Roadmap for the Emerging Field of Cancer Neuroscience.

Mounting evidence indicates that the nervous system plays a central role in cancer pathogenesis. In turn, cancers and cancer therapies can alter nervous system form and function. This Commentary seeks to describe the burgeoning field of "cancer neuroscience" and encourage multidisciplinary collaboration for the study of cancer-nervous system interactions.

Machine learning approaches reveal subtle differences in breathing and sleep fragmentation in Phox2b-derived astrocytes ablated mice.

Modern neurophysiology research requires the interrogation of high-dimensionality data sets. Machine learning and artificial intelligence (ML/AI) workflows have permeated into nearly all aspects of daily life in the developed world but have not been implemented routinely in neurophysiological analyses. The power of these workflows includes the speed at which they can be deployed, their availability of open-source programming languages, and the objectivity permitted in their data analysis. We used classification-based algorithms, including random forest, gradient boosted machines, support vector machines, and neural networks, to test the hypothesis that the animal genotypes could be separated into their genotype based on interpretation of neurophysiological recordings. We then interrogate the models to identify what were the major features utilized by the algorithms to designate genotype classification. By using raw EEG and respiratory plethysmography data, we were able to predict which recordings came from genotype class with accuracies that were significantly improved relative to the no information rate, although EEG analyses showed more overlap between groups than respiratory plethysmography. In comparison, conventional methods where single features between animal classes were analyzed, differences between the genotypes tested using baseline neurophysiology measurements showed no statistical difference. However, ML/AI workflows successfully were capable of providing successful classification, indicating that interactions between features were different in these genotypes. ML/AI workflows provide new methodologies to interrogate neurophysiology data. However, their implementation must be done with care so as to provide high rigor and reproducibility between laboratories. We provide a series of recommendations on how to report the utilization of ML/AI workflows for the neurophysiology community.NEW & NOTEWORTHY ML/AI classification workflows are capable of providing insight into differences between genotypes for neurophysiology research. Analytical techniques utilized in the neurophysiology community can be augmented by implementing ML/AI workflows. Random forest is a robust classification algorithm for respiratory plethysmography data. Utilization of ML/AI workflows in neurophysiology research requires heightened transparency and improved community research standards.

Acute exposure to low-level light at night is sufficient to induce neurological changes and depressive-like behavior.

The advent and wide-spread adoption of electric lighting over the past century has profoundly affected the circadian organization of physiology and behavior for many individuals in industrialized nations; electric lighting in homes, work environments, and public areas have extended daytime activities into the evening, thus, increasing night-time exposure to light. Although initially assumed to be innocuous, chronic exposure to light at night (LAN) is now associated with increased incidence of cancer, metabolic disorders, and affective problems in humans. However, little is known about potential acute effects of LAN. To determine whether acute exposure to low-level LAN alters brain function, adult male, and female mice were housed in either light days and dark nights (LD; 14 h of 150 lux:10 h of 0 lux) or light days and low level light at night (LAN; 14 h of 150 lux:10 h of 5 lux). Mice exposed to LAN on three consecutive nights increased depressive-like responses compared to mice housed in dark nights. In addition, female mice exposed to LAN increased central tendency in the open field. LAN was associated with reduced hippocampal vascular endothelial growth factor-A (VEGF-A) in both male and female mice, as well as increased VEGFR1 and interleukin-1ß mRNA expression in females, and reduced brain derived neurotrophic factor mRNA in males. Further, LAN significantly altered circadian rhythms (activity and temperature) and circadian gene expression in female and male mice, respectively. Altogether, this study demonstrates that acute exposure to LAN alters brain physiology and can be detrimental to well-being in otherwise healthy individuals.

Time-Restricted Feeding Alters the Innate Immune Response to Bacterial Endotoxin.

