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PDL1 is a protein that induces immunosuppression by binding to PD1 expressed on immune cells. In line with historical studies, we found that membrane-bound PD1 expression was largely restricted to immune cells; PD1 was not detectable at either the mRNA or protein level in peripheral neurons using single neuron qPCR, immunolabeling and flow cytometry. However, we observed widespread expression of PDL1 in both sensory and sympathetic neurons that could have important implications for patients receiving immunotherapies targeting this pathway that include unexpected autonomic and sensory related effects. While signaling pathways downstream of PD1 are well established, little to no information is available regarding the intracellular signaling downstream of membrane-bound PDL1 (also known as reverse signaling). Here, we administered soluble PD1 to engage neuronally expressed PDL1 and found that PD1 significantly reduced nocifensive behaviors evoked by algogenic capsaicin. We used calcium imaging to examine the underlying neural mechanism of this reduction and found that exogenous PD1 diminished TRPV1-dependent calcium transients in dissociated sensory neurons. Furthermore, we observed a reduction in membrane expression of TRPV1 following administration of PD1. Exogenous PD1 had no effect on pain-related behaviors in sensory neuron specific PDL1 knockout mice. These data indicate that neuronal PDL1 activation is sufficient to modulate sensitivity to noxious stimuli and as such, may be an important homeostatic mechanism for regulating acute nociception.

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR2) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR2-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared with matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR2in NaV1.8-positive neurons (Par2Nav1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1.Par2Nav1.8andLgmndeletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR2-dependent hyperexcitability of trigeminal neurons from WT female mice.Par2deletion, LI-1, and inhibitors of adenylyl cyclase or protein kinase A (PKA) prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N terminus of PAR2at Asn30↓Arg31, proximal to the canonical trypsin activation site. Lgmn activated PAR2by biased mechanisms in HEK293 cells to induce Ca2+mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not β-arrestin recruitment or PAR2endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR2by biased mechanisms that evoke cancer pain.SIGNIFICANCE STATEMENTOral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR2) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared with matched normal oral tissue. Lgmn evokes pain-like behavior through PAR2. Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR2. Inhibitors of adenylyl cyclase and protein kinase A (PKA) prevented the effects of Lgmn. Lgmn activated PAR2to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not β-arrestin recruitment or PAR2endocytosis. Thus, Lgmn is a biased agonist of PAR2that evokes cancer pain.

Head and neck squamous cell carcinoma are highly innervated by peripheral sensory neurons. Local neurotransmitter release (e.g., calcitonin gene-related peptide (CGRP)) from sensory neurons innervating cancer is linked to tumorigenesis. CGRP-immunoreactive nerve presence comprised 9.53±1.9% of total nerve area across 11 HNSCC patients. A syngeneic tongue tumor transplant mouse model of oral cancer and a global Calca knockout mouse (CGRP

Oral cancer patients report sensitivity to spicy foods and liquids. The mechanism responsible for chemosensitivity induced by oral cancer is not known. We simulate oral cancer-induced chemosensitivity in a xenograft oral cancer mouse model using two-bottle choice drinking and conditioned place aversion assays. An anatomic basis of chemosensitivity is shown in increased expression of TRPV1 in anatomically relevant trigeminal ganglion (TG) neurons in both the xenograft and a carcinogen (4-nitroquinoline 1-oxide)-induced oral cancer mouse models. The percent of retrograde labeled TG neurons that respond to TRPV1 agonist, capsaicin, is increased along with the magnitude of response as measured by calcium influx, in neurons from the cancer models. To address the possible mechanism of TRPV1 sensitivity in tongue afferents, we study the role of PAR

Evolutionarily the nervous system and immune cells have evolved to communicate with each other to control inflammation and host responses against injury. Recent findings in neuroimmune communication demonstrate that these mechanisms extend to cancer initiation and progression. Lymphoid structures and tumors, which are often associated with inflammatory infiltrate, are highly innervated by multiple nerve types (e.g. sympathetic, parasympathetic, sensory). Recent preclinical and clinical studies demonstrate that targeting the nervous system could be a therapeutic strategy to promote anti-tumor immunity while simultaneously reducing cancer-associated neurological symptoms, such as chronic pain, fatigue, and cognitive impairment. Sympathetic nerve activity is associated with physiological or psychological stress, which can be induced by tumor development and cancer diagnosis. Targeting the stress response through suppression of sympathetic activity or activation of parasympathetic activity has been shown to drive activation of effector T cells and inhibition of myeloid derived suppressor cells within the tumor. Additionally, there is emerging evidence that sensory nerves may regulate tumor growth and metastasis by promoting or inhibiting immunosuppression in a tumor-type specific manner. Since neural effects are often tumor-type specific, further study is required to optimize clinical therapeutic strategies. This review examines the emerging evidence that neuroimmune communication can regulate anti-tumor immunity as well as contribute to development of cancer-related neurological symptoms.

