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Murine tumors are indispensable model systems in preclinical immuno-oncology research. While immunologic heterogeneity is well-known to be an important factor that can influence treatment outcome, there is a severe paucity of data concerning the nature of this heterogeneity in murine tumor models. Using serial sectioning methodology combined with IHC analysis and whole-slide image analysis, the depth-dependent variation in immune-cell abundance in tumor specimens was investigated at single-cell resolution. Specifically, intra- and intertumor variability in cell density of nine immune-cell biomarkers was quantified in multiple murine tumor models. The analysis showed that intertumor variability was typically the dominant source of variation in measurements of immune-cell densities. Statistical power analysis revealed the effect of group size and variance in immune-cell density on the predictive power of detecting a statistically meaningful fold-change in immune-cell density. Intertumor variability in the ratio of immune-cell densities showed distinct patterns in select tumor models and revealed the existence of strong correlations between select biomarker pairs. Furthermore, the relative proportion of immune cells at different depths across tumor samples was preserved in some but not all tumor models, thereby revealing the existence of compositional heterogeneity. Taken together, these results reveal novel insights into the nature of immunologic heterogeneity, which is not accessible through typical omics approaches.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the β1 subunit. After confirming that human and rodent kidney predominately express AMPK β1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Several chemicals and pharmaceuticals increase the incidence of hemangiosarcomas (HSAs) in mice, but the relevance to humans is uncertain. Recently, canine HSAs were identified as a powerful tool for investigating the pathogenesis of human HSAs. To characterize the cellular phenotype of canine HSAs, we evaluated immunoreactivity and/or messenger RNA (mRNA) expression of markers for hematopoietic stem cells (HSCs), endothelial cells (ECs), a tumor suppressor protein, and a myeloid marker in canine HSAs. Neoplastic canine cells expressed EC markers and a myeloid marker, but expressed HSC markers less consistently. The canine tumor expression results were then compared to previously published immunoreactivity results for these markers in human and mouse HSAs. There are 2 noteworthy differences across species: (1) most human HSAs had HSC marker expression, indicating that they were comprised of tumor cells that were less differentiated than those in canine and mouse tumors; and (2) human and canine HSAs expressed a late-stage EC maturation marker, whereas mouse HSAs were negative, suggesting that human and canine tumors may retain greater differentiation potential than mouse tumors. These results indicate that HSA development is variable across species and that caution is necessary when discussing translation of carcinogenic risk from animal models to humans.

Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3(-/-)) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3(-/-) mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3(-/-) mice. Gpat3(-/-) enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3(-/-) mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism.

Small vessel vasculitis is a life-threatening condition and patients typically present with renal and pulmonary injury. Disease pathogenesis is associated with neutrophil accumulation, activation, and oxidative damage, the latter being driven in large part by myeloperoxidase (MPO), which generates hypochlorous acid among other oxidants. MPO has been associated with vasculitis, disseminated vascular inflammation typically involving pulmonary and renal microvasculature and often resulting in critical consequences. MPO contributes to vascular injury by 1) catabolizing nitric oxide, impairing vasomotor function; 2) causing oxidative damage to lipoproteins and endothelial cells, leading to atherosclerosis; and 3) stimulating formation of neutrophil extracellular traps, resulting in vessel occlusion and thrombosis. Here we report a selective 2-thiouracil mechanism-based MPO inhibitor (PF-1355 [2-(6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide) and demonstrate that MPO is a critical mediator of vasculitis in mouse disease models. A pharmacokinetic/pharmacodynamic response model of PF-1355 exposure in relation with MPO activity was derived from mouse peritonitis. The contribution of MPO activity to vasculitis was then examined in an immune complex model of pulmonary disease. Oral administration of PF-1355 reduced plasma MPO activity, vascular edema, neutrophil recruitment, and elevated circulating cytokines. In a model of anti-glomerular basement membrane disease, formerly known as Goodpasture disease, albuminuria and chronic renal dysfunction were completely suppressed by PF-1355 treatment. This study shows that MPO activity is critical in driving immune complex vasculitis and provides confidence in testing the hypothesis that MPO inhibition will provide benefit in treating human vasculitic diseases.

