In-vivo Tracking Research Articles

In-utero transfer of decidualized endometrial stromal cells increases the frequency of regulatory T cells and normalizes the abortion rate in the CBA/J × DBA/2 abortion model.

Failure to adequate decidualization leads to adverse pregnancy outcomes including pregnancy loss. Although there are plenty of reports underscoring immune dysfunction as the main cause of abortion in CBA/J females mated with DBA/2 males (CBA/J × DBA/2), little is known about the potential role of impaired endometrial decidualization. Endometrial stromal cells (ESCs) from CBA/J mice were in-vitro decidualized, and the proteome profile of the secretome was investigated by membrane-based array. CBA/J mice were perfused In-utero with either decidualized ESCs (C×D/D), undecidualized ESCs (C×D/ND), or PBS (C×D/P) 12 days before mating with DBA/2 males. Control mice were not manipulated and were mated with male DBA/2 (C×D) or Balb/c (C×B) mice. On day 13.5 of pregnancy, reproductive parameters were measured. In-vivo tracking of EdU-labeled ESCs was performed using fluorescence microscopy. The frequency of regulatory T cells (Tregs) in paraaortic/renal and inguinal lymph nodes was measured by flow cytometry. The proliferation of pregnant CBA/J splenocytes in response to stimulation with DBA/2 splenocytes was assessed by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometry. In C×D/D mice, the resorption rate was reduced to match that seen in the C×B group. Intrauterine perfused ESCs appeared in uterine stroma after 2 days, which remained there for at least 12 days. There was no difference in the number of implantation sites and embryo weight across all groups. The frequency of Tregs in the inguinal lymph nodes was similar across all groups, but it increased in the paraaortic/renal lymph nodes of C×D/D mice to the level found in C×B mice. No significant changes were observed in the proliferation of splenocytes from pregnant C×D/D compared to those of the C×D group in response to stimulation with DBA/2 splenocytes. Decidualization of ESCs was associated with a profound alteration in ESC secretome exemplified by alteration in proteins involved in extracellular matrix (ECM) remodeling, response to inflammation, senescence, and immune cell trafficking. Our results showed that the deficiency of Tregs is not the primary driver of abortion in the CBA/J × DBA/2 model and provided evidence that impaired endometrial decidualization probably triggers endometrial immune dysfunction and abortion in this model.

Ultralow-dose irradiation enables engraftment and intravital tracking of disease initiating niches in clonal hematopoiesis

Recent advances in imaging suggested that spatial organization of hematopoietic cells in their bone marrow microenvironment (niche) regulates cell expansion, governing progression, and leukemic transformation of hematological clonal disorders. However, our ability to interrogate the niche in pre-malignant conditions has been limited, as standard murine models of these diseases rely largely on transplantation of the mutant clones into conditioned mice where the marrow microenvironment is compromised. Here, we leveraged live-animal microscopy and ultralow dose whole body or focal irradiation to capture single cells and early expansion of benign/pre-malignant clones in the functionally preserved microenvironment. 0.5 Gy whole body irradiation (WBI) allowed steady engraftment of cells beyond 30 weeks compared to non-conditioned controls. In-vivo tracking and functional analyses of the microenvironment showed no change in vessel integrity, cell viability, and HSC-supportive functions of the stromal cells, suggesting minimal inflammation after the radiation insult. The approach enabled in vivo imaging of Tet2+/− and its healthy counterpart, showing preferential localization within a shared microenvironment while forming discrete micro-niches. Notably, stationary association with the niche only occurred in a subset of cells and would not be identified without live imaging. This strategy may be broadly applied to study clonal disorders in a spatial context.

Magnetic Cell Targeting for Cardiovascular Tissue Engineering.

There is a critical need for novel approaches to translate cell therapy and regenerative medicine to clinical practice. Magnetic cell targeting with site specificity has started to open avenues in these fields as a potential therapeutic platform. Magnetic targeting is gaining popularity in the field of biomedicine due to its ability to concentrate and retain at a target site while minimizing deleterious effects at off-target sites. It is regarded as a relatively straightforward and safe approach for a wide range of therapeutic applications. This review discusses the latest advancements and approaches in magnetic cell targeting using endocytosed and surface-bound magnetic nanoparticles as well as invivo tracking using magnetic resonance imaging (MRI). The most common form of magnetic nanoparticles is superparamagnetic iron oxide nanoparticles (SPION). The biodegradable and biocompatible properties of these magnetically responsive particles and capacity for rapid endocytosis into cells make them a breakthrough in targeted therapy. This review further discusses specific applications of magnetic targeting approaches in cardiovascular tissue engineering including myocardial regeneration, therapeutic angiogenesis, and endothelialization of implantable cardiovascular devices.