Simple Summary Oral cancer is often deadly and severely painful. Because this form of cancer pain cannot be adequately treated with current medications including opiates, new treatment approaches are needed. Cathepsin S, a lysosomal cysteine protease may play a role in oral cancer pain through a protease-activated receptor-2 (PAR2)-dependent mechanism. We undertook a series of experiments to define the role of cathepsin S in oral cancer pain. We compared cathepsin S activity in human oral cancer tumors versus patient-matched normal tissue; a human oral cancer cell line versus a benign dysplastic oral keratinocyte cell line; and in an orthotopic xenograft tongue cancer mouse model versus normal controls in mice. We localized cathepsin S in macrophages and carcinoma cells in human oral cancers. The injection of cathepsin S caused nociception in a mouse model while the injection of oral cancer cells in which the gene for cathepsin S is deleted generated less nociception. Our findings will lay the foundations for clinical trials of cathepsin S inhibitors for treating oral cancer pain. Abstract Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.

Head and neck squamous cell carcinoma (HNSCC) causes more severe pain and psychological stress than other types of cancer. Despite clinical evidence linking pain, stress, and cancer progression, the underlying relationship between pain and sympathetic neurotransmission in oral cancer is unknown. We found that human HNSCC tumors and mouse tumor tissue are innervated by peripheral sympathetic and sensory nerves. Moreover, β-adrenergic 1 and 2 receptors (β-ARs) are overexpressed in human oral cancer cell lines, and norepinephrine treatment increased β-AR2 protein expression as well as cancer cell proliferation in vitro. We have recently demonstrated that inhibition of tumor necrosis factor alpha (TNFα) signaling reduces oral cancer-induced nociceptive behavior. Norepinephrine-treated cancer cell lines secrete more TNFα which, when applied to tongue-innervating trigeminal neurons, evoked a larger Ca 2+ transient; TNF-TNFR inhibitor blocked the increase in the evoked Ca 2+ transient. Using an orthotopic xenograft oral cancer model, we found that mice demonstrated significantly less orofacial cancer-induced nociceptive behavior during systemic β-adrenergic inhibitory treatment with propranolol. Furthermore, chemical sympathectomy using guanethidine led to a significant reduction in tumor size and nociceptive behavior. We infer from these results that sympathetic signaling modulates oral cancer pain through TNFα secretion and tumorigenesis. Further investigation of the role of neurocancer communication in cancer progression and pain is warranted.

The demographics of head and neck cancer (HNC) survivors are changing, contributing to a growing number of survivors and a greater length of survivorship. Curative treatment involves intense multimodal therapy, which contributes to both short-term toxicities and long-term treatment-related effects. Delivering high-quality, relevant cancer survivorship care is a growing national priority. Various survivorship models and tools, such as survivorship care plans, have been utilized in an attempt to enhance care and optimize outcomes. However, an essential, yet understudied, component of high-quality survivorship care is the identification and management of late and long-term treatment-related effects. In this article, we will describe the current advancements in survivorship care as well as the research related to late and long-term treatment effects. While there is a growing body of literature that describes the prevalence of treatment-related effects and their impact on quality of life, more work is needed. Research that investigates the interplay of these complex treatment effects, the biological mechanisms that contribute to their variability, and interventions designed to mitigate them are desperately needed. While de-intensification offers the potential to alleviate these effects for future survivors, we need clinically meaningful assessment tools and therapies to provide the survivors we evaluate and treat daily. Targeted patient-reported outcomes and objective measures validated through clinical research are needed to help us systematically identify and treat late and long-term effects. In order to tailor and optimize the care we provide to our HNC survivors, we will need to leverage these tools as well as the expertise of all members of our multidisciplinary survivorship teams.

