Nociceptor neurons affect cancer immunosurveillance.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems1-5. Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)-one such nociceptor-produced neuropeptide-directly increases the exhaustion of cytotoxic CD8+ T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8+ T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8+ T cells, Ramp1-/- CD8+ T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1-deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1-expressing CD8+ T cells were more exhausted than their RAMP1-negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8+ T cells.

Tumor-associated genetic amplifications impact extracellular vesicle miRNA cargo and their recruitment of nerves in head and neck cancer.

Cancer neuroscience is an emerging field of cancer biology focused on defining the interactions and relationships between the nervous system, developing malignancies, and their environments. Our previous work demonstrates that small extracellular vesicles (sEVs) released by head and neck squamous cell carcinomas (HNSCCs) recruit loco-regional nerves to the tumor. sEVs contain a diverse collection of biological cargo, including microRNAs (miRNAs). Here, we asked whether two genes commonly amplified in HNSCC, CCND1, and PIK3CA, impact the sEV miRNA cargo and, subsequently, sEV-mediated tumor innervation. To test this, we individually overexpressed these genes in a syngeneic murine HNSCC cell line, purified their sEVs, and tested their neurite outgrowth activity on dorsal root ganglia (DRG) neurons in vitro. sEVs purified from Ccnd1-overexpressing cells significantly increased neurite outgrowth of DRG compared to sEVs from parental or Pik3ca over-expressing cells. When implanted into C57BL/6 mice, Ccnd1 over-expressing tumor cells promoted significantly more tumor innervation in vivo. qPCR analysis of sEVs shows that increased expression of Ccnd1 altered the packaging of miRNAs (miR-15-5p, miR-17-5p, and miR-21-5p), many of which target transcripts important in regulating axonogenesis. These data indicate that genetic amplifications harbored by malignancies impose changes in sEV miRNA cargo, which can influence tumorc innervation.

Multiple divergent haplotypes express completely distinct sets of class I MHC genes in zebrafish.

The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines; however, histocompatibility in zebrafish remains poorly understood. We examined a set of diverse zebrafish class I major histocompatibility complex (MHC) genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching has been shown to improve engraftment after hematopoietic transplantation. Using flanking gene polymorphisms, we identified six distinct chromosome 19 haplotypes. We describe several novel class I U lineage genes and characterize their sequence properties, expression, and haplotype distribution. Altogether, ten full-length zebrafish class I genes were analyzed, mhc1uba through mhc1uka. Expression data and sequence properties indicate that most are candidate classical genes. Several substitutions in putative peptide anchor residues, often shared with deduced MHC molecules from additional teleost species, suggest flexibility in antigen binding. All ten zebrafish class I genes were uniquely assigned among the six haplotypes, with dominant or codominant expression of one to three genes per haplotype. Interestingly, while the divergent MHC haplotypes display variable gene copy number and content, the different genes appear to have ancient origin, with extremely high levels of sequence diversity. Furthermore, haplotype variability extends beyond the MHC genes to include divergent forms of psmb8. The many disparate haplotypes at this locus therefore represent a remarkable form of genomic region configuration polymorphism. Defining the functional MHC genes within these divergent class I haplotypes in zebrafish will provide an important foundation for future studies in immunology and transplantation.

Tumor-infiltrating nerves functionally alter brain circuits and modulate behavior in a mouse model of head-and-neck cancer.

Cancer patients often experience changes in mental health, prompting an exploration into whether nerves infiltrating tumors contribute to these alterations by impacting brain functions. Using a male mouse model for head and neck cancer, we utilized neuronal tracing techniques and show that tumor-infiltrating nerves indeed connect to distinct brain areas via the ipsilateral trigeminal ganglion. The activation of this neuronal circuitry led to behavioral alterations represented by decreased nest-building, increased latency to eat a cookie, and reduced wheel running. Tumor-infiltrating nociceptor neurons exhibited heightened activity, as indicated by increased calcium mobilization. Correspondingly, the specific brain regions receiving these neural projections showed elevated cFos and delta FosB expression in tumor-bearing mice, alongside markedly intensified calcium responses compared to non-tumor-bearing counterparts. The genetic elimination of nociceptor neurons in tumor-bearing mice led to decreased brain Fos expression and mitigated the behavioral alterations induced by the presence of the tumor. While analgesic treatment successfully restored behaviors involving oral movements to normalcy in tumor-bearing mice, it did not have a similar therapeutic effect on voluntary wheel running. This discrepancy points towards an intricate relationship, where pain is not the exclusive driver of such behavioral shifts. Unraveling the interaction between the tumor, infiltrating nerves, and the brain is pivotal to developing targeted interventions to alleviate the mental health burdens associated with cancer. Significance Statement: Head and neck cancers are infiltrated by sensory nerves which connect to a pre-existing circuit that includes areas in the brain. Neurons within this circuit are altered and mediate modifications in behavior.

