Imaging PARP with [18F]rucaparib in pancreatic cancer models.

PURPOSE: Rucaparib, an FDA-approved PARP inhibitor, is used as a single agent in maintenance therapy to provide promising treatment efficacy with an acceptable safety profile in various types of BRCA-mutated cancers. However, not all patients receive the same benefit from rucaparib-maintenance therapy. A predictive biomarker to help with patient selection for rucaparib treatment and predict clinical benefit is therefore warranted. With this aim, we developed [18F]rucaparib, an 18F-labelled isotopologue of rucaparib, and employed it as a PARP-targeting agent for cancer imaging with PET. Here, we report the in vitro and in vivo evaluation of [18F]rucaparib in human pancreatic cancer models. METHOD: We incorporated the positron-emitting 18F isotope into rucaparib, enabling its use as a PET imaging agent. [18F]rucaparib binds to the DNA damage repair enzyme, PARP, allowing direct visualisation and measurement of PARP in cancerous models before and after PARP inhibition or other genotoxic cancer therapies, providing critical information for cancer diagnosis and therapy. Proof-of-concept evaluations were determined in pancreatic cancer models. RESULTS: Uptake of [18F]rucaparib was found to be mainly dependent on PARP1 expression. Induction of DNA damage increased PARP expression, thereby increasing uptake of [18F]rucaparib. In vivo studies revealed relatively fast blood clearance of [18F]rucaparib in PSN1 tumour-bearing mice, with a tumour uptake of 5.5 ± 0.5%ID/g (1 h after i.v. administration). In vitro and in vivo studies showed significant reduction of [18F]rucaparib uptake by addition of different PARP inhibitors, indicating PARP-selective binding. CONCLUSION: Taken together, we demonstrate the potential of [18F]rucaparib as a non-invasive PARP-targeting imaging agent for pancreatic cancers.

Feed Restriction Modulates Growth, Gut Morphology and Gene Expression in Zebrafish.

A reduction in daily caloric or nutrient intake has been observed to promote health benefits in mammals and other vertebrates. Feed Restriction (FR), whereby the overall food intake of the organism is reduced, has been explored as a method to improve metabolic and immune health, as well as to optimize productivity in farming. However, less is known regarding the molecular and physiological consequences of FR. Using the model organism, Danio rerio, we investigated the impact of a short-term (month-long) FR on growth, gut morphology and gene expression. Our data suggest that FR has minimal effects on the average growth rates, but it may affect weight and size heterogeneity in a sex-dependent manner. In the gut, we observed a significant reduction in gut circumference and generally lower mucosal heights, whereas other parameters remained unchanged. Gene Ontology (GO), EuKaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified numerous metabolic, reproductive, and immune response pathways that were affected by FR. These results broaden our understanding of FR and contribute towards growing knowledge of its effects on vertebrate health.

Development and Evaluation of an 18F-Radiolabeled Monocyclam Derivative for Imaging CXCR4 Expression.

In humans, C-X-C chemokine receptor type 4 (CXCR4) is a protein that is encoded by the CXCR4 gene and binds the ligand CXCL12 (also known as SDF-1). The CXCR4-CXCL12 interaction in cancer elicits biological activities that result in tumor progression and has accordingly been the subject of significant investigation for detection and treatment of the disease. Peptidic antagonists have been labeled with a variety of radioisotopes for the detection of CXCR4, but the methodology utilizing small molecules has predominantly used radiometals. We report here the development of a 18F-radiolabeled cyclam-based small molecule radioprobe, [18F]MCFB, for imaging CXCR4 expression. The IC50 value of [19F]MCFB for CXCR4 was similar to that of AMD3465 (111.3 and 89.8 nM, respectively). In vitro binding assays show that the tracer depicted a differential CXCR4 expression, which was blocked in the presence of AMD3465, demonstrating the specificity of [18F]MCFB. Positron emission tomography (PET) imaging studies showed a distinct uptake of the radioprobe in lymphoma and breast cancer xenografts. High liver and kidney uptakes were seen with [18F]MCFB, leading us to further examine the basis of its pharmacokinetics in relation to the tracer's cationic nature and thus the role of organic cation transporters (OCTs). Substrate competition following the intravenous injection of metformin led to a marked decrease in the urinary excretion of [18F]MCFB, with moderate changes observed in other organs, including the liver. Our results suggest involvement of OCTs in the renal elimination of the tracer. In conclusion, the 18F-radiolabeled monocyclam, [18F]MCFB, has potential to detect tumor CXCR4 in nonhepatic tissues.

Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding.

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics.

Correction: Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding.

[This corrects the article DOI: 10.1371/journal.pgen.1005954.].

Systematic discovery of novel ciliary genes through functional genomics in the zebrafish.

Cilia are microtubule-based hair-like organelles that play many important roles in development and physiology, and are implicated in a rapidly expanding spectrum of human diseases, collectively termed ciliopathies. Primary ciliary dyskinesia (PCD), one of the most prevalent of ciliopathies, arises from abnormalities in the differentiation or motility of the motile cilia. Despite their biomedical importance, a methodical functional screen for ciliary genes has not been carried out in any vertebrate at the organismal level. We sought to systematically discover novel motile cilia genes by identifying the genes induced by Foxj1, a winged-helix transcription factor that has an evolutionarily conserved role as the master regulator of motile cilia biogenesis. Unexpectedly, we find that the majority of the Foxj1-induced genes have not been associated with cilia before. To characterize these novel putative ciliary genes, we subjected 50 randomly selected candidates to a systematic functional phenotypic screen in zebrafish embryos. Remarkably, we find that over 60% are required for ciliary differentiation or function, whereas 30% of the proteins encoded by these genes localize to motile cilia. We also show that these genes regulate the proper differentiation and beating of motile cilia. This collection of Foxj1-induced genes will be invaluable for furthering our understanding of ciliary biology, and in the identification of new mutations underlying ciliary disorders in humans.

Cilia-driven fluid flow as an epigenetic cue for otolith biomineralization on sensory hair cells of the inner ear.

Ciliary motility is necessary for many developmental and physiological processes in animals. In zebrafish, motile cilia are thought to be required for the deposition of otoliths, which comprise crystals of protein and calcium carbonate, on hair cells of the inner ear. The identity of the motile cilia and their role in otolith biogenesis, however, remain controversial. Here, we show that the ear vesicle differentiates numerous motile cilia, the spatial distribution of which changes as a function of the expression pattern of the ciliogenic gene foxj1b. By contrast, the hair cells develop immotile kinocilia that serve as static tethers for otolith crystallization. In ears devoid of all cilia, otoliths can form but they are of irregular shapes and sizes and appear to attach instead to the hair cell apical membranes. Moreover, overproduction of motile cilia also disrupts otolith deposition through sustained agitation of the precursor particles. Therefore, the correct spatial and temporal distribution of the motile cilia is crucial for proper otolith formation. Our findings support the view that the hair cells express a binding factor for the otolith precursors, while the motile cilia ensure that the precursors do not sediment prematurely and are efficiently directed towards the hair cells. We also provide evidence that the kinocilia are modified motile cilia that depend on Foxj1b for their differentiation. We propose that in hair cells, a Foxj1b-dependent motile ciliogenic program is altered by the proneural Atoh proteins to promote the differentiation of immotile kinocilia.

Reactivation of low avidity tumor-specific CD8+ T cells associates with immunotherapeutic efficacy of anti-PD-1.

