Increased proliferation of late-born retinal progenitor cells by gestational lead exposure delays rod and bipolar cell differentiation.

PURPOSE: Studies of neuronal development in the retina often examine the stages of proliferation, differentiation, and synaptic development, albeit independently. Our goal was to determine if a known neurotoxicant insult to a population of retinal progenitor cells (RPCs) would affect their eventual differentiation and synaptic development. To that end, we used our previously published human equivalent murine model of low-level gestational lead exposure (GLE). Children and animals with GLE exhibit increased scotopic electroretinogram a- and b-waves. Adult mice with GLE exhibit an increased number of late-born RPCs, a prolonged period of RPC proliferation, and an increased number of late-born rod photoreceptors and rod and cone bipolar cells (BCs), with no change in the number of late-born Müller glial cells or early-born neurons. The specific aims of this study were to determine whether increased and prolonged RPC proliferation alters the spatiotemporal differentiation and synaptic development of rods and BCs in early postnatal GLE retinas compared to control retinas. METHODS: C57BL/6N mouse pups were exposed to lead acetate via drinking water throughout gestation and until postnatal day 10, which is equivalent to the human gestation period for retinal neurogenesis. RT-qPCR, immunohistochemical analysis, and western blots of well-characterized, cell-specific genes and proteins were performed at embryonic and early postnatal ages to assess rod and cone photoreceptor differentiation, rod and BC differentiation and synaptic development, and Müller glial cell differentiation. RESULTS: Real-time quantitative PCR (RT-qPCR) with the rod-specific transcription factors Nrl, Nr2e3, and Crx and the rod-specific functional gene Rho, along with central retinal confocal studies with anti-recoverin and anti-rhodopsin antibodies, revealed a two-day delay in the differentiation of rod photoreceptors in GLE retinas. Rhodopsin immunoblots supported this conclusion. No changes in glutamine synthetase gene or protein expression, a marker for late-born Müller glial cells, were observed in the developing retinas. In the retinas from the GLE mice, anti-PKCα, -Chx10 (Vsx2) and -secretagogin antibodies revealed a two- to three-day delay in the differentiation of rod and cone BCs, whereas the expression of the proneural and BC genes Otx2 and Chx10, respectively, increased. In addition, confocal studies of proteins associated with functional synapses (e.g., vesicular glutamate transporter 1 [VGluT1], plasma membrane calcium ATPase [PMCA], transient receptor potential channel M1 [TRPM1], and synaptic vesicle glycoprotein 2B [SV2B]) revealed a two-day delay in the formation of the outer and inner plexiform layers of the GLE retinas. Moreover, several markers revealed that the initiation of the differentiation and intensity of the labeling of early-born cells in the retinal ganglion cell and inner plexiform layers were not different in the control retinas. CONCLUSIONS: Our combined gene, confocal, and immunoblot findings revealed that the onset of rod and BC differentiation and their subsequent synaptic development is delayed by two to three days in GLE retinas. These results suggest that perturbations during the early proliferative stages of late-born RPCs fated to be rods and BCs ultimately alter the coordinated time-dependent progression of rod and BC differentiation and synaptic development. These GLE effects were selective for late-born neurons. Although the molecular mechanisms are unknown, alterations in soluble neurotrophic factors and/or their receptors are likely to play a role. Since neurodevelopmental delays and altered synaptic connectivity are associated with neuropsychiatric and behavioral disorders as well as cognitive deficits, future work is needed to determine if similar effects occur in the brains of GLE mice and whether children with GLE experience similar delays in retinal and brain neuronal differentiation and synaptic development.

The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases.