Engineered E. coli for Long-Term Oral Enzyme Delivery.

Intestinal flora shows excellent affinity in the gut, and the adhesive property is borrowed for oral drug delivery. A facile strategy for bacteria engineering has been successfully developed by introducing metal-organic framework (MOF) mineralization. The MOF exoskeleton serves as an extendable platform for accommodating various cargoes with good Escherichia coli morphology maintained. The artificial exoskeleton surrounding E. coli is employed for encapsulating macromolecules as a therapeutic cargo, maintaining good bioactivity with high immobilization efficiency (60%) after systematic optimization of the MOF precursor. Leveraging the natural affinity of E. coli in the gut, the in-vivo tracking of MOF-engineered E. coli in the gastrointestinal tract confirmed excellent adhesion to the GI mucosa and a 17.9-fold increase in the gut retention half-time, demonstrating significant advantages in retention capability. In comparison, the control group without E. coli equipment resulted in quick gut passage. Furthermore, the artificially engineered E. coli serves as an effective carrier for macromolecules without notable oral toxicity, as evidenced by biocompatibility evaluations in cells and animals. Overall, the MOF-engineered E. coli provides an extendable platform for loading on-demand cargoes in versatile therapeutic functions with promising clinical transnationality for long-term applications.

Enhanced anti-tumor efficacy with multi-transgene armed mesenchymal stem cells for treating peritoneal carcinomatosis

BackgroundMesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime.MethodsIn this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking.ResultsFirstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively.ConclusionsTaken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC.

Magnetic particle image scanner based on asymmetric core-filled electromagnetic actuator

Monitoring the distribution of magnetic nanoparticles (MNPs) in the vascular system is an important task for the advancement of precision therapeutics and drug delivery. Despite active targeting using active motilities, it is required to visualize the position and concentration of carriers that reach the target, to promote the development of this technology. In this work, a feasibility study is presented on a tomographic scanner that allows monitoring of the injected carriers quantitatively in a relatively short interval. The device is based on a small-animal-scale asymmetric magnetic platform integrated with magnetic particle imaging technology. An optimized isotropic field-free region (FFR) generation method using a magnetic manipulation system (MMS) is derived and numerically investigated. The in-vitro and in-vivo tracking performances are demonstrated with a high position accuracy of approximately 1 mm. A newly proposed tracking method was developed, specialized in vascular system, with quick scanning time (about 1s). In this paper, the primary function of the proposed system is to track magnetic particles using a magnetic manipulation system. Through this, proposed method enables the conventional magnetic actuation systems to upgrade the functionalities of both manipulation and localization of magnetic objects.

Cross-organ single-cell transcriptome profiling reveals macrophage and dendritic cell heterogeneity in zebrafish

Tissue-resident macrophages (TRMs) and dendritic cells (DCs) are highly heterogeneous and essential for immunity, tissue regeneration, and homeostasis maintenance. Here, we comprehensively profile the heterogeneity of TRMs and DCs across adult zebrafish organs via single-cell RNA sequencing. We identify two macrophage subsets: pro-inflammatory macrophages with potent phagocytosis signatures and pro-remodeling macrophages with tissue regeneration signatures in barrier tissues, liver, and heart. In parallel, one conventional dendritic cell (cDC) population with prominent antigen presentation capacity and plasmacytoid dendritic cells (pDCs) featured by anti-virus properties are also observed in these organs. Remarkably, in addition to a single macrophage/microglia population with potent phagocytosis capacity, a pDC population and two distinct cDC populations are identified in the brain. Finally, we generate specific reporter lines for invivo tracking of macrophage and DC subsets. Our study depicts the landscape of TRMs and DCs and creates valuable tools for in-depth study of these cells in zebrafish.

