A new macrocyclic system 2,2'-(12-amino-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid (ATRIDAT) was designed for coordinating metals in +2 and +3 oxidation states particularly Ga(III), for PET imaging. ATRIDAT was conjugated to d-biotin for pretargeting via biotin-avidin interaction. This model provides high tumor targeting efficiency and stability to biotinidase activity leading to modest signal amplification at the tumor site. Cyclization of triethylenetetramine with protected diethylamino malonate resulted in the formation of 13 membered diamide ring. d-Biotin was then anchored on the pendant amine rendering α-methyne carbon to the biotinamide bond which blocks the biotinidase enzyme activity. Biotinidase stability assay showed remarkable stability toward the action of biotinidase with ∼95% remaining intact after treatment following 4 h. Binding affinity experiments such as HABA assay, competitive displacement studies with d-biotin and CD showed high binding affinity of the molecule with avidin in nanomolar range. Biotin conjugate was successfully radiolabeled withGa(III) with radiolabeling efficiency of ∼70% and then purified to get 99.9% radiochemical yield. IC of the compound was found to be 2.36 mM in HEK cell line and 0.82 mM in A549 as assessed in MTT assay. In biodistribution studies, the major route of excretion was found to be renal. Significant uptake of 4.15 ± 0.35% was observed in tumor in the avidin pretreated mouse at 1 h. μPET images also showed a high tumor to muscle ratio of 26.8 and tumor to kidney ratio of 1.74 at 1 h post-injection after avidin treatment.

We investigated the potential of DTPA-bis(Methionine), a target specific amino acid based probe for detection of L-type amino acid transporters (LAT1) known to over express in proliferating tumours using multimodality imaging. The ligand, DTPA-bis(Met) was readily converted to lanthanide complexes and was found capable of targeting cancer cells using multimodality imaging. DTPA-bis(Met) complexes were synthesized and characterized by mass spectroscopy. MR longitudinal relaxivity, r₁ = 4.067 ± 0.31 mM⁻¹s⁻¹ and transverse relaxivity, r₂ = 8.61 ± 0.07 mM⁻¹s⁻¹ of Gd(III)-DTPA-bis(Met) were observed at pH 7.4 at 7 T. Bright, localized fluorescence of Eu(III)-DTPA-bis(Met) was observed with standard microscopy and displacement studies indicated ligand functionality. K(D) value determined for Eu(III)-DTPA-bis(Met) on U-87 MG cells was found to be 17.3 pM and showed appreciable fluorescence within the cells. Radio HPLC showed a radiochemical purity more than 95% (specific activity = 400-500 MBq/μmol, labelling efficiency 78 %) for ⁶⁸Ga(III)-DTPA-bis(Met). Pre-treatment of xenografted U-87 MG athymic mice with ⁶⁸Ga(III)-DTPA-bis(Met) following unlabelled L-methionine administration reduced tumour uptake by 10-folds in Micro PET. These data support the specific binding of ⁶⁸Ga(III)-DTPA-bis(Met) to the LAT1 transporter. To summarize, this agent possesses high stability in biological environment and exhibits effective interaction with its LAT1 transporters giving high accumulation in tumour area, excellent tumour/non-tumour ratio and low non-specific retention in vivo.

Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers. The redox processes ensuing from GGT-mediated metabolism of extracellular GSH are implicated in critical aspects of tumor cell biology. Reportedly, Glutathione monoethyl ester (GSHMe) is a substrate of GGT, which has been used for its rapid transport over glutathione. Exploring GGT to be an important target, a homobivalent peptide system, DT(GSHMe)2 was designed to target GGT-over expressing tumors for diagnostic purposes. DT(GSHMe)2 was synthesized, characterized and preclinically evaluated in vitro using toxicity, cell binding assays and time dependent experiments. Stable and defined radiochemistry with 99mTc and 68Ga was optimized for high radiochemical yield. In vivo biodistribution studies were conducted for different time points along with scintigraphic studies of radiolabeled DT(GSHMe)2 on xenografted tumor models. For further validation, in silico docking studies were performed on GGT (hGGT1, P19440). Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration. Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min. Quantitative estimation of GGT expression from western blot experiments showed substantial expression with 41.6 ± 7.07 % IDV for tumor. Small animal micro PET (Positron Emission Tomography)/CT(Computed Tomography) coregistered images depicted significantly high uptake of DT(GSHMe)2 at the BMG-1 tumor site. ROI analysis showed high tumor to contra lateral muscle ratio of 9.33 in PET imaging studies. Avid accumulation of radiotracer was observed at tumor versus inflammation site at 2 h post i.v. injection in an Ehrlich Ascites tumor (EAT) mice model, showing evident specificity for tumor. We propose DT(GSHMe)2 to be an excellent candidate for prognostication and tumor imaging using PET/SPECT.

