Characterizing Intra-Tumor and Inter-Tumor Variability of Immune Cell Infiltrates in Murine Syngeneic Tumors.

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.

An in vitro alveolar epithelial cell model recapitulates LRRK2 inhibitor-induced increases in lamellar body size observed in preclinical models.

Alveolar type II (ATII) epithelial cells contain lamellar bodies (LBs) which synthesize and store lung surfactants. In animals, the inhibition or knockout of leucine-rich repeat kinase 2 (LRRK2) causes abnormal enlargement of LBs in ATII cells. This effect of LRRK2 inhibition in lung is largely accepted as being mediated directly through blocking of the kinase function; however, downstream consequences in the lung remain unknown. In this work we established an in vitro alveolar epithelial cell (AEC) model that recapitulates the in vivo phenotype of ATII cells and developed an assay to quantify changes in LB size in response to LRRK2 inhibitors. Culture of primary human AECs at the air-liquid interface on matrigel and collagen-coated transwell inserts in the presence of growth factors promoted the LB formation and apical microvilli and induced expression of LRRK2 and ATII cell markers. Treatment with a selective LRRK2 inhibitor resulted in pharmacological reduction of phospho-LRRK2 and a significant increase in LB size; effects previously reported in lungs of non-human primates treated with LRRK2 inhibitor. In summary, our human in vitro AEC model recapitulates the abnormal lung findings observed in LRRK2-perturbed animals and holds the potential for expanding current understanding of LRRK2 function in the lung.

Glutamate dehydrogenase as a biomarker for mitotoxicity; insights from furosemide hepatotoxicity in the mouse.

Glutamate dehydrogenase (GLDH) is a liver-specific biomarker of hepatocellular damage currently undergoing qualification as a drug development tool. Since GLDH is located within the mitochondrial matrix, it has been hypothesized that it might also be useful in assessing mitotoxicity as an initiating event during drug-induced liver injury. According to this hypothesis, hepatocyte death that does not involve primary mitochondrial injury would result in release of intact mitochondria into circulation that could be removed by high speed centrifugation and result in lower GLDH activity measured in spun serum vs un-spun serum. A single prior study in mice has provided some support for this hypothesis. We sought to repeat and extend the findings of this study. Accordingly, mice were treated with the known mitochondrial toxicant, acetaminophen (APAP), or with furosemide (FS), a toxicant believed to cause hepatocyte death through mechanisms not involving mitotoxicity as initiating event. We measured GLDH levels in fresh plasma before and after high speed centrifugation to remove intact mitochondria. We found that both APAP and FS treatments caused substantial hepatocellular necrosis that correlated with plasma alanine aminotransferase (ALT) and GLDH elevations. The plasma GLDH activity in both the APAP- and FS- treated mice was not affected by high-speed centrifugation. Interestingly, the ratio of GLDH:ALT was 5-fold lower during FS compared to APAP hepatotoxicity. Electron microscopy confirmed that both APAP- and FS-treatments had resulted in mitochondrial injury. Mitochondria within vesicles were only observed in the FS-treated mice raising the possibility that mitophagy might account for reduced release of GLDH in the FS-treated mice. Although our results show that plasma GLDH is not clinically useful for evaluating mitotoxicity, the GLDH:ALT ratio as a measure of mitophagy needs to be further studied.

No Evidence of Neurogenesis in Adult Rat Sympathetic Ganglia Following Guanethidine-Induced Neuronal Loss.

The potential for neurogenesis in the cranial (superior) cervical ganglia (SCG) of the sympathetic nervous system was evaluated. Eleven consecutive daily doses of guanethidine (100 mg/kg/d) were administered intraperitoneally to rats in order to destroy postganglionic sympathetic neurons in SCG. Following the last dose, animals were allowed to recover 1, 3, or 6 months. Right and left SCG from guanethidine-treated and age-matched, vehicle-treated control rats were harvested for histopathologic, morphometric, and stereologic evaluations. Both morphometric and stereologic evaluations confirmed neuron loss following guanethidine treatment. Morphometric analysis revealed a 50% to 60% lower number of tyrosine hydroxylase (TH)-positive neurons per unit area of SCG at both 3 and 6 months of recovery, compared to ganglia of age-matched controls, with no evidence of restoration of neuron density between 3 and 6 months. Reductions in TH-positive neurons following guanethidine treatment were corroborated by unbiased stereology of total hematoxylin and eosin-stained neuron numbers in SCG. Stereologic analyses revealed that total neuron counts were lower by 37% at 3 months of recovery when compared to age-matched vehicle controls, again with no obvious restoration between 3 and 6 months. Thus, no evidence was found that postganglionic neurons of the sympathetic nervous system in the adult rat have a neurogenic capacity.

Design and Synthesis of Clinical Candidate PF-06751979: A Potent, Brain Penetrant, ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Lacking Hypopigmentation.

A major challenge in the development of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer's disease is the alignment of potency, drug-like properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.

High resolution molecular and histological analysis of renal disease progression in ZSF1 fa/faCP rats, a model of type 2 diabetic nephropathy.

ZSF1 rats exhibit spontaneous nephropathy secondary to obesity, hypertension, and diabetes, and have gained interest as a model system with potentially high translational value to progressive human disease. To thoroughly characterize this model, and to better understand how closely it recapitulates human disease, we performed a high resolution longitudinal analysis of renal disease progression in ZSF1 rats spanning from early disease to end stage renal disease. Analyses included metabolic endpoints, renal histology and ultrastructure, evaluation of a urinary biomarker of fibrosis, and transcriptome analysis of glomerular-enriched tissue over the course of disease. Our findings support the translational value of the ZSF1 rat model, and are provided here to assist researchers in the determination of the model's suitability for testing a particular mechanism of interest, the design of therapeutic intervention studies, and the identification of new targets and biomarkers for type 2 diabetic nephropathy.