Cancer cells secrete pronociceptive mediators that sensitize adjacent sensory neurons and cause pain. Identification and characterization of these mediators could pinpoint novel targets for cancer pain treatment. In this study, we identified candidate genes in cancer cell lines that encode for secreted or cell surface proteins that may drive nociception. To undertake this work, we used an acute cancer pain mouse model, transcriptomic analysis of publicly available human tumor-derived cell line data, and a literature review. Cancer cell line supernatants were assigned a phenotype based on evoked nociceptive behavior in an acute cancer pain mouse model. We compared gene expression data from nociceptive and nonnociceptive cell lines. Our analyses revealed differentially expressed genes and pathways; many of the identified genes were not previously associated with cancer pain signaling. Epidermal growth factor receptor (EGFR) and disintegrin metalloprotease domain 17 (ADAM17) were identified as potential targets among the differentially expressed genes. We found that the nociceptive cell lines contained significantly more ADAM17 protein in the cell culture supernatant compared to nonnociceptive cell lines. Cytoplasmic EGFR was present in almost all (>90%) tongue primary afferent neurons in mice. Monoclonal antibody against EGFR, cetuximab, inhibited cell line supernatant-induced nociceptive behavior in an acute oral cancer pain mouse model. We infer from these data that ADAM17-EGFR signaling is involved in cancer mediator-induced nociception. The differentially expressed genes and their secreted protein products may serve as candidate therapeutic targets for oral cancer pain and warrant further evaluation.

Cancer invading into nerves, termed perineural invasion (PNI), is associated with pain. Here, we show that oral cancer patients with PNI report greater spontaneous pain and mechanical allodynia compared with patients without PNI, suggesting that unique mechanisms drive PNI-induced pain. We studied the impact of PNI on peripheral nerve physiology and anatomy using a murine sciatic nerve PNI model. Mice with PNI exhibited spontaneous nociception and mechanical allodynia. Perineural invasion induced afterdischarge in A high-threshold mechanoreceptors (HTMRs), mechanical sensitization (ie, decreased mechanical thresholds) in both A and C HTMRs, and mechanical desensitization in low-threshold mechanoreceptors. Perineural invasion resulted in nerve damage, including axon loss, myelin damage, and axon degeneration. Electrophysiological evidence of nerve injury included decreased conduction velocity, and increased percentage of both mechanically insensitive and electrically unexcitable neurons. We conclude that PNI-induced pain is driven by nerve injury and peripheral sensitization in HTMRs.

Oral cancer is very painful and impairs a patient’s ability to eat, talk, and drink. Mediators secreted from oral cancer can excite and sensitize sensory neurons inducing pain. Cancer mediators can also activate Schwann cells, the peripheral glia that regulates neuronal function and repair. The contribution of Schwann cells to oral cancer pain is unclear. We hypothesize that the oral cancer mediator TNFα activates Schwann cells, which further promotes cancer progression and pain. We demonstrate that TNFα is overexpressed in human oral cancer tissues and correlates with increased self-reported pain in patients. Antagonizing TNFα reduces oral cancer proliferation, cytokine production, and nociception in mice with oral cancer. Oral cancer or TNFα alone increases Schwann cell activation (measured by Schwann cell proliferation, migration, and activation markers), which can be inhibited by neutralizing TNFα. Cancer- or TNFα-activated Schwann cells release pro-nociceptive mediators such as TNFα and nerve growth factor (NGF). Activated Schwann cells induce nociceptive behaviors in mice, which is alleviated by blocking TNFα. Our study suggests that TNFα promotes cancer proliferation, progression, and nociception at least partially by activating Schwann cells. Inhibiting TNFα or Schwann cell activation might serve as therapeutic approaches for the treatment of oral cancer and associated pain.

Neuro-glial activation is a recently identified hallmark of growing cancers. Targeting tumor hyperinnervation in preclinical and small clinical trials has yielded promising antitumor effects, highlighting the need of systematic analysis of neural influences in cancer (NIC). Here, we outline the strategies translating these findings from bench to the clinic.

Bone cancer metastasis is extremely painful and decreases the quality of life of the affected patients. Available pharmacological treatments are not able to sufficiently ameliorate the pain, and as patients with cancer are living longer, new treatments for pain management are needed. Decitabine (5-aza-2'-deoxycytidine), a DNA methyltransferases inhibitor, has analgesic properties in preclinical models of postsurgical and soft-tissue oral cancer pain by inducing an upregulation of endogenous opioids. In this study, we report that daily treatment with decitabine (2 µg/g, intraperitoneally) attenuated nociceptive behavior in the 4T1-luc2 mouse model of bone cancer pain. We hypothesized that the analgesic mechanism of decitabine involved activation of the endogenous opioid system through demethylation and reexpression of the transcriptionally silenced endothelin B receptor gene, Ednrb. Indeed, Ednrb was hypermethylated and transcriptionally silenced in the mouse model of bone cancer pain. We demonstrated that expression of Ednrb in the cancer cells lead to release of β-endorphin in the cell supernatant, which reduced the number of responsive dorsal root ganglia neurons in an opioid-dependent manner. Our study supports a role of demethylating drugs, such as decitabine, as unique pharmacological agents targeting the pain in the cancer microenvironment.

