In vitro and preclinical systematic dose-effect studies of Auger electron- and beta particle-emitting radionuclides and external beam radiation for cancer treatment.

PURPOSE: Despite a rise in clinical use of radiopharmaceutical therapies, the biological effects of radionuclides and their relationship with absorbed radiation dose are poorly understood. Here, we set out to define this relationship for Auger electron-emitters [99mTc]TcO4 and [123I]I, and ß--particle-emitter [188Re]ReO4. Studies were carried out using genetically-modified cells that permitted direct radionuclide comparisons. METHODS AND MATERIALS: Triple-negative MDA-MB-231 breast cancer cells, expressing the human sodium/iodide symporter (hNIS) and green fluorescent protein (GFP; MDA-MB-231.hNIS-GFP) were used. In vitro radiotoxicity of [99mTc]TcO4, [123I]I and [188Re]ReO4 was determined using clonogenic assays. Radionuclide uptake, efflux, and subcellular location were used to calculate nuclear-absorbed doses using the Medical Internal Radiation Dose formalism. In vivo studies were performed using female NSG mice bearing orthotopic MDA-MB-231.hNIS-GFP tumors and compared to X-ray-treated (12.6-15 Gy) and untreated cohorts. Absorbed dose per unit activity in tumors and NIS-expressing organs were extrapolated to reference human adult models using OLINDA/EXM®. RESULTS: [99mTc]TcO4- and [123I]I reduced the survival fraction only in hNIS-expressing cells, whereas [188Re]ReO4 reduced survival fraction in hNIS-expressing and parental cells. [123I]I required 2.4-fold and 1.5-fold lower decays/cell to achieve 37% survival compared to [99mTc]TcO4- and [188Re]ReO4, respectively, following 72 hours incubation. Additionally, [99mTc]TcO4-, [123I]I and [188Re]ReO4 had superior cell killing effectiveness in vitro compared to X-rays. In vivo, X-ray led to a greater median survival compared to [188Re]ReO4 and [123I]I (54 days versus 45 and 43 days, respectively). Unlike the X-ray cohort, no metastases were visualized in the radionuclide-treated cohorts. Extrapolated human absorbed doses of [188Re]ReO4 to a 1 g tumor were 13.8-fold and 11.2-fold greater than for [123I]I in female and male models, respectively. CONCLUSIONS: This work reports reference dose-effect data using cell and tumor models for [99mTc]TcO4, [123I]I, and [188Re]ReO4, for the first time. We further demonstrate the tumor controlling effects of [123I]I, and [188Re]ReO4 in comparison to EBRT.

Multi-scale in vivo imaging of tumour development using a germline conditional triple-reporter system.

Imaging reporter genes are indispensable for visualising biological processes in living subjects, particularly in cancer research where they have been used to observe tumour development, cancer cell dissemination, and treatment response. Engineering reporter genes into the germline frequently involves single imaging modality reporters operating over limited spatial scales. To address these limitations, we developed an inducible triple-reporter mouse model (Rosa26LSL - NRL) that integrates reporters for complementary imaging modalities, flfluorescence, bioluminescence and positron emission tomography (PET), along with inducible Cre-lox functionality for precise spatiotemporal control of reporter expression. We demonstrated robust reporter inducibility across various tissues in the Rosa26LSL - NRL mouse, facilitating effective tracking and characterisation of tumours in liver and lung cancer mouse models. We precisely pinpointed tumour location using multimodal whole-body imaging which guided in situ lung microscopy to visualise cell-cell interactions within the tumour microenvironment. The triple-reporter system establishes a robust new platform technology for multi-scale investigation of biological processes within whole animals, enabling tissue-specific and sensitive cell tracking, spanning from the whole-body to cellular scales.

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers.

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.

Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer.

BACKGROUND: Small interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. METHODS: PEG12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. RESULTS: Our results showed that PEG12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. CONCLUSION: In conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

Combined Vorinostat and Chloroquine Inhibit Sodium-Iodide Symporter Endocytosis and Enhance Radionuclide Uptake In Vivo.

