Targeting CD47 with monoclonal antibody (mAb) therapy activates tumor-associated macrophages (TAMs). Quantitative imaging methods are important for identifying responders to this novel immunotherapy. The purpose of our study was to investigate whether the metabolic activity of osteosarcomas on 18F-FDG PET/CT changes after CD47 mAb treatment. Twenty female BALB/c mice with intratibial murine K7M2 osteosarcomas, twenty female NOD scid gamma (NSG) mice with intratibial human 143B tumors, and twenty male NSG mice with intratibial human MG63.3 tumors were treated with either phosphate-buffered saline (PBS) or murine/human CD47 mAb (n = 10 per arm) and underwent either 18F-FDG PET/CT or ferumoxytol-enhanced MRI (n = 5 per group). Differences in tumor metabolic activity (%ID/g max), tumor T2* relaxation times, TAM (%F4/80), and M1 macrophage polarization (%CD80+) between PBS and CD47 mAb-treated mice were estimated from linear regression. The tumor %ID/g max of CD47 mAb-treated K7M2 tumors (6.58 ± 2.42) was not significantly different compared to PBS-treated K7M2 tumors (8.04 ± 2.91; p = 0.17). Similarly, the tumor %ID/g max of CD47 mAb-treated 143B tumors (9.12 ± 1.68) and MG63.3 tumors (5.44 ± 1.99) were not significantly different compared to PBS-treated 143B tumors (9.38 ±  2.32; p = 0.32 ) and MG63.3 tumors (6.02 ±  0.63, p = 0.79 ). By comparison, K7M2 tumors, 143B tumors, and MG63.3 tumors all demonstrated significantly shorter T2* relaxation times after CD47 mAb treatment compared to PBS treatment (all p < 0.001). All tumors exhibited significantly higher TAM (%F4/80+) and M1 macrophage polarization (%CD80+) after CD47 mAb treatment compared to PBS treatment (all p < 0.05). The metabolic activity of osteosarcomas on 18F-FDG PET/CT does not show significant changes after CD47 mAb treatment. This lowers the risk of observing pseudoprogression and misinterpreting drug-induced inflammation, simplifying routine clinical scan interpretation.

B7-H4 is an inhibitory immune checkpoint molecule that is upregulated in various cancers and correlates with advanced tumor stages and poor clinical outcomes. This study aimed to develop an immunoPET radiotracer for noninvasive assessment of B7-H4 expression in tumors and tumor-associated macrophages (TAM) and to evaluate the radiotracer potential to monitor therapeutic responses. We generated a B7-H4-targeted immunoPET imaging tracer by radiolabeling the anti-B7-H4 monoclonal antibody (2H9) with [89Zr], yielding [89Zr]Zr-DFO-2H9, and assessed its biodistribution in prostate cancer xenografts to quantitatively measure B7-H4 expression in vivo. In vitro binding studies confirmed the retained immunoreactivity and specificity for B7-H4. Radiochemical purity was verified using size exclusion chromatography. In vivo evaluation of [89Zr]Zr-DFO-2H9 was first performed in immunodeficient nude mice bearing subcutaneous DU145 human prostate tumors, with longitudinal PET imaging conducted over 7 days postinjection, followed by terminal biodistribution analysis. [89Zr]Zr-DFO-2H9 demonstrated a good tumor-binding profile and specificity in DU145 tumor xenografts. To distinguish PET signals from tumor cells versus macrophages, immunocompetent C57BL/6 mice bearing syngeneic TRAMP-C2 prostate tumors were divided into three cohorts and treated with PBS (control), cold anti-B7-H4 mAb (for B7-H4 blockade), or clodronate liposome (for macrophage depletion). In TRAMP-C2 tumors, the PET signal was significantly reduced in both the B7-H4 blocked and macrophage-depleted group compared to controls. Immunohistochemistry revealed that B7-H4 expression differences among TRAMP-C2 treatment groups were not as clearly distinguishable as those observed in vivo via PET imaging. Multiplexed immunofluorescence staining of macrophage markers indicated that infiltrating TAMs were the major contributors to B7-H4-specific PET signals within the tumor stroma. Collectively, these results show that [89Zr]Zr-DFO-2H9 binds B7-H4 with high affinity and specificity and reflects changes in TAM levels in vivo. The new radiotracer shows promise for detecting B7-H4 positive tumors and TAM levels, profiling the immune microenvironment, and monitoring macrophage-targeted immunotherapies.