PURPOSE: The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. METHODS: mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. RESULTS: The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor inner segments. The combined results indicate that glycolysis is regulated by the compartmental expression of hexokinase 2, pyruvate kinase M1, and pyruvate kinase M2 in photoreceptors, whereas the inner retinal neurons exhibit a lower capacity for glycolysis and aerobic glycolysis. Expression of nucleoside diphosphate kinase, mitochondria-associated adenylate kinase, and several mitochondria-associated creatine kinase isozymes was highest in the outer retina, whereas expression of cytosolic adenylate kinase and brain creatine kinase was higher in the cones, horizontal cells, and amacrine cells indicating the diversity of ATP-buffering strategies among retinal neurons. Based on the antibody intensities and the COX and LDH activity, Müller glial cells (MGCs) had the lowest capacity for glycolysis, aerobic glycolysis, and OXPHOS. However, they showed high expression of glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate thiokinase, GABA transaminase, and ~P transferring kinases. This suggests that MGCs utilize TCA cycle anaplerosis and cataplerosis to generate GTP and ~P transferring kinases to produce ATP that supports MGC energy requirements. CONCLUSIONS: Our comprehensive and integrated results reveal that the adult mouse retina expresses numerous isoforms of ATP synthesizing, regulating, and buffering genes; expresses differential cellular and compartmental levels of glycolytic, OXPHOS, TCA cycle, and ~P transferring kinase proteins; and exhibits differential layer-by-layer LDH and COX activity. New insights into cell-specific and compartmental ATP and GTP production, as well as utilization and buffering strategies and their relationship with known retinal and cellular functions, are discussed. Developing therapeutic strategies for neuroprotection and treating retinal deficits and degeneration in a cell-specific manner will require such knowledge. This work provides a platform for future research directed at identifying the molecular targets and proteins that regulate these processes.

Axitinib inhibits retinal and choroidal neovascularization in in vitro and in vivo models.

Age-related Macular Degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly in developed countries. Neovascular/exudative (wet) AMD is the aggressive form of AMD and can involve choroidal neovascularization and vascular leakage. Anti-vascular endothelial growth factor (anti-VEGF) medications have significantly improved treatment of wet-AMD. However, only approximately 40% of patients obtain full benefit from anti-VEGF therapy and the medications are given by intravitreal injection. Axitinib, a small molecule multi-receptor tyrosine kinase inhibitor used for the treatment of advanced renal cell carcinoma, is taken orally and inhibits VEGF activity by blocking VEGF receptors. Axitinib also has the advantage of blocking platelet derived growth factor (PDGF) receptors which play a role in neovascularization. Using in vitro human retinal microvascular endothelial cells (HRMVECs), human brain vascular pericytes (HBVRs), 3D co-culture vessel sprout assay, and in vivo laser induced rat choroidal neovascularization (CNV) models, the effect of axitinib on neovascularization was evaluated. Axitinib inhibited neovascularization better than anti-VEGF and/or anti-hPDGF-B mAb in the in vitro models demonstrating that combined inhibition of both VEGF and PDGF pathways may be synergistic in treating wet-AMD. Additionally, axitinib showed good efficacy at a low dose (0.875 mg/day) in laser-induced CNV model in rats. In conclusion our data shows that axitinib, an inhibitor of VEGF and PDGF-B pathways may be useful in ameliorating wet-AMD therapy.

Axitinib and crizotinib combination therapy inhibits bone loss in a mouse model of castration resistant prostate cancer.

BACKGROUND: Castration resistant prostate cancer (CRPC) is a leading cause of cancer-related deaths in men. The primary cause of mortality and morbidity in patients is bone metastases and remodeling resulting in osteoblastic and osteolytic lesions. Recently, cabozantinib, a multi-kinase inhibitor (VEGFR2 and c-MET inhibitor), was shown to have efficacy on bone lesions in patients. In this study we tested multi-kinase inhibitors: axitinib (VEGFR inhibitor) and crizotinib (c-MET inhibitor) in a combination trial in mice models. METHODS: VCaP-Luc cells were grown as subcutaneous implants in intact and castrated NOD-SCID-gamma (NSG) mice to confirm the androgen dependency. For bone metastasis model two cohorts of NSG mice (castrated and intact) received orthotopic injection of VCaP-Luc cells into the bone marrow cavity of left tibia. Mice were monitored weekly for tumor growth using bioluminescence imaging. Animals were randomized into 4 groups based on the tumor bioluminescence signal: vehicle, crizotinib alone, axitinib alone, crizotinib and axitinib in combination. Animals were imaged weekly by in vivo 2-D X-ray imaging to monitor bone remodeling. At the end of the study animals were euthanized and both tibias were extracted for ex vivo high-resolution 3-D micro-computed tomography (µCT) imaging. RESULTS: Subcutaneous model showed that androgen stimulation may be helpful but not essential for the growth of VCaP-Luc cells. VCaP-Luc cells grown intra-tibially in intact animals caused extensive remodeling of bone with mixed osteoblastic (bone formation) and osteolytic (bone matrix dissolution) lesions. The osteoblastic lesions were predominant and at times extended beyond the tibial shaft into the surrounding tissue. In contrast, only osteolytic lesions were prominent throughout the study in castrated animals. Treatment with crizotinib alone reduced the osteolytic lesions in castrated animals. Axitinib alone reduced the osteoblastic lesions in the intact animals. Combination therapy with axitinib and crizotinib remarkably inhibited the tibial remodeling by VCaP-Luc cells which resulted in a significant reduction of both osteoblastic and osteolytic lesions. CONCLUSION: Our data show that combined inhibition of c-MET and VEGFR can be beneficial for treatment of metastatic bone disease in CRPC and that the drugs act on two different stages of the disease.