Borrelia burgdorferi Engages Mammalian Type I IFN Responses via the cGAS-STING Pathway.

Borrelia burgdorferi, the etiologic agent of Lyme disease, is a spirochete that modulates numerous host pathways to cause a chronic, multisystem inflammatory disease in humans. B. burgdorferi infection can lead to Lyme carditis, neurologic complications, and arthritis because of the ability of specific borrelial strains to disseminate, invade, and drive inflammation. B. burgdorferi elicits type I IFN (IFN-I) responses in mammalian cells and tissues that are associated with the development of severe arthritis or other Lyme-related complications. However, the innate immune sensors and signaling pathways controlling IFN-I induction remain unclear. In this study, we examined whether intracellular nucleic acid sensing is required for the induction of IFN-I to B. burgdorferi. Using fluorescence microscopy, we show that B. burgdorferi associates with mouse and human cells in culture, and we document that internalized spirochetes colocalize with the pattern recognition receptor cyclic GMP-AMP synthase (cGAS). Moreover, we report that IFN-I responses in mouse macrophages and murine embryonic fibroblasts are significantly attenuated in the absence of cGAS or its adaptor stimulator of IFN genes (STING), which function to sense and respond to intracellular DNA. Longitudinal invivo tracking of bioluminescent B. burgdorferi revealed similar dissemination kinetics and borrelial load in C57BL/6J wild-type, cGAS-deficient, or STING-deficient mice. However, infection-associated tibiotarsal joint pathology and inflammation were modestly reduced in cGAS-deficient compared with wild-type mice. Collectively, these results indicate that the cGAS-STING pathway is a critical mediator of mammalian IFN-I signaling and innate immune responses to B. burgdorferi.

Generation of AAVS1-EGFP reporter cell lines from an isogenic pair of trisomy 21 and euploid human iPSCs

Human mouse chimeric models are valuable tools to develop in-vivo disease models. However, in-vivo detection of human cells limits their analysis. To facilitate in-vivo modeling of Down syndrome (DS), we generated a stable AAVS1-EGFP isogenic pair of DS human iPSCs by zinc finger mediated genetic engineering of the AAVS1 locus. These lines overcome the limitation of reporter human iPSCs generated using random integration, which may not express reporter gene in all tissues due to heterochromatin-induced gene silencing. These reporter cell lines provide a valuable tool to facilitate in-vivo tracking of the graft cell integration, differentiation, and distinction from host cells.

Longitudinal trimodal imaging of midbrain-associated network degeneration in Parkinson’s disease

The prevailing network perspective of Parkinson’s disease (PD) emerges not least from the ascending neuropathology traceable in histological studies. However, whether longitudinal in vivo correlates of network degeneration in PD can be observed remains unresolved. Here, we applied a trimodal imaging protocol combining 18F-fluorodeoxyglucose (FDG)- and 18F-fluoro-L-Dopa- (FDOPA)-PET with resting-state functional MRI to assess longitudinal changes in midbrain metabolism, striatal dopamine depletion and striatocortical dysconnectivity in 17 well-characterized PD patients. Whole-brain (un)paired-t-tests with focus on midbrain or striatum were performed between visits and in relation to 14 healthy controls (HC) in PET modalities. Resulting clusters of FDOPA-PET comparisons provided volumes for seed-based functional connectivity (FC) analyses between visits and in relation to HC. FDG metabolism in the left midbrain decreased compared to baseline along with caudatal FDOPA-uptake. This caudate cluster exhibited a longitudinal FC decrease to sensorimotor and frontal areas. Compared to healthy subjects, dopamine-depleted putamina indicated stronger decline in striatocortical FC at follow-up with respect to baseline. Increasing nigrostriatal deficits and striatocortical decoupling were associated with deterioration in motor scores between visits in repeated-measures correlations. In summary, our results demonstrate the feasibility of in-vivo tracking of progressive network degeneration using a multimodal imaging approach. Specifically, our data suggest advancing striatal and widespread striatocortical dysfunction via an anterior-posterior gradient originating from a hypometabolic midbrain cluster within a well-characterized and only mild to moderately affected PD cohort during a relatively short period.