Oral cancer is often painful and lethal. Oral cancer progression and pain may result from shared pathways that involve unresolved inflammation and elevated levels of pro-inflammatory cytokines. Resolvin D-series (RvDs) are endogenous lipid mediators derived from omega-3 fatty acids that exhibit pro-resolution and anti-inflammatory actions. These mediators have recently emerged as a novel class of therapeutics for diseases that involve inflammation; the specific roles of RvDs in oral cancer and associated pain are not defined. The present study investigated the potential of RvDs (RvD1 and RvD2) to treat oral cancer and alleviate oral cancer pain. We found down-regulated mRNA levels of GPR18 and GPR32 (which code for receptors RvD1 and RvD2) in oral cancer cells. Both RvD1 and RvD2 inhibited oral cancer proliferation in vitro. Using two validated mouse oral squamous cell carcinoma xenograft models, we found that RvD2, the more potent anti-inflammatory lipid mediator, significantly reduced tumor size. The mechanism of this action might involve suppression of IL-6, C-X-C motif chemokine 10 (CXCL10), and reduction of tumor necrosis. RvD2 generated short-lasting analgesia in xenograft cancer models, which coincided with decreased neutrophil infiltration and myeloperoxidase activity. Using a cancer supernatant model, we demonstrated that RvD2 reduced cancer-derived cytokines/chemokines (TNF-α, IL-6, CXCL10, and MCP-1), cancer mediator-induced CD11b+Ly6G− myeloid cells, and nociception. We infer from our results that manipulation of the endogenous pro-resolution pathway might provide a novel approach to improve oral cancer and cancer pain treatment.

Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.

Patients with oral cancer report severe pain during function. Inflammation plays a role in the oral cancer microenvironment; however, the role of immune cells and associated secretion of inflammatory mediators in oral cancer pain has not been well defined. In this study, we used 2 oral cancer mouse models: a cell line supernatant injection model and the 4-nitroquinoline-1-oxide (4NQO) chemical carcinogenesis model. We used the 2 models to study changes in immune cell infiltrate and orofacial nociception associated with oral squamous cell carcinoma (oSCC). Oral cancer cell line supernatant inoculation and 4NQO-induced oSCC resulted in functional allodynia and neuronal sensitization of trigeminal tongue afferent neurons. Although the infiltration of immune cells is a prominent component of both oral cancer models, our use of immune-deficient mice demonstrated that oral cancer-induced nociception was not dependent on the inflammatory component. Furthermore, the inflammatory cytokine, tumor necrosis factor alpha (TNFα), was identified in high concentration in oral cancer cell line supernatant and in the tongue tissue of 4NQO-treated mice with oSCC. Inhibition of TNFα signaling abolished oral cancer cell line supernatant-evoked functional allodynia and disrupted T-cell infiltration. With these data, we identified TNFα as a prominent mediator in oral cancer-induced nociception and inflammation, highlighting the need for further investigation in neural-immune communication in cancer pain.

Nanoparticle-mediated drug delivery is especially useful for targets within endosomes because of the endosomal transport mechanisms of many nanomedicines within cells. Here, we report the design of a pH-responsive, soft polymeric nanoparticle for the targeting of acidified endosomes to precisely inhibit endosomal signalling events leading to chronic pain. In chronic pain, the substance P (SP) neurokinin 1 receptor (NK(1)R) redistributes from the plasma membrane to acidified endosomes, where it signals to maintain pain. Therefore, the NK(1)R in endosomes provides an important target for pain relief. The pH-responsive nanoparticles enter cells by clathrin- and dynamin-dependent endocytosis and accumulate in NK(1)R-containing endosomes. Following intrathecal injection into rodents, the nanoparticles, containing the FDA-approved NK(1)R antagonist aprepitant, inhibit SP-induced activation of spinal neurons and thus prevent pain transmission. Treatment with the nanoparticles leads to complete and persistent relief from nociceptive, inflammatory and neuropathic nociception and offers a much-needed nonopioid treatment option for chronic pain.

