Neuronal distribution in colorectal cancer: associations with clinicopathological parameters and survival.

Over the last years, insights in the cancer neuroscience field increased rapidly and a potential role for neurons in colorectal carcinogenesis has been recognized. However, knowledge on the neuronal distribution, subtypes, origin and associations with clinicopathological characteristics in human studies is sparse. In this study, colorectal tumor tissues from the Netherlands Cohort Study on diet and cancer (n=490) and an in-cohort validation population (n=529) were immunohistochemically stained for the pan-neuronal markers neurofilament (NF) and protein gene product 9.5 (PGP9.5) to study the association between neuronal marker expression and clinicopathological characteristics. In addition, tumor and healthy colon tissue were stained for neuronal subtype markers and their immunoreactivity in colorectal cancer (CRC) stroma was analyzed. NF and PGP9.5 positive nerve fibers were found within the tumor stroma and were mostly characterized by the neuronal subtype markers vasoactive intestinal protein (VIP) and neuronal nitric oxide synthase (nNOS), suggesting that inhibitory neurons are the most prominent neuronal subtype in CRC. NF and PGP9.5 protein expression were not consistently associated with tumor stage, sublocation, differentiation grade and median survival. NF immunoreactivity was associated with a worse CRC-specific survival in the study cohort (p=0.025), independent of other prognostic factors (HR=2.31; 95% CI 1.33-4.03; p=0.003), but these results were not observed in the in-cohort validation group. PGP9.5 on the other hand, was associated with a worse CRC-specific survival in the in-cohort validation (p=0.046) but not in the study population. This effect disappeared in multivariate analyses (HR=0.81; 95% CI 0.50-1.32; p=0.393) indicating that this effect was dependent on other prognostic factors. This study demonstrates that the tumor stroma of CRC patients mainly harbors inhibitory neurons and that NF as a single marker is significantly associated with a poorer CRC-specific survival in the study cohort but necessitates future validation.

Differences in enteric neuronal density in the NSE-Noggin mouse model across institutes.

The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However, in our hands NSE-Noggin mice did not present with a hyperganglionic phenotype. NSE-Noggin mice were phenotyped based on fur appearance, genotyped and DNA sequenced to demonstrate transgene and intact NSE-Noggin-IRES-EGFP construct presence, and RNA expression of Noggin was shown to be upregulated. Positive EGFP staining in the plexus of NSE-Noggin mice also confirmed Noggin protein expression. Myenteric plexus preparations of the colon were examined to quantify both the overall density of enteric neurons and the proportions of enteric neurons expressing specific subtype markers. The total number of enteric neurons in the colonic myenteric plexus of transgenic mice did not differ significantly from wild types, nor did the proportion of calbindin, calretinin, or serotonin immunoreactive myenteric neurons. Possible reasons as to why the hyperinnervated phenotype could not be observed in contrast with original studies using this mouse model are discussed, including study design, influence of microbiota, and other environmental variables.

Promoter hypermethylation of neural-related genes is compatible with stemness in solid cancers.

BACKGROUND: DNA hypermethylation is an epigenetic feature that modulates gene expression, and its deregulation is observed in cancer. Previously, we identified a neural-related DNA hypermethylation fingerprint in colon cancer, where most of the top hypermethylated and downregulated genes have known functions in the nervous system. To evaluate the presence of this signature and its relevance to carcinogenesis in general, we considered 16 solid cancer types available in The Cancer Genome Atlas (TCGA). RESULTS: All tested cancers showed significant enrichment for neural-related genes amongst hypermethylated genes. This signature was already present in two premalignant tissue types and could not be explained by potential confounders such as bivalency status or tumor purity. Further characterization of the neural-related DNA hypermethylation signature in colon cancer showed particular enrichment for genes that are overexpressed during neural differentiation. Lastly, an analysis of upstream regulators identified RE1-Silencing Transcription factor (REST) as a potential mediator of this DNA methylation signature. CONCLUSION: Our study confirms the presence of a neural-related DNA hypermethylation fingerprint in various cancers, of genes linked to neural differentiation, and points to REST as a possible regulator of this mechanism. We propose that this fingerprint indicates an involvement of DNA hypermethylation in the preservation of neural stemness in cancer cells.