In vivo real-time imaging reveals megalin as the aminoglycoside gentamicin transporter into cochlea whose inhibition is otoprotective

Aminoglycosides (AGs) are commonly used antibiotics that cause deafness through the irreversible loss of cochlear sensory hair cells (HCs). How AGs enter the cochlea and then target HCs remains unresolved. Here, we performed time-lapse multicellular imaging of cochlea in live adult hearing mice via a chemo-mechanical cochleostomy. The invivo tracking revealed that systemically administered Texas Red-labeled gentamicin (GTTR) enters the cochlea via the stria vascularis and then HCs selectively. GTTR uptake into HCs was completely abolished in transmembrane channel-like protein 1 (TMC1) knockout mice, indicating mechanotransducer channel-dependent AG uptake. Blockage of megalin, the candidate AG transporter in the stria vascularis, by binding competitor cilastatin prevented GTTR accumulation in HCs. Furthermore, cilastatin treatment markedly reduced AG-induced HC degeneration and hearing loss invivo. Together, our invivo real-time tracking of megalin-dependent AG transport across the blood-labyrinth barrier identifies new therapeutic targets for preventing AG-induced ototoxicity.

In-vivo tracking of harmonic nanoparticles: a study based on a TIGER widefield microscope [Invited

In vivo tracking of harmonic nanoparticles (HNPs) in living animals is a technique not yet exploited, despite the great potential offered by these markers, due to a lack of an appropriate tool. The main drawback is the necessity to excite nonlinear effects in the millimeter area in a widefield mode with a sufficient signal to noise ratio. Our approach to this problem consists in a redesign of the laser space parameters in a region of high energy per pulse and low repetition rate in the kHz regime, in counter-trend with the actual microscope research technology. We realise this by means of a regeneratively amplified fs-laser system, creating an easy alignable and reproducible Tunable hIGh EneRgy (TIGER) widefield microscope. This one is successfully applied for HNPs tracking in the blood flow of the heart system of a Drosophila larvae, a powerful platform to study socially relevant diseases, such as congenital heart defects in human beings. It is possible to follow nonlinear emitting marker in a remarkable field-of-view of up to 1.5 × 1.5 mm2 at 70 frame per seconds. The impact of the energy per pulse, the pulse repetition rate as well as of the photon energy on the SNR is determined and the optimum setup conditions are deduced. At the same time, wavelengths of fundamental and harmonic pulses are carefully considered and tailored to match the transmission fingerprint of the Drosophila larvae. Our findings clearly demonstrate the large impact of precise pulse parameter management in the view of the optical features of the sample, the optical setup and the photosensitivity of the detector. A step-by-step instruction for more general use of the technique is described, opening the path for addressing biological research questions that require far-field imaging at high frame rates with exceedingly high spatial and temporal precision.

Neurochemical Differences in Spinocerebellar Ataxia Type 14 and 1

Autosomal-dominant spinocerebellar ataxias (SCA) are neurodegenerative diseases characterized by progressive ataxia. Here, we report on neurometabolic alterations in spinocerebellar ataxia type 1 (SCA1; SCA-ATXN1) and 14 (SCA14; SCA-PRKCG) assessed by non-invasive 1H magnetic resonance spectroscopy. Three Tesla 1H magnetic resonance spectroscopy was performed in 17 SCA14, 14 SCA1 patients, and in 31 healthy volunteers. We assessed metabolites in the cerebellar vermis, right cerebellar hemisphere, pons, prefrontal, and motor cortex. Additionally, clinical characteristics were obtained for each patient to correlate them with metabolites. In SCA14, metabolic changes were restricted to the cerebellar vermis compared with widespread neurochemical alterations in SCA1. In SCA14, total N-acetylaspartate (tNAA) was reduced in the vermis by 34%. In SCA1, tNAA was reduced in the vermis (24%), cerebellar hemisphere (26%), and pons (25%). SCA14 patients showed 24% lower glutamate+glutamine (Glx) and 46% lower γ-aminobutyric acid (GABA) in the vermis, while SCA1 patients showed no alterations in Glx and GABA. SCA1 revealed a decrease of aspartate (Asp) in the vermis (62%) and an elevation in the prefrontal cortex (130%) as well as an elevation of myo-inositol (Ins) in the cerebellar hemisphere (51%) and pons (46%). No changes of Asp and Ins were detected in SCA14. Beyond, glucose (Glc) was increased in the vermis of both SCA14 (155%) and SCA1 (247%). 1H magnetic resonance spectroscopy revealed differing neurochemical profiles in SCA1 and SCA14 and confirmed metabolic changes that may be indicative for neuronal loss and dysfunctional energy metabolism. Therefore, 1H magnetic resonance spectroscopy represents a helpful tool for in-vivo tracking of disease-specific pathophysiology.