PURPOSE: Patients with aggressive thyroid cancer are frequently failed by the central therapy of ablative radioiodide (RAI) uptake, due to reduced plasma membrane (PM) localization of the sodium/iodide symporter (NIS). We aimed to understand how NIS is endocytosed away from the PM of human thyroid cancer cells, and whether this was druggable in vivo. EXPERIMENTAL DESIGN: Informed by analysis of endocytic gene expression in patients with aggressive thyroid cancer, we used mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake, and NanoBRET to understand the mechanisms of NIS endocytosis in transformed cell lines and patient-derived human primary thyroid cells. Systemic drug responses were monitored via 99mTc pertechnetate gamma counting and gene expression in BALB/c mice. RESULTS: We identified an acidic dipeptide within the NIS C-terminus that mediates binding to the σ2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine (CQ) modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor vorinostat/SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence. CONCLUSIONS: NIS internalization is specifically druggable in vivo. Our data, therefore, provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer. See related commentary by Lechner and Brent, p. 1220.

Genetic Engineering of Therapeutic Cells with the Sodium Iodide Symporter (NIS) to Enable Noninvasive In Vivo Therapy Tracking.

Noninvasive long-term imaging of therapeutic cells in preclinical models can be achieved through introducing a reporter gene into the cells of interest. Despite important recent developments such as gene editing, cell engineering based on lentiviruses remains a mainstream tool for gene transfer applicable to a variety of different cell types.In this chapter, we describe how to use lentivirus-based genetic engineering to render different candidate cell therapies in vivo traceable by radionuclide imaging. We illustrate this reporter gene technology using the sodium iodide symporter (NIS), which is compatible with both positron emission tomography (PET) and single-photon emission computed tomography (SPECT). For preclinical experimentation, we fused NIS with a suitable fluorescent protein such as monomeric GFP or RFP to streamline cell line generation and downstream analyses of ex vivo tissue samples. We present protocols for reporter gene engineering of human cardiac progenitor cells, regulatory T cells, and effector T cells as well as for the characterization experiments required to validate NIS-fluorescent protein reporter function in these candidate therapeutic cells.

Spatiotemporal quantitative microRNA-155 imaging reports immune-mediated changes in a triple-negative breast cancer model.

Introduction: MicroRNAs are small non-coding RNAs and represent key players in physiology and disease. Aberrant microRNA expression is central to the development and progression of cancer, with various microRNAs proposed as potential cancer biomarkers and drug targets. There is a need to better understand dynamic microRNA expression changes as cancers progress and their tumor microenvironments evolve. Therefore, spatiotemporal and non-invasive in vivo microRNA quantification in tumor models would be highly beneficial. Methods: We developed an in vivo microRNA detector platform in which the obtained signals are positively correlated to microRNA presence, and which permitted stable expression in cancer cells as needed for long-term experimentation in tumor biology. It exploits a radionuclide-fluorescence dual-reporter for quantitative in vivo imaging of a microRNA of choice by radionuclide tomography and fluorescence-based downstream ex vivo tissue analyses. We generated and characterized breast cancer cells stably expressing various microRNA detectors and validated them in vitro. Results: We found the microRNA detector platform to report on microRNA presence in cells specifically and accurately, which was independently confirmed by real-time PCR and through microRNA modulation. Moreover, we established various breast tumor models in animals with different levels of residual immune systems and observed microRNA detector read-outs by imaging. Applying the detector platform to the progression of a triple-negative breast cancer model, we found that miR-155 upregulation in corresponding tumors was dependent on macrophage presence in tumors, revealing immune-mediated phenotypic changes in these tumors as they progressed. Conclusion: While applied to immunooncology in this work, this multimodal in vivo microRNA detector platform will be useful whenever non-invasive quantification of spatiotemporal microRNA changes in living animals is of interest.