Pharmaceuticals and chemicals produce hemangiosarcomas (HS) in mice, often by nongenotoxic, proliferative mechanisms. A mode-of-action (MOA) for hemangiosarcoma was proposed based on information presented at an international workshop (Cohen et al., Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18.). Five key elements of the MOA were articulated and included hypoxia, macrophage activation, increased angiogenic growth factors, dysregulated angiogenesis/erythropoiesis, and endothial cell proliferation. The goal of the current study was to add to the weight-of-evidence for the proposed MOA by assessing these key elements with 3 different compounds of varying potency for HS induction: fenretinide (high), troglitazone (intermediate), and elmiron (low). Multiple endpoints, including hypoxia (hyproxyprobe, transcriptomics), endothelial cell (EC) proliferation, and clinical and anatomic pathology, were assessed after 2, 4, and 13-weeks of treatment in B6C3F1 mice. All 3 compounds demonstrated strong evidence for dysregulated erythropoiesis (decrease in RBC and a failure to increase reticulocytes) and macrophage activation (4- to 11-fold increases); this pattern of hematological changes in mice might serve as an early biomarker to evaluate EC proliferation in suspected target organs for potential HS formation. Fenretinide demonstrated all 5 key elements, while troglitazone demonstrated 4 and elmiron demonstrated 3. Transcriptomics provided support for the 5 elements of the MOA, but was not any more sensitive than hypoxyprobe immunohistochemistry for detecting hypoxia. The overall transcriptional evidence for the key elements of the proposed MOA was also consistent with the potency of HS induction. These data, coupled with the previous work with 2-butoxyethanol and pregablin, increase the weight-of-evidence for the proposed MOA for HS formation.

Treatment-induced epididymal inflammation and granuloma formation is only an occasional problem in preclinical drug development, but it can effectively terminate the development of that candidate molecule. Screening for backup molecules without that toxicity must be performed in animals (generally rats) that requires at least 2 to 3 weeks of in vivo exposure, a great deal of specially synthesized candidate compound, and histologic examination of the target tissues. We instead hypothesized that these treatments induced proinflammatory gene expression, and so used mixed-cell cultures from the rat epididymal tubule to monitor the induction of proinflammatory cytokines. Cells were exposed for 24 hr and then cytotoxicity was evaluated with the MTS assay and mRNA levels of Interleukin-6 (IL-6) and growth-related oncogene (GRO) were measured. We found that compounds that were more toxic in vivo stimulated a greater induction of IL-6 and GRO mRNA levels in vitro. By relating effective concentrations in vitro with the predicted C(eff), we could rank compounds by their propensity to induce inflammation in rats in vivo. This method allowed the identification of several compounds with very low inflammatory induction in vitro. When tested in rats, the compounds produced small degrees of inflammation at an acceptable margin (approximately 20×), and have progressed into further development.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the

The phosphodiesterases (PDEs) are a superfamily of enzymes that regulate spatio-temporal signaling by the intracellular second messengers cAMP and cGMP. PDE2A is expressed at high levels in the mammalian brain. To advance our understanding of the role of this enzyme in regulation of neuronal signaling, we here describe the distribution of PDE2A in the rat brain. PDE2A mRNA was prominently expressed in glutamatergic pyramidal cells in cortex, and in pyramidal and dentate granule cells in the hippocampus. Protein concentrated in the axons and nerve terminals of these neurons; staining was markedly weaker in the cell bodies and proximal dendrites. In addition, in both hippocampus and cortex, small populations of non-pyramidal cells, presumed to be interneurons, were strongly immunoreactive. PDE2A mRNA was expressed in medium spiny neurons in neostriatum. Little immunoreactivity was observed in cell bodies, whereas dense immunoreactivity was found in the axon tracts of these neurons and their terminal regions in globus pallidus and substantia nigra pars reticulata. Immunostaining was dense in the medial habenula, but weak in other diencephalic regions. In midbrain and hindbrain, immunostaining was restricted to discrete regions of the neuropil or clusters of cell bodies. These results suggest that PDE2A may modulate cortical, hippocampal and striatal networks at several levels. Preferential distribution of PDE2A into axons and terminals of the principal neurons suggests roles in regulation of axonal excitability or transmitter release. The enzyme is also in forebrain interneurons, and in mid- and hindbrain neurons that may modulate forebrain networks and circuits.