TUMOR-INFILTRATING NOCICEPTOR NEURONS PROMOTE IMMUNOSUPPRESSION.

Nociceptor neurons impact tumor immunity. Removing nociceptor neurons reduced myeloid-derived suppressor cell (MDSCs) tumor infiltration in mouse models of head and neck carcinoma and melanoma. Carcinoma-released small extracellular vesicles (sEVs) attract nociceptive nerves to tumors. sEV-deficient tumors fail to develop in mice lacking nociceptor neurons. Exposure of dorsal root ganglia (DRG) neurons to cancer sEVs elevated expression of Substance P, IL-6 and injury-related neuronal markers while treatment with cancer sEVs and cytotoxic CD8 T-cells induced an immunosuppressive state (increased exhaustion ligands and cytokines). Cancer patient sEVs enhanced DRG responses to capsaicin, indicating increased nociceptor sensitivity. Conditioned media from DRG and cancer cell co-cultures promoted expression of MDSC markers in primary bone marrow cells while DRG conditioned media together with cancer sEVs induced checkpoint expression on T-cells. Our findings indicate that nociceptor neurons facilitate CD8+ T cell exhaustion and enhance MDSC infiltration. Targeting nociceptor-released IL-6 emerges as a novel strategy to disrupt harmful neuro-immune interactions in cancer and enhance anti-tumor immunity.

Tumor-infiltrating nerves functionally alter brain circuits and modulate behavior in a mouse model of head-and-neck cancer.

Cancer patients often experience changes in mental health, prompting an exploration into whether nerves infiltrating tumors contribute to these alterations by impacting brain functions. Using a mouse model for head and neck cancer and neuronal tracing, we show that tumor-infiltrating nerves connect to distinct brain areas. The activation of this neuronal circuitry altered behaviors (decreased nest-building, increased latency to eat a cookie, and reduced wheel running). Tumor-infiltrating nociceptor neurons exhibited heightened calcium activity and brain regions receiving these neural projections showed elevated Fos as well as increased calcium responses compared to non-tumor-bearing counterparts. The genetic elimination of nociceptor neurons decreased brain Fos expression and mitigated the behavioral alterations induced by the presence of the tumor. While analgesic treatment restored nesting and cookie test behaviors, it did not fully restore voluntary wheel running indicating that pain is not the exclusive driver of such behavioral shifts. Unraveling the interaction between the tumor, infiltrating nerves, and the brain is pivotal to developing targeted interventions to alleviate the mental health burdens associated with cancer.

A lot of cancer survivors experience a decline in mental health, persisting often decades after successful treatment. Many factors contribute to this reduced mental well-being, including the physical, emotional and financial stresses they experience. Scientists think that the increased prevalence of mental health disorders among cancer patients and survivors may also be linked to the cancer itself. Previous research has shown that most tumors, in particular in melanomas, cervical and ovarian cancers, and head and neck cancers, contain sensory nerves that sense thermal, mechanical and chemical changes and so alert an organism about a potential danger, such as extreme temperature, pressure, changes in pH or inflammation. To investigate whether these nerves contribute to the worsened mental health of cancer patients, Barr, Walz et al. studied male mice with tumors growing in their mouths, mimicking the disease of patients with head and neck cancers. The mice with tumors exhibited several altered behaviors linked to their well-being, suggesting that they had reduced overall health compared to mice without tumors. For example, they were less inclined to build nests, accept treats or run on a wheel. Next, Barr, Walz et al. injected a fluorescent dye into the tumors to label the nerves inside the cancerous growths. Fluorescence microscopy and imaging studies revealed that, days later, the dye had traveled to multiple regions of the brain, indicating that the nerves in the tumors had connected to a preexisting nerve circuit that included these brain regions. Further experiments revealed that the nerve cells in these brain regions were more active in mice with tumors and had different functional properties compared to mice without tumors. Removing the connecting nerves either genetically or with a drug improved all the behaviors of the mice with tumors. Treating the mice with painkillers also improved some but not all of their behaviors, indicating that pain is not the exclusive driver of such behavioral shifts. These two experiments suggest that the nerves from the tumors relay information about pain to the brain and contribute to reduced well-being of the mice. Further studies will test whether these tumor-brain connections also contribute to behavioral changes in mice with other types of cancer. The data suggest that disrupting the neural connections between a tumor and the brain may improve the mental health of patients with cancer, but more research is needed to establish this link.