Radiolabelling of Polyclonally Expanded Human Regulatory T Cells (Treg) with 89Zr-oxine for Medium-Term In Vivo Cell Tracking.

Regulatory T cells (Tregs) are a promising candidate cell therapy to treat autoimmune diseases and aid the longevity of transplanted solid organs. Despite increasing numbers of clinical trials using human Treg therapy, important questions pertaining to their in vivo fate, distribution, and function remain unanswered. Treg accumulation in relevant tissues was found to be crucial for Treg therapy efficacy, but existing blood-borne biomarkers are unlikely to accurately reflect the tissue state. Non-invasive Treg tracking by whole-body imaging is a promising alternative and can be achieved by direct radiolabelling of Tregs and following the radiolabelled cells with positron emission tomography (PET). Our goal was to evaluate the radiolabelling of polyclonal Tregs with 89Zr to permit their in vivo tracking by PET/CT for longer than one week with current preclinical PET instrumentation. We used [89Zr]Zr(oxinate)4 as the cell-labelling agent and achieved successful radiolabelling efficiency of human Tregs spanning 0.1-11.1 Bq 89Zr/Treg cell, which would be compatible with PET tracking beyond one week. We characterized the 89Zr-Tregs, assessing their phenotypes, and found that they were not tolerating these intracellular 89Zr amounts, as they failed to survive or expand in a 89Zr-dose-dependent manner. Even at 0.1 Bq 89Zr per Treg cell, while 89Zr-Tregs remained functional as determined by a five-day-long effector T cell suppression assay, they failed to expand beyond day 3 in vitro. Moreover, PET imaging revealed signs of 89Zr-Treg death after adoptive transfer in vivo. In summary, 89Zr labelling of Tregs at intracellular radioisotope amounts compatible with cell tracking over several weeks did not achieve the desired outcomes, as 89Zr-Tregs failed to expand and survive. Consequently, we conclude that indirect Treg labelling is likely to be the most effective alternative method to satisfy the requirements of this cell tracking scenario.

Radiolabelling Pt-based quadruplex DNA binders via click chemistry.

Guanine-rich sequences of DNA and RNA can fold into intramolecular tetra-helical assemblies known as G-quadruplexes (G4). Their formation in vivo has been associated to a range of biological functions and therefore they have been identified as potential drug targets. Consequently, a broad range of small molecules have been developed to target G4s. Amongst those are metal complexes with Schiff base ligands. Herein, we report the functionalisation of one of these well-established G4 DNA binders (based on a square planar platinum(II)-salphen complex) with two different radiolabelled complexes. An 111In-conjugate was successfully used to assess its in vivo distribution in a mouse tumour model using single-photon emission computed tomography (SPECT) imaging. These studies highlighted the accumulation of this Pt-salphen-111In conjugate in the tumour.

Generation of In Vivo Traceable Hepatocyte-Like Cells from Human iPSCs.

In this chapter, we describe a protocol for differentiation of human-induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) and their transduction with a lentivirus for gene transfer. Here, we engineer them to express the human sodium iodide symporter, which can be exploited as a radionuclide reporter gene, thereby enabling these cells to be tracked in vivo by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. Differentiation of HLCs from iPSCs involves three steps: induction of iPSCs to definitive endoderm, differentiation to a hepatic progenitor cell population, and maturation of immature HLCs. Once proliferation of hepatic progenitors has ceased and an immature HLC population is generated, lentiviral transduction can be performed. The immature hepatic gene expression profile/morphology at the stage of transduction will be compatible with further maturation following transgene expression either in vitro or in vivo, with expression of the transgene retained. We detail how transgenic cells can be imaged in vivo. While we provide a protocol for the NIS reporter gene, the cell engineering aspects of this protocol are transferable for use with other (reporter) genes if desired.

PET Imaging of Small Extracellular Vesicles via [89Zr]Zr(oxinate)4 Direct Radiolabeling.