Oral cancer patients report severe function-induced pain; severity is greater in females. We hypothesize that a neutrophil-mediated endogenous analgesic mechanism is responsible for sex differences in nociception secondary to oral squamous cell carcinoma (SCC). Neutrophils isolated from the cancer-induced inflammatory microenvironment contain β-endorphin protein and are identified by the Ly6G+ immune marker. We previously demonstrated that male mice with carcinogen-induced oral SCC exhibit less nociceptive behavior and a higher concentration of neutrophils in the cancer microenvironment compared to female mice with oral SCC. Oral cancer cells secrete granulocyte colony stimulating factor (G-CSF), a growth factor that recruits neutrophils from bone marrow to the cancer microenvironment. We found that recombinant G-CSF (rG-CSF, 5 μg/mouse, intraperitoneal) significantly increased circulating Ly6G+ neutrophils in the blood of male and female mice within 24 h of administration. In an oral cancer supernatant mouse model, rG-CSF treatment increased cancer-recruited Ly6G+ neutrophil infiltration and abolished orofacial nociceptive behavior evoked in response to oral cancer supernatant in both male and female mice. Local naloxone treatment restored the cancer mediator-induced nociceptive behavior. We infer that rG-CSF-induced Ly6G+ neutrophils drive an endogenous analgesic mechanism. We then evaluated the efficacy of chronic rG-CSF administration to attenuate oral cancer-induced nociception using a tongue xenograft cancer model with the HSC-3 human oral cancer cell line. Saline-treated male mice with HSC-3 tumors exhibited less oral cancer-induced nociceptive behavior and had more β-endorphin protein in the cancer microenvironment than saline-treated female mice with HSC-3 tumors. Chronic rG-CSF treatment (2.5 μg/mouse, every 72 h) increased the HSC-3 recruited Ly6G+ neutrophils, increased β-endorphin protein content in the tongue and attenuated nociceptive behavior in female mice with HSC-3 tumors. From these data, we conclude that neutrophil-mediated endogenous opioids warrant further investigation as a potential strategy for oral cancer pain treatment.

Designer receptors exclusively activated by designer drugs (DREADDs) are an advanced experimental tool that could potentially provide a novel approach to pain management. In particular, expression of an inhibitory (Gi-coupled) DREADD in nociceptors might enable ligand-dependent analgesia. To test this possibility, TRPV1-cre mice were used to restrict expression of Gi-DREADDs to predominantly C-fibers. Whereas baseline heat thresholds in both male and female mice expressing Gi-DREADD were normal, 1 mg/kg clozapine-N-oxide (CNO) produced a significant 3 h increase in heat threshold that returned to baseline by 5 h after injection. Consistent with these behavioral results, CNO decreased action potential firing in isolated sensory neurons from Gi-DREADD mice. Unexpectedly, however, the expression of Gi-DREADD in sensory neurons caused significant changes in voltage-gated Ca2+and Na+currents in the absence of CNO, as well as an increase in Na+channel (NaV1.7) expression. Furthermore, CNO-independent excitatory and inhibitory second-messenger signaling was also altered in these mice, which was associated with a decrease in the analgesic effect of endogenous inhibitory G-protein-coupled receptor activation. These results highlight the potential of this exciting technology, but also its limitations, and that it is essential to identify the underlying mechanisms for any observed behavioral phenotypes.SIGNIFICANCE STATEMENTDREADD technology is a powerful tool enabling manipulation of activity and/or transmitter release from targeted cell populations. The purpose of this study was to determine whether inhibitory DREADDs in nociceptive afferents could be used to produce analgesia, and if so, how. DREADD activation produced a ligand-dependent analgesia to heatin vivoand a decrease in neuronal firing at the single-cell level. However, we observed that expression of Gi-DREADD also causes ligand-independent changes in ion channel activity and second-messenger signaling. These findings highlight both the potential and the limitations of this exciting technology as well as the necessity to identify the mechanisms underlying any observed phenotype.