Single-Cell Evolutionary Analysis Reveals Drivers of Plasticity and Mediators of Chemoresistance in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is often a heterogeneous tumor, where dynamic regulation of key transcription factors can drive multiple populations of phenotypically different cells which contribute differentially to tumor dynamics. This tumor is characterized by a very low 2-year survival rate, high rates of metastasis, and rapid acquisition of chemoresistance. The heterogeneous nature of this tumor makes it difficult to study and to treat, as it is not clear how or when this heterogeneity arises. Here we describe temporal, single-cell analysis of SCLC to investigate tumor initiation and chemoresistance in both SCLC xenografts and an autochthonous SCLC model. We identify an early population of tumor cells with high expression of AP-1 network genes that are critical for tumor growth. Furthermore, we have identified and validated the cancer testis antigens (CTA) PAGE5 and GAGE2A as mediators of chemoresistance in human SCLC. CTAs have been successfully targeted in other tumor types and may be a promising avenue for targeted therapy in SCLC. IMPLICATIONS: Understanding the evolutionary dynamics of SCLC can shed light on key mechanisms such as cellular plasticity, heterogeneity, and chemoresistance.

Functional neuronal circuits promote disease progression in cancer.

The molecular and functional contributions of intratumoral nerves to disease remain largely unknown. We localized synaptic markers within tumors suggesting that these nerves form functional connections. Consistent with this, electrophysiological analysis shows that malignancies harbor significantly higher electrical activity than benign disease or normal tissues. We also demonstrate pharmacologic silencing of tumoral electrical activity. Tumors implanted in transgenic animals lacking nociceptor neurons show reduced electrical activity. These data suggest that intratumoral nerves remain functional at the tumor bed. Immunohistochemical staining demonstrates the presence of the neuropeptide, Substance P (SP), within the tumor space. We show that tumor cells express the SP receptor, NK1R, and that ligand/receptor engagement promotes cellular proliferation and migration. Our findings identify a mechanism whereby intratumoral nerves promote cancer progression.

Neural regulations of the tumor microenvironment.

The identification of nerves in the tumor microenvironment has ushered in a new area of research in cancer biology. Numerous studies demonstrate the presence of various types of peripheral nerves (sympathetic, parasympathetic, sensory) within the tumor microenvironment; moreover, an increased density of nerves in the tumor microenvironment correlates with worse prognosis. In this review, we address the current understanding of nerve-mediated alterations of the tumor microenvironment and how they impact disease through a variety of processes, including direct nerve-cancer cell communication, alteration of the infiltrative immune population, and alteration of stromal components.

Intra-Tumoral Nerve-Tracing in a Novel Syngeneic Model of High-Grade Serous Ovarian Carcinoma.

Dense tumor innervation is associated with enhanced cancer progression and poor prognosis. We observed innervation in breast, prostate, pancreatic, lung, liver, ovarian, and colon cancers. Defining innervation in high-grade serous ovarian carcinoma (HGSOC) was a focus since sensory innervation was observed whereas the normal tissue contains predominantly sympathetic input. The origin, specific nerve type, and the mechanisms promoting innervation and driving nerve-cancer cell communications in ovarian cancer remain largely unknown. The technique of neuro-tracing enhances the study of tumor innervation by offering a means for identification and mapping of nerve sources that may directly and indirectly affect the tumor microenvironment. Here, we establish a murine model of HGSOC and utilize image-guided microinjections of retrograde neuro-tracer to label tumor-infiltrating peripheral neurons, mapping their source and circuitry. We show that regional sensory neurons innervate HGSOC tumors. Interestingly, the axons within the tumor trace back to local dorsal root ganglia as well as jugular-nodose ganglia. Further manipulations of these tumor projecting neurons may define the neuronal contributions in tumor growth, invasion, metastasis, and responses to therapeutics.

Reduced interleukin 1A gene expression in the dorsolateral prefrontal cortex of individuals with PTSD and depression.

The inflammatory system has been implicated in the pathophysiology of a variety of psychiatric conditions. Individuals with PTSD, depression, and other fear- and anxiety-related disorders exhibit alterations in peripheral circulating inflammatory markers, suggesting dysregulation of the inflammatory system. The relationship between inflammation and PTSD has been investigated almost exclusively in the periphery, and has not been extensively explored in human postmortem brain tissue. Interleukins (ILs) represent a subtype of cytokines and are key signaling proteins in the immune and inflammatory systems. Based on prior research implicating IL signaling in PTSD and depression, we performed a preliminary investigation of IL gene expression in a region of the cortex involved in emotion regulation and PTSD, the dorsolateral prefrontal cortex (dlPFC), using tissue from the newly established VA National PTSD Brain Bank. Gene expression analyses were conducted on post-mortem tissue from the dlPFC from 50 donors: 13 controls, 12 PTSD cases, and 25 depressed cases. RNA was extracted from frozen dlPFC tissue, reverse transcribed to cDNA, and quantitative polymerase chain reaction (qPCR) was performed to assess gene expression of IL1A, IL1B, IL6, IL8, IL10, IL13, and IL15. We found a multiple-testing corrected significant decrease in IL1A expression in the dlPFC for PTSD and depression cases compared to controls (p < 0.005) with age at death, sex, race and RNA integrity number (RIN) included as covariates. To our knowledge this finding is the first demonstration of altered IL expression in brain tissue from deceased individuals with histories of PTSD and/or depression.