Antibodies (Abs) and their fragments can be labeled with PET radioisotope (immunoPET) for in vivo diagnostic imaging. Compared to the conventional FDG-PET, immunoPET can be designed to target in vivo cancer-specific antigen expression levels for various tumors and metastasis, which makes immunoPET (iPET) a powerful technique for molecular imaging and therapy monitoring. However, achieving the optimal dose to minimize radioisotope toxicity without compromising the visualization of the smallest tumor is challenging. To find an ultra-minimal tracer dose, we have developed a novel iPET with an intact rituximab Ab labeled with 64Cu to image human CD20 (hCD20) in a transgenic mouse model for non-Hodgkin’s lymphoma (NHL) imaging. Using phantom and in vivo mouse models, we optimized the minimal dose that can be administered in a mouse using a high-specific iPET tracer prepared from 64Cu-rituximab. A phantom study was used to characterize the scanner capability and limit for imaging using low doses. An ultra-minimal dose administered in a mouse model showed good image quality with high signal-to-noise ratio without compromising quantitative accuracy. The phantom study with below 50 μCi dose level indicated a slight increase in variability due to reduced dose specifically for target regions with lower uptakes (<3:1 ratio) relative to the background. In vivo study performed with four groups of mice (n = 3), each group injected with ~90, ~50, ~25, and ~10 μCi showed a linear increase of tracer uptake measured as percentage injected dose per gram (%ID/g). This tracer has shown high specific uptake in the spleen, where most B-cells are engineered to express hCD20. The study demonstrated that the lowest dose threshold limit for 64Cu-antibody-based iPET was about 25 μCi while achieving a high-quality image and quantitative accuracy.

Glioblastoma (GBM), a lethal primary adult malignancy, is difficult to treat because of the restrictive nature of the blood–brain barrier (BBB), blood-tumor barrier (BTB), and the immunosuppressive tumor microenvironment (TME). Since pulsed focused ultrasound (pFUS) is currently used to improve therapeutic deliveries across these barriers, this study aims to characterize the impact of pFUS on the TME proteomics upon opening the BBB and BTB. We utilized MRI-guided, pFUS with ultrasound contrast microbubbles (termed ‘pFUS’ herein) to selectively and transiently open the BBB and BTB investigating proteomic modifications in the TME. Utilizing an orthotopically-allografted mouse GL26 GBM model (Ccr2RFP/wt − Cx3cr1GFP/wt), pFUS’s effect on glioma proteomics was evaluated using a Luminex 48-plex assay. pFUS treated tumors exhibited increases in pro-inflammatory cytokines, chemokines, and trophic factors (CCTFs). Proteomic changes in tumors tend to peak at 24 h after single pFUS session (1x), with levels then plateauing or declining over the subsequent 24 h. Tumors receiving three pFUS sessions (3x) showed elevated CCTFs levels peaking as early as 6 h after the third session. pFUS together with microbubbles induces a sterile inflammatory response in the TME of a mouse GBM tumor. Moreover, this proinflammatory shift can be sustained and perhaps primed for more rapid responses upon multiple sessions of pFUS. These findings raise the intriguing potential that pFUS-induced BBB and BTB opening may not only be effective in facilitating the therapeutic agent delivery, but also be harnessed to modify the TME to assist immunotherapies in overcoming immune evasion in GBM.