An important entraining signal for the endogenous circadian clock, independent of light, is food intake. The circadian and immune systems are linked; forced desynchrony of the circadian clock via nighttime light exposure or genetic ablation of core clock components impairs immune function. The timing of food intake affects various aspects of the circadian clock, but its effects on immune function are unknown. We tested the hypothesis that temporal desynchrony of food intake alters innate immune responses. Adult male Swiss Webster mice were provided with food during the night, the day, or ad libitum for 4 wk, followed by administration of LPS prior to the onset of either the active phase (zeitgeber time [ZT]12: Experiment 1) or the inactive phase (ZT0: Experiment 2). Three hours after LPS administration, blood was collected, and serum was tested for bacteria-killing capacity against Escherichia coli, as a functional assay of immune function. Additionally, cytokine expression was examined in the serum (protein), spleen, and hypothalamus (mRNA). Day-fed mice suppressed bacteria-killing capacity and serum cytokine responses to LPS during the active phase (ZT12). Night-fed mice increased bactericidal capacity, as well as serum and hypothalamic mRNA responses of certain proinflammatory cytokines during the active phase. Only day-fed mice enhanced serum cytokine responses when LPS challenge occurred during the inactive phase (ZT0); this did not result in enhanced bactericidal capacity. These data suggest that mistimed feeding has functional relevance for immune function and provide further evidence for the integration of the circadian, metabolic, and immune systems.

Photoperiodic regulation of behavior: Peromyscus as a model system.

Winter and summer present vastly different challenges to animals living outside of the tropics. To survive and reproduce, individuals must anticipate seasonal environmental changes and adjust physiology and behavior accordingly. Photoperiod (day length) offers a relatively 'noise free' environmental signal that non-tropical animals use to tell the time of year, and whether winter is approaching or receding. In some cases, photoperiodic signals may be fine-tuned by other proximate cues such as food availability or temperature. The pineal hormone, melatonin, is a primary physiological transducer of the photoperiodic signal. It tracks night length and provokes changes in physiology and behavior at appropriate times of the year. Because of their wide latitudinal distribution, Peromyscus has been well studied in the context of photoperiodic regulation of physiology and behavior. Here, we discuss how photoperiodic signals are transduced by pineal melatonin, how melatonin acts on target tissues, and subsequent consequences for behavior. Using a life-history paradigm involving trade-offs between the immune and reproductive systems, specific emphasis is placed on aggression, metabolism, and cognition. We discuss future directions including examining the effects of light pollution on photoperiodism, genetic manipulations to test the role of specific genes in the photoperiodic response, and using Peromyscus to test evolutionary theories of aging.

The role of PHOX2B-derived astrocytes in chemosensory control of breathing and sleep homeostasis.

KEY POINTS: The embryonic PHOX2B-progenitor domain generates neuronal and glial cells which together are involved in chemosensory control of breathing and sleep homeostasis. Ablating PHOX2B-derived astrocytes significantly contributes to secondary hypoxic respiratory depression as well as abnormalities in sleep homeostasis. PHOX2B-derived astrocyte ablation results in axonal pathologies in the retrotrapezoid nucleus. ABSTRACT: We identify in mice a population of ∼800 retrotrapezoid nucleus (RTN) astrocytes derived from PHOX2B-positive, OLIG3-negative progenitor cells, that interact with PHOX2B-expressing RTN chemosensory neurons. PHOX2B-derived astrocyte ablation during early life results in adult-onset O2 chemoreflex deficiency. These animals also display changes in sleep homeostasis, including fragmented sleep and disturbances in delta power after sleep deprivation, all without observable changes in anxiety or social behaviours. Ultrastructural evaluation of the RTN demonstrates that PHOX2B-derived astrocyte ablation results in features characteristic of degenerative neuro-axonal dystrophy, including abnormally dilated axon terminals and increased amounts of synapses containing autophagic vacuoles/phagosomes. We conclude that PHOX2B-derived astrocytes are necessary for maintaining a functional O2 chemosensory reflex in the adult, modulate sleep homeostasis, and are key regulators of synaptic integrity in the RTN region, which is necessary for the chemosensory control of breathing. These data also highlight how defects in embryonic development may manifest as neurodegenerative pathology in an adult.