Applying deep learning to segmentation of murine lung tumors in pre-clinical micro-computed tomography.

Lung cancer remains a leading cause of cancer-related death, but scientists have made great strides in developing new treatments recently, partly owing to the use of genetically engineered mouse models (GEMMs). GEMM tumors represent a translational model that recapitulates human disease better than implanted models because tumors develop spontaneously in the lungs. However, detection of these tumors relies on in vivo imaging tools, specifically micro-Computed Tomography (micro-CT or µCT), and image analysis can be laborious with high inter-user variability. Here we present a deep learning model trained to perform fully automated segmentation of lung tumors without the interference of other soft tissues. Trained and tested on 100 3D µCT images (18,662 slices) that were manually segmented, the model demonstrated a high correlation to manual segmentations on the testing data (r2=0.99, DSC=0.78) and on an independent dataset (n = 12 3D scans or 2328 2D slices, r2=0.97, DSC=0.73). In a comparison against manual segmentation performed by multiple analysts, the model (r2=0.98, DSC=0.78) performed within inter-reader variability (r2=0.79, DSC=0.69) and close to intra-reader variability (r2=0.99, DSC=0.82), all while completing 5+ hours of manual segmentations in 1 minute. Finally, when applied to a real-world longitudinal study (n = 55 mice), the model successfully detected tumor progression over time and the differences in tumor burden between groups induced with different virus titers, aligning well with more traditional analysis methods. In conclusion, we have developed a deep learning model which can perform fast, accurate, and fully automated segmentation of µCT scans of murine lung tumors.

Type I interferon regulation by USP18 is a key vulnerability in cancer.

Precise regulation of Type I interferon signaling is crucial for combating infection and cancer while avoiding autoimmunity. Type I interferon signaling is negatively regulated by USP18. USP18 cleaves ISG15, an interferon-induced ubiquitin-like modification, via its canonical catalytic function, and inhibits Type I interferon receptor activity through its scaffold role. USP18 loss-of-function dramatically impacts immune regulation, pathogen susceptibility, and tumor growth. However, prior studies have reached conflicting conclusions regarding the relative importance of catalytic versus scaffold function. Here, we develop biochemical and cellular methods to systematically define the physiological role of USP18. By comparing a patient-derived mutation impairing scaffold function (I60N) to a mutation disrupting catalytic activity (C64S), we demonstrate that scaffold function is critical for cancer cell vulnerability to Type I interferon. Surprisingly, we discovered that human USP18 exhibits minimal catalytic activity, in stark contrast to mouse USP18. These findings resolve human USP18's mechanism-of-action and enable USP18-targeted therapeutics.

Volumetric imaging of optically cleared and fluorescently labeled animal tissue (VIOLA) for quantifying the 3D biodistribution of nanoparticles at cellular resolution in tumor tissue.