Reduction of Squalene Epoxidase by Cholesterol Accumulation Accelerates Colorectal Cancer Progression and Metastasis

Squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol biosynthesis, is suggested as a proto-oncogene. Paradoxically, SQLE is degraded by excess cholesterol, and low SQLE is associated with aggressive colorectal cancer (CRC). Therefore, we studied the functional consequences of SQLE reduction in CRC progression. Gene and protein expression data and clinical features of CRCs were obtained from public databases and 293 human tissues, analyzed by immunohistochemistry. Invitro studies showed underlying mechanisms of CRC progression mediated by SQLE reduction. Mice were fed a 2% high-cholesterol or a control diet before and after cecum implantation of SQLE genetic knockdown/control CRC cells. Metastatic dissemination and circulating cancer stem cells were demonstrated by invivo tracking and flow cytometry analysis, respectively. Invitro studies showed that SQLE reduction helped cancer cells overcome constraints by inducing the epithelial-mesenchymal transition required to generate cancer stem cells. Surprisingly, SQLE interacted with GSK3β and p53. Active GSK3β contributes to the stability of SQLE, thereby increasing cell cholesterol content, whereas SQLE depletion disrupted the GSK3β/p53 complex, resulting in a metastatic phenotype. This was confirmed in a spontaneous CRC metastasis mice model, where SQLE reduction, by a high-cholesterol regimen or genetic knockdown, strikingly promoted CRC aggressiveness through the production of migratory cancer stem cells. We showed that SQLE reduction caused by cholesterol accumulation aggravates CRC progression via the activation of the β-catenin oncogenic pathway and deactivation of the p53 tumor suppressor pathway. Our findings provide new insights into the link between cholesterol and CRC, identifying SQLE as a key regulator in CRC aggressiveness and a prognostic biomarker.

Abstract LB-024: Development of a non-invasive clinically applicable real time cell tracking platform for evaluating cell-based cancer therapy using magnetic resonance imaging (MRI)

Abstract While advancement in chimeric antigen receptors (CARs) has greatly improved the anti-tumor activity of immune cell-based therapies utilizing modified T lymphocytes (CAR-T) and natural killer cells (CAR-NK), there remains a critical unmet need for diagnostic technologies to help evaluate their biological performance effectively and efficiently after injecting into the body.The ability to better understand the biodistribution of injected cells and devise dosing strategies are key determinants to the success or failure of cell therapy. However, clinicians are limited by the available technology for real time in-vivo tracking of cell therapy products in clinical settings. Here we present a clinically applicable novel magnetic agent that can effectively label a variety of cell types used in cancer cell therapy development including T cells, NK cells, and neural stem cells (NSCs), and when combined with the 3D ultrashort echo time Cones (3D UTE-Cones) sequence, can enable quantitative assessment of cell therapy products using MRI. In this study, we optimized loading of our tracking agent into T cells, NK cells, and NSCs, evaluated the morphology and viability of the labeled cells, and confirmed internalization of the labeling agent by Prussian blue staining and TEM. The in vitro MRI properties of the labeled cells were quantitated by the 3D UTE-Cones sequence. Ex vivo MRI analysis was performed by injecting labeled cells into harvested mouse livers and brains to ascertain their MRI visibility in tissues using a clinical MRI scanner. We confirmed that our magnetic labeling agent efficiently labeled each cell line tested, and the morphology or viability of the labeled cells were unaffected. The MRI detectability of labeled cells were confirmed by in vitro MRI phantoms. Further, using quantitative susceptibility mapping (QSM), T1, and T2* mapping based on 3D UTE-Cones sequences, we found near perfect correlations between cell density and MRI properties. By applying the parameters established in the MRI phantoms, we were able to detect and quantity the number of labeled cells injected into mouse livers and brains ex vivo. In summary, we have developed a clinically applicable imaging-based platform that empowers cell therapy researchers to better understand the results of their biodistribution and dosing studies through real-time cell tracking and quantitative assessments using MRI. Citation Format: Johnny C. Akers, Zhao Wei, Hyungseok Jang, Eric Chang, Jiang Du, Mya S. Thu. Development of a non-invasive clinically applicable real time cell tracking platform for evaluating cell-based cancer therapy using magnetic resonance imaging (MRI) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-024.