BACKGROUND: CD8+ T cells are a highly diverse population of cells with distinct phenotypic functions that can influence immunotherapy outcomes. Further insights on the roles of CD8+ specificities and TCR avidity of naturally arising tumor-specific T cells, where both high and low avidity T cells recognizing the same peptide-major histocompatibility complex (pMHC) coexist in the same tumor, are crucial for understanding T cell exhaustion and resistance to PD-1 immunotherapy. METHODS: CT26 models were treated with anti-PD-1 on days 3, 6 and 9 following subcutaneous tumor implantation generating variable responses during early tumor development. Tetramer staining was performed to determine the frequency and avidity of CD8+ T cells targeting the tumor-specific epitope GSW11 and confirmed with tetramer competition assays. Functional characterization of high and low avidity GSW11-specific CD8+ T cells was conducted using flow cytometry and bulk RNA-seq. In vitro cytotoxicity assays and in vivo adoptive transfer experiments were performed to determine the cytotoxicity of high and low avidity populations. RESULTS: Treatment success with anti-PD-1 was associated with the preferential expansion of low avidity (Tetlo) GSW11-specific CD8+ T cells with Vß TCR expressing clonotypes. High avidity T cells (Tethi), if present, were only found in progressing PD-1 refractory tumors. Tetlo demonstrated precursor exhausted or progenitor T cell phenotypes marked by higher expression of Tcf-1 and T-bet, and lower expression of the exhaustion markers CD39, PD-1 and Eomes compared with Tethi, whereas Tethi cells were terminally exhausted. Transcriptomics analyses showed pathways related to TCR signaling, cytotoxicity and oxidative phosphorylation were significantly enriched in Tetlo found in both regressing and progressing tumors compared with Tethi, whereas genes related to DNA damage, apoptosis and autophagy were downregulated. In vitro studies showed that Tetlo exhibits higher cytotoxicity than Tethi. Adoptive transfer of Tetlo showed more effective tumor control than Tethi, and curative responses were achieved when Tetlo was combined with two doses of anti-PD-1. CONCLUSIONS: Targeting subdominant T cell responses with lower avidity against pMHC affinity neoepitopes showed potential for improving PD-1 immunotherapy. Future interventions may consider expanding low avidity populations via vaccination or adoptive transfer.

Interferon-γ couples CD8+ T cell avidity and differentiation during infection.

Effective responses to intracellular pathogens are characterized by T cell clones with a broad affinity range for their cognate peptide and diverse functional phenotypes. How T cell clones are selected throughout the response to retain a breadth of avidities remains unclear. Here, we demonstrate that direct sensing of the cytokine IFN-γ by CD8+ T cells coordinates avidity and differentiation during infection. IFN-γ promotes the expansion of low-avidity T cells, allowing them to overcome the selective advantage of high-avidity T cells, whilst reinforcing high-avidity T cell entry into the memory pool, thus reducing the average avidity of the primary response and increasing that of the memory response. IFN-γ in this context is mainly provided by virtual memory T cells, an antigen-inexperienced subset with memory features. Overall, we propose that IFN-γ and virtual memory T cells fulfil a critical immunoregulatory role by enabling the coordination of T cell avidity and fate.

[123I]CC1: A PARP-Targeting, Auger Electron-Emitting Radiopharmaceutical for Radionuclide Therapy of Cancer.