Nanoparticle (NP) technology holds significant promise to mediate targeted drug delivery to specific organs in the body. Understanding the 3D biodistribution of NPs in heterogeneous environments such as the tumor tissue can provide crucial information on efficacy, safety and potential clinical outcomes. Here we present a novel end-to-end workflow, VIOLA, which makes use of tissue clearing methodology in conjunction with high resolution imaging and advanced 3D image processing to quantify the spatiotemporal 3D biodistribution of fluorescently labeled ACCURIN® NPs. Specifically, we investigate the spatiotemporal biodistribution of NPs in three different murine tumor models (CT26, EMT6, and KPC-GEM) of increasing complexity and translational relevance. We have developed new endpoints to characterize NP biodistribution at multiple length scales. Our observations reveal that the macroscale NP biodistribution is spatially heterogeneous and exhibits a gradient with relatively high accumulation at the tumor periphery that progressively decreases towards the tumor core in all the tumor models. Microscale analysis revealed that NP extravasation from blood vessels increases in a time dependent manner and plateaus at 72 h post injection. Volumetric analysis and pharmacokinetic modeling of NP biodistribution in the vicinity of the blood vessels revealed that the local NP density exhibits a distance dependent spatiotemporal biodistribution which provide insights into the dynamics of NP extravasation in the tumor tissue. Our data represents a comprehensive analysis of NP biodistribution at multiple length scales in different tumor models providing unique insights into their spatiotemporal dynamics. Specifically, our results show that NPs exhibit a dynamic equilibrium with macroscale heterogeneity combined with microscale homogeneity.

Efficacy and Imaging-Enabled Pharmacodynamic Profiling of KRAS G12C Inhibitors in Xenograft and Genetically Engineered Mouse Models of Cancer.

KRAS is one of the most commonly mutated oncogenes in lung, colorectal, and pancreatic cancers. Recent clinical trials directly targeting KRAS G12C presented encouraging results for a large population of non-small cell lung cancer (NSCLC), but resistance to treatment is a concern. Continued exploration of new inhibitors and preclinical models is needed to address resistance mechanisms and improve duration of patient responses. To further enable the development of KRAS G12C inhibitors, we present a preclinical framework involving translational, non-invasive imaging modalities (CT and PET) and histopathology in a conventional xenograft model and a novel KRAS G12C knock-in mouse model of NSCLC. We utilized an in-house developed KRAS G12C inhibitor (Compound A) as a tool to demonstrate the value of this framework in studying in vivo pharmacokinetic/pharmacodynamic (PK/PD) relationship and anti-tumor efficacy. We characterized the Kras G12C-driven genetically engineered mouse model (GEMM) and identify tumor growth and signaling differences compared to its Kras G12D-driven counterpart. We also find that Compound A has comparable efficacy to sotorasib in the Kras G12C-driven lung tumors arising in the GEMM, but like observations in the clinic, some tumors inevitably progress on treatment. These findings establish a foundation for evaluating future KRAS G12C inhibitors that is not limited to xenograft studies and can be applied in a translationally relevant mouse model that mirrors human disease progression and resistance.

Mouse lung automated segmentation tool for quantifying lung tumors after micro-computed tomography.

Unlike the majority of cancers, survival for lung cancer has not shown much improvement since the early 1970s and survival rates remain low. Genetically engineered mice tumor models are of high translational relevance as we can generate tissue specific mutations which are observed in lung cancer patients. Since these tumors cannot be detected and quantified by traditional methods, we use micro-computed tomography imaging for longitudinal evaluation and to measure response to therapy. Conventionally, we analyze microCT images of lung cancer via a manual segmentation. Manual segmentation is time-consuming and sensitive to intra- and inter-analyst variation. To overcome the limitations of manual segmentation, we set out to develop a fully-automated alternative, the Mouse Lung Automated Segmentation Tool (MLAST). MLAST locates the thoracic region of interest, thresholds and categorizes the lung field into three tissue categories: soft tissue, intermediate, and lung. An increase in the tumor burden was measured by a decrease in lung volume with a simultaneous increase in soft and intermediate tissue quantities. MLAST segmentation was validated against three methods: manual scoring, manual segmentation, and histology. MLAST was applied in an efficacy trial using a Kras/Lkb1 non-small cell lung cancer model and demonstrated adequate precision and sensitivity in quantifying tumor growth inhibition after drug treatment. Implementation of MLAST has considerably accelerated the microCT data analysis, allowing for larger study sizes and mid-study readouts. This study illustrates how automated image analysis tools for large datasets can be used in preclinical imaging to deliver high throughput and quantitative results.

Automated monitoring of respiratory rate as a novel humane endpoint: A refinement in mouse metastatic lung cancer models.