Abstract LB-023: Dynamic real time in vivo CAR T cell tracking: Clinical and preclinical studies using a novel dual PET-MR imaging agent

Abstract Objective: The aim is to demonstrate dynamic in-vivo tracking of CAR T cell therapy for treatment of solid tumors using Cu-64 labeled superparamagnetic iron oxide nanoparticles (SPION) as novel dual PET-MR imaging agent. Methodology: Cu-64 SPION: Cu-64 radioisotope is bound to silica coated SPION using enhanced electrolysis plating techniques with tin and palladium seeding. Preclinical Model: Mouse splenic T cells were activated with anti-CD3, anti-CD28 & cultured with IL-2 and IL-7, prior to being transduced with second generation anti-Her-2 CAR (scFv-CD28-CD3ζ). 5 x 105 E0771-hHER2 breast tumor cells were implanted subcutaneously into male C57Bl/6-human HER2 transgenic mice. 107 labeled CAR T or control T cells (Cu-64 5-8 MBq) were injected into tail vein. Clinical Model: Activated T cells using antibody CD3 (OKT3) & IL-2 are transduced with retroviral vector constructs encoding for chimeric T-cell receptor specific for Lewis Y antigen. Modified T-cells are further expanded ex-vivo and reinfused. 3 x 108 CAR T cells were labeled with Cu-64 (200 - 300 MBq). Labeling of CAR T cells with Cu-64 SPION: Transfecting agent protamine sulphate facilitated cellular uptake of Cu-64 SPION within cells. Functional assays: 51Chromium release, cytometric bead array and cell viability showed that labeling process did not affect CAR T cell cytotoxicity, cytokine secretion (TNFα and IFN-γ) and viability. CAR T Cell Tracking: Scanning was performed using clinical grade dual PET-MR scanner. Preliminary Data: In this clinical trial (HREC/16/PMCC/30) patients are being enrolled for first in human in vivo study to determine how many cells distribute to solid tumor sites within first few days of CAR T cell therapy. This is first data that demonstrates that CAR-T cells can be consistently and efficiently labeled (≤60%) with cell viability (≥85%) and at sensitivity comparable to detecting at least z cells at tumor site using clinical grade dual PET-MR scanner. SUVmean values provides insight into individual response to therapy. The observed increase in SUVmax over time specifies localization of CAR T cells at tumor sites. Clinical data at early time point showed moderate uptake in lungs posterior basal segments without increased activity over next few days, thus suggesting transient process. Mild, diffuse bone marrow and relatively intense uptake in the liver and spleen suggests margination of cells to the reticulo-endothelial system. Distinct PET signal suggests localization of labeled cells in the secondary tumor sites. Little background uptake in important organs such as brain and heart indicate the safety profile of imaging agent. Absence of signal in bladder indicates hepatobiliary excretion, which may allow re-absorption from GI tract and re-circulation. Distinct PET signal within tumor in preclinical studies confirms trafficking of CAR T cells to tumor site as compared to controls. A negative contrast in the liver on T2 weighted MRI in both the preclinical and clinical studies. Preliminary Conclusion:This is first in human in vivo study to show CAR T cell distribution in real time and provides insight into individual responses of tumors to therapy. CAR T cell functionality largely remain unchanged due to labeling process. The preliminary findings indicate that labeled cells traffic to tumor sites in first few hours of infusion and remain persistent for extended period. Citation Format: Ritu Singla, Dominic Wall, Samuel Anderson, Nicholas Zia, James C. Korte, Lucy Kravets, Gerard McKiernan, Jeanne Butler, Amanda Gammilonghi, Jyoti Arora, Ben Solomon, Rodney Hicks, Timothy Cain, Phillip Darcy, Carleen Cullinane, Paul Neeson, Rajesh Ramanathan, Ravi Shukla, Vipul Bansal, Simon Harrison. Dynamic real time in vivo CAR T cell tracking: Clinical and preclinical studies using a novel dual PET-MR imaging agent [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-023.