Molecular Mechanisms of Cancer-Induced Sleep Disruption.

Sleep is essential for health. Indeed, poor sleep is consistently linked to the development of systemic disease, including depression, metabolic syndrome, and cognitive impairments. Further evidence has accumulated suggesting the role of sleep in cancer initiation and progression (primarily breast cancer). Indeed, patients with cancer and cancer survivors frequently experience poor sleep, manifesting as insomnia, circadian misalignment, hypersomnia, somnolence syndrome, hot flushes, and nightmares. These problems are associated with a reduction in the patients' quality of life and increased mortality. Due to the heterogeneity among cancers, treatment regimens, patient populations and lifestyle factors, the etiology of cancer-induced sleep disruption is largely unknown. Here, we discuss recent advances in understanding the pathways linking cancer and the brain and how this leads to altered sleep patterns. We describe a conceptual framework where tumors disrupt normal homeostatic processes, resulting in aberrant changes in physiology and behavior that are detrimental to health. Finally, we discuss how this knowledge can be leveraged to develop novel therapeutic approaches for cancer-associated sleep disruption, with special emphasis on host-tumor interactions.

Enduring effects of perinatal nicotine exposure on murine sleep in adulthood.

The long-term consequences of early life nicotine exposure are poorly defined. Approximately 8-10% of women report smoking during pregnancy, and this may promote aberrant development in the offspring. To this end, we investigated potential enduring effects of perinatal nicotine exposure on murine sleep and affective behaviors in adulthood (~13-15 wk of age) in C57Bl6j mice. Mothers received a water bottle containing 200 µg/ml nicotine bitartrate dihydrate in 2% wt/vol saccharin or pH-matched 2% saccharin with 0.2% (vol/vol) tartaric acid throughout pregnancy and before weaning. Upon reaching adulthood, offspring were tested in the open field and elevated plus maze, as well as the forced swim and sucrose anhedonia tests. Nicotine-exposed male (but not female) mice had reduced mobility in the open field, but no differences were observed in anxiety-like or depressive-like responses. Upon observing this male-specific phenotype, we further assessed sleep-wake states via wireless EEG/EMG telemetry. Following baseline recording, we assessed whether mice exposed to nicotine altered their homeostatic response to 5 h of total sleep deprivation and whether nicotine influenced responses to a powerful somnogen [i.e., lipopolysaccharides (LPS)]. Males exposed to perinatal nicotine decreased the percent time spent awake and increased time in non-rapid eye movement (NREM) sleep, without changes to REM sleep. Nicotine-exposed males also displayed exaggerated responses (increased time asleep and NREM spectral power) to sleep deprivation. Nicotine-exposed animals additionally had blunted EEG slow-wave responses to LPS administration. Together, our data suggest that perinatal nicotine exposure has long-lasting effects on normal sleep and homeostatic sleep processes into adulthood.

Neuroendocrine control of photoperiodic changes in immune function.

Seasonal variation in immune function putatively maximizes survival and reproductive success. Day length (photoperiod) is the most potent signal for time of year. Animals typically organize breeding, growth, and behavior to adapt to spatial and temporal niches. Outside the tropics individuals monitor photoperiod to support adaptations favoring survival and reproductive success. Changes in day length allow anticipation of seasonal changes in temperature and food availability that are critical for reproductive success. Immune function is typically bolstered during winter, whereas reproduction and growth are favored during summer. We provide an overview of how photoperiod influences neuronal function and melatonin secretion, how melatonin acts directly and indirectly to govern seasonal changes in immune function, and the manner by which other neuroendocrine effectors such as glucocorticoids, prolactin, thyroid, and sex steroid hormones modulate seasonal variations in immune function. Potential future research avenues include commensal gut microbiota and light pollution influences on photoperiodic responses.

Light at night, clocks and health: from humans to wild organisms.