In oncology research, while xenograft tumor models are easily visualized and humane endpoints can be clearly defined, metastatic tumor models are often based on more subjective clinical observations as endpoints. This study aimed at identifying objective non-invasive criteria for predicting imminent distress and mortality in metastatic lung tumor-bearing mice. BALB/c and C57BL/6 mice were inoculated with CT26 or B16F10 cells, respectively. The mice were housed in Vium smart cages to continuously monitor and stream respiratory rate and locomotion for up to 28 days until scheduled euthanasia or humane endpoint criteria were met. Body weight and body temperature were measured during the study. On days 11, 14, 17 and 28, lungs of subsets of animals were microCT imaged in vivo to assess lung metastasis progression and then euthanized for lung microscopic evaluations. Beginning at day 21, most tumor-bearing animals developed increased respiratory rates followed by decreased locomotion 1-2 days later, compared with the baseline values. Increases in respiratory rate did not correlate to surface tumor nodule counts or lung weight. Body weight measurement did not show significant changes from days 14-28 in either tumor-bearing or control animals. We propose that increases in respiratory rate (1.3-1.5 X) can be used to provide an objective benchmark to signal the need for increased clinical observations or euthanasia. Adoption of this novel humane endpoint criterion would allow investigators time to collect tissue samples prior to spontaneous morbidity or death and significantly reduce the distress of mice in the terminal stages of these metastatic lung tumor models.

Targeting and pharmacology of an anti-IL13Rα2 antibody and antibody-drug conjugate in a melanoma xenograft model.

IL13Rα2 is a cell surface tumor antigen that is overexpressed in multiple tumor types. Here, we studied biodistribution and targeting potential of an anti-IL13Rα2 antibody (Ab) and anti-tumor activity of anti-IL13Rα2-antibody-drug conjugate (ADC). The anti-IL13Rα2 Ab was labeled with fluorophore AF680 or radioisotope 89Zr for in vivo tracking using fluorescence molecular tomography (FMT) or positron emission tomography (PET) imaging, respectively. Both imaging modalities showed that the tumor was the major uptake site for anti-IL13Rα2-Ab, with peak uptake of 5-8% ID and 10% ID/g as quantified from FMT and PET, respectively. Pharmacological in vivo competition with excess of unlabeled anti-IL13Rα2-Ab significantly reduced the tumor uptake, indicative of antigen-specific tumor accumulation. Further, FMT imaging demonstrated similar biodistribution and pharmacokinetic profiles of an auristatin-conjugated anti-IL13Rα2-ADC as compared to the parental Ab. Finally, the anti-IL13Rα2-ADC exhibited a dose-dependent anti-tumor effect on A375 xenografts, with 90% complete responders at a dose of 3 mg/kg. Taken together, both FMT and PET showed a favorable biodistribution profile for anti-IL13Rα2-Ab/ADC, along with antigen-specific tumor targeting and excellent therapeutic efficacy in the A375 xenograft model. This work shows the great potential of this anti-IL13Rα2-ADC as a targeted anti-cancer agent.

Preclinical Evaluation of 89Zr-Df-IAB22M2C PET as an Imaging Biomarker for the Development of the GUCY2C-CD3 Bispecific PF-07062119 as a T Cell Engaging Therapy.

PURPOSE: A sensitive and specific imaging biomarker to monitor immune activation and quantify pharmacodynamic responses would be useful for development of immunomodulating anti-cancer agents. PF-07062119 is a T cell engaging bispecific antibody that binds to CD3 and guanylyl cyclase C, a protein that is over-expressed by colorectal cancers. Here, we used 89Zr-Df-IAB22M2C (89Zr-Df-Crefmirlimab), a human CD8-specific minibody to monitor CD8+ T cell infiltration into tumors by positron emission tomography. We investigated the ability of 89Zr-Df-IAB22M2C to track anti-tumor activity induced by PF-07062119 in a human CRC adoptive transfer mouse model (with injected activated/expanded human T cells), as well as the correlation of tumor radiotracer uptake with CD8+ immunohistochemical staining. PROCEDURES: NOD SCID gamma mice bearing human CRC LS1034 tumors were treated with four different doses of PF-07062119, or a non-targeted CD3 BsAb control, and imaged with 89Zr-Df-IAB22M2C PET at days 4 and 9. Following PET/CT imaging, mice were euthanized and dissected for ex vivo distribution analysis of 89Zr-Df-IAB22M2C in tissues on days 4 and 9, with additional data collected on day 6 (supplementary). Data were analyzed and reported as standard uptake value and %ID/g for in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were evaluated by immunohistochemistry for CD8+ T cells. RESULTS: The results demonstrated substantial mean uptake of 89Zr-Df-IAB22M2C (%ID/g) in PF-07062119-treated tumors, with significant increases in comparison to non-targeted BsAb-treated controls, as well as PF-07062119 dose-dependent responses over time of treatment. A moderate correlation was observed between tumor tissue radioactivity uptake and CD8+ cell density, demonstrating the value of the imaging agent for non-invasive assessment of intra-tumoral CD8+ T cells and the mechanism of action for PF-07062119. CONCLUSION: Immune-imaging technologies for quantitative cellular measures would be a valuable biomarker in immunotherapeutic clinical development. We demonstrated a qualification of 89Zr-IAB22M2C PET to evaluate PD responses (mice) to a novel immunotherapeutic.