Exosomes or small extracellular vesicles (sEVs) are increasingly gaining attention for their potential as drug delivery systems and biomarkers of disease. Therefore, it is important to understand their in vivo biodistribution using imaging techniques that allow tracking over time and at the whole-body level. Positron emission tomography (PET) allows short- and long-term whole-body tracking of radiolabeled compounds in both animals and humans and with excellent quantification properties compared to other nuclear imaging techniques. In this report, we explored the use of [89Zr]Zr(oxinate)4 (a cell and liposome radiotracer) for direct and intraluminal radiolabeling of several types of sEVs, achieving high radiolabeling yields. The radiosynthesis and radiolabeling protocols were optimized for sEV labeling, avoiding sEV damage, as demonstrated using several characterizations (cryoEM, nanoparticle tracking analysis, dot blot, and flow cytometry) and in vitro techniques. Using pancreatic cancer sEVs (PANC1) in a healthy mouse model, we showed that it is possible to track 89Zr-labeled sEVs in vivo using PET imaging for at least up to 24 h. We also report differential biodistribution of intact sEVs compared to intentionally heat-damaged sEVs, with significantly reduced spleen uptake for the latter. Therefore, we conclude that 89Zr-labeled sEVs using this method can reliably be used for in vivo PET tracking and thus allow efficient exploration of their potential as drug delivery systems.

Relationship of In Vitro Toxicity of Technetium-99m to Subcellular Localisation and Absorbed Dose.

Auger electron-emitters increasingly attract attention as potential radionuclides for molecular radionuclide therapy in oncology. The radionuclide technetium-99m is widely used for imaging; however, its potential as a therapeutic radionuclide has not yet been fully assessed. We used MDA-MB-231 breast cancer cells engineered to express the human sodium iodide symporter-green fluorescent protein fusion reporter (hNIS-GFP; MDA-MB-231.hNIS-GFP) as a model for controlled cellular radionuclide uptake. Uptake, efflux, and subcellular location of the NIS radiotracer [99mTc]TcO4- were characterised to calculate the nuclear-absorbed dose using Medical Internal Radiation Dose formalism. Radiotoxicity was determined using clonogenic and γ-H2AX assays. The daughter radionuclide technetium-99 or external beam irradiation therapy (EBRT) served as controls. [99mTc]TcO4- in vivo biodistribution in MDA-MB-231.hNIS-GFP tumour-bearing mice was determined by imaging and complemented by ex vivo tissue radioactivity analysis. [99mTc]TcO4- resulted in substantial DNA damage and reduction in the survival fraction (SF) following 24 h incubation in hNIS-expressing cells only. We found that 24,430 decays/cell (30 mBq/cell) were required to achieve SF0.37 (95%-confidence interval = [SF0.31; SF0.43]). Different approaches for determining the subcellular localisation of [99mTc]TcO4- led to SF0.37 nuclear-absorbed doses ranging from 0.33 to 11.7 Gy. In comparison, EBRT of MDA-MB-231.hNIS-GFP cells resulted in an SF0.37 of 2.59 Gy. In vivo retention of [99mTc]TcO4- after 24 h remained high at 28.0% ± 4.5% of the administered activity/gram tissue in MDA-MB-231.hNIS-GFP tumours. [99mTc]TcO4- caused DNA damage and reduced clonogenicity in this model, but only when the radioisotope was taken up into the cells. This data guides the safe use of technetium-99m during imaging and potential future therapeutic applications.

Chimeric antigen receptor-modified human regulatory T cells that constitutively express IL-10 maintain their phenotype and are potently suppressive.