First in-human study of in vivo imaging of ex vivo labeled CAR T cells with dual PET-MR.

3557 Background: This is a first in human in-vivo biodistribution of ex-vivo labelled CAR T cells assessed in a cohort of patients. Cells were labelled with novel Cu-64 labelled superparamagnetic iron oxide nanoparticles (SPION) and infused IV into patients with solid tumors & tracked using clinical dual PET-MR. The study validates the clinical translation of CAR T cell in-vivo tracking in real time. Methods: Cu-64 radioisotope was bound to silica coated SPION using electrolysis plating with tin & palladium seeding. Cellular uptake of Cu-64 SPION was facilitated with a transfecting agent. Functional assays including 51Chromium release, cytometric bead array demonstrated that labelling process did not affect cytotoxicity & cytokine secretion (TNFα & IFN-g). T cells were transduced with retroviral vector constructs encoding for second-generation chimeric T-cell receptor specific for carbohydrate Lewis Y antigen. Modified T-cells were expanded ex-vivo & were labelled with Cu-64 (~300 MBq) prior to re-infusion (3 x108 labelled cells). Scanning is performed with Siemens 3T dual PET-MR scanner. Results: In this first in human in-vivo study (HREC/16/PMCC/30) a cohort of patients received ex-vivo labelled CAR T cells to determine how many labelled cells distribute to solid tumor sites within 3-5 days. Our results demonstrate that cells can be efficiently labelled (≤60%) with high cell viability (≥85%) at a sensitivity sufficient to detect labelled cells at tumor site for up to 5 days. An observed trend in SUVmean & SUVmax provided insight into efficacy & individual response to therapy. Early time points showed moderate uptake of labelled cells in lungs posterior basal segments without increased activity over next few days, suggesting a transient process. Mild, diffuse bone marrow & relatively intense uptake of labelled cells in liver & spleen suggests margination of cells to reticulo-endothelial system. Distinct PET signal at some of the tumor sites at 24 h suggests antigen specific localization & time taken to reach these sites. Excretion via hepatobiliary indicated reabsorption from GI tract & re-circulation of labelled cells. Minimal uptake in brain & heart supported safety profile of labeling agent. Conclusions: This is first in human in-vivo study to provide highly valuable visual and dynamic data in real time and provides insight into individual responses to therapy. CAR T cell functionality largely remain unchanged due to labeling process. The findings indicate that labelled cells traffic to tumor sites at later time points & remain persistent for extended period of time.

First in Human Study of In-vivo Imaging of Ex-Vivo Labelled CAR T Cells with Dual PET-MR

Background & Aim This is a first in human in-vivo biodistribution of ex-vivo labelled CAR T cells assessed in a cohort of patients. Cells were labelled with novel Cu-64 labelled superparamagnetic iron oxide nanoparticles (SPION) and infused IV into patients with solid tumors & tracked using clinical dual PET-MR. The study validates the clinical translation of CAR T cell in-vivo tracking in real time. Methods, Results & Conclusion Methods Cu-64 radioisotope was bound to silica coated SPION using electrolysis plating with tin & palladium seeding. Cellular uptake of Cu-64 SPION was facilitated with a transfecting agent. Functional assays including 51Chromium release, cytometric bead array demonstrated that labelling process did not affect cytotoxicity & cytokine secretion (TNFα & IFN-g). T cells were transduced with retroviral vector constructs encoding for second-generation chimeric T-cell receptor specific for carbohydrate Lewis Y antigen. Modified T-cells were expanded ex-vivo & were labelled with Cu-64 (∼300 MBq) prior to re-infusion (3 × 108 labelled cells). Scanning is performed with Siemens 3T dual PET-MR scanner. Results In this first in human in-vivo study (HREC/16/PMCC/30) a cohort of patients received ex-vivo labelled CAR T cells to determine how many labelled cells distribute to various body organs and lesions within first 3-5 days. Our results demonstrate that both fresh (N =3) and thawed cells (N =3) can be efficiently labelled in the range of 40% - 80% with high cell viability (≥90%). An observed trend in SUVmean provided insight into efficacy & individual response to therapy. Early time points showed moderate uptake of labelled cells in lungs posterior basal segments without increased activity over next few days, suggesting a transient process. Mild, diffuse bone marrow & relatively intense uptake of labelled cells in liver & spleen suggests margination of cells to reticulo-endothelial system. Excretion via hepatobiliary indicated reabsorption from GI tract & re-circulation of labelled cells. The initial data shows that labelled cell may remain with in the system for upto 10-15 days. Minimal uptake in brain & heart supported safety profile of labeling agent. Conclusions This is first in human in-vivo study to provide highly valuable visual and dynamic data in real time and provides insight into individual responses to therapy. CAR T cell functionality largely remain unchanged due to labeling process. The findings indicate that labelled cells remain persistent for extended period of time.