Objective To characterize glioma preoperatively using quantitative 99mTc-methionine SPECT and comparison with MR-perfusion/spectroscopy and histopatholgical/Ki-67 scoring. Methods Twenty-nine patients (21M: 8F; mean age 42.3 ± 10.5 years) with clinical and radiological suspicion of glioma assessed by 99mTc-MDM/SPECT and ceMRI. Additionally, 12/29 patients underwent dynamic susceptibility contrast-enhanced (DSCE) MRI and magnetic resonance spectroscopy (MRS) examination. Three patients with benign pathologies were recruited as controls. Histopathological tumor analysis was done in all ( n = 29) the patients, and the Ki-67 index was evaluated in 20/29 patients. The target-to-nontarget (T/NT) methionine tumor uptake ratios, normalized cerebral blood volume (nCBV) and metabolites [choline/N-acetyl aspartate (Cho/NAA), Cho/creatine (Cr), Cr/NAA and Cr/Cho) ratios were measured in tumor areas. Results On histopathological analysis, 26/29 patients had glioma (G IV-13; G III-04; G II-09). The mean T/NT ratio in G-II was significantly lower (2.46 ± 2.3) than in G-III (7.13 ± 2.2) and G-IV (5.16 ± 1.2). However, the mean ratio was highest (15.9 ± 6.8) in meningioma (n=3). The T/NT cutoff ratio of 3.08 provided 100% sensitivity, 87.5% specificity for discriminating high-grade glioma (HGG) from low-grade glioma (LGG) disease. Likewise, the nCBV cutoff of 2.43 offered 100% sensitivity and 80% specificity. Only the Cho/NAA cutoff value of greater than 3.34 provided reasonable sensitivity and specificity of 85.7% and 80.0% respectively for this differentiation. T/NT ratio correlated significantly with nCBV and Cho/NAA, Cho/Cr ratios but not with Ki-67. Conclusion Quantitative 99mTc-MDM -SPECT provided high sensitivity and specificity to differentiate HGG versus LGG preoperatively and demonstrated a potential role for the differential diagnosis of glial versus nonglial tumors.

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Objective: The elevated choline transporters (ChT), choline kinase (ChK), choline uptake, and phosphorylation in certain tumor cells have influenced the development of radiolabeled choline derivatives as diagnostic probes for imaging cell membrane proliferation. We, therefore, aimed to develop a choline-based moiety for imaging choline kinase-overexpressed tumors by single-photon emission tomography (SPECT). A novel choline-based diagnostic probe was synthesized and evaluated preclinically in various ChT- and ChK-overexpressed tumor models for SPECT imaging applications. Methods: The synthesis of diethylenetriaminepentaacetic acid-bis-choline ethylamine [DTPA-bis(ChoEA)] featured the conjugation of dimethylaminoethanol to a bifunctional chelator DTPA anhydride. [ 99m Tc]Tc-DTPA-bis(ChoEA) was prepared, and its in vivo characteristics were evaluated in BALB/c mice and tumor-xenografted PC3, A549, and HCT116 athymic mouse models. The in vitro parameters, including cell binding and cytotoxicity, were assessed in PC3, A549, and HCT116 cell lines. To evaluate the specificity of the radioprobe, competitive binding studies were performed. Small-animal SPECT/CT diagnostic imaging was performed for in vivo evaluation. The mouse biodistribution data was further investigated to estimate the radiation dose in humans. Results: In silico studies suggested high binding with enhanced specificity. A standard radiolabeling procedure using stannous chloride as a reducing agent showed a labeling yield of 99.5 ± 0.5%. The in silico studies suggested high binding with enhanced specificity. [ 99m Tc]Tc-DTPA-bis(ChoEA) showed high in vitro stability and specificity. The pharmacokinetic studies of [ 99m Tc]Tc-DTPA-bis(ChoEA) in mice showed an increased tumor-to-background ratio after few minutes of intravenous administration. The first-in-human trial was also conducted. The effective dose was estimated to be 0.00467 mSv/MBq (4.67 mSv/GBq), resulting in a radiation dose of up to 1.73 mSv for the 370 MBq injection of [ 99m Tc]Tc-DTPA-bis(ChoEA). Conclusions: The synthesized radioprobe [ 99m Tc]Tc-DTPA-bis(ChoEA) accumulates specifically in choline kinase-overexpressed tumors with a high signal-to-noise ratio. The preclinical and first-in-man data suggested that [ 99m Tc]Tc-DTPA-bis(ChoEA) could potentially be used as a diagnostic SPECT tracer in the monitoring and staging of cancer.