Background: B7-H4 is a cell surface ligand overexpressed by tumors to inhibit T cell functions and evade the immune system. B7-H4 is minimally expressed in normal tissues but is highly expressed by various cancer cells and tumor-associated macrophages (TAM). Despite its importance as an immune checkpoint inhibitor, no imaging techniques specifically targeting B7-H4 have been established. To close this gap, we sought to assess the ability of a monoclonal antibody (mAb) based immunoPET radiotracer to visualize B7-H4 in human and murine prostate cancer models. Methods: Anti-B7-H4 mAb clone 2H9 was functionally characterized for binding to the human and mouse B7-H4 protein. The antibody was conjugated with chelator p-SCN-Bn-Deferoxamine (DFO) and labeled with radioisotope Zirconium-89 (89Zr) to obtain immunoPET tracer 89Zr-2H9-mAb. The biolayer interferometry method was used to test the binding kinetics of DFO-2H9-mAb compared to that of parental 2H9 mAb. A group of six athymic nude mice with human DU145 prostate tumor xenograft underwent MicroPET imaging after tail vein injection of ~150uCi 89Zr-2H9-mAb or non-binding 89Zr-Isotype-mAb. Next, immunocompetent C57BL/6J mice with TRAMP-C2 tumors each were injected with either PBS (n=8), cold 2H9 mAb (10mg/kg) to block B7-H4 (n=6), or chlodronate liposome (15mg/kg) to cause total macrophage depletion (n=6), followed by 89Zr-2H9-mAb MicroPET imaging. An ex vivo biodistribution assay was performed after 144 hr post radiotracer injection. Tumor radiotracer binding, quantified as a percentage injected dose per gram (%ID/g), was compared between different experimental groups using two-way ANOVA with Bonferroni or Tukey corrections. Results: Immunoconjugation yielded a 2.59 +/- 0.08 chelator-to-antibody ratio, and the binding of DFO conjugated 2H9-mAb was similar to that of parental 2H9 mAb, with unaffected affinity in targeting B7-H4 protein moiety. The radiochemical purity of 89Zr-2H9-mAb tracer was yielded >95% with an average specific activity of 5uCi/ug antibody. DU145 tumor xenografts demonstrated significantly stronger radiotracer binding at 24, 48, 72, 96, and 120 hr than the non-binding isotype control group. In TRAMP-C2 tumor xenografts, the radiotracer binding in B7-H4 blocked tumors was significantly lower than in the non-blocked PBS-injected group. Macrophage depletion resulted in a significant decrease in tumor binding compared to the control group. 89Zr-2H9-mAb could efficiently distinguish tumors with high sensitivity, showing a high correlation between PET imaging and bio-distribution. More importantly, the immunohistochemistry of the harvested tumor revealed no significant difference between the three groups, as discernible through in vivo PET imaging. Conclusion: This study highlights the potential of B7-H4 immunoPET imaging for monitoring immunotherapy response. With the emerging potential of B7-H4 blocking as an immunotherapeutic, immunoPET imaging could be readily expanded to patient stratification and therapy monitoring. B7-H4 imaging could augment our understanding of B7-H4 dynamics in response to various therapeutic interventions in clinical trials. The new B7-H4 immunoPET probe is, in principle, clinically translatable. ### Competing Interest Statement The authors have declared no competing interest.

The authors regret that the Acknowledgments in the article were incomplete. The full Acknowledgments are as follows: This work was supported by National Institutes of Health Grants R01 CA217953-01 and R01 NS102194. We acknowledge the core facility ISI-MR co-funded by the MEYS CR (LM2023050 Czech‐BioImaging) for the support in AIF estimation The authors would like to apologise for any inconvenience caused. Molecular Identity Changes of Tumor-Associated Macrophages and Microglia After Magnetic Resonance Imaging–Guided Focused Ultrasound–Induced Blood–Brain Barrier Opening in a Mouse Glioblastoma ModelUltrasound in Medicine and BiologyVol. 49Issue 5PreviewAn orthotopically allografted mouse GL26 glioma model (Ccr2RFP/wt–Cx3cr1GFP/wt) was used to evaluate the effect of transient, focal opening of the blood–brain barrier (BBB) on the composition of tumor-associated macrophages and microglia (TAMs). BBB opening was induced by magnetic resonance imaging (MRI)–guided focused ultrasound (MRgFUS) combined with microbubbles. CX3CR1-GFP cells and CCR2-RFP cells in brain tumors were quantified in microscopic images. Tumors in animals treated with a single session of MRgFUS did not exhibit significant changes in cell numbers when compared with tumors in animals not receiving FUS. Full-Text PDF