Dual-modality imaging of endothelial progenitor cells transplanted after ischaemic photothrombotic stroke

AimsStroke is a refractory cerebral blood circulation disorder. Endothelial progenitor cells (EPCs) participate in the repair and regeneration of vascular injury through the combination of cell replacement and bystander effects. Here, we evaluated the biological function of EPCs in treating a mouse model of cerebral ischaemic stroke, using dual-mode bioluminescence and magnetic resonance imaging to trace EPCs in vivo. Main methodsWe constructed a viral vector with a luciferase-enhanced green fluorescent protein (Luc-eGFP) reporter gene for bioluminescence imaging (BLI) detection, and simultaneously synthesized the magnetic resonance imaging (MRI) contrast agent, nano-sized superparamagnetic iron oxide (USPIO), to co-label human umbilical cord blood-derived EPCs (hEPCs). The labelled hEPCs were transplanted into mice with stroke, and the biological behaviours of the cells in-vivo were studied using BLI and MRI, and methods of molecular biology and histology. Key findingsComparing the two cell transplantation routes by BLI confirmed that many cells transplanted via the left ventricular route homed to ischaemic brain tissue. The dual-modality-imaging showed the prognosis of in-vivo tracking cells after transplantation in ischaemic tissues at different time points. Histological staining and neurological function scores confirmed that EPC transplantation can improve the symptoms of nerve deficit in the mouse stroke model. Histological staining revealed that cell transplantation can lead to recovery of neurological function after stroke, via various processes. These include reduced blood brain barrier permeability, recovery of white matter and of myelin, and the enhancement of neuroneogenesis. SignificanceDual-modality imaging revealed EPCs as potential candidates for the treatment of ischaemic stroke.

A novel model of autosomal recessive polycystic kidney questions the role of the fibrocystin C-terminus in disease mechanism

Autosomal recessive polycystic kidney disease (OMIM 263200) is a serious condition of the kidney and liver caused by mutations in a single gene, PKHD1. This geneencodes fibrocystin/polyductin (FPC, PD1), a large protein shown by invitro studies to undergo Notch-like processing. Its cytoplasmic tail, reported to include a ciliary targeting sequence, a nuclear localization signal, and a polycystin-2 binding domain, is thought to traffic to the nucleus after cleavage. We now report a novel mouseline with a triple HA-epitope "knocked-in" to the C-terminus along with lox P sites flanking exon 67, which encodes most of the C-terminus (Pkhd1Flox67HA). The triple HA-epitope has no functional effect as assayed by phenotype and allows invivo tracking of Fibrocystin. We used the HA tag to identify previously predicted Fibrocystin cleavage products in tissue. In addition, we found that Polycystin-2 fails to co-precipitate with Fibrocystin in kidney samples. Immunofluorescence studies with anti-HA antibodies demonstrate that Fibrocystin is primarily present in a sub-apical location the in kidney, biliary duct, and pancreatic ducts, partially overlapping with the Golgi. In contrast to previous studies, the endogenous protein in the primary cilia was not detectable in mouse tissues. After Cre-mediated deletion, homozygous Pkhd1Δ67 mice are completely normal. Thus, Pkhd1Flox67HA is a valid model to track Pkhd1-derived products containing the C-terminus. Significantly, exon 67 containing the nuclear localization signal and the polycystin-2 binding domain is not essential for Fibrocystin function in our model.