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There is increasing evidence showing the heterogeneity of microglia activation in neuroinflammatory and neurodegenerative diseases. It has been hypothesized that pro‐inflammatory microglia are detrimental and contribute to disease progression, while anti‐inflammatory microglia play a role in damage repair and remission. The development of therapeutics targeting the deleterious glial activity and modulating it into a regenerative phenotype relies heavily upon a clearer understanding of the microglia dynamics during disease progression and the ability to monitor therapeutic outcome in vivo. To that end, molecular imaging techniques are required to assess microglia dynamics and study their role in disease progression as well as to evaluate the outcome of therapeutic interventions. Positron emission tomography (PET) is such a molecular imaging technique, and provides unique capabilities for non‐invasive quantification of neuroinflammation and has the potential to discriminate between microglia phenotypes and define their role in the disease process. However, several obstacles limit the possibility for selective in vivo imaging of microglia phenotypes mainly related to the poor characterization of specific targets that distinguish the two ends of the microglia activation spectrum and lack of suitable tracers. PET tracers targeting translocator protein 18 kDa (TSPO) have been extensively explored, but despite the success in evaluating neuroinflammation they failed to discriminate between microglia activation statuses. In this review, we highlight the current knowledge on the microglia phenotypes in the major neuroinflammatory and neurodegenerative diseases. We also discuss the current and emerging PET imaging targets, the tracers and their potential in discriminating between the pro‐ and anti‐inflammatory microglia activation states., The development of therapeutics targeting the deleterious glial activity and modulating it into a regenerative phenotype heavily relies on a better understanding of the microglia dynamics during disease progression and the ability to monitor therapeutic outcome in vivo. In this review, we will highlight the current knowledge on the microglia phenotypes in the major neuroinflammatory and neurodegenerative diseases. We will also discuss the current and emerging PET imaging targets, the tracers, and their potential in discriminating between the pro‐ and anti‐inflammatory microglia activation states.

Introduction The assessment of ex vivo biodistribution is the preferred method for quantification of radiotracers biodistribution in preclinical models, but is not in line with current ethics on animal research. PET imaging allows for noninvasive longitudinal evaluation of tracer distribution in the same animals, but systemic comparison with ex vivo biodistribution is lacking. Our aim was to evaluate the potential of preclinical PET imaging for accurate tracer quantification, especially in tumor models. Methods NEMA NU 4-2008 phantoms were filled with 11C, 68Ga, 18F, or 89Zr solutions and scanned in Mediso nanoPET/CT and PET/MR scanners until decay. N87 tumor-bearing mice were i.v. injected with either [18F]FDG (~ 14 MBq), kept 50 min under anesthesia followed by imaging for 20 min, or with [89Zr]Zr-DFO-NCS-trastuzumab (~ 5 MBq) and imaged 3 days post-injection for 45 min. After PET acquisition, animals were killed and organs of interest were collected and measured in a γ-counter to determine tracer uptake levels. PET data were reconstructed using TeraTomo reconstruction algorithm with attenuation and scatter correction and regions of interest were drawn using Vivoquant software. PET imaging and ex vivo biodistribution were compared using Bland–Altman plots. Results In phantoms, the highest recovery coefficient, thus the smallest partial volume effect, was obtained with 18F for both PET/CT and PET/MR. Recovery was slightly lower for 11C and 89Zr, while the lowest recovery was obtained with 68Ga in both scanners. In vivo, tumor uptake of the 18F- or 89Zr-labeled tracer proved to be similar irrespective whether quantified by either PET/CT and PET/MR or ex vivo biodistribution with average PET/ex vivo ratios of 0.8–0.9 and a deviation of 10% or less. Both methods appeared less congruent in the quantification of tracer uptake in healthy organs such as brain, kidney, and liver, and depended on the organ evaluated and the radionuclide used. Conclusions Our study suggests that PET quantification of 18F- and 89Zr-labeled tracers is reliable for the evaluation of tumor uptake in preclinical models and a valuable alternative technique for ex vivo biodistribution. However, PET and ex vivo quantification require fully described experimental and analytical procedures for reliability and reproducibility.

The identification of molecular drivers of disease and the compelling rise of biotherapeutics have impacted clinical care but have also come with challenges. Such therapeutics include peptides, monoclonal antibodies, antibody fragments and nontraditional binding scaffolds, activatable antibodies, bispecific antibodies, immunocytokines, antibody-drug conjugates, enzymes, polynucleotides, and therapeutic cells, as well as alternative drug carriers such as nanoparticles. Drug development is expensive, attrition rates are high, and efficacy rates are lower than desired. Almost all these drugs, which in general have a long residence time in the body, can stably be labeled with 89Zr for whole-body PET imaging and quantification. Although not restricted to monoclonal antibodies, this approach is called 89Zr-immuno-PET. This review summarizes the state of the art of the technical aspects of 89Zr-immuno-PET and illustrates why it has potential for steering the design, development, and application of biologic drugs. Appealing showcases are discussed to illustrate what can be learned with this emerging technology during preclinical and especially clinical studies about biologic drug formats and disease targets. In addition, an overview of ongoing and completed clinical trials is provided. Although 89Zr-immuno-PET is a young tool in drug development, its application is rapidly expanding, with first clinical experiences giving insight on why certain drug-target combinations might have better perspectives than others.

Purpose Almost all radiolabellings of antibodies with 89Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of 89Zr in bones due to instability of the resulting metal complex. DFO*-NCS and the squaramide ester of DFO, DFOSq, are novel analogues that gave more stable 89Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents. Methods Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with 89Zr via DFO*-NCS, DFOSq, DFO-NCS or DFO*Sq. Stability in vitro was compared at 37 °C in serum (7 days), in formulation solution (24 h ± chelator challenges) and in vivo with N87 and A431 tumour-bearing mice. Finally, to demonstrate the practical benefit of more stable complexation for the accurate detection of bone metastases, [89Zr]Zr-DFO*-NCS and [89Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially. Results [89Zr]Zr-DFO*-NCS-trastuzumab and [89Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [89Zr]Zr-DFO counterparts under all conditions. While tumour uptake was similar for all conjugates, bone uptake was lower for DFO* conjugates. Lower bone uptake for DFO* conjugates was confirmed using a second xenograft model: A431 combined with cetuximab. Finally, in the intratibial BT-474 bone metastasis model, the DFO* conjugates provided superior detection of tumour-specific signal over the DFO conjugates. Conclusion DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical 89Zr-immuno-PET.

The P2Y12receptor (P2Y12R) is uniquely expressed on microglia in the brain, and its expression level directly depends on the microglial activation state. Therefore, P2Y12R provides a promising imaging marker for distinguishing the pro- and anti-inflammatory microglial phenotypes, both of which play crucial roles in neuroinflammatory diseases. In this study, three P2Y12R antagonists were selected from the literature, radiolabeled with carbon-11 or fluorine-18, and evaluated in healthy Wistar rats. Brain imaging was performed with and without blocking of efflux transportersP-glycoprotein and breast cancer resistance protein using tariquidar. Low brain uptake in healthy rats was observed for all tracers at baseline conditions, whereas blocking of efflux transporters resulted in a strong (6–7 fold) increase in brain uptake for both of them. Binding of the most promising tracer, [18F]3, was further evaluated by in vitro autoradiography on rat brain sections, ex vivo metabolite studies, and in vivo P2Y12R blocking studies. In vitro binding of [18F]3on rat brain sections indicated high P2Y12R targeting with approximately 70% selective and specific binding. At 60 min post-injection, over 95% of radioactivity in the brain accounted for an intact tracer. In blood plasma, still 40% intact tracer was found, and formed metabolites did not enter the brain. A moderate P2Y12R blocking effect was observed in vivo by positron emission tomography (PET) imaging with [18F]3(p= 0.04). To conclude, three potential P2Y12R PET tracers were obtained and analyzed for P2Y12R targeting in the brain. Unfortunately, the brain uptake appeared low. Future work will focus on the design of P2Y12R inhibitors with improved physicochemical characteristics to reduce efflux transport and increase brain penetration.

Introduction Parathyroid hyperplasia is a disease characterized by overactive parathyroid glands secreting increased levels of parathyroid hormone. Surgical removal of the parathyroid glands is the standard treatment but requires precise pre-operative localization of the glands. However, currently available imaging modalities show limited sensitivity. Since positron emission tomography (PET) is a molecular imaging technique with high accuracy and sensitivity, our aim was to develop a new PET tracer for overactive parathyroid glands imaging by radiolabelling cinacalcet, a drug binding to the calcium-sensing receptor of the parathyroid glands. Methods [18F]Cinacalcet was synthesized by copper-catalysed [18F]trifluoromethylation of a boronic acid precursor using high molar activity [18F]fluoroform. Ex vivo biodistribution and metabolism were evaluated in 12 healthy male Wistar rats at 5, 15, 45 and 90 min. PET scans were performed at baseline and after blocking with NPS R-568. Results [18F]Cinacalcet was obtained in an overall radiosynthesis time of 1 h with a radiochemical purity of 98 ± 1%, a radiochemical yield of 8 ± 4% (overall, n = 7, corrected for decay) and a molar activity of 40 ± 11 GBq/μmol (n = 7, at EOS). The ex vivo biodistribution showed uptake in the thyroid and parathyroid glands as well as in other glands such as adrenals, salivary glands and pancreas. The tracer was rapidly cleared from the blood via liver and kidneys and showed fast metabolism. PET images confirmed uptake in the target organ. However, in a blocking study with NPS R-568 specific binding of [18F]cinacalcet to the CaSR could not be confirmed. Conclusions [18F]Cinacalcet was successfully synthesized. First in vivo experiments in healthy rats showed uptake of the tracer in the target organ and fast metabolism, encouraging further in vivo evaluation of this tracer.

Neutrophilic inflammation correlates with severe tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb). Granulomas are lesions that form in TB, and a PET probe for following neutrophil recruitment to granulomas could predict disease progression. We tested the formyl peptide receptor 1 (FPR1)-targeting peptide FLFLF in Mtb-infected macaques. Preliminary studies in mice demonstrated specificity for neutrophils. In macaques, (64)Cu-FLFLF was retained in lung granulomas and analysis of lung granulomas identified positive correlations between (64)Cu-FLFLF and neutrophil and macrophage numbers (R(2) = 0.8681 and 0.7643, respectively), and weaker correlations for T cells and B cells (R(2) = 0.5744 and 0.5908, respectively), suggesting that multiple cell types drive (64)Cu-FLFLF avidity. By PET/CT imaging, we found that granulomas retained (64)Cu-FLFLF but with less avidity than the glucose analog (18)F-FDG. These studies suggest that neutrophil-specific probes have potential PET/CT applications in TB, but important issues need to be addressed before they can be used in nonhuman primates and humans.

Background Non-invasive imaging of the activation status of microglia and the ability to identify a pro- or anti-inflammatory environment can provide valuable insights not only into pathogenesis of neuro-inflammatory and neurodegenerative diseases but also the monitoring of the efficacy of immunomodulatory therapies. P2X7R is highly expressed on pro-inflammatory microglia and [11C]SMW139, a specific P2X7R tracer for positron emission tomography imaging, showed good pharmacokinetics, stability, and brain permeability in vivo. Our objective was to evaluate the potential of [11C]SMW139 for PET imaging of neuroinflammation in vivo in the experimental autoimmune encephalomyelitis (EAE) model. Methods We induced EAE in Lewis rats by immunization with MBP 69-88 in complete Freund’s adjuvant (CFA). We determined the affinity of [11C]SMW139 to human and rat P2X7R using saturation binding assay. Using this tracer, PET imaging was performed at the peak of disease and in the recovery phase. In vivo blocking experiments were conducted to validate the specific brain uptake of the tracer. Immunohistochemistry staining and autoradiography were performed to evaluate the level of neuroinflammation and validate the specific binding of [11C]SMW139. Results [11C]SMW139 showed good affinity for the rat P2X7R with a Kd of 20.6 ± 1.7 nM. The uptake of [11C]SMW139 was significantly higher in EAE animals at the peak of disease compared to the recovery phase but not in CFA control animals. The amplitude of increase of [11C]SMW139 uptake showed significant positive correlation with clinical scores mainly in the spinal cord (Pearson = 0.75, Spearman = 0.76; p 7R antagonist JNJ-47965567 blocked the uptake of [11C]SMW139 in the spinal cord, cerebellum, and brain stem, demonstrating specific accumulation of the tracer. P-glycoprotein blocking with tariquidar (30 mg/kg) did not affect tracer penetration in the brain showing that [11C]SMW139 is not a Pgp substrate. Conclusion Our data shows that [11C]SMW139 is a promising PET tracer for imaging neuroinflammation and evaluating the dynamics of pro-inflammatory microglia in the brain. This can provide crucial insights into the role of microglia in disease progression and enables the development of novel treatment strategies aimed at modulating the immune response in order to promote neuroprotection.

The choroid plexus (CP) is a key regulator of the central nervous system (CNS) homeostasis through its secretory, immunological and barrier properties. Accumulating evidence suggests that the CP plays a pivotal role in the pathogenesis of multiple sclerosis (MS), but the underlying mechanisms remain largely elusive. To get a comprehensive view on the role of the CP in MS, we studied transcriptomic alterations of the human CP in progressive MS and non-neurological disease controls using RNA sequencing. We identified 17 genes with significantly higher expression in progressive MS patients relative to that in controls. Among them is the newly described long non-coding RNA HIF1A-AS3. Next to that, we uncovered disease-affected pathways related to hypoxia, secretion and neuroprotection, while only subtle immunological and no barrier alterations were observed. In an ex vivo CP explant model, a subset of the upregulated genes responded in a similar way to hypoxic conditions. Our results suggest a deregulation of the Hypoxia-Inducible Factor (HIF)-1 pathway in progressive MS CP. Importantly, cerebrospinal fluid levels of the hypoxia-responsive secreted peptide PAI-1 were higher in MS patients with high disability relative to those with low disability. These findings provide for the first time a complete overview of the CP transcriptome in health and disease, and suggest that the CP environment becomes hypoxic in progressive MS patients, highlighting the altered secretory and neuroprotective properties of the CP under neuropathological conditions. Together, these findings provide novel insights to target the CP and promote the secretion of neuroprotective factors into the CNS of progressive MS patients.

Objective: The goal of this research was to evaluate c(RGDyK) conjugated to phosphonate-based cross-bridged chelators using Cu-free click chemistry in the 4T1 mouse mammary tumor bone metastasis model in comparison with (64)Cu-CB-TE2A-c(RGDyK), which previously showed selective binding to integrin αvβ3 on osteoclasts. Experimental: Two phosphonate-based cross-bridged chelators (CB-TE1A1P and CB-TE1K1P) were conjugated to c(RGDyK) through bio-orthogonal strain-promoted alkyne–azide cycloaddition. In vitro and in vivo evaluation of the (64)Cu-labeled TE1A1P-DBCO-c(RGDyK) (AP-c(RGDyK)), TE1K1P-PEG4-DBCO-c(RGDyK) (KP-c(RGDyK)), and CB-TE2A-c(RGDyK) were compared in the 4T1 mouse model of bone metastasis. The affinities of the unconjugated and chelator-c(RGDyK) analogs for αvβ3 integrin were determined using a competitive-binding assay. For in vivo evaluation, BALB/c mice were injected with 1 × 10(5) 4T1/Luc cells in the left ventricle. Formation of metastases was monitored by bioluminescence imaging (BLI) followed by small-animal PET/CT 2 h postinjection of radiotracers. Results: The chelator–peptide conjugates showed similar affinity to integrin αvβ3, in the low nM range. PET imaging demonstrated a higher uptake in bones having metastases for all (64)Cu-labeled c(RGDyK) analogs compared with bones in nontumor-bearing mice. The correlation between uptake of (64)Cu-AP-c(RGDyK) and (64)Cu-KP-c(RGDyK) in bones with metastases based on PET/CT imaging, and osteoclast number based on histomorphometry, was improved over the previously investigated (64)Cu-CB-TE2A-c(RGDyK). Conclusion: These data suggest that the phosphonate chelator conjugates of c(RDGyK) peptides are promising PET tracers suitable for imaging tumor-associated osteoclasts in bone metastases.

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory / protective state of microglia for the development of novel PET tracers. Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues. Results: Here we provide evidence that P2RY12 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-11 labeled tracer targeting P2RY12, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient. Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases.

Positron emission tomography and computed tomography imaging (PET/CT) is an increasingly valuable tool for diagnosing tuberculosis (TB). The glucose analog [18F]fluoro-2-deoxy-2-d-glucose ([18F]-FDG) is commonly used in PET/CT that is retained by metabolically active inflammatory cells in granulomas, but lacks specificity for particular cell types. A PET probe that could identify recruitment and differentiation of different cell populations in granulomas would be a useful research tool and could improve TB diagnosis and treatment. We used the Mycobacterium-antigen murine inflammation model and macaques with TB to identify [64Cu]-labeled CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A), a high affinity peptidomimetic ligand for very late Ag-4 (VLA-4; also called integrin α4β1) binding cells in granulomas, and compared [64Cu]-LLP2A with [18F]-FDG over the course of infection. We found that [64Cu]-LLP2A retention was driven by macrophages and T cells, with less contribution from neutrophils and B cells. In macaques, granulomas had higher [64Cu]-LLP2A uptake than uninfected tissues, and immunohistochemical analysis of granulomas with known [64Cu]-LLP2A uptake identified significant correlations between LLP2A signal and macrophage and T cell numbers. The same cells coexpressed integrin α4 and β1, further supporting that macrophages and T cells drive [64Cu]-LLP2A avidity in granulomas. Over the course of infection, granulomas and thoracic lymph nodes experienced dynamic changes in affinity for both probes, suggesting metabolic changes and cell differentiation or recruitment occurs throughout granuloma development. These results indicate [64Cu]-LLP2A is a PET probe for VLA-4, which when used in conjunction with [18F]-FDG, may be a useful tool for understanding granuloma biology in TB.

Very late antigen-4 (VLA-4; also known as integrin α(4)β(1)) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radionuclide therapy (TRT). (177)Lu-DOTA-PEG(4)-LLP2A ((177)Lu-LLP2A) is a TRT that shows high affinity for VLA-4 and high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of (177)Lu-LLP2A, alone and combined with immune checkpoint inhibitors (ICIs) (anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies), in B16F10 tumor–bearing mice. Methods: Tumor cells (1 × 10(6)) were implanted subcutaneously in C57BL/6 mice. After 8–10 d, the mice were randomized into 8 groups. (177)Lu-LLP2A was injected intravenously on day 8 or 9 (single dose), and ICI antibodies were administered intraperitoneally in 3 doses. Tumor growth was monitored over time via calipers. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3–scrambled LLP2A was injected in tumor-bearing mice, and tumors were collected 4 h after injection and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results: TRT alone showed efficacy comparable to the dual-ICI anti-PD-1 + anti-CTLA-4 or anti-PD-L1 + anti-CTLA-4, whereas TRT + ICIs significantly enhanced survival. TUNEL staining showed that the highest levels of apoptosis were in the TRT + ICI groups. In addition to targeting tumor cells, TRT also bound immune cells in the tumor microenvironment. Flow cytometry data showed that the tumors consisted of about 77% tumor cells and fibroblasts (CD45-negative/CD49d-positive) and about 23% immune cells (CD45-positive/CD49d-positive) and that immune cells expressed higher levels of VLA-4. Cy3-LLP2A and CD49d colocalized with macrophages (CD68), T cells (CD8, CD4), and B cells (CD19). Immunohistochemical analysis identified a significant colocalization of Cy3-LLP2A and CD68. Conclusion: Combination treatment with TRT + ICIs targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.

Background The protein cross-linking enzyme tissue transglutaminase (TG2; EC 2.3.2.13) is associated with the pathogenesis of various diseases, including cancer. Recently, the synthesis and initial evaluation of two high-potential radiolabelled irreversible TG2 inhibitors were reported by us. In the present study, these two compounds were evaluated further in a breast cancer (MDA-MB-231) tumour xenograft model for imaging active tissue transglutaminase in vivo. Results The metabolic stability of [11C]1 and [18F]2 in SCID mice was comparable to the previously reported stability in Wistar rats. Quantitative real-time polymerase chain reaction analysis on MDA-MB-231 cells and isolated tumours showed a high level of TG2 expression with very low expression of other transglutaminases. PET imaging showed low tumour uptake of [11C]1 (approx. 0.5 percentage of the injected dose per gram (%ID/g) at 40–60 min p.i.) and with relatively fast washout. Tumour uptake for [18F]2 was steadily increasing over time (approx. 1.7 %ID/g at 40–60 min p.i.). Pretreatment of the animals with the TG2 inhibitor ERW1041E resulted in lower tumour activity concentrations, and this inhibitory effect was enhanced using unlabelled 2. Conclusions Whereas the TG2 targeting potential of [11C]1 in this model seems inadequate, targeting of TG2 using [18F]2 was achieved. As such, [18F]2 could be used in future studies to clarify the role of active tissue transglutaminase in disease.

The P2X7 receptor plays a significant role in microglial activation, and as a potential drug target, the P2X7 receptor is also an interesting target in positron emission tomography. The current study aimed at the development and evaluation of a potent tracer targeting the P2X7 receptor, to which end four adamantanyl benzamide analogues with high affinity for the human P2X7 receptor were labelled with carbon-11. All four analogues could be obtained in excellent radiochemical yield and high radiochemical purity and molar activity, and all analogues entered the rat brain. [11C]SMW139 showed the highest metabolic stability in rat plasma, and showed high binding to the hP2X7 receptor in vivo in a hP2X7 receptor overexpressing rat model. Although no significant difference in binding of [11C]SMW139 was observed between post mortem brain tissue of Alzheimer’s disease patients and that of healthy controls in in vitro autoradiography experiments, [11C]SMW139 could be a promising tracer for P2X7 receptor imaging using positron emission tomography, due to high receptor binding in vivo in the hP2X7 receptor overexpressing rat model. However, further investigation of both P2X7 receptor expression and binding of [11C]SMW139 in other neurological diseases involving microglial activation is warranted.

Targeting macrophages for therapeutic and diagnostic purposes is an attractive approach applicable to multiple diseases. Here, we present a theranostic nanoemulsion platform for simultaneous delivery of an anti-inflammatory drug (celecoxib) to macrophages and monitoring of macrophage migration patterns by optical imaging, as measurement of changes in inflammation. The anti-inflammatory effect of the theranostic nanoemulsions was evaluated in a mouse inflammation model induced with complete Freund's adjuvant (CFA). Nanoemulsions showed greater accumulation in the inflamed vs. control paw, with histology confirming their specific localization in CD68 positive macrophages expressing cyclooxygenase-2 (COX-2) compared to neutrophils. With a single dose administration of the celecoxib-loaded theranostic, we observed a reduction in fluorescence in the paw with time, corresponding to a reduction in macrophage infiltration. Our data strongly suggest that delivery of select agents to infiltrating macrophages can potentially lead to new treatments of inflammatory diseases where macrophage behavior changes are monitored in vivo.

Two potent SP1–7 peptidomimetics have been successfully radiolabeled via [11C]CO2-fixation with excellent yields, purity, and molar activity. l-[11C]SP1–7-peptidomimetic exhibited promising ex vivo biodistribution profile. Metabolite analysis showed that l-[11C]SP1–7-peptidomimetic is stable in brain and spinal cord, whereas rapid metabolic degradation occurs in rat plasma. Metabolic stability can be significantly improved by substituting l-Phe for d-Phe, preserving 70% more of intact tracer and resulting in better brain and spinal cord tracer retention. Positron emission tomography (PET) scanning confirmed moderate brain (1.5 SUV; peak at 3 min) and spinal cord (1.0 SUV; peak at 10 min) uptake for l- and d-[11C]SP1–7-peptidomimetic. A slight decrease in SUV value was observed after pretreatment with natural peptide SP1–7 in spinal cord for l-[11C]SP1–7-peptidomimetic. On the contrary, blocking using cold analogues of l- and d-[11C]tracers did not reduce the tracers’ brain and spinal cord exposure. In summary, PET scanning of l- and d-[11C]SP1–7-peptidomimetics confirms rapid blood–brain barrier and blood–spinal-cord barrier penetration. Therefore, further validation of these two tracers targeting SP1–7 is needed in order to define a new PET imaging target and select its most appropriate radiopharmaceutical.

Background Microglia are major players in the pathogenesis of multiple sclerosis (MS) and may play a dual role in disease progression. The activation status of microglia in vivo is highly dynamic and occurs as a continuum, with the pro-inflammatory and anti-inflammatory phenotypes on either end of this spectrum. Little is known about in vivo dynamics of microglia phenotypes in MS due to the lack of diagnostic tools. Positron emission tomography (PET) imaging is a powerful non-invasive technique that allows real-time imaging of microglia activation phenotypes in the central nervous system, depending on the availability of selective PET tracers. Our objective is to investigate and characterize the expression of the purinergic receptors P2Y12R and P2X7R as potential targets for PET tracer development and subsequent PET imaging in order to evaluate the dynamics of microglia status in vivo. Methods We used immunohistochemical analysis to explore the expression of P2Y12R and P2X7R in experimental autoimmune encephalomyelitis (EAE) post-mortem tissues and different stages of well-characterized MS lesions. We evaluated by quantitative real-time polymerase chain reaction the expression of P2Y12R and P2X7R in human polarized microglia, and we performed autoradiography binding assay with radiolabeled P2Y12R and P2X7R antagonists using MS and rat EAE tissues. Results Here, we demonstrate that P2X7R is associated with a pro-inflammatory phenotype of human microglia in vitro, and is highly expressed in microglia in MS lesions as well as during the peak of EAE. In contrast, P2Y12R was associated with an anti-inflammatory phenotype in human microglia in vitro and was expressed at lower levels in active inflammatory MS lesions compared to normal-appearing white matter (NAWM) and similarly in EAE, while its expression increased in the remission phase of EAE. Binding of radiolabeled tracers specific for P2Y12R and P2X7R on ex vivo tissues validated the value of these receptors as PET imaging targets for microglia phenotypes in vivo. Conclusion Our results suggest that P2Y12R and P2X7R are excellent targets for PET imaging to discriminate distinct microglia phenotypes in MS. Ultimately, this may provide insight into the role of microglia in disease progression and monitor novel treatment strategies to alter microglia phenotype. Electronic supplementary material The online version of this article (10.1186/s12974-017-1034-z) contains supplementary material, which is available to authorized users.

Previously, we have shown that conjugation of a palmitic chain via a thioester bond to a cysteine residue in weakly or nonencephalitogenic or neuritogenic peptides markedly enhances their ability to induce autoimmune disease in an MHC class II–restricted manner. From those studies, however, it was not clear whether thiopalmitoylation of the peptides was merely enhancing their disease-inducing potential or whether the lipid was itself playing a pathogenic role. To investigate this further, we have now tested the effects of thiopalmitoylation on MHC class II–restricted altered peptide ligands (APLs), which are normally protective in experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. We hypothesized that if thiopalmitoylation of a peptide merely enhances its innate potential, then thiopalmitoylated APLs (S-palmAPLs) should show enhanced protective effects. Alternatively, if thiopalmitoylation itself can make a peptide pathogenic, then S-palmAPLs should have decreased therapeutic potential. We synthesized APLs and corresponding S-palmAPLs and showed that the S-palmAPLs were much more effective than the nonconjugated APL at inhibiting the development of experimental autoimmune encephalomyelitis. This was due to several features of the S-palmAPL:S-palmAPL–primed cells show an enhanced ability to proliferate and produce the anti-inflammatory cytokine, IL-10, in vitro. Furthermore, the bioavailability of S-palmAPL was greatly enhanced, compared with the nonpalmitoylated APL, and S-palm APL was taken up more rapidly into dendritic cells and channeled into the MHC class II processing pathway. These results show that thiopalmitoylation of MHC class II–restricted peptides is a simple way to enhance their effects in vivo and could have wide therapeutic application.

Spliced X-box binding protein-1 (XBP1s) is a major effector of the unfolded protein response (UPR) signaling. We previously reported that human XBP1s in bone marrow stromal cells plays an essential role in regulating stromal support of multiple myeloma bone disease (MMBD). Here we report that human XBP1s (hXBP1s) is a physiological & direct phosphorylation substrate of JNKs. This phosphorylation greatly increases hXBP1s protein degradation by the E3 ubiquitin ligase β-TrCP. Mutation of the phosphorylation site of JNKs to alanine largely prevents JNK phosphorylation of XBP1s & JNK-induced protein degradation of hXBP1s. Further, we found that bone marrow stromal cells (BMSCs) harboring this XBP1s mutant are resistant to JNK phosphorylation and provided significantly greater support of MM growth and MM-induced bone destruction both in vitro & in vivo, compared with counterparts expressing WT hXBP1s. Finally, we observed that JNK-mediated phosphorylation of hXBP1s is associated with favorable therapeutic responses of MM patients to combination therapy of bortezomib, &/or dexamethasone &/or thalidomide, MM drugs known to stimulate JNK. These results suggest p-hXBP1sJNK is a potential predictive biomarker for MM patients' response to the drug treatment. Taken together, our study provides the first evidence that hXBP1s is a novel physiological & direct target of JNK phosphorylation and demonstrates the pathophysiological significance of this event in the hXBP1s-mediated bone microenvironment support of MM growth and MM-induced bone destruction. Citation Format: Risheng Chen, Guoshuang Xu, Wissam Beaino, Kai Lui, Xuemei Zeng, Nathan Yates, Rong Chong, Konstas Verdelis, G. David Roodman, Denise Toscani, Nicola Giuliani, Yan Lin, Carolyn J. Anderson, Hongjiao Ouyang. JNKs-mediated phosphorylation of human stromal xbp1s regulates multiple myeloma growth & osteolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-360.

Thiopalmitoylation (i.e., the covalent attachment of palmitic acid via a thioester linkage to cysteine residues in the polypeptide backbone) is a common post-translational modification of proteins. Several proteins that have been identified as putative autoantigens in a variety of T-cell mediated autoimmune diseases are thiopalmitoylated, and thus, we have hypothesized that endogenous thiopalmitoylated peptides released during tissue breakdown may play a role in the development and chronicity of autoimmune diseases. To investigate this, we have studied the effect of thiopalmitoylation on the immunogenic and neuritogenic properties of P0, the major peripheral nervous system (PNS) myelin protein, which is thiopalmitoylated at cysteine 153, and described as a candidate autoantigen in Guillain-Barre syndrome (GBS), a human inflammatory demyelinating disease of the PNS. This paper describes the synthesis of palmitoylated peptide P0(180-199) and P0(152-171) by on-resin acylation using specific cysteine side-chain protecting groups: Mmt (labile in diluted acid) and StBu (labile in the presence of tributylphosphine). Our results show that the thiol protecting group had to be adjusted to the peptide sequence: Mmt was efficiently used for P0(180-199) thioacylation, but it was not suitable for thiopalmitoylation of P0(152-171) because of a premature deprotection of the Boc protecting group on the epsilon-NH(2) Lys in the presence of 2% TFA, leading to dipalmitoylation. Palmitoylated P0(152-171) was successfully obtained by using StBu as the thiol protecting group. We could show by circular dichroism that palmitoylation has no influence on the structuration of the peptide in solution but palmitoylation increased the stability of the peptide in the presence of serum. Using EAN (experimental autoimmune neuritis), the rat model of GBS, we have compared the immunological properties of palm and non-palm P0 peptides and showed that thiopalmitoylation has indeed a great influence on their neuritogenic and immunogenic properties. This study provides further support for our hypothesis concerning the role of thiopalmitoylation in the development and chronicity of inflammatory demyelinating diseases and confirms that thiopalmitoylation of peptides may provide a simple means to increase MHC class II restricted responses.

Very-late-antigen-4 (VLA-4, α4β1 integrin, CD49d/CD29) is a transmembrane adhesion receptor that plays an important role in cancer and immune responses. Enhanced VLA-4 expression has been observed in multiple myeloma (MM) cells and surrounding stroma. VLA-4 conformational activation has been associated with MM pathogenesis. VLA-4 is a promising MM imaging and therapeutic biomarker. Methods: Specificity of 64Cu-LLP2A (64Cu-CB-TE1A1P-PEG4-LLP2A), a high-affinity VLA-4 peptidomimetic–based radiopharmaceutical, was evaluated in α4 knock-out mice and by competitive blocking in wild-type tumor-bearing mice. 64Cu-LLP2A PET/CT (static and dynamic) imaging was conducted in C57BL6/KaLwRij mice bearing murine 5TGM1-GFP syngeneic tumors generated after intravenous injection via the tail. Blood samples were collected for serum protein electrophoresis. Bone marrow and splenic cells extracted from tumor-bearing and control mice (n = 3/group) were coincubated with the optical analog LLP2A-Cy5 and mouse B220, CD4, Gr1, and Mac1 antibodies and analyzed by fluorescence-activated cell sorting. Human radiation dose estimates for 64Cu-LLP2A were extrapolated from mouse biodistribution data (6 time points, 0.78 MBq/animal, n = 4/group). Ten formalin-fixed paraffin-embedded bone marrow samples from deceased MM patients were stained with LLP2A-Cy5. Results:64Cu-LLP2A and LLP2A-Cy5 demonstrated high specificity for VLA-4–positive mouse 5TGM1-GFP myeloma and nonmalignant inflammatory host cells such as T cells and myeloid/monocytic cells. Ex vivo flow cytometric analysis supported a direct effect of myeloma on increased VLA-4 expression in host hematopoietic microenvironmental elements. SUVs and the number of medullar lesions detected by 64Cu-LLP2A PET corresponded with increased monoclonal (M) protein (g/dL) in tumor-bearing mice over time (3.29 ± 0.58 at week 0 and 9.97 ± 1.52 at week 3). Dynamic PET with 64Cu-LLP2A and 18F-FDG demonstrated comparable SUV in the prominent lesions in the femur. Human radiation dose estimates indicated urinary bladder wall as the dose-limiting organ (0.200 mGy/MBq), whereas the dose to the red marrow was 0.006 mGy/MBq. The effective dose was estimated to be 0.017 mSv/MBq. Seven of the ten human samples displayed a high proportion of cells intensely labeled with LLP2A-Cy5 probe. Conclusion:64Cu-LLP2A and LLP2A-Cy5 demonstrated binding specificity for VLA-4 in an immune-competent murine MM model. 64Cu-LLP2A displayed favorable dosimetry for human studies and is a potential imaging candidate for overexpressed VLA-4.

Malignant melanoma is a highly aggressive cancer, and the incidence of this disease is increasing worldwide at an alarming rate. Despite advances in the treatment of melanoma, patients with metastatic disease still have a poor prognosis and low survival rate. New strategies, including targeted radiotherapy, would provide options for patients who become resistant to therapies such as BRAF inhibitors. Very late antigen-4 (VLA-4) is expressed on melanoma tumor cells in higher levels in more aggressive and metastatic disease and may provide an ideal target for drug delivery and targeted radiotherapy. In this study, we evaluated (177)Lu- and (68)Ga-labeled DOTA-PEG4-LLP2A as a VLA-4-targeted radiotherapeutic with a companion PET agent for diagnosis and monitoring metastatic melanoma treatment. DOTA-PEG4-LLP2A was synthesized by solid-phase synthesis. The affinity of (177)Lu- and (68)Ga-labeled DOTA-PEG4-LLP2A to VLA-4 was determined in B16F10 melanoma cells by saturation binding and competitive binding assays, respectively. Biodistribution of the LLP2A conjugates was determined in C57BL/6 mice bearing B16F10 subcutaneous tumors, while PET/CT imaging was performed in subcutaneous and metastatic models. (177)Lu-DOTA-PEG4-LLP2A showed high affinity to VLA-4 with a Kd of 4.1 ± 1.5 nM and demonstrated significant accumulation in the B16F10 melanoma tumor after 4 h (31.5 ± 7.8%ID/g). The tumor/blood ratio of (177)Lu-DOTA-PEG4-LLP2A was highest at 24 h (185 ± 26). PET imaging of metastatic melanoma with (68)Ga-DOTA-PEG4-LLP2A showed high uptake in sites of metastases and correlated with bioluminescence imaging of the tumors. These data demonstrate that (177)Lu-DOTA-PEG4-LLP2A has potential as a targeted therapeutic for treating melanoma as well as other VLA-4-expressing tumors. In addition, (68)Ga-DOTA-PEG4-LLP2A is a readily translatable companion PET tracer for imaging of metastatic melanoma.

Melanoma is a malignant tumor derived from epidermal melanocytes and is known for its therapeutic resistance, aggressive clinical behavior, and predisposition for late metastasis. Very late antigen 4 (VLA-4) is a transmembrane non-covalent heterodimer (also called integrin alpha 4 beta 1) overexpressed in melanoma tumors that plays an important role in tumor growth, angiogenesis and metastasis by promoting adhesion and migration of cancer cells. There has been increasing interest in targeting this receptor for cancer imaging and therapy. Here we evaluated analogs of the high VLA-4 binding peptidomimetic ligand, LLP2A, for PET-CT imaging (labeled with Ga-68; positron emitter, half-life = 68 min) and targeted radiotherapy (labeled with Lu-177; beta minus emitter, half-life = 6.7 d). The DOTA-PEG4-LLP2A analog was labeled with Ga-68 and Lu-177, and the two agents showed high affinity to VLA-4 in B16F10 mouse melanoma cells (IC50 = 1.56 nM and Kd = 4.1 nM, respectively). B16F10 subcutaneous tumors in C57/BL6 ...

Melanoma is a malignant tumor derived from epidermal melanocytes, and it is known for its aggressiveness, therapeutic resistance, and predisposition for late metastasis. Very late antigen-4 (VLA-4; also called integrin α4β1) is a transmembrane noncovalent heterodimer overexpressed in melanoma tumors that plays an important role in tumor growth, angiogenesis, and metastasis by promoting adhesion and migration of cancer cells. In this study, we evaluated 2 conjugates of a high-affinity VLA-4 peptidomimetic ligand, LLP2A, for PET/CT imaging in a subcutaneous and metastatic melanoma tumor.LLP2A was conjugated to 1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) (CB-TE1A1P) and 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA) chelators for (68)Ga and (64)Cu labeling. The conjugates were synthesized by solid-phase peptide synthesis, purified by reversed-phase high-performance liquid chromatography, and verified by liquid chromatography mass spectrometry. Saturation and competitive binding assays with B16F10 melanoma cells determined the affinity of the compounds for VLA-4. The biodistributions of the LLP2A conjugates were evaluated in murine B16F10 subcutaneous tumor-bearing C57BL/6 mice. Melanoma metastasis was induced by intracardiac injection of B16F10 cells. PET/CT imaging was performed at 2, 4, and 24 h after injection for the (64)Cu tracers and 1 h after injection for the (68)Ga tracer.(64)Cu-labeled CB-TE1A1P-PEG4-LLP2A and NODAGA-PEG4-LLP2A showed high affinity to VLA-4, with a comparable dissociation constant (0.28 vs. 0.23 nM) and receptor concentration (296 vs. 243 fmol/mg). The tumor uptake at 2 h after injection was comparable for the 2 probes, but (64)Cu-CB-TE1A1P-PEG4-LLP2A trended toward higher uptake than (64)Cu-NODAGA-PEG4-LLP2A (16.9 ± 2.2 vs. 13.4 ± 1.7 percentage injected dose per gram, P = 0.07). Tumor-to-muscle and tumor-to-blood ratios from biodistribution and PET/CT images were significantly higher for (64)Cu-CB-TE1A1P-PEG4-LLP2A than (64)Cu-NODAGA-PEG4-LLP2A (all P values0.05). PET/CT imaging of metastatic melanoma with (68)Ga-NODAGA-PEG4-LLP2A and (64)Cu-NODAGA-PEG4-LLP2A showed high uptake of the probes at the site of metastasis, correlating with the bioluminescence imaging of the tumor.These data demonstrate that (64)Cu-labeled CB-TE1A1P/NODAGA LLP2A conjugates and (68)Ga-labeled NODAGA-LLP2A are excellent imaging agents for melanoma and potentially other VLA-4-positive tumors. (64)Cu-CB-TE1A1P-PEG4-LLP2A had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a PET imaging agent for melanoma.

Owing to its cytotoxicity, free copper is chelated by protein side chains and does not exist in vivo. Several chaperones transport copper to various cell compartments, but none have been identified that traffic copper to the nucleus. Copper-64 decays by β + and β − emission, allowing positron emission tomography and targeted radionuclide therapy for cancer. Because the delivery of 64Cu to the cell nucleus may enhance the therapeutic effect of copper radiopharmaceuticals, elucidation of the pathway(s) involved in transporting copper to the tumor cell nucleus is important for optimizing treatment. We identified Atox1 as one of the proteins that binds copper in the nucleus. Mouse embryonic fibroblast cells, positive and negative for Atox1, were used to determine the role of Atox1 in 64Cu transport to the nucleus. Mouse embryonic fibroblast Atox1+/+ cells accumulated more 64Cu in the nucleus than did Atox1−/− cells. HCT 116 colorectal cancer cells expressing p53 (+/+) and not expressing p53 (−/−) were used to evaluate the role of this tumor suppressor protein in 64Cu transport. In cells treated with cisplatin, the uptake of 64Cu in the nucleus of HCT 116 p53+/+ cells was greater than that in HCT 116 p53−/− cells. Atox1 expression increased in HCT 116 p53+/+ and p53−/− cells treated with cisplatin; however, Atox1 localized to the nuclei of p53+/+ cells more than in the p53−/− cells. The data presented here indicate that Atox1 is involved in copper transport to the nucleus, and cisplatin affects nuclear transport of 64Cu in HCT 116 cells by upregulating the expression and the nuclear localization of Atox1.

Introduction: Melanoma is a malignant tumor derived from epidermal melanocytes, and it is known for its therapeutic resistance, aggressive clinical behavior, and predisposition for late metastasis. Integrin α4β1 is a transmembrane non-covalent heterodimer overexpressed in melanoma tumors that plays an important role in tumor growth, angiogenesis and metastasis by promoting adhesion and migration of cancer cells. There has been increasing interest in targeting this receptor for cancer imaging and therapy. In this study we evaluated three conjugates of a peptidomimetic ligand, LLP2A known to have high binding affinity for α4β1 for PET-CT imaging and potential radiotherapy. Methods: LLP2A was conjugated to three different chelators, CB-TE1A1P, NODAGA and DOTA, for 68Ga and 64Cu labeling. The conjugates were synthesized by solid phase peptide synthesis, purified by RP-HPLC and verified by LC-MS mass spectrometry. 68Ga and 64Cu labeling was done in acetate buffer pH 4 for 68Ga and pH 6.5 for 64Cu at 70°C for 20 min. High Specific activity (1 mCi/µg) and radiopurity (>98%) were achieved. Saturation binding and competitive binding assays with B16F10 melanoma cells determined the binding affinity of the compounds. The biodistributions of the LLP2A conjugates were evaluated in B16F10 subcutaneous tumor bearing C57BL/6 mice. PET-CT imaging was performed at 2, 4 and 24 h post-injection for the 64Cu tracers and 1h post-injection for 68Ga tracer. Results: The competitive binding assay indicated that Ga-NODAGA-PEG4-LLP2A had higher affinity (IC50 = 0.68, Ki = 0.11 nM) compared to Ga-DOTA-PEG4-LLP2A (IC50 = 9.37, Ki = 1.56 nM); however, biodistribution showed similar tumor uptake for 68Ga-NODAGA-PEG4-LLP2A and 68Ga-DOTA-PEG4-LLP2A (8.7 ± 1.3 %ID/g and 9.1 ± 0.9 %ID/g respectively) but less renal and liver retention for the DOTA conjugate. Both 64Cu-labeled CB-TE1A1P-PEG4-LLP2A and NODAGA-PEG4-LLP2A showed high affinity to α4β1 integrin with a comparable Kd (0.28 nM vs 0.23 nM) and Bmax (296 fmol/mg vs 243 fmol/mg). The tumor uptake at 2 h post-injection was comparable for the two probes but 64Cu-CB-TE1A1P-PEG4-LLP2A had higher uptake compared to 64Cu-NODAGA-PEG4-LLP2A (16.9 ± 2.2 %ID/g vs13.4 ± 1.7 %ID/g). Tumor to muscle and tumor to blood ratios from biodistribution and PET-CT images were significantly higher for 64Cu-CB-TE1A1P-PEG4-LLP2A compared to 64Cu-NODAGA-PEG4-LLP2A. In addition, liver and kidney uptake was 2-fold lower for 64Cu-CB-TE1A1P-PEG4-LLP2A. Conclusion: These data demonstrate that 68Ga-labeled DOTA, NODAGA and 64Cu-labeled CB-TE1A1P, NODAGA LLP2A conjugates are excellent imaging agents for melanoma or other α4β1-positive tumors, with 64Cu-CB-TE1A1P-PEG4-LLP2A being the best. These data also suggest the potential for radiotherapy and radiotheranostics using radioisotopes like Lu-177, Y-90 and Cu-67. Citation Format: Wissam Beaino, Carolyn J. Anderson. Gallium-68- and Copper-64-labeled LLP2A conjugates for PET-CT imaging of integrin α4β1 in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4923. doi:10.1158/1538-7445.AM2014-4923