PDE10A is a newly identified cAMP/cGMP phosphodiesterase for which mRNA is highly expressed in the mammalian striatum. In the present study, PDE10A protein and mRNA expression throughout the rat brain were determined, using a monoclonal antibody (24F3.F11) for Western blot and immunohistochemical analyses and an antisense riboprobe for in situ hybridization. High levels of mRNA are observed in most of the neuronal cell bodies of striatal complex (caudate n, n. accumbens and olfactory tubercle), indicating that PDE10A is expressed by the striatal medium spiny neurons. PDE10A-like immunoreactivity is dense throughout the striatal neuropil, as well as in the internal capsule, globus pallidus, and substantia nigra. These latter regions lack significant expression of PDE10A mRNA. Thus, PDE10A is transported throughout the dendritic tree and down the axons to the terminals of the medium spiny neurons. These data suggest a role for PDE10A in regulating activity within both the striatonigral and striatopallidal pathways. In addition, PDE10A immunoreactivity and mRNA are found at lower levels in the hippocampal pyramidal cell layer, dentate granule cell layer and throughout the cortex and cerebellar granule cell layer. Immunoreactivity is detected only in cell bodies in these latter regions. This more restricted subcellular localization of PDE10A outside the striatum suggests a second, distinct function for the enzyme in these regions.

The ATP-binding cassette transporter A1 (ABCA1) encodes a membrane protein that promotes cholesterol and phospholipid efflux from cells. Mutations in ABCA1 lead to HDL deficiency and tissue accumulation of macrophages in patients with homozygous Tangier disease. In this study, we examined whether the complete absence of ABCA1 or selected inactivation in macrophages is accompanied by an increase in atherosclerotic lesion progression in hypercholesterolemic apolipoprotein E–deficient (apoE −/− ) mice and LDLR receptor–deficient (LDLr −/− ) mice. The absence of ABCA1 led to reduced plasma cholesterol levels in both the apoE −/− and LDLr −/− mice, along with severe skin xanthomatosis characterized by marked foamy macrophages and cholesterol ester accumulation. However, the complete absence of ABCA1 did not affect the development, progression, or composition of atherosclerotic lesions in either the LDLr −/− or the apoE −/− mice fed a chow or atherogenic diet. In contrast, bone marrow transplantation studies demonstrated that the selective inactivation of ABCA1 in macrophages markedly increased atherosclerosis and foam cell accumulation in apoE −/− . Taken together, these findings demonstrate that the complete absence of ABCA1 has a major impact on plasma lipoprotein homeostasis, and the proposed antiatherogenic effect resulting from ABCA1 deficiency is compensated by a less atherogenic profile. ABCA1 deficiency in macrophages, however, demonstrates the antiatherogenic properties of ABCA1 independent of plasma lipids and HDL levels.

Treatment-induced epididymal inflammation and granuloma formation is only an occasional problem in preclinical drug development, but it can effectively terminate the development of that candidate molecule. Screening for backup molecules without that toxicity must be performed in animals (generally rats) that requires at least 2 to 3 weeks of in vivo exposure, a great deal of specially synthesized candidate compound, and histologic examination of the target tissues. We instead hypothesized that these treatments induced proinflammatory gene expression, and so used mixed-cell cultures from the rat epididymal tubule to monitor the induction of proinflammatory cytokines. Cells were exposed for 24 hr and then cytotoxicity was evaluated with the MTS assay and mRNA levels of Interleukin-6 (IL-6) and growth-related oncogene (GRO) were measured. We found that compounds that were more toxic in vivo stimulated a greater induction of IL-6 and GRO mRNA levels in vitro. By relating effective concentrations in vitro with the predicted C(eff), we could rank compounds by their propensity to induce inflammation in rats in vivo. This method allowed the identification of several compounds with very low inflammatory induction in vitro. When tested in rats, the compounds produced small degrees of inflammation at an acceptable margin (approximately 20×), and have progressed into further development.

Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3β. Here we show that tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus/hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases.