Poly(adenosine diphosphate ribose) polymerase (PARP) has emerged as an effective therapeutic strategy against cancer that targets the DNA damage repair enzyme. PARP-targeting compounds radiolabeled with an Auger electron-emitting radionuclide can be trapped close to damaged DNA in tumor tissue, where high ionizing potential and short range lead Auger electrons to kill cancer cells through the creation of complex DNA damage, with minimal damage to surrounding normal tissue. Here, we report on [123I]CC1, an 123I-labeled PARP inhibitor for radioligand therapy of cancer. Methods: Copper-mediated 123I iododeboronation of a boronic pinacol ester precursor afforded [123I]CC1. The level and specificity of cell uptake and the therapeutic efficacy of [123I]CC1 were determined in human breast carcinoma, pancreatic adenocarcinoma, and glioblastoma cells. Tumor uptake and tumor growth inhibition of [123I]CC1 were assessed in mice bearing human cancer xenografts (MDA-MB-231, PSN1, and U87MG). Results: In vitro and in vivo studies showed selective uptake of [123I]CC1 in all models. Significantly reduced clonogenicity, a proxy for tumor growth inhibition by ionizing radiation in vivo, was observed in vitro after treatment with as little as 10 Bq [123I]CC1. Biodistribution at 1 h after intravenous administration showed PSN1 tumor xenograft uptake of 0.9 ± 0.06 percentage injected dose per gram of tissue. Intravenous administration of a relatively low amount of [123I]CC1 (3 MBq) was able to significantly inhibit PSN1 xenograft tumor growth but was less effective in xenografts that expressed less PARP. [123I]CC1 did not cause significant toxicity to normal tissues. Conclusion: Taken together, these results show the potential of [123I]CC1 as a radioligand therapy for PARP-expressing cancers.

Clinical Translation of Neutrophil Imaging and Its Role in Cancer.

Neutrophils are the first line of defense against pathogens and abnormal cells. They regulate many biological processes such as infections and inflammation. Increasing evidence demonstrated a role for neutrophils in cancer, where different subpopulations have been found to possess both pro- or anti-tumorigenic functions in the tumor microenvironment. In this review, we discuss the phenotypic and functional diversity of neutrophils in cancer, their prognostic significance, and therapeutic relevance in human and preclinical models. Molecular imaging methods are increasingly used to probe neutrophil biology in vivo, as well as the cellular changes that occur during tumor progression and over the course of treatment. This review will discuss the role of neutrophil imaging in oncology and the lessons that can be drawn from imaging in infectious diseases and inflammatory disorders. The major factors to be considered when developing imaging techniques and biomarkers for neutrophils in cancer are reviewed. Finally, the potential clinical applications and the limitations of each method are discussed, as well as the challenges for future clinical translation.

MRI techniques for immunotherapy monitoring.

MRI is a widely available clinical tool for cancer diagnosis and treatment monitoring. MRI provides excellent soft tissue imaging, using a wide range of contrast mechanisms, and can non-invasively detect tissue metabolites. These approaches can be used to distinguish cancer from normal tissues, to stratify tumor aggressiveness, and to identify changes within both the tumor and its microenvironment in response to therapy. In this review, the role of MRI in immunotherapy monitoring will be discussed and how it could be utilized in the future to address some of the unique clinical questions that arise from immunotherapy. For example, MRI could play a role in identifying pseudoprogression, mixed response, T cell infiltration, cell tracking, and some of the characteristic immune-related adverse events associated with these agents. The factors to be considered when developing MRI imaging biomarkers for immunotherapy will be reviewed. Finally, the advantages and limitations of each approach will be discussed, as well as the challenges for future clinical translation into routine clinical care. Given the increasing use of immunotherapy in a wide range of cancers and the ability of MRI to detect the microstructural and functional changes associated with successful response to immunotherapy, the technique has great potential for more widespread and routine use in the future for these applications.

Radiofluorination of a highly potent ATM inhibitor as a potential PET imaging agent.