Clinical trials of Treg therapy in transplantation are currently entering phases IIa and IIb, with the majority of these employing polyclonal Treg populations that harbor a broad specificity. Enhancing Treg specificity is possible with the use of chimeric antigen receptors (CARs), which can be customized to respond to a specific human leukocyte antigen (HLA). In this study, we build on our previous work in the development of HLA-A2 CAR-Tregs by further equipping cells with the constitutive expression of interleukin 10 (IL-10) and an imaging reporter as additional payloads. Cells were engineered to express combinations of these domains and assessed for phenotype and function. Cells expressing the full construct maintained a stable phenotype after transduction, were specifically activated by HLA-A2, and suppressed alloresponses potently. The addition of IL-10 provided an additional advantage to suppressive capacity. This study therefore provides an important proof-of-principle for this cell engineering approach for next-generation Treg therapy in transplantation.

Validation of the plasmid study to relate DNA damaging effects of radionuclides to those from external beam radiotherapy.

INTRODUCTION: The biological consequences of absorbed radiation doses are ill-defined for radiopharmaceuticals, unlike for external beam radiotherapy (EBRT). A reliable assay that assesses the biological consequences of any radionuclide is much needed. Here, we evaluated the cell-free plasmid DNA assay to determine the relative biological effects of radionuclides such as Auger electron-emitting [67Ga]GaCl3 or [111In]InCl3 compared to EBRT. METHODS: Supercoiled pBR322 plasmid DNA (1.25 or 5 ng/µL) was incubated with 0.5 or 1 MBq [67Ga]GaCl3 or [111In]InCl3 for up to 73 h or was exposed to EBRT (137Cs; 5 Gy/min; 0-40 Gy). The induction of relaxed and linear plasmid DNA, representing single and double strand breaks, respectively, was assessed by gel electrophoresis. Chelated forms of 67Ga were also investigated using DOTA and THP. Topological conversion rates for supercoiled-to-relaxed (ksrx) or relaxed-to-linear (krlx) DNA were obtained by fitting a kinetic model. RESULTS: DNA damage increased both with EBRT dose and incubation time for [67Ga]GaCl3 and [111In]InCl3. Damage caused by [67Ga]GaCl3 decreased when chelated. [67Ga]GaCl3 proved more damaging than [111In]InCl3; 1.25 ng/µL DNA incubated with 0.5 MBq [67Ga]GaCl3 for 2 h led to a 70% decrease of intact plasmid DNA as opposed to only a 19% decrease for [111In]InCl3. For both EBRT and radionuclides, conversion rates were slower for 5 ng/µL than 1.25 ng/µL plasmid DNA. DNA damage caused by 1 Gy EBRT was the equivalent to damage caused by 0.5 MBq unchelated [67Ga]GaCl3 and [111In]InCl3 after 2.05 ± 0.36 and 9.3 ± 0.77 h of incubation, respectively. CONCLUSIONS: This work has highlighted the power of the plasmid DNA assay for a rapid determination of the relative biological effects of radionuclides compared to external beam radiotherapy. It is envisaged this approach will enable the systematic assessment of imaging and therapeutic radionuclides, including Auger electron-emitters, to further inform radiopharmaceutical design and application.

A preclinical pipeline to evaluate migrastatics as therapeutic agents in metastatic melanoma.

BACKGROUND: Metastasis is a hallmark of cancer and responsible for most cancer deaths. Migrastatics were defined as drugs interfering with all modes of cancer cell invasion and thus cancers' ability to metastasise. First anti-metastatic treatments have recently been approved. METHODS: We used bioinformatic analyses of publicly available melanoma databases. Experimentally, we performed in vitro target validation (including 2.5D cell morphology analysis and mass spectrometric analysis of RhoA binding partners), developed a new traceable spontaneously metastasising murine melanoma model for in vivo validation, and employed histology (haematoxylin/eosin and phospho-myosin II staining) to confirm drug action in harvested tumour tissues. RESULTS: Unbiased and targeted bioinformatic analyses identified the Rho kinase (ROCK)-myosin II pathway and its various components as potentially relevant targets in melanoma. In vitro validation demonstrated redundancy of several RhoGEFs upstream of RhoA and confirmed ROCK as a druggable target downstream of RhoA. The anti-metastatic effects of two ROCK inhibitors were demonstrated through in vivo melanoma metastasis tracking and inhibitor effects also confirmed ex vivo by digital pathology. CONCLUSIONS: We proposed a migrastatic drug development pipeline. As part of the pipeline, we provide a new traceable spontaneous melanoma metastasis model for in vivo quantification of metastasis and anti-metastatic effects by non-invasive imaging.