Gemtuzumab ozogamicin (Mylotarg, GO) and inotuzumab ozogamicin (IO) are antibody-drug conjugates (ADCs) comprised of different humanized monoclonal antibodies (against CD33 and CD22 antigen, respectively) and of the same acid-labile linker and calicheamicin payload. GO and IO are developed for the treatment of acute myeloid leukemia and acute lymphoblastic leukemia, respectively. Adverse events reported with these 2 drugs in patients include thrombocytopenia and liver toxicity, which is characterized by increases in serum aminotransferases and bilirubin along with occasional cases of sinusoidal obstruction syndrome (SOS). The platelet and liver effects were seen in patients with conjugates targeting unrelated antigens and are likely target-independent. Since the specific mechanisms of these toxicities remain elusive, an investigative study was performed in cynomolgus monkeys with a non-binding ADC containing the same linker and payload as GO and IO, PF-0259, with the objectives to investigate the mechanism for thrombocytopenia, characterize the liver injury and identify potential safety biomarkers. Cynomolgus monkeys were dosed intravenously with PF-0259 at 6 mg/m2/dose once every 3 weeks for up to 3 doses and were necropsied 48 hours after the 1st administration (Day 3) or 3 weeks after the 3rd administration (Day 63). PF-0259 induced acute thrombocytopenia (up to 86% reduction in platelet count) in monkeys with nadirs on Days 3-4. There was no indication of effects on megakaryocytes in bone marrow or activation of platelets in peripheral blood. Microscopic evaluation of liver samples from animals necropsied on Day 3 demonstrated midzonal degeneration and loss of sinusoidal endothelial cells (SECs) associated with marked platelet accumulation in sinusoids. Liver histopathology on Day 63 showed variable endothelial cell recovery and progression to a combination of sinusoidal capillarization and sinusoidal dilation/hepatocellular atrophy, consistent with early SOS. Among biomarkers evaluated, there were early and sustained increases in serum hyaluronic acid (HA) that correlated well with AST levels and liver microscopic changes, suggesting that HA could be a sensitive diagnostic marker of the liver microvascular injury. In conclusion, this work has demonstrated that target-independent damage to liver SECs was responsible for acute thrombocytopenia (through platelet sequestration in the liver) and development of early SOS in monkeys. The translation of these observations to humans has not been evaluated. We further hypothesize that this toxicity mechanism may operate for other types of non-calicheamicin based ADCs in patients where adverse events of thrombocytopenia, increased liver enzymes and liver microvascular disorders (including nodular regenerative hyperplasia) have been observed. Citation Format: Magali Guffroy, Hadi Falahatpisheh, Kathleen Biddle, John Kreeger, Leslie Obert, Karen Walters, Richard Goldstein, Germaine Boucher, Timothy Coskran, William Reagan, Danielle Sullivan, Sharon Sokolowski, Richard Giovanelli, Hans-Peter Gerber, Martin Finkelstein, Nasir Khan. Liver microvascular injury and thrombocytopenia of antibody-calicheamicin conjugates: mechanism and monitoring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-202.

Phosphodiesterase 5A (PDE5A) inhibitors improve functional recovery after middle cerebral artery occlusion (MCA-o) in rats. We used the PDE5A inhibitor 3-(4-(2-hydroxyethyl)piperazin-1-yl)-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one hydrochloride (PF-5) to determine the timing, duration, and degree of inhibition that yields maximum efficacy. We also investigated the localization of PDE5A to determine the tissues and cells that would be targets for PDE5 inhibition and that may mediate efficacy. Nearly complete inhibition of PDE5A, starting 24 h after MCA-o and continued for 7 days, resulted in nearly complete recovery of sensorimotor function that was sustained for 3 months. Delaying administration until 72 h after MCA-o resulted in equivalent efficacy, whereas delaying treatment for 14 days was ineffective. Treatment for 7 days was equivalently efficacious to 28 or 84 days of treatment, whereas treatment for 1 day was less effective. In the normal forebrain, PDE5A immunoreactivity was prominent in smooth muscle of meningeal arteries and a few smaller blood vessels, with weak staining in a few widely scattered cortical neurons and glia. At 24 and 48 h after MCA-o, the number and intensity of blood vessel staining increased in the infarcted cortex and striatum. PDE5A immunoreactivity also was increased at 48 h in putative microglia in penumbra, whereas there was no change in staining of the scattered cortical neurons. Given the window for efficacy and the PDE5A distribution, we hypothesize that efficacy results from an effect on vasculature, and perhaps modulation of microglial function, both of which may facilitate recovery of neuronal function.

A monoclonal antibody directed against the amino terminal of rat phosphodiesterase 10A (PDE10A) was used to localize PDE10A in multiple central nervous system (CNS) and peripheral tissues from mouse, rat, dog, cynomolgus macaque, and human. PDE10A immunoreactivity is strongly expressed in the CNS of these species with limited expression in peripheral tissues. Within the brain, strong immunoreactivity is present in both neuronal cell bodies and neuropil of the striatum, in striatonigral and striatopallidal white matter tracks, and in the substantia nigra and globus pallidus. Outside the brain, PDE10A immunoreactivity is less intense, and distribution is limited to few tissues such as the testis, epididymal sperm, and enteric ganglia. These data demonstrate that PDE10A is an evolutionarily conserved phosphodiesterase highly expressed in the brain but with restricted distribution in the periphery in multiple mammalian species. (J Histochem Cytochem 54:1205-1213, 2006)