The impact of peripheral neurons on carcinogenesis is an understudied process in head and neck cancer. Peripheral nerve innervation is increased in preclinical models of oral cancer; sympathectomy resulted in smaller, less invasive cancers. Local neurotransmitter release from peripheral neurons innervating cancer has been linked to cancer growth and immune suppression. We hypothesize that local neurotransmitter release from trigeminal sensory neurons (TGNs) and sympathetic postganglionic neurons promotes cancer proliferation and suppresses the immune response. We used real-time PCR to determine RNA expression in human oral cancer cell lines and a colorimetric cell proliferation assay to quantify cancer cell growth in response to the sympathetic and sensory neurotransmitters. Oral cancer cell lines, HSC-3 and SCC-4, overexpress the norepinephrine (NE) receptor, adrenergic receptor beta 2 (ADRβ2) and sensory neurotransmitter receptors, receptor activity modifying protein1 (RAMP1) and neurokinin1 receptor (NK1R) compared to the non-tumorigenic cell line, HaCaT. Treatment with sympathetic (NE) and sensory (CGRP, substance p) neurotransmitters for 48 hours significantly increased cell proliferation in HSC-3 (>2-fold) and SCC-4 (>1.5-fold) cell lines, but had no effect on HaCaT cell proliferation. Using optical clearing and immunohistochemistry, we observed increased PGP9.5+ neuronal fiber density in the microenvironment using high-resolution microscopy in a tongue oral cancer xenograft mouse model. In vitro we measured a 44.6 ± 1.14% increase in TGN sprouting after 48-hour exposure to conditioned media from the HSC-3, compared to conditioned media from HaCaT. Lastly, HSC-3 cell culture supernatant injection (50 µl) into the tongue of C57Bl/6 female mice resulted in infiltration of CD45+ immune cells (9.10 ± 1.2 % of total live cells). Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to a 50 µl injection of HSC-3 supernatant into the tongue resulted in significantly more CD45+ immune cell infiltration (21.66 ± 3.4 %) compared to HSC-3 supernatant without pharmacologic nerve block. These results together suggest neurotransmitter-induced increase in oral cancer cell proliferation and neurotransmitter-induced suppression of the cancer-evoked immune response. Understanding the nerve-cancer interaction might improve strategies to treat head ad neck cancer by targeting peripheral neurons in the cancer microenvironment. Citation Format: Megan A. Atherton, Marci L. Nilsen, Nicole N. Scheff. Peripheral neurons invade oral squamous cell carcinoma microenvironment and drive tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-050.

Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR2), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR2-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR2, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR2, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR2-dependent hyperexcitability of nociceptors, and PAR2 association with β-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR2 A cholestanol-conjugated PAR2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.

Persistent inflammation results in an increase in the amplitude and duration of depolarization-evoked Ca2+transients in putative nociceptive afferents. Previous data indicated that these changes were the result of neither increased neuronal excitability nor an increase in the amplitude of depolarization. Subsequent data also ruled out an increase in voltage-gated Ca2+currents and recruitment of Ca2+-induced Ca2+release. Parametric studies indicated that the inflammation-induced increase in the duration of the evoked Ca2+transient required a relatively large and long-lasting increase in the concentration of intracellular Ca2+implicating the Na+/Ca2+exchanger (NCX), a major Ca2+extrusion mechanism activated with high intracellular Ca2+loads. The contribution of NCX to the inflammation-induced increase in the evoked Ca2+transient in rat sensory neurons was tested using fura-2 AM imaging and electrophysiological recordings. Changes in NCX expression and protein were assessed with real-time PCR and Western blot analysis, respectively. An inflammation-induced decrease in NCX activity was observed in a subpopulation of putative nociceptive neurons innervating the site of inflammation. The time course of the decrease in NCX activity paralleled that of the inflammation-induced changes in nociceptive behavior. The change in NCX3 in the cell body was associated with a decrease in NCX3 protein in the ganglia, an increase in the peripheral nerve (sciatic) yet no change in the central root. This single response to inflammation is associated with changes in at least three different segments of the primary afferent, all of which are likely to contribute to the dynamic response to persistent inflammation.