Dynamic contrast-enhanced MR imaging (DCE-MRI) can assess the integrity of the blood brain barrier (BBB) and has been used in GBM patients to determine glioma grade, predict prognosis, evaluate treatment response, and differentiate treatment-induced effect from recurrence. The volume transfer constant Ktrans is the most frequently used metric in tumor assessment. Based on previous studies that a higher WHO grade of brain tumor was associated with greater impairments of immunity and that Ktrans value was associated with the pathological grading, the relationship between differential composition of immune cells in GBM tissue and dynamic changes in Ktrans mapping was anticipated in this study. The present study utilized an orthotopic allograft model of GBM in which mouse GL26 cells are implanted into Ccr2RFP/wtCx3cr1GFP/wt mice on a C57 background. The brain tumors exhibited heterogenous Ktrans values with the coefficients of variation (CV) above 75%, or relatively homogeneous Ktrans maps with CV values below 50%. The Ktrans values of homogeneous tumors ranged between 0.02/min-0.32/min with a median value of 0.10/min. The immune cell composition defined by quantitative immunohistochemistry and cell sorting was compared between the tumors with Ktrans values above 0.10/min (higher Ktrans) or below 0.10/min (lower Ktrans). Histological analysis showed that tumors with higher Ktrans values exhibited greater numbers of CCR2pos cells (257.60 ± 16.42/mm2 vs 203.23 ± 12.20/mm2, p = 0.04) and an increased ratio of CCR2pos cells to CX3CR1pos cells (1.20 ± 0.02 vs 0.38 ± 0.04, p = 0.001), the numbers of CX3CR1pos cells did not differ significantly based on Ktrans values (219.70 ± 16.20/mm2 vs 250.38 ± 21.20/mm2, p = 0.19). Flowcytometry analysis showed that tumors with higher Ktrans values (above 0.1/min) were associated with greater numbers of both overall monocytes (54.93 ± 6.81% vs 29.75 ± 3.54%, p = 0.01) and inflammatory monocytes (72.38 ± 1.49% vs 59.52 ± 2.44%, p = 0.001). In contrast, tumors with lower Ktrans values (below 0.1/min) exhibited greater numbers of patrolling monocytes (75.65 ± 4.14% vs 63 ± 6.94%, p = 0.05). In the tumors with lower Ktrans values, all three types of tumor associated cells, including patrolling monocytes, inflammatory monocytes, and microglia cells possessed a higher proportion of cells at pro-inflammatory status (41.77 ± 6.13% vs 25.06 ± 6.72%, p = 0.05; 27.50 ± 2.11% vs 20.62 ± 1.87%, p = 0.03; and 55.80 ± 9.88% vs 31.12 ± 7.31%, p = 0.05), inflammatory monocytes showed fewer anti-inflammatory cells (1.25 ± 0.62% vs 3.16 ± 3.56%, p = 0.04). Taken together, differences in Ktrans values were associated with differential immune cell phenotypes and polarizations. Ktrans mapping may therefore represent a novel approach for defining the immune status of GBM.

Purpose: The aim of this study was to develop a positron emission tomography (PET) radiotracer for measuring pyruvate kinase M2 (PKM2) with improved physicochemical and pharmacokinetic properties compared to [18F] DASA-23.Experimental design: First, we synthesized [18F]DASA-10 and tested its uptake and retention compared to [18F] DASA-23 in human and mouse glioma cell lines. We then confirmed the specificity of [18F]DASA-10 by transiently modulating the expression of PKM2 in DU145 and HeLa cells. Next, we determined [18F]DASA-10 pharmacokinetics in healthy nude mice using PET imaging and subsequently assessed the ability of [18F]DASA10 versus [18F]DASA-23 to enable in vivo detection of intracranial gliomas in syngeneic C6 rat models of glioma. Results: [18F]DASA-10 demonstrated excellent cellular uptake and retention with values significantly higher than [18F]DASA-23 in all cell lines and timepoints investigated. [18F]DASA-10 showed a 73 % and 65 % reduced uptake respectively in DU145 and HeLa cells treated with PKM2 siRNA as compared to control siRNA treated cells. [18F]DASA-10 showed favorable biodistribution and pharmacokinetic properties and a significantly improved tumor-to-brain ratio in rat C6 glioma models relative to [18F]DASA-23 (3.2 & PLUSMN; 0.8 versus 1.6 & PLUSMN; 0.3, p = 0.01).Conclusion: [18F]DASA-10 is a new PET radiotracer for molecular imaging of PKM2 with potential to overcome the prior limitations observed with [18F]DASA-23. [18F]DASA-10 shows promise for clinical translation to enable imaging of brain malignancies owing to its low background signal in the healthy brain.