Selective Activation of AMPK ß1-Containing Isoforms Improves Kidney Function in a Rat Model of Diabetic Nephropathy.

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 ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, 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.

Liver Microvascular Injury and Thrombocytopenia of Antibody-Calicheamicin Conjugates in Cynomolgus Monkeys-Mechanism and Monitoring.

Purpose: Adverse reactions reported in patients treated with antibody-calicheamicin conjugates such as gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin include thrombocytopenia and sinusoidal obstruction syndrome (SOS). The objective of this experimental work was to investigate the mechanism for thrombocytopenia, characterize the liver injury, and identify potential safety biomarkers.Experimental Design: Cynomolgus monkeys were dosed intravenously at 6 mg/m2/dose once every 3 weeks with a nonbinding antibody-calicheamicin conjugate (PF-0259) containing the same linker-payload as gemtuzumab ozogamicin and inotuzumab ozogamicin. Monkeys were necropsied 48 hours after the first administration (day 3) or 3 weeks after the third administration (day 63).Results: PF-0259 induced acute thrombocytopenia (up to 86% platelet reduction) with nadirs on days 3 to 4. There was no indication of effects on megakaryocytes in bone marrow or activation of platelets in peripheral blood. Microscopic evaluation of liver 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 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 serum aspartate aminotransferase and liver microscopic changes, suggesting that HA may be a sensitive diagnostic marker of the liver microvascular injury.Conclusions: These data support the conclusion that target-independent damage to liver SECs may be responsible for acute thrombocytopenia (through platelet sequestration in liver sinusoids) and development of SOS. Clin Cancer Res; 23(7); 1760-70. ©2016 AACR.

The development of subcutaneous sarcomas in rodents exposed to peroxisome proliferators agonists: hypothetical mechanisms of action and de-risking attitude.

Peroxisome proliferator-activated receptors (PPARs) represent therapeutic targets for the management of type 2 diabetes mellitus and dyslipidemia. Rodent carcinogenicity studies have revealed a link between γ and dual γ/α PPAR agonist treatment and the increased incidence of subcutaneous (SC) liposarcomas/fibrosarcomas or hemangiosarcomas, but very little has been reported for potent and selective PPARα agonists. We present a mode of action framework for the development of SC mesenchymal tumors in rodents given PPAR agonists. (1) Tumor promotion results from pharmacologically mediated recruitment (proliferation and differentiation), thermogenesis and adipogenesis of stromovascular cells, and subsequent generation of oxidative free radicals. (2) Tumor initiation consists of chemotype-driven mitochondrial dysfunction causing uncontrolled oxidative stress and permanent DNA damage. Promotion is characterized by enhanced adipogenesis in the SC adipose tissue, where the baseline PPARγ expression and responsiveness to PPARγ ligands is the highest, and by thermogenesis through expression of the uncoupling protein 1 (UCP-1) and the PPARγ co-activator 1 α (PGC-1α), two factors more highly expressed in brown versus white adipose tissue. Initiation is supported by the demonstration of mitochondrial uncoupling and OXPHOS Complexes dysfunction (Complexes III, IV and V) by compounds associated with increased incidences of sarcomas (muraglitazar and troglitazone), but not others lacking malignant tumor effects (pioglitazone, rosiglitazone).

Renal dysplasia in Beagle dogs: four cases.

Anomalies of renal development comprise abnormalities in the amount of renal tissue (agenesis and hypoplasia); anomalies of renal position, form, and orientation; and renal dysplasia. There are previous reports of canine renal dysplasia in different breeds but none in the Beagle breed. This is the first report of renal dysplasia in this breed of dog. Morphologic descriptions of the range of microscopic features observed in four cases of renal dysplasia from preclinical studies in laboratory Beagle dogs are presented (including persistent primitive mesenchyme, persistence of metanephric ducts, asynchronous differentiation of nephrons, and atypical tubular epithelium), along with a basis for the classification of the lesion.

Increased cholesterol deposition, expression of scavenger receptors, and response to chemotactic factors in Abca1-deficient macrophages.

OBJECTIVE: Studies in bone marrow transplanted from ATP-binding cassette transporter A1 (ABCA1)-deficient mice into normal mice provides direct evidence that the absence of leukocyte ABCA1 exerts a marked proatherogenic effect independent of changes in plasma lipids, suggesting that ABCA1 plays a key role in the regulation of cholesterol homeostasis and function of macrophages. Therefore, we examined whether the absence of ABCA1 affects the morphology, properties, and functional activities of macrophages that could be related to the development of atherosclerosis. METHODS AND RESULTS: We conducted a series of experiments in macrophages isolated from Abca1-deficient and wild-type mice and compared several of their properties that are thought to be related to the development of atherosclerosis. Macrophages isolated from Abca1-deficient mice have an increase in cholesterol content, expression of scavenger receptors, and secretion of chemokines, growth factors, and cytokines, resulting in an increased ability to respond to a variety of chemotactic factors. CONCLUSIONS: Our studies indicate that the absence of ABCA1 leads to significant changes in the morphology, properties, and functional activities of macrophages. These changes, together with the proinflammatory condition present in ABCA1-deficient mice and increased reactivity of macrophages to chemotactic factors, play a key role in the development and progression of atherosclerosis.