The increasing use of electric lights has modified the natural light environment dramatically, posing novel challenges to both humans and wildlife. Indeed, several biomedical studies have linked artificial light at night to the disruption of circadian rhythms, with important consequences for human health, such as the increasing occurrence of metabolic syndromes, cancer and reduced immunity. In wild animals, light pollution is associated with changes in circadian behaviour, reproduction and predator-prey interactions, but we know little about the underlying physiological mechanisms and whether wild species suffer the same health problems as humans. In order to fill this gap, we advocate the need for integrating ecological studies in the field, with chronobiological approaches to identify and characterize pathways that may link temporal disruption caused by light at night and potential health and fitness consequences.

Cytotoxic chemotherapy increases sleep and sleep fragmentation in non-tumor-bearing mice.

Sleep disruption ranks among the most common complaints of breast cancer patients undergoing chemotherapy. Because of the complex interactions among cancer, treatment regimens, and life-history traits, studies to establish a causal link between chemotherapy and sleep disruption are uncommon. To investigate how chemotherapy acutely influences sleep, adult female c57bl/6 mice were ovariectomized and implanted with wireless biotelemetry units. EEG/EMG biopotentials were collected over the course of 3days pre- and post-injection of 13.5mg/kg doxorubicin and 135mg/kg cyclophosphamide or the vehicle. We predicted that cyclophosphamide+doxorubicin would disrupt sleep and increase central proinflammatory cytokine expression in brain areas that govern vigilance states (i.e., hypothalamus and brainstem). The results largely support these predictions; a single chemotherapy injection increased NREM and REM sleep during subsequent active (dark) phases; this induced sleep was fragmented and of low quality. Mice displayed marked increases in low theta (5-7Hz) to high theta (7-10Hz) ratios following chemotherapy treatment, indicating elevated sleep propensity. The effect was strongest during the first dark phase following injection, but mice displayed disrupted sleep for the entire 3-day duration of post-injection sleep recording. Vigilance state timing was not influenced by treatment, suggesting that acute chemotherapy administration alters sleep homeostasis without altering sleep timing. qPCR analysis revealed that disrupted sleep was accompanied by increased IL-6 mRNA expression in the hypothalamus. Together, these data implicate neuroinflammation as a potential contributor to sleep disruption after chemotherapy.

The cytokine receptor Fn14 is a molecular brake on neuronal activity that mediates circadian function in vivo.

To survive, organisms must adapt to a staggering diversity of environmental signals, ranging from sensory information to pathogenic infection, across the lifespan. At the same time, organisms intrinsically generate biological oscillations, such as circadian rhythms, without input from the environment. While the nervous system is well-suited to integrate extrinsic and intrinsic cues, how the brain balances these influences to shape biological function system-wide is not well understood at the molecular level. Here, we demonstrate that the cytokine receptor Fn14, previously identified as a mediator of sensory experience-dependent synaptic refinement during brain development, regulates neuronal activity and function in adult mice in a time-of-day-dependent manner. We show that a subset of excitatory pyramidal (PYR) neurons in the CA1 subregion of the hippocampus increase Fn14 expression when neuronal activity is heightened. Once expressed, Fn14 constrains the activity of these same PYR neurons, suggesting that Fn14 operates as a molecular brake on neuronal activity. Strikingly, differences in PYR neuron activity between mice lacking or expressing Fn14 were most robust at daily transitions between light and dark, and genetic ablation of Fn14 caused aberrations in circadian rhythms, sleep-wake states, and sensory-cued and spatial memory. At the cellular level, microglia contacted fewer, but larger, excitatory synapses in CA1 in the absence of Fn14, suggesting that these brain-resident immune cells may dampen neuronal activity by modifying synaptic inputs onto PYR neurons. Finally, mice lacking Fn14 exhibited heightened susceptibility to chemically induced seizures, implicating Fn14 in disorders characterized by hyperexcitation, such as epilepsy. Altogether, these findings reveal that cytokine receptors that mediates inflammation in the periphery, such as Fn14, can also play major roles in healthy neurological function in the adult brain downstream of both extrinsic and intrinsic cues.

Next Directions in the Neuroscience of Cancers Arising outside the CNS.

SUMMARY: The field of cancer neuroscience has begun to define the contributions of nerves to cancer initiation and progression; here, we highlight the future directions of basic and translational cancer neuroscience for malignancies arising outside of the central nervous system.

Sleep Disruption and Cancer: Chicken or the Egg?

Sleep is a nearly ubiquitous phenomenon across the phylogenetic tree, highlighting its essential role in ensuring fitness across evolutionary time. Consequently, chronic disruption of the duration, timing, or structure of sleep can cause widespread problems in multiple physiological systems, including those that regulate energy balance, immune function, and cognitive capacity, among others. Many, if not all these systems, become altered throughout the course of cancer initiation, growth, metastatic spread, treatment, and recurrence. Recent work has demonstrated how changes in sleep influence the development of chronic diseases, including cancer, in both humans and animal models. A common finding is that for some cancers (e.g., breast), chronic disruption of sleep/wake states prior to disease onset is associated with an increased risk for cancer development. Additionally, sleep disruption after cancer initiation is often associated with worse outcomes. Recently, evidence suggesting that cancer itself can affect neuronal circuits controlling sleep and wakefulness has accumulated. Patients with cancer often report difficulty falling asleep, difficulty staying asleep, and severe fatigue, during and even years after treatment. In addition to the psychological stress associated with cancer, cancer itself may alter sleep homeostasis through changes to host physiology and via currently undefined mechanisms. Moreover, cancer treatments (e.g., chemotherapy, radiation, hormonal, and surgical) may further worsen sleep problems through complex biological processes yet to be fully understood. This results in a "chicken or the egg" phenomenon, where it is unclear whether sleep disruption promotes cancer or cancer reciprocally disrupts sleep. This review will discuss existing evidence for both hypotheses and present a framework through which the interactions between sleep and cancer can be dissociated and causally investigated.

Neuropeptides in Cancer: Friend and Foe?

Neuropeptides are small regulatory molecules found throughout the body, most notably in the nervous, cardiovascular, and gastrointestinal systems. They serve as neurotransmitters or hormones in the regulation of diverse physiological processes. Cancer cells escape normal growth control mechanisms by altering their expression of growth factors, receptors, or intracellular signals, and neuropeptides have recently been recognized as mitogens in cancer growth and development. Many neuropeptides and their receptors exist in multiple subtypes, coupling with different downstream signaling pathways and playing distinct roles in cancer progression. The consideration of neuropeptide/receptor systems as anticancer targets is already leading to new biological and diagnostic knowledge that has the potential to enhance the understanding and treatment of cancer. In this review, recent discoveries regarding neuropeptides in a wide range of cancers, emphasizing their mechanisms of action, signaling cascades, regulation, and therapeutic potential, are discussed. Current technologies used to manipulate and analyze neuropeptides/receptors are described. Applications of neuropeptide analogs and their receptor inhibitors in translational studies and radio-oncology are rapidly increasing, and the possibility for their integration into therapeutic trials and clinical treatment appears promising.

Cancer as a tool for preclinical psychoneuroimmunology.

Cancer represents a novel homeostatic challenge to the host system. How the brain senses and responds to changes in peripheral physiology elicited by tumor growth is a largely untapped area of research. This is especially relevant given the widespread prevalence of systemic problems that people with various types of cancer experience. These include disruptions in sleep/wake cycles, cognitive function, depression, and changes in appetite/food intake, among others. Critically, many of these problems are evident prior to diagnosis, indicating that their etiology is potentially distinct from the effects of cancer treatment or the stress of a cancer diagnosis. Psychoneuroimmunology (PNI) is well equipped to tackle these types of problems, as it uses approaches from multiple disciplines to understand how specific stimuli (endogenous and environmental) are transduced into neural, endocrine, and immune signals that ultimately regulate health and behavior. In this article, I first provide a brief historical perspective of cancer and PNI, introduce the idea of cancer as a systemic homeostatic challenge, and provide examples from preclinical literature supporting this hypothesis. Given the rise of advanced tools in neuroscience (e.g., calcium imaging), we can now monitor and manipulate genetically defined neural circuits over the extended time scales necessary to disentangle distal communication between peripheral tumors and the brain.

Cancer as a homeostatic challenge: the role of the hypothalamus.

The initiation, progression, and metastatic spread of cancer elicits diverse changes in systemic physiology. In this way, cancer represents a novel homeostatic challenge to the host system. Here, we discuss how the hypothalamus, a critical brain region involved in homeostasis senses, integrates and responds to cancer-induced changes in physiology. Through this lens, cancer-associated changes in behavior (e.g., sleep disruption) and physiology (e.g., glucocorticoid dysregulation) can be viewed as the result of an inability to re-establish homeostasis. We provide examples at each level (receptor sensing, integration of systemic signals, and efferent regulatory pathways) of how homeostatic organization becomes disrupted across different cancers. Finally, we lay out predictions of this hypothesis and highlight outstanding questions that aim to guide further work in this area.

Peripheral Lipopolyssacharide Rapidly Silences REM-Active LHGABA Neurons.

Immune factors (e.g., cytokines, chemokines) can alter the activity of neuronal circuits to promote "sickness behavior," a suite of adaptive actions that organisms exhibit in response to infection/injury in order to maximize their chances of recovery (i.e., return to homeostasis). This includes drastic alterations in sleep/wake states, locomotor activity, and food intake, among other behaviors. Despite the ample evidence highlighting interactions between the brain and systemic immunity, studies on how immune challenges alter the activity of genetically defined cell populations controlling arousal states are scarce. As the lateral hypothalamus (LH) serves a major integrative function in behavioral arousal, food intake, and monitoring and responding to changes in systemic physiology, we investigated how GABAergic neurons within this brain region alter their activity across normal sleep/wake states and in response to a peripheral immune challenge with bacterial endotoxin [lipopolysaccharides (LPS)]. Using fiber photometry (GCaMP6s Ca2+ signal) in tandem with electroencephalogram (EEG)/EMG recordings to determine arousal states, we observed that population activity of GABAergic neurons in the lateral hypothalamus (LHGABA) is highest during rapid-eye-movement sleep (REM), and this activity changes drastically across spontaneous arousal state transitions, with the lowest activity observed during non-REM sleep. Upon intraperitoneal LPS challenge, LHGABA neurons rapidly decrease their activity in tandem with elimination of REM sleep behavior (characteristic of cytokine-induced sickness). Together, these data suggest that peripheral immune challenges can rapidly (in < 40 min) alter subcortical neuronal circuits controlling arousal states. Additionally, we demonstrate that fiber photometry offers a sensitive and cell-type specific tool that can be applied to study the neuronal substrates of sickness behavior.

Neuroendocrine and Behavioral Consequences of Hyperglycemia in Cancer.

A hallmark of cancer is the disruption of cellular metabolism during the course of malignant growth. Major focus is now on how these cell-autonomous processes propagate to the tumor microenvironment and, more generally, to the entire host system. This chain of events can have major consequences for a patient's health and wellbeing. For example, metabolic "waste" produced by cancer cells activates systemic inflammatory responses, which can interfere with hepatic insulin receptor signaling and glucose homeostasis. Research is just now beginning to understand how these processes occur, and how they contribute to systemic symptoms prevalent across cancers, including hyperglycemia, fatigue, pain, and sleep disruption. Indeed, it is only recently that we have begun to appreciate that the brain does not play a passive role in responding to cancer-induced changes in physiology. In this review, we provide a brief discussion of how oncogene-directed metabolic reprogramming disrupts host metabolism, with a specific emphasis on cancer-induced hyperglycemia. We further discuss how the brain senses circulating glucose concentrations and how this process goes awry as a response to distant neoplastic growth. Finally, as glucose-sensing neurons control diverse aspects of physiology and behavior, we link cancer-induced changes in energy balance to neuroendocrine and behavioral consequences for the host organism.