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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.

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.

A model of para-aminophenol (PAP) nephrotoxicity in Sprague–Dawley rats was utilized to characterize potential indicators of toxicity in the kidney and in biofluids, and to chronicle the progression of acute renal injury. Rats were administered PAP at a low or high dose and examined terminally at 6, 24 and 48 hours (4 animals/group with matching controls). Acute tubular necrosis was observed in the medullary rays (low and high doses) and the outer stripe of outer medulla (high dose only) as early as 6 hours postdosing. Starting at 24 hours, regeneration of the tubular epithelium was evident in both low and high dose studies. Associated with the tubular lesions, we observed elevation of urinary α-glutathione S-transferase levels, an indicator of proximal tubular injury. By immunohistochemistry of the kidney, decreased γ-glutamylcysteine synthetase expression correlated with tubular injury, especially at high dose, whereas elevation of vimentin, osteopontin, and Ki-67 expression was concurrent with tubular regeneration. Clusterin and kidney injury molecule-1 displayed expression patterns characteristic of both renal injury and regeneration. Taken together, this study provided insight into the progression of nephrotoxicity, and allowed the evaluation of potential urinary and tissue protein biomarkers that could complement the early detection of acute tubular injury.

Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; K i

The fibroblast growth factor receptors (FGFR) play a major role in angiogenesis and are desirable targets for the development of therapeutics. Groups of Wistar Han rats were dosed orally once daily for 4 days with a small molecule pan-FGFR inhibitor (5mg/kg) or once daily for 6 days with a small molecule MEK inhibitor (3mg/kg). Serum phosphorous and FGF23 levels increased in all rats during the course of the study. Histologically, rats dosed with either drug exhibited multifocal, multiorgan soft tissue mineralization. Expression levels of the sodium phosphate transporter Npt2a and the vitamin D-metabolizing enzymes Cyp24a1 and Cyp27b1 were modulated in kidneys of animals dosed with the pan-FGFR inhibitor. Both inhibitors decreased ERK phosphorylation in the kidneys and inhibited FGF23-induced ERK phosphorylation in vitro in a dose-dependent manner. A separate cardiovascular outcome study was performed to monitor hemodynamics and cardiac structure and function of telemetered rats dosed with either the pan-FGFR inhibitor or MEK inhibitor for 3 days. Both compounds increased blood pressure (~+ 17 mmHg), decreased heart rate (~-75 bpm), and modulated echocardiography parameters. Our data suggest that inhibition of FGFR signaling following administration of either pan-FGFR inhibitor or MEK inhibitor interferes with the FGF23 pathway, predisposing animals to hyperphosphatemia and a tumoral calcinosis-like syndrome in rodents.