Integrin αvß8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy.

αvß8 integrin, a key activator of transforming growth factor ß (TGF-ß), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvß8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvß8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of ß8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-ß and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvß8 integrin as a promising target for cancer immunotherapy.

Molecular imaging reveals biodistribution of P-cadherin LP-DART bispecific and trafficking of adoptively transferred T cells in mouse xenograft model.

P-cadherin-LP-DART is a bispecific antibody targeting P-cadherin expressed on the tumor cells and CD3 on the T-cells. Previously we demonstrated the development and efficacy of P-cadherin-LP-DART in in vitro and in vivo models. Here, we evaluated the three pillars: exposure, targeting specificity and pharmacodynamic modulation for P-cadherin-LP-DART using fluorescence molecular tomography (FMT). Bispecific antibodies and T-cells were conjugated with a near-infrared fluorophores: VivoTag®680XL (VT680) and CellVue®NIR815 (CV815), respectively. In vitro binding and cytotoxic T-lymphocyte assay demonstrated that P-cadherin-LP-DART significantly retained its properties after VT680 conjugation. In vivo FMT imaging was performed to determine the bispecific biodistribution and T-cell trafficking in HCT-116 xenograft model. Peak tumor exposure (2.71%ID) was observed at 96 hr post-injection with measurable quantity even at 240 hr (1.46%ID) (Pillar 1). P-cadherin-LP-DART accumulation in tumor was 20-25 fold higher compared to Control-LP-DART demonstrating the targeting specificity (Pillar 2). Imaging after engraftment of CV815 labeled T-cells showed P-cadherin-LP-DART mediated T-cell trafficking in tumors (Pillar 3). This study harnessed the multichannel capability of FMT and demonstrated the targeting of drug and trafficking of T cells to tumors, simultaneously. Our results show the impact of molecular imaging in demonstrating three pillars of pharmacology, longitudinally and non-invasively.

Low-level human equivalent gestational lead exposure produces sex-specific motor and coordination abnormalities and late-onset obesity in year-old mice.

BACKGROUND: Low-level developmental lead exposure is linked to cognitive and neurological disorders in children. However, the long-term effects of gestational lead exposure (GLE) have received little attention. OBJECTIVES: Our goals were to establish a murine model of human equivalent GLE and to determine dose-response effects on body weight, motor functions, and dopamine neurochemistry in year-old offspring. METHODS: We exposed female C57BL/6 mice to water containing 0, 27 (low), 55 (moderate), or 109 ppm (high) of lead from 2 weeks prior to mating, throughout gestation, and until postnatal day 10 (PN10). Maternal and litter measures, blood lead concentrations ([BPb]), and body weights were obtained throughout the experiment. Locomotor behavior in the absence and presence of amphetamine, running wheel activity, rotarod test, and dopamine utilization were examined in year-old mice. RESULTS: Peak [BPb] were < 1, < or = 10, 24-27, and 33-42 microg/dL in control, low-, moderate- and high-dose GLE groups at PN0-10, respectively. Year-old male but not female GLE mice exhibited late-onset obesity. Similarly, we observed male-specific decreased spontaneous motor activity, increased amphetamine-induced motor activity, and decreased rotarod performance in year-old GLE mice. Levels of dopamine and its major metabolite were altered in year-old male mice, although only forebrain utilization increased. GLE-induced alterations were consistently larger in low-dose GLE mice. CONCLUSIONS: Our novel results show that GLE produced permanent male-specific deficits. The nonmonotonic dose-dependent responses showed that low-level GLE produced the most adverse effects. These data reinforce the idea that lifetime measures of dose-response toxicant exposure should be a component of the neurotoxic risk assessment process.