MicroRNAs are critical regulators of cancer initiation, progression, and dissemination. Extensive evidence suggests that the inhibition of over-expressed oncogenic miRNA function can be a robust strategy for anticancer therapy. However, in vivo targeted delivery of miRNA therapeutics to various types of cancers remains a major challenge. Inspired by their natural synthesis and cargo delivery capabilities, researchers have exploited tumor cell-derived extracellular vesicles (TEVs) for the cancer-targeted delivery of therapeutics and theranostics. Here, we investigate a TEV-based nanoplatform for multimodal miRNA delivery and phototherapy treatments as well as the magnetic resonance imaging of cancer. We demonstrated loading of anti-miR-21 that blocks the function of endogenous oncogenic miR-21 over-expressed in cancer cells into and subsequent delivery by TEVs derived from 4T1 cells. We also produced Cy5-anti-miR-21-loaded TEVs from two other cancer cell lines (HepG2 and SKBR3) and confirmed their robust homologous and heterologous transfection efficiency and intracellular Cy5-anti-miR-21 delivery. Additionally, TEV-mediated anti-miR-21 delivery attenuated doxorubicin (DOX) resistance in breast cancer cells with a 3-fold higher cell kill efficiency than in cells treated with DOX alone. We then investigated TEVs as a biomimetic source for the functionalization of gold iron oxide nanoparticles (GIONs) and demonstrated nanotheranostic properties of TEV-GIONs in vitro. TEV-GIONs demonstrated excellent T2 contrast in in vitro magnetic resonance (MR) imaging and resulted in efficient photothermal effect in 4T1 cells. We also evaluated the biodistribution and theranostic property of anti-miR-21 loaded TEV-GIONs in vivo by labeling with indocyanine green near-infrared dye. We further validated the tumor specific accumulation of TEV-GIONs using MR imaging. Our findings demonstrate that the distribution pattern of the TEV-anti-miR-21-GIONs correlated well with the tumor-targeting capability as well as the activity and efficacy obtained in response to doxorubicin combination treatments. TEVs and TEV-GIONs are promising nanotheranostics for future applications in cancer molecular imaging and therapy.

Chronic innate immune activation is a key hallmark of many neurological diseases and is known to result in the upregulation of GPR84 in myeloid cells (macrophages, microglia, and monocytes). As such, GPR84 can potentially serve as a sensor of proinflammatory innate immune responses. To assess the utility of GPR84 as an imaging biomarker, we synthesized 11C-MGX-10S and 11C-MGX-11Svia carbon-11 alkylation for use as positron emission tomography (PET) tracers targeting this receptor. In vitro experiments demonstrated significantly higher binding of both radiotracers to hGPR84-HEK293 cells than that of parental control HEK293 cells. Co-incubation with the GPR84 antagonist GLPG1205 reduced the binding of both radiotracers by >90%, demonstrating their high specificity for GPR84 in vitro. In vivo assessment of each radiotracer via PET imaging of healthy mice illustrated the superior brain uptake and pharmacokinetics of 11C-MGX-10S compared to 11C-MGX-11S. Subsequent use of 11C-MGX-10S to image a well-established mouse model of systemic and neuro-inflammation revealed a high PET signal in affected tissues, including the brain, liver, lung, and spleen. In vivo specificity of 11C-MGX-10S for GPR84 was confirmed by the administration of GLPG1205 followed by radiotracer injection. When compared with 11C-DPA-713-an existing radiotracer used to image innate immune activation in clinical research studies-11C-MGX-10S has multiple advantages, including its higher binding signal in inflamed tissues in the CNS and periphery and low background signal in healthy saline-treated subjects. The pronounced uptake of 11C-MGX-10S during inflammation, its high specificity for GPR84, and suitable pharmacokinetics strongly support further investigation of 11C-MGX-10S for imaging GPR84-positive myeloid cells associated with innate immune activation in animal models of inflammatory diseases and human neuropathology.