Nerves in gastrointestinal cancer: from mechanism to modulations.

Maintenance of gastrointestinal health is challenging as it requires balancing multifaceted processes within the highly complex and dynamic ecosystem of the gastrointestinal tract. Disturbances within this vibrant environment can have detrimental consequences, including the onset of gastrointestinal cancers. Globally, gastrointestinal cancers account for ~19% of all cancer cases and ~22.5% of all cancer-related deaths. Developing new ways to more readily detect and more efficiently target these malignancies are urgently needed. Whereas members of the tumour microenvironment, such as immune cells and fibroblasts, have already been in the spotlight as key players of cancer initiation and progression, the importance of the nervous system in gastrointestinal cancers has only been highlighted in the past few years. Although extrinsic innervations modulate gastrointestinal cancers, cells and signals from the gut's intrinsic innervation also have the ability to do so. Here, we shed light on this thriving field and discuss neural influences during gastrointestinal carcinogenesis. We focus on the interactions between neurons and components of the gastrointestinal tract and tumour microenvironment, on the neural signalling pathways involved, and how these factors affect the cancer hallmarks, and discuss the neural signatures in gastrointestinal cancers. Finally, we highlight neural-related therapies that have potential for the management of gastrointestinal cancers.

The gut brain in a dish: Murine primary enteric nervous system cell cultures.

BACKGROUND: The enteric nervous system (ENS) is an extensive neural network embedded in the wall of the gastrointestinal tract that regulates digestive function and gastrointestinal homeostasis. The ENS consists of two main cell types; enteric neurons and enteric glial cells. In vitro techniques allow simplified investigation of ENS function, and different culture methods have been developed over the years helping to understand the role of ENS cells in health and disease. PURPOSE: This review focuses on summarizing and comparing available culture protocols for the generation of primary ENS cells from adult mice, including dissection of intestinal segments, enzymatic digestions, surface coatings, and culture media. In addition, the potential of human ENS cultures is also discussed.

Intestinal multicellular organoids to study colorectal cancer.

Modeling colorectal cancer (CRC) using organoids has burgeoned in the last decade, providing enhanced in vitro models to study the development and possible treatment options for this type of cancer. In this review, we describe both normal and CRC intestinal organoid models and their utility in the cancer research field. Besides highlighting studies that develop epithelial CRC organoid models, i.e. organoids without tumor microenvironment (TME) cellular components, we emphasize on the need for TME in CRC modeling, to help reduce translational disparities in this area. Also, we discuss the utilization of CRC organoids derived from pluripotent stem cells, as well as their potential to be used in cancer research. Finally, limitations and challenges in the current CRC organoids field, are discussed.

Redox Imbalance in Idiopathic Pulmonary Fibrosis: A Role for Oxidant Cross-Talk Between NADPH Oxidase Enzymes and Mitochondria.

Significance: Idiopathic pulmonary fibrosis (IPF) is a progressive age-related lung disease with a median survival of only 3 years after diagnosis. The pathogenic mechanisms behind IPF are not clearly understood, and current therapeutic approaches have not been successful in improving disease outcomes. Recent Advances: IPF is characterized by increased production of reactive oxygen species (ROS), primarily by NADPH oxidases (NOXes) and mitochondria, as well as altered antioxidant defenses. Recent studies have identified the NOX isoform NOX4 as a key player in various important aspects of IPF pathology. In addition, mitochondrial dysfunction is thought to enhance pathological features of IPF, in part by increasing mitochondrial ROS (mtROS) production and altering cellular metabolism. Recent findings indicate reciprocal interactions between NOX enzymes and mitochondria, which affect regulation of NOX activity as well as mitochondrial function and mtROS production, and collectively promote epithelial injury and profibrotic signaling. Critical Issues and Future Directions: The precise molecular mechanisms by which ROS from NOX or mitochondria contribute to IPF pathology are not known. This review summarizes the current knowledge with respect to the various aspects of ROS imbalance in the context of IPF and its proposed roles in disease development, with specific emphasis on the importance of inappropriate NOX activation, mitochondrial dysfunction, and the emerging evidence of NOX-mitochondria cross-talk as important drivers in IPF pathobiology.