Spatiotemporal regulation of ATP and Ca2+ dynamics in vertebrate rod and cone ribbon synapses.

PURPOSE: In conventional neurons, Ca2+ enters presynaptic terminals during an action potential and its increased local concentration triggers transient exocytosis. In contrast, vertebrate photoreceptors are nonspiking neurons that maintain sustained depolarization and neurotransmitter release from ribbon synapses in darkness and produce light-dependent graded hyperpolarizing responses. Rods transmit single photon responses with high fidelity, whereas cones are less sensitive and exhibit faster response kinetics. These differences are likely due to variations in presynaptic Ca2+ dynamics. Metabolic coupling and cross-talk between mitochondria, endoplasmic reticulum (ER), plasma membrane Ca2+ ATPase (PMCA), and Na+-Ca2+ exchanger (NCX) coordinately control presynaptic ATP production and Ca2+ dynamics. The goal of our structural and functional studies was to determine the spatiotemporal regulation of ATP and Ca2+ dynamics in rod spherules and cone pedicles. METHODS: Central retina tissue from C57BL/6 mice was used. Laser scanning confocal microscopy (LSCM) experiments were conducted on fixed-frozen vertical sections. Primary antibodies were selected for their tissue/cellular specificity and ability to recognize single, multiple or all splice variants of selected isoforms. Electron microscopy (EM) and 3-D electron tomography (ET) studies used our standard procedures on thin- and thick-sectioned retinas, respectively. Calibrated fluo-3-Ca2+ imaging experiments of dark- and light-adapted rod and cone terminals in retinal slices were conducted. RESULTS: Confocal microscopy showed that mitochondria, ER, PMCA, and NCX1 exhibited distinct retinal lamination patterns and differential distribution in photoreceptor synapses. Antibodies for three distinct mitochondrial compartments differentially labeled retinal areas with high metabolic demand: rod and cone inner segments, previously undescribed cone juxtanuclear mitochondria and the two plexiform layers. Rod spherule membranes uniformly and intensely stained for PMCA, whereas the larger cone pedicles preferentially stained for NCX1 at their active zones and PMCA near their mitochondria. EM and ET revealed that mitochondria in rod spherules and cone pedicles differed markedly in their number, location, size, volume, and total cristae surface area, and cristae junction diameter. Rod spherules had one large ovoid mitochondrion located near its active zone, whereas cone pedicles averaged five medium-sized mitochondria clustered far from their active zones. Most spherules had one ribbon synapse, whereas pedicles contained numerous ribbon synapses. Fluo-3 imaging studies revealed that during darkness rod spherules maintained a lower [Ca2+] than cone pedicles, whereas during light adaptation pedicles rapidly lowered their [Ca2+] below that observed in spherules. CONCLUSIONS: These findings indicate that ATP demand and mitochondrial ATP production are greater in cone pedicles than rod spherules. Rod spherules employ high affinity/low turnover PMCA and their mitochondrion to maintain a relatively low [Ca2+] in darkness, which increases their sensitivity and signal-to-noise ratio. In contrast, cone pedicles utilize low affinity/high turnover NCX to rapidly lower their high [Ca2+] during light adaptation, which increases their response kinetics. Spatiotemporal fluo-3-Ca2+ imaging results support our immunocytochemical results. The clustering of cone pedicle mitochondria likely provides increased protection from Ca2+ overload and permeability transition. In summary, these novel studies reveal that several integrated cellular and subcellular components interact to regulate ATP and Ca2+ dynamics in rod and cone synaptic terminals. These results should provide a greater understanding of in vivo photoreceptor synaptic terminal exocytosis/endocytosis, Ca2+ overload and therapies for retinal degenerations.

Assessment of near-infrared fluorophores to study the biodistribution and tumor targeting of an IL13 receptor α2 antibody by fluorescence molecular tomography.

Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores (ex/em: 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo/ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo/ex vivo FMT imaging showed 17-34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.

Editor's Highlight: Plasma miR-183/96/182 Cluster and miR-124 are Promising Biomarkers of Rat Retinal Toxicity.