18 F-FDG PET/CT use in functional assessment of the testes: A systematic review.

INTRODUCTION: Our study analysed previous studies employing positron emission tomography with co-registered computer tomography (PET/CT) in andrological patient evaluation and assessed the differences in 2-[18 F]F-fluoro-2'-deoxyglucose (FDG) uptake between three groups: healthy testes, benign and malignant testicular pathology. METHODS: Medline and Embase were systematically searched for studies involving FDG-PET/CT imaging of testes with results expressed as mean standardised uptake value (SUVmean ). A one-way ANOVA was used to compare SUVmean between three groups. All papers assessing andrological parameters were pooled to compare fertility data. RESULTS: Seventeen studies, including three relating to fertility diagnosis, with a total of 830 patients, were included in the review. One-way ANOVA showed a statistical difference between mean values of tracer SUVmean in healthy and malignant testes (Dif. = -2.77, 95% CI = -4.32 to 1.21, p < 0.01) as well as benign and malignant (Dif. = -2.95, 95% CI = -4.33 to -1.21, p < 0.01) but no difference between healthy and benign (Dif. = 0.19, 95% CI = -0.96 to 1.33, p = 0.90). There is some evidence to suggest that FDG uptake and testicular volume are positively correlated to total sperm count, sperm concentration and sperm motility and that germ cells are likely to account for the majority of testicular FDG accumulation. CONCLUSION: Our findings indicate that malignant testicular lesions demonstrate a significantly higher FDG uptake than benign testicular lesions or healthy testes. Some evidence also suggests that FDG-PET could visualise metabolic activity and thus spermatogenesis; however more studies are required to determine whether FDG-PET could also be used to diagnose infertility. Further studies should focus on correlating both sex hormone-serum levels and semen analysis results with imaging data.

Options for imaging cellular therapeutics in vivo: a multi-stakeholder perspective.

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics.

Spatiotemporal in vivo tracking of polyclonal human regulatory T cells (Tregs) reveals a role for innate immune cells in Treg transplant recruitment.

Regulatory T cells (Tregs) are emerging as a new cell-based therapy in solid organ transplantation. Adoptive transfer of Tregs has been shown preclinically to protect from graft rejection, and the safety of Treg therapy has been demonstrated in clinical trials. Despite these successes, the in vivo distribution and persistence of adoptively transferred Tregs remained elusive, which hampers clinical translation. Here we isolated human Tregs using a GMP-compatible protocol and lentivirally transduced them with the human sodium iodide symporter to render them traceable in vivo by radionuclide imaging. Engineered human Tregs were characterized for phenotype, survival, suppressive capacity, and reporter function. To study their trafficking behavior, they were subsequently administered to humanized mice with human skin transplants. Traceable Tregs were quantified in skin grafts by non-invasive nano-single-photon emission computed tomography (nanoSPECT)/computed tomography (CT) for up to 40 days, and the results were validated ex vivo. Using this approach, we demonstrated that Treg trafficking to skin grafts was regulated by the presence of recipient Gr-1+ innate immune cells. We demonstrated the utility of radionuclide reporter gene-afforded quantitative Treg in vivo tracking, addressing a fundamental need in Treg therapy development and offering a clinically compatible methodology for future Treg therapy imaging in humans.

WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion.

Melanoma is a highly aggressive tumour that can metastasize very early in disease progression. Notably, melanoma can disseminate using amoeboid invasive strategies. We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells. Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation. Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures. As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive. This pattern is further enhanced in metastatic lesions. We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy.