Using bioluminescence to image gene expression and spontaneous behavior in freely moving mice.

Bioluminescence imaging (BLI) of gene expression in live animals is a powerful method for monitoring development, tumor growth, infections, healing, and other progressive, long-term biological processes. BLI remains an effective approach for reducing the number of animals needed to monitor dynamic changes in gene activity because images can be captured repeatedly from the same animals. When examining these ongoing changes, it is sometimes necessary to remove rhythmic effects on the bioluminescence signal caused by the circadian clock's daily modulation of gene expression. Furthermore, BLI using freely moving animals remains limited because the standard procedures can alter normal behaviors. Another obstacle with conventional BLI of animals is that luciferin, the firefly luciferase substrate, is usually injected into mice that are then imaged while anesthetized. Unfortunately, the luciferase signal declines rapidly during imaging as luciferin is cleared from the body. Alternatively, mice are imaged after they are surgically implanted with a pump or connected to a tether to deliver luciferin, but stressors such as this surgery and anesthesia can alter physiology, behavior, and the actual gene expression being imaged. Consequently, we developed a strategy that minimizes animal exposure to stressors before and during sustained BLI of freely moving unanesthetized mice. This technique was effective when monitoring expression of the Per1 gene that serves in the circadian clock timing mechanism and was previously shown to produce circadian bioluminescence rhythms in live mice. We used hairless albino mice expressing luciferase that were allowed to drink luciferin and engage in normal behaviors during imaging with cooled electron-multiplying-CCD cameras. Computer-aided image selection was developed to measure signal intensity of individual mice each time they were in the same posture, thereby providing comparable measurements over long intervals. This imaging procedure, performed primarily during the animal's night, is compatible with entrainment of the mouse circadian timing system to the light cycle while allowing sampling at multi-day intervals to monitor long-term changes. When the circadian expression of a gene is known, this approach provides an effective alternative to imaging immobile anesthetized animals and can removing noise caused by circadian oscillations and body movements that can degrade data collected during long-term imaging studies.

Assume It Will Break: Parental Perspectives on Negative Communication Experiences in Pediatric Oncology.

PURPOSE: Communication breakdowns in pediatric oncology can have negative consequences for patients and families. A detailed analysis of these negative encounters will support clinicians in anticipating and responding to communication breakdowns. METHODS: Semistructured interviews with 80 parents of children with cancer across three academic medical centers during treatment, survivorship, or bereavement. We analyzed transcripts using semantic content analysis. RESULTS: Nearly all parents identified negative communication experiences (n = 76). We identified four categories of contributors to negative experiences: individual (n = 68), team (n = 26), organization (n = 46), and greater health care system (n = 8). These experiences involved a variety of health care professionals across multiple specialties. Parents reported 12 personal consequences of communication breakdowns: emotional distress (n = 65), insufficient understanding (n = 48), decreased trust or confidence (n = 37), inconvenience (n = 36), medical harm (n = 23), decreased self-confidence (n = 17), decreased emotional support (n = 13), decreased engagement (n = 9), false hope (n = 9), decreased hope (n = 7), financial insult (n = 7), and decreased access to resources (n = 3). We identified five categories of supportive responses from clinicians: exploring (n = 8), acknowledging (n = 17), informing (n = 27), adapting (n = 27), and advocating (n = 18). Parents often increased their own advocacy on behalf of their child (n = 47). Parents also identified the need for parental engagement in finding solutions (n = 12). Finally, one parent suggested that clinicians should assume that communication will fail and develop contingency plans in advance. CONCLUSION: Communication breakdowns in pediatric oncology negatively affect parents and children. Clinicians should plan for communication breakdowns and respond by exploring, acknowledging, informing, adapting, advocating, and engaging parents in finding solutions.

C3HeB/FeJ Mice mimic many aspects of gene expression and pathobiological features of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

DNA Alkylating Agent Protects Against Spontaneous Hepatocellular Carcinoma Regardless of O6-Methylguanine-DNA Methyltransferase Status.

Hepatocellular carcinoma is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study hepatocellular carcinoma tumorigenesis because they mimic human hepatocellular carcinoma with delayed onset, male gender bias, approximately 50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O(6)-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild-type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN; 0.025 µmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase-deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU; 25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12 to 15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild-type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared with O(6)-alkylguanine lesions. Together, these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing hepatocellular carcinoma.