Retinal toxicity is one of the leading causes of attrition in drug development, and drug-induced retinal toxicity remains an issue in both drug discovery and postmarketed drugs. Derisking strategies to help with early identification of retinal injury utilizing a predictive retinal miRNA biomarker would greatly benefit decision-making in drug discovery programs, ultimately reducing attrition due to retinal toxicity. Our previous work demonstrated elevation of circulating retina-enriched miRNAs in a retinal toxicity model. To further validate our previous observation, 2 additional rat retinal injury models were utilized in this study: NaIO3-induced retinal injury and laser-induced choroidal neovascularization (CNV) injury model. Following induction of retina tissue injuries, circulating miR-183/96/182 cluster (miR-183 cluster), and miR-124 was investigated, as well as evaluations using an electroretinogram (ERG) and histopathology analysis. Data revealed that circulating miR-183/96/182 cluster was significantly increased (2- to 15-fold) compared with baseline/control in both laser-induced CNV and NaIO3-induced retinal injury models. Moreover, the severity of the retinal injury evaluated by ERG and histopathology correlated highly with elevation of these retina-enriched miRNAs in plasma. MiR-124 was also significantly increased in comparison with baseline/control by ∼25-fold postrepeat-doses of 30 mg/kg NaIO3 treatment. Increased level of these plasma miRNA biomarkers appeared to be dose- and time-dependent upon NaIO3 or laser treatment. The results suggest that the retina-enriched miRNAs (miR-183/96/182 cluster and miR-124) could serve as convenient and predictive biomarkers of retinal toxicity in drug development.

Biodistribution and Targeting of Anti-5T4 Antibody-Drug Conjugate Using Fluorescence Molecular Tomography.

Understanding a drug's whole-body biodistribution and tumor targeting can provide important information regarding efficacy, safety, and dosing parameters. Current methods to evaluate biodistribution include in vivo imaging technologies like positron electron tomography and single-photon emission computed tomography or ex vivo quantitation of drug concentrations in tissues using autoradiography and standard biochemical assays. These methods use radioactive compounds or are cumbersome and do not give whole-body information. Here, for the first time, we show the utility of fluorescence molecular tomography (FMT) imaging to determine the biodistribution and targeting of an antibody-drug conjugate (ADC). An anti-5T4-antibody (5T4-Ab) and 5T4-ADC were conjugated with a near-infrared (NIR) fluorophore VivoTag 680XL (VT680). Both conjugated compounds were stable as determined by SEC-HPLC and plasma stability studies. Flow cytometry and fluorescence microscopy studies showed that VT680-conjugated 5T4-ADC specifically bound 5T4-expressing cells in vitro and also exhibited a similar cytotoxicity profile as the unconjugated 5T4-ADC. In vivo biodistribution and tumor targeting in an H1975 subcutaneous xenograft model demonstrated no significant differences between accumulation of VT680-conjugated 5T4-Ab or 5T4-ADC in either normal tissues or tumor. In addition, quantitation of heart signal from FMT imaging showed good correlation with the plasma pharmacokinetic profile suggesting that it (heart FMT imaging) may be a surrogate for plasma drug clearance. These results demonstrate that conjugation of VT680 to 5T4-Ab or 5T4-ADC does not change the behavior of native biologic, and FMT imaging can be a useful tool to understand biodistribution and tumor-targeting kinetics of antibodies, ADCs, and other biologics. Mol Cancer Ther; 15(10); 2530-40. ©2016 AACR.

Comprehensive bioimaging with fluorinated nanoparticles using breathable liquids.

Fluorocarbons are lipophobic and non-polar molecules that exhibit remarkable biocompatibility, with applications in liquid ventilation and synthetic blood. The unique properties of these compounds have also enabled mass spectrometry imaging of tissues where the fluorocarbons act as a Teflon-like coating for nanostructured surfaces to assist in desorption/ionization. Here we report fluorinated gold nanoparticles (f-AuNPs) designed to facilitate nanostructure imaging mass spectrometry. Irradiation of f-AuNPs results in the release of the fluorocarbon ligands providing a driving force for analyte desorption. The f-AuNPs allow for the mass spectrometry analysis of both lipophilic and polar (central carbon) metabolites. An important property of AuNPs is that they also act as contrast agents for X-ray microtomography and electron microscopy, a feature we have exploited by infusing f-AuNPs into tissue via fluorocarbon liquids to facilitate multimodal (molecular and anatomical) imaging.