, correction for blood glucose level will make SUV a more robust outcome measure of MRGlu.