Cancer cells secrete pro-nociceptive mediators into the microenvironment that sensitize sensory neurons and cause pain. We sought to identify protein encoding genes that drive nociception. We used a preclinical mouse model, transcriptomic analysis, and a proteomic literature review to undertake this work. Cancer cell line supernatant was assigned a phenotype based on evoked nociceptive behavior in a cancer pain mouse model. Mice received 50μl injections of supernatant in the tongue for 3 days followed by orofacial behavior assessment with a dolognawmeter 1 hour after the third injection. Data from the NCBI Gene Expression Omnibus was used to performdifferential gene expression and pathway analysis. We identified proteins potentially responsible for nociception using a literature review of the nociceptive cell lines. Mediator targets produced by these analyses were validated with the cancer pain mouse model. We identified 4 nociceptive and 4 non-nociceptive cell lines. We subsequently identified 1760 differentially expressed genes (DEG) and 14 significantly perturbed pathways by comparing the nociceptive and non-nociceptive cell lines. Thirty of 1760 DEG were reported in the literature to yield over-expressed proteins secreted or anchored to the plasma membrane in at least one of the nociceptive cell lines tested. For these 30 DEG, epidermal growth factor receptor (EGFR) was the most commonly cited druggable target (Drug–Gene Product Interaction Database). To determine if EGFR signaling is involved in cancer supernatant-induced nociception, we administered either EGFR antagonist, Cetuximab, or saline followed by nociceptive cell line supernatant injected into the mouse tongue. Mice that received saline exhibited significantly longer gnaw-time following injection of cancer supernatant (48.8±4.5%) compared to mice that received Cetuximab (13.8±11.8%). DEG and their secreted protein products may serve as therapeutic targets for cancer pain and warrant further evaluation.

Tongue cancer patients suffer severe, chronic pain driven by oral cancer-induced activation of sensory neurons innervating the oral cavity. However, the impact of sensory neurotransmission on oral carcinogenesis is understudied. Local neurotransmitter release from sensory neurons innervating the cancer has been linked to cancer growth and immune suppression. We hypothesize that oral cancer-induced neurotransmitter release from trigeminal sensory neurons (TGNs) promotes oral cancer proliferation and suppresses the immune response. To investigate sensory neuron subtypes that may impact cancer in the oral cavity, we characterized TGNs that innervate the tongue using a retrograde tracer, DiI, and immunohistochemistry for neuropeptides. We used a colorimetric MTS assay kit to quantify cancer cell proliferation in response to supernatant from TGN cultures. Using flow cytometry, we tested whether pharmacologic inhibition of peripheral sensory neurons impacted inflammation in response to oral cancer. We found that a majority of TGNs innervating the tongue are peptidergic and express neurotransmitters calcitonin-gene related peptide (CGRP, 64.3±13.2%) and substance P (SP, 44.1± 9.9%). Oral cancer cell line, HSC-3, exposed to TGN culture supernatant for 48 hours had a 2.00±0.04-fold increase in proliferation compared to Dulbecco's Modified Eagle Medium (DMEM) cell culture media. HSC-3 cell culture supernatant injection (50µl) into the tongue of C57Bl/6 female mice resulted in infiltration of CD45+ immune cells (9.10±1.2% of total live cells). Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to a 50µl injection of HSC-3 supernatant into the tongue resulted in significantly more CD45+ immune cell infiltration (21.66±3.4%) compared to HSC-3 supernatant without pharmacologic nerve block. These results suggest neurotransmitter-induced increase in oral cancer cell proliferation and neurotransmitter-induced suppression of the cancer-evoked immune response. Understanding the nerve-cancer interaction may improve strategies to treat oral cancer and pain by targeting sensory neurons.

Approximately 20% of the adult population suffers from migraine. This debilitating pain disorder is three times more prevalent in women than in men. To begin to evaluate the underlying mechanisms that may contribute to this sex difference, we tested the hypothesis that there is a sex difference in the inflammatory mediator (IM)-induced sensitization of dural afferents. Acutely dissociated retrogradely labeled dural afferents from adult Sprague-Dawley rats were examined with whole cell patch-clamp recordings. Baseline passive and active electrophysiological properties of dural afferents from both sexes were comparable. However, while IM-induced increases in the excitability of dural afferents from male and female rats were also comparable, the proportion of dural afferents from female rats sensitized by IM (∼100%) was significantly greater than that of dural afferents from male rats (∼50%). This appeared to be due to differences downstream of IM receptors, as tetrodotoxin-resistant sodium current was increased by IM in a majority of male dural afferents (13/14). These data indicate that there are both quantitative and qualitative differences in the IM-induced sensitization of dural afferents that may contribute to the sex difference in the manifestation of migraine.