An orthotopically allografted mouse GL26 glioma model (Ccr2RFP/wt-Cx3cr1GFP/wt) was used to evaluate the effect of transient, focal opening of the blood-brain barrier (BBB) on the composition of tumor-associated macrophages and microglia (TAMs). BBB opening was induced by magnetic resonance imaging (MRI)-guided focused ultrasound (MRgFUS) combined with microbubbles. CX3CR1-GFP cells and CCR2-RFP cells in brain tumors were quantified in microscopic images. Tumors in animals treated with a single session of MRgFUS did not exhibit significant changes in cell numbers when compared with tumors in animals not receiving FUS. However, tumors that received two or three sessions of MRgFUS had significantly increased amounts of both CX3CR1-GFP and CCR2-RFP cells. The effect of MRgFUS on immune cell composition was also characterized and quantified using flow cytometry. Glioma implantation resulted in increased amounts of lymphocytes, monocytes and neutrophils in the brain parenchyma. Tumors administered MRgFUS exhibited increased numbers of monocytes and monocyte-derived TAMs. In addition, MRgFUS-treated tumors exhibited more CD80+ cells in monocytes and microglia. In summary, transient, focal opening of the BBB using MRgFUS combined with microbubbles can activate the homing and differentiation of monocytes and induce a shift toward a more pro-inflammatory status of the immune environment in glioblastoma.

Cellular senescence has been implicated in age-related pathophysiologies, including osteoarthritis (OA). The targeted removal of senescent cells can ameliorate the development of OA. To advance our understanding of the role of senescent cells in OA and monitor novel senolytic therapies, there is a pressing need for imaging biomarkers that can detect senescence in arthritic joints. Senescent cells overexpress β-galactosidase. Our objective was to detect senescent cells in knee joints of small (mice) and large (pigs) animal models using a novel β-galactosidase-based radiotracer, [18F]-PyGal, using PET imaging. We hypothesized that senescent cells will demonstrate increased [18F]-PyGal radiotracer uptake compared to viable cells, in vitro and in vivo. Triplicate samples of murine primary chondrocytes incubated with 400 nM doxorubicin (doxo) to induce senescence, followed by incubation with 20 µM [18F]-PyGal radiotracer for one hour and PET-CT imaging. Non-doxo-exposed cells served as controls. Next, senescence was induced in the left knee joint of 30 C57BL/6 mice (5, 12, and 23-month-old mice, both sexes) by intra-articular doxorubicin injection. The contralateral knee served as control. All knees underwent PET/CT and PET/MRI imaging at 1 hour after intravenous injection of 250uCi [18F]-PyGal. The radiotracer signal of senescent and viable joints was measured using operator defined regions of interest (ROI; %ID/g) and compared with a student's t-test. Next, senescent mesenchymal stromal cells (MSC) or viable controls were implanted into twelve cartilage defects of six knees of three Yucatan pigs. 24 hours later, 250uCi [18F]-PyGal was injected into the knee joint, followed by integrated PET-MRI. The standardized uptake value (SUV) of senescent and viable cell implants was compared with a t-test. IL-6, CXCL5 and β-galactosidase immunostains served as reference standards for all experiments. Compared to untreated controls, doxorubicin-exposed chondrocytes demonstrated significantly increased expression of IL6 (Control: 18.39±5.10 pg/mL; senescence 58.85±1.43pg/mL, p<0.005), CXCL5 (Control 153.8162±38.14pg/mL, senescence:1163.24±169.09pg/mL p<0.005) and beta-gal (Control 9.16±0.7%, Senescence 90±2%, p<0.0005). Senescent cells exhibited a 2.28-fold higher uptake of [18F]-PyGal compared to controls (Control 1754±247.22, Senescence 3704.23±570.76, p=0.005). In 12-month-old mice, senescent knees demonstrated significantly higher retention of [18F]-PyGal (0.47±0.18 %ID/g, 0.47±0.12 %ID/g), compared to control knees (0.22±0.03%ID/g, 0.29±0.08, p=0.02, p=0.03 female and male respectively). Accordingly senescent knees demonstrated significantly increased p-16 staining, p-21 staining, and β-gal staining compared to normal controls. In the large animal model, cartilage defects implanted with senescent cells showed significantly higher [18F]-PyGal uptake (39.835±7.3 SUVmax) compared to control knees (19.69±0.69 SUVmax, p<0.05). Histopathologic correlations of large animal studies are ongoing. [18F]-PyGal can detect senescent cells in knee joints of small (mice) and large (pigs) animal models using a novel β-galactosidase-based radiotracer, [18F]-PyGal and PET imaging. Senescent cells demonstrated significantly increased [18F]-PyGal radiotracer uptake compared to viable cells, in vitro and in vivo, in small and large animal models of OA.