PURPOSE: Ataxia telangiectasia mutated (ATM) is a key mediator of the DNA damage response, and several ATM inhibitors (ATMi) are currently undergoing early phase clinical trials for the treatment of cancer. A radiolabelled ATMi to determine drug pharmacokinetics could assist patient selection in a move towards more personalised medicine. The aim of this study was to synthesise and investigate the first 18F-labelled ATM inhibitor [18F]1 for non-invasive imaging of ATM protein and ATMi pharmacokinetics. METHODS: Radiofluorination of a confirmed selective ATM inhibitor (1) was achieved through substitution of a nitro-precursor with [18F]fluoride. Uptake of [18F]1 was assessed in vitro in H1299 lung cancer cells stably transfected with shRNA to reduce expression of ATM. Blocking studies using several non-radioactive ATM inhibitors assessed binding specificity to ATM. In vivo biodistribution studies were performed in wild-type and ATM-knockout C57BL/6 mice using PET/CT and ex vivo analysis. Uptake of [18F]1 in H1299 tumour xenografts was assessed in BALB/c nu/nu mice. RESULTS: Nitro-precursor 2 was synthesised with an overall yield of 12%. Radiofluorination of 2 achieved radiochemically pure [18F]1 in 80 ± 13 min with a radiochemical yield of 20 ± 13% (decay-corrected) and molar activities up to 79.5 GBq/µmol (n = 11). In vitro, cell-associated activity of [18F]1 increased over 1 h, and retention of [18F]1 dropped to 50% over 2 h. [18F]1 uptake did not correlate with ATM expression, but could be reduced significantly with an excess of known ATM inhibitors, demonstrating specific binding of [18F]1 to ATM. In vivo, fast hepatobiliary clearance was observed with tumour uptake ranging 0.13-0.90%ID/g after 1 h. CONCLUSION: Here, we report the first radiofluorination of an ATM inhibitor and its in vitro and in vivo biological evaluations, revealing the benefits but also some limitations of 18F-labelled ATM inhibitors.

Correlation between molar activity, injection mass and uptake of the PARP targeting radiotracer [18F]olaparib in mouse models of glioma.

PURPOSE: Radiopharmaceuticals targeting poly(ADP-ribose) polymerase (PARP) have emerged as promising agents for cancer diagnosis and therapy. PARP enzymes are expressed in both cancerous and normal tissue. Hence, the injected mass, molar activity and potential pharmacological effects are important considerations for the use of radiolabelled PARP inhibitors for diagnostic and radionuclide therapeutic applications. Here, we performed a systematic evaluation by varying the molar activity of [18F]olaparib and the injected mass of [TotalF]olaparib to investigate the effects on tumour and normal tissue uptake in two subcutaneous human glioblastoma xenograft models. METHODS: [18F]Olaparib uptake was evaluated in the human glioblastoma models: in vitro on U251MG and U87MG cell lines, and in vivo on tumour xenograft-bearing mice, after administration of [TotalF]olaparib (varying injected mass: 0.04-8.0 µg, and molar activity: 1-320 GBq/µmol). RESULTS: Selective uptake of [18F]olaparib was demonstrated in both models. Tumour uptake was found to be dependent on the injected mass of [TotalF]olaparib (µg) but not the molar activity. An injected mass of 1 µg resulted in the highest tumour uptake (up to 6.9 ± 1.3%ID/g), independent of the molar activity. In comparison, both the lower and higher injected masses of [TotalF]olaparib resulted in lower relative tumour uptake (%ID/g; P < 0.05). Ex vivo analysis of U87MG xenograft sections showed that the heterogeneity in [18F]olaparib intratumoural uptake correlated with PARP1 expression. Substantial upregulation of PARP1-3 expression was observed after administration of [TotalF]olaparib (> 0.5 µg). CONCLUSION: Our findings show that the injected mass of [TotalF]olaparib has significant effects on tumour uptake. Moderate injected masses of PARP inhibitor-derived radiopharmaceuticals may lead to improved relative tumour uptake and tumour-to-background ratio for cancer diagnosis and radionuclide therapy.

In Vivo Cell Tracking Using PET: Opportunities and Challenges for Clinical Translation in Oncology.

Cell therapy is a rapidly evolving field involving a wide spectrum of therapeutic cells for personalised medicine in cancer. In vivo imaging and tracking of cells can provide useful information for improving the accuracy, efficacy, and safety of cell therapies. This review focuses on radiopharmaceuticals for the non-invasive detection and tracking of therapeutic cells using positron emission tomography (PET). A range of approaches for imaging therapeutic cells is discussed: Direct ex vivo labelling of cells, in vivo indirect labelling of cells by utilising gene reporters, and detection of specific antigens expressed on the target cells using antibody-based radiopharmaceuticals (immuno-PET). This review examines the evaluation of PET imaging methods for therapeutic cell tracking in preclinical cancer models, their role in the translation into patients, first-in-human studies, as well as the translational challenges involved and how they can be overcome.

The emerging role of cell surface receptor and protein binding radiopharmaceuticals in cancer diagnostics and therapy.

Targeting specific cell membrane markers for both diagnostic imaging and radionuclide therapy is a rapidly evolving field in cancer research. Some of these applications have now found a role in routine clinical practice and have been shown to have a significant impact on patient management. Several molecular targets are being investigated in ongoing clinical trials and show promise for future implementation. Advancements in molecular biology have facilitated the identification of new cancer-specific targets for radiopharmaceutical development.

Multiparametric MRI of early tumor response to immune checkpoint blockade in metastatic melanoma.

BACKGROUND: Immune checkpoint inhibitors are now standard of care treatment for many cancers. Treatment failure in metastatic melanoma is often due to tumor heterogeneity, which is not easily captured by conventional CT or tumor biopsy. The aim of this prospective study was to investigate early microstructural and functional changes within melanoma metastases following immune checkpoint blockade using multiparametric MRI. METHODS: Fifteen treatment-naïve metastatic melanoma patients (total 27 measurable target lesions) were imaged at baseline and following 3 and 12 weeks of treatment on immune checkpoint inhibitors using: T2-weighted imaging, diffusion kurtosis imaging, and dynamic contrast-enhanced MRI. Treatment timepoint changes in tumor cellularity, vascularity, and heterogeneity within individual metastases were evaluated and correlated to the clinical outcome in each patient based on Response Evaluation Criteria in Solid Tumors V.1.1 at 1 year. RESULTS: Differential tumor growth kinetics in response to immune checkpoint blockade were measured in individual metastases within the same patient, demonstrating significant intertumoral heterogeneity in some patients. Early detection of tumor cell death or cell loss measured by a significant increase in the apparent diffusivity (Dapp) (p<0.05) was observed in both responding and pseudoprogressive lesions after 3 weeks of treatment. Tumor heterogeneity, as measured by apparent diffusional kurtosis (Kapp), was consistently higher in the pseudoprogressive and true progressive lesions, compared with the responding lesions throughout the first 12 weeks of treatment. These preceded tumor regression and significant tumor vascularity changes (Ktrans, ve, and vp) detected after 12 weeks of immunotherapy (p<0.05). CONCLUSIONS: Multiparametric MRI demonstrated potential for early detection of successful response to immune checkpoint inhibitors in metastatic melanoma.

Intravital Imaging of Adoptive T-Cell Morphology, Mobility and Trafficking Following Immune Checkpoint Inhibition in a Mouse Melanoma Model.

Efficient T-cell targeting, infiltration and activation within tumors is crucial for successful adoptive T-cell therapy. Intravital microscopy is a powerful tool for the visualization of T-cell behavior within tumors, as well as spatial and temporal heterogeneity in response to immunotherapy. Here we describe an experimental approach for intravital imaging of adoptive T-cell morphology, mobility and trafficking in a skin-flap tumor model, following immune modulation with immune checkpoint inhibitors (ICIs) targeting PD-L1 and CTLA-4. A syngeneic model of ovalbumin and mCherry-expressing amelanotic mouse melanoma was used in conjunction with adoptively transferred OT-1+ cytotoxic T-cells expressing GFP to image antigen-specific live T-cell behavior within the tumor microenvironment. Dynamic image analysis of T-cell motility showed distinct CD8+ T-cell migration patterns and morpho-dynamics within different tumor compartments in response to ICIs: this approach was used to cluster T-cell behavior into four groups based on velocity and meandering index. The results showed that most T-cells within the tumor periphery demonstrated Lévy-like trajectories, consistent with tumor cell searching strategies. T-cells adjacent to tumor cells had reduced velocity and appeared to probe the local environment, consistent with cell-cell interactions. An increased number of T-cells were detected following treatment, traveling at lower mean velocities than controls, and demonstrating reduced displacement consistent with target engagement. Histogram-based analysis of immunofluorescent images from harvested tumors showed that in the ICI-treated mice there was a higher density of CD31+ vessels compared to untreated controls and a greater infiltration of T-cells towards the tumor core, consistent with increased cellular trafficking post-treatment.

Detection limit of 89Zr-labeled T cells for cellular tracking: an in vitro imaging approach using clinical PET/CT and PET/MRI.

PURPOSE: Tracking cells in vivo using imaging can provide non-invasive information to understand the pharmacology, efficacy, and safety of novel cell therapies. Zirconium-89 (t1/2 = 78.4 h) has recently been used to synthesize [89Zr]Zr(oxinate)4 for cell tracking using positron emission tomography (PET). This work presents an in vitro approach to estimate the detection limit for in vivo PET imaging of Jurkat T cells directly labeled with [89Zr]Zr(oxinate)4 utilizing clinical PET/CT and PET/MRI. METHODS: Jurkat T cells were labeled with varying concentrations of [89Zr]Zr(oxinate)4 to generate different cell-specific activities (0.43-31.91 kBq/106 cells). Different concentrations of labeled cell suspensions (104, 105, and 106 cells) were seeded on 6-well plates and into a 3 × 3 cubic-well plate with 1 cm3 cubic wells as a gel matrix. Plates were imaged on clinical PET/CT and PET/MRI scanners for 30 min. The total activity in each well was determined by drawing volumes of interest over each well on PET images. The total cell-associated activity was measured using a well counter and correlated with imaging data. Simulations for non-specific signal were performed to model the effect of non-specific radioactivity on detection. RESULTS: Using this in vitro model, the lowest cell number that could be visualized on 6-well plate images was 6.8 × 104, when the specific activity was 27.8 kBq/106 cells. For the 3 × 3 cubic-well, a plate of 3.3 × 104 cells could be detected on images with a specific activity of 15.4 kBq/106 cells. CONCLUSION: The results show the feasibility of detecting [89Zr]Zr(oxinate)4-labeled Jurkat T cells on clinical PET systems. The results provide a best-case scenario, as in vivo detection using PET/CT or PET/MRI will be affected by cell number, specific activity per cell, the density of cells within the target volume, and non-specific signal. This work has important implications for cell labeling studies in patients, particularly when using radiosensitive cells (e.g., T cells), which require detection of low cell numbers while minimizing radiation dose per cell.

Correlating Radiomic Features of Heterogeneity on CT with Circulating Tumor DNA in Metastatic Melanoma.

Clinical imaging methods, such as computed tomography (CT), are used for routine tumor response monitoring. Imaging can also reveal intratumoral, intermetastatic, and interpatient heterogeneity, which can be quantified using radiomics. Circulating tumor DNA (ctDNA) in the plasma is a sensitive and specific biomarker for response monitoring. Here we evaluated the interrelationship between circulating tumor DNA mutant allele fraction (ctDNAmaf), obtained by targeted amplicon sequencing and shallow whole genome sequencing, and radiomic measurements of CT heterogeneity in patients with stage IV melanoma. ctDNAmaf and radiomic observations were obtained from 15 patients with a total of 70 CT examinations acquired as part of a prospective trial. 26 of 39 radiomic features showed a significant relationship with log(ctDNAmaf). Principal component analysis was used to define a radiomics signature that predicted ctDNAmaf independent of lesion volume. This radiomics signature and serum lactate dehydrogenase were independent predictors of ctDNAmaf. Together, these results suggest that radiomic features and ctDNAmaf may serve as complementary clinical tools for treatment monitoring.