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Immunotherapy may be promising for the treatment of some GBM patients, however, there is a need for non-invasive neuroimaging techniques to predict immunotherapeutic responses. The effectiveness of most immunotherapeutic strategies requires T-cells activation. Therefore, we aimed to evaluate an early marker of T-cell activation, CD69, for its use as an imaging biomarker of response to immunotherapy for GBM. Herein, we performed CD69 immunostaining on human and mouse T-cells following in vitro activation and post immune-checkpoint inhibitors (ICI) in an orthotopic syngeneic mouse glioma model. CD69 expression on tumor-infiltrating leukocytes was assessed using single-cell RNA sequence (scRNA-seq) data from recurrent GBM patients receiving ICI. Radiolabeled CD69 antibody (Ab) positron emission tomography/computed tomography (PET/CT) imaging (CD69 immuno-PET) was performed on GBM-bearing mice longitudinally to quantify CD69 and its association with survival following immunotherapy. We show CD69 expression is upregulated upon T-cell activation and on tumor-infiltrating lymphocytes (TILs) in response to immunotherapy. Similarly, scRNA-seq data demonstrated elevated CD69 on TILs from ICI-treated recurrent GBM patients as compared with TILs from control cohorts. CD69 immuno-PET studies showed a significantly higher tracer uptake in the tumors of ICI-treated mice compared with controls. Importantly, we observed a positive correlation between survival and CD69 immuno-PET signals in immunotherapy-treated animals and established a trajectory of T-cell activation by virtue of CD69-immuno-PET measurements. Our study supports the potential use of CD69 immuno-PET as an immunotherapy response assessment imaging tool for GBM patients.

<p>Immuno-PET of ⁸⁹Zr-DFO-CD69 Ab visualizes the TME after ICI treatment in a GBM mouse model. Mice were inoculated with GL261 cells, and evaluated by immuno-PET of ⁸⁹Zr-DFO-CD69 Ab, comparing the treatment (ICI) group to the control group (represented by orange and blue dots, respectively). <b>A,</b> Schematic showing timeline of tumor inoculation, ICI treatment, tail vein injection of ⁸⁹Zr-DFO-CD69 Ab, immuno-PET, and BioD. <b>B,</b> Representative coronal head images from immuno-PET of ⁸⁹Zr-DFO-CD69 Ab of ICI-treated and control mice at designated time points. Scales show SUVs of PET (SUV; colored) and CT (HU; gray). <b>C</b> and <b>D,</b> Comparison between ICI-treated mice and control at designated time points of tumor-specific regions’ SUV_max (C) and SUV_max TBR (D). <b>E,</b> Representative coronal-ventral and sagittal 3D whole-body MIP PET images of ⁸⁹Zr-DFO-CD69 Ab of control and ICI-treated mice acquired 6 days after tracer administration. The scale shows SUVs of PET. <b>F,</b> Day 6 BioD results of blood- and tumor site–associated radioactivity, assessed as %ID/g. In C, D, and F, bars show means ± SEM. Scattered dots represent individual mice. Shown are representative data of three independent experiments, <i>n</i> = 5 per group. C and D, Multiple unpaired <i>t</i> test with Welch correction. F, Two-way ANOVA with multiple comparison test (*, <i>P</i> < 0.05; **, <i>P</i> <0.01; ***, <i>P</i> <0.001). IP, intraperitoneal.</p>