Most oral cancer patients suffer severe, chronic pain promoted by oral cancer-induced changes in sensory nerves innervating the oral cavity. However, the impact of sensory neurons on carcinogenesis beyond nociceptive signaling is an understudied problem in oral cancer. We hypothesize that oral cancer and sensory neurons interact, such that, oral cancer induces sensitization, sprouting and plasticity in neurons, and efferent neuronal activity promotes oral carcinogenesis. Using an oral cancer subcutaneous xenograft model, we tested the impact of peripheral nerve presence on squamous cell carcinoma tumor growth in the mouse hindpaw. Mice that received a sciatic nerve axotomy one week prior to HSC-3 oral cancer cell inoculation developed significantly smaller tumors (28.23 ± 6.6 % of total paw area) compared to mice that received sham surgery (50.71 ± 2.48 %). These data suggest that peripheral neuron innervation supports oral cancer growth. Additionally, neuropeptides are released from primary afferent neurons and suppression of the immune response has been linked to peripheral neurotransmission. Using flow cytometry, we tested whether inhibition of peripheral sensory neurons impacted inflammation in response to oral cancer. Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to injection of supernatant from HSC-3 oral cancer cells resulted in significantly more CD45+ immune cell infiltration (21.66 ± 3.4 %) into the tongue compared to HSC-3 supernatant with intact nerve function (9.10 ± 1.2 %). These results suggest neuron-mediated suppression of cancer-evoked inflammation and support the premise that neurons and neuronal activity modulates the oral cancer microenvironment. High-resolution imaging of neuronal innervation into tongue cancer and its remodeling in response to pathological changes is challenging due to dense neuronal innervation in the tongue. We utilized a recently developed optical clearing protocol (CLARITY) that preserves protein integrity and three-dimensional structure of tissue and high-resolution microscopy to visualize and characterize sensory neuron innervation in thick 1 mm tongue sections from an orthotopic xenograft mouse model of oral cancer. In a preliminary study of neuronal innervation in a tongue HSC-3 xenograft model, we observed that PGP9.5+ neuronal fiber density was greater in the cancer environment and diminished with distance from the cancer. These data suggest that oral cancer induces plasticity and sprouting of innervating peripheral neurons. A better understanding of the nerve-cancer interaction has potential for development of new therapeutic strategies to treat oral cancer and oral cancer pain by targeting sensory neurons. Citation Format: Nicole N. Scheff, Richard Klares, Brian L. Schmidt. Characterization and functional consequences of neuronal invasion in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1057.

Object The authors used a rat model to assess spinal cord compression following an incomplete spinal cord injury (SCI). Methods Incomplete SCI was created in the thoracic spinal cord in a novel application of a rodent spinal cord compression model. A moderate impaction force was applied instantaneously to the spinal cord and was followed by 0 seconds, 10 seconds, 30 seconds, or 5 minutes of continued compression (termed “dwell”). The different groups were assessed by behavioral testing with the Basso, Beattie, Bresnahan locomotor rating scale, and with histological injury quantification and morphometrical analysis. Results Compression after the SCI resulted in worsened Basso, Beattie, Bresnahan scale scores; however, the duration of compression was not significant. Compression did not significantly affect the percentage of spared total tissue, percent spared total white matter, or percent spared total gray matter. Percent spared tissue at the epicenter of injury was statistically worsened by compression but not in a time-dependent manner. Conclusions The authors' results suggest that spinal cord compression after the initial injury is an additional mechanism by which SCI worsens, and that the mechanism of this injury occurs rapidly. These data, however, do not support duration of compression as a significant variable.

Transdermal systems are attractive methods of drug administration specifi cally when treating patients for drug addiction. Current systems however are defi cient in therapies that allow variable fl ux values of drug, such as nicotine for smoking cessation or complex dosing regimens using clonidine when treating opioid withdrawal symptoms. Through the use of functionalized carbon nanotube (CNT) membranes, drug delivery to the skin can be controlled by applying a small electrical bias to create a programmable drug delivery system. Clearly, a transdermal patch system that can be tailored to an individual's needs will increase patient compliance as well as provide much more effi cient therapy. The purpose of this paper is to discuss the applicability of using carbon nanotube membranes in transdermal systems for treatment of drug abuse. Introductionabuse, whether alcohol, tobacco, prescription or illicit, continues to burden society, economics, medical care systems, and the family dynamic. The World Health Organization (WHO) estimates that globally 185 million people are illicit drug users and an astounding 1.3 billion individuals are smokers. 1 Smoking related deaths, estimated at nearly 5 million worldwide, continue to be the leading preventable cause of death. Additionally, in the United States alone, $167 billion in annual health-related economic loss can be attributed to smoking. 2 Although there are roughly only 200,000 deaths reported to be directly associated with illicit drug use, the detriment to family and society, and the economic costs associated with crime, law enforcement, medical care, and drug treatment and rehabilitation, are immense. 3,4 While