Imaging has become an invaluable tool in preclinical research for its capability to non-invasively detect and monitor disease and assess treatment response. With the increased use of preclinical imaging, large volumes of image data are being generated requiring critical data management tools. Due to proprietary issues and continuous technology development, preclinical images, unlike DICOM-based images, are often stored in an unstructured data file in company-specific proprietary formats. This limits the available DICOM-based image management database to be effectively used for preclinical applications. A centralized image registry and management tool is essential for advances in preclinical imaging research. Specifically, such tools may have a high impact in generating large image datasets for the evolving artificial intelligence applications and performing retrospective analyses of previously acquired images. In this study, a web-based server application is developed to address some of these issues. The application is designed to reflect the actual experimentation workflow maintaining detailed records of both individual images and experimental data relevant to specific studies and/or projects. The application also includes a web-based 3D/4D image viewer to easily and quickly view and evaluate images. This paper briefly describes the initial implementation of the web-based application.

The development of gene delivery vehicles with high organ specificity when administered systemically is a critical goal for gene therapy. We combine optical and positron emission tomography (PET) imaging of 1) reporter genes and 2) capsid tags to assess the temporal and spatial distribution and transduction of adeno-associated viruses (AAVs). AAV9 and two engineered AAV vectors (PHP.eB and CAP-B10) that are noteworthy for maximizing blood-brain barrier transport were compared. CAP-B10 shares a modification in the 588 loop with PHP.eB, but also has a modification in the 455 loop, added with the goal of reducing off-target transduction. PET and optical imaging revealed that the additional modifications retained brain receptor affinity. In the liver, the accumulation of AAV9 and the engineered AAV capsids was similar (∼15% of the injected dose per cc and not significantly different between capsids at 21 h). However, the engineered capsids were primarily internalized by Kupffer cells rather than hepatocytes, and liver transduction was greatly reduced. PET reporter gene imaging after engineered AAV systemic injection provided a non-invasive method to monitor AAV-mediated protein expression over time. Through comparison with capsid tagging, differences between brain localization and transduction were revealed. In summary, AAV capsids bearing imaging tags and reporter gene payloads create a unique and powerful platform to assay the pharmacokinetics, cellular specificity and protein expression kinetics of AAV vectors in vivo, a key enabler for the field of gene therapy.

Surgery can be highly effective for treating certain cases of drug resistant epilepsy. The current study tested a novel, non-invasive, surgical strategy for treating seizures in a rat model of temporal lobe epilepsy. The surgical approach uses magnetic resonance-guided, low-intensity focused ultrasound (MRgFUS) in combination with intravenous microbubbles to open the blood-brain barrier (BBB) in a transient and focal manner. During the period of BBB opening, a systemically administered neurotoxin (Quinolinic Acid: QA) that is normally impermeable to the BBB gains access to a targeted area in the brain, destroying neurons where the BBB has been opened. This strategy is termed Precise Intracerebral Non-invasive Guided Surgery (PING). Spontaneous recurrent seizures induced by pilocarpine were monitored behaviorally prior to and after PING or under control conditions. Seizure frequency in untreated animals or animals treated with MRgFUS without QA exhibited expected seizure rate fluctuations frequencies between the monitoring periods. In contrast, animals treated with PING targeting the intermediate-temporal aspect of the hippocampus exhibited substantial reductions in seizure frequency, with convulsive seizures being eliminated entirely in two animals. These findings suggest that PING could provide a useful alternative to invasive surgical interventions for treating drug resistant epilepsy, and perhaps for treating other neurological disorders in which aberrant neural circuitries play a role.

Polymeric nanocarriers (PNCs) can be used to deliver therapeutic microRNAs (miRNAs) to solid cancers. However, the ability of these nanocarriers to specifically target tumors remains a challenge. Alternatively, extracellular vesicles (EVs) derived from tumor cells show homotypic affinity to parent cells, but loading sufficient amounts of miRNAs into EVs is difficult. Here, it is investigated whether uPAR-targeted delivery of nanococktails containing PNCs loaded with therapeutic antimiRNAs, and coated with uPA engineered extracellular vesicles (uPA-eEVs) can elicit synergistic antitumor responses. The uPA-eEVs coating on PNCs increases natural tumor targeting affinities, thereby enhancing the antitumor activity of antimiRNA nanococktails. The systemic administration of uPA-eEV-PNCs nanococktail shows a robust tumor tropism, which significantly enhances the combinational antitumor effects of antimiRNA-21 and antimiRNA-10b, and leads to significant tumor regression and extension of progression free survival for syngeneic 4T1 tumor-bearing mice. In addition, the uPA-eEV-PNCs-antimiRNAs nanococktail plus low dose doxorubicin results in a synergistic antitumor effect as evidenced by inhibition of tumor growth, reduction of lung metastases, and extension of survival of 4T1 tumor-bearing mice. The targeted combinational nanococktail strategy could be readily translated to the clinical setting by using autologous cancer cells that have flexibility for ex vivo expansion and genetic engineering.

Advanced multipurpose cell imaging systems along with integrated rapid quantitation software can enhance and expedite cancer cell culture studies in a variety of applications. Though accurate cell culture studies are an important and necessary component of nearly all cancer biomarker detection and therapy studies, the methods we currently use are of low-throughput, time consuming, and lack accuracy. Hence, it is important to improve several features of the assays to increase the accuracy of their quantitative outputs in most studies. In general, we perform cell culture analysis semimanually by counting a small aliquot of suspended cells using a hemocytometer or viewing a small area of cells on a plate using a bright-field microscope, and then extrapolate the counts or observations to estimate the values for the total numbers of cells. The fundamental problem with this process lies in using techniques, such as extrapolation, which inherently introduces intrasample variability while collecting the cells by enzymatic trypsinization for these assays that are affecting cell growth and other downstream assessments. Fluorescence (FL) microscopy-based assays are also used to image and count cells for various applications, including cell viability, proliferation, apoptosis, cell death, transfection efficiency, protein expression, stem cell properties, colony formation, cytotoxicity, drug dose-response, and treatment efficacy studies. These methods are not optimal for many researches, as they require real-time visualization under a microscope plus manual analysis to determine the final results. Owing to long exposure times for cells under fluorescent light of a microscope, the cells may be exposed to suboptimal conditions that affect cell growth, and with occasional photobleaching of the expressed FL probes. Alternatively, the use of cell imaging systems that integrate both advanced bright-field and FL imaging for cell counting and quantification can be useful. In this protocol, we discuss the advantages of a high-throughput cell imaging system using a whole-plate imaging format when used in various bioimaging studies by highlighting a few applications of the system. The system is designed to fundamentally improve the accuracy and time of cell culture analysis while also allowing us to perform the assay without trypsinization, thus avoiding the need to replicate multiple wells for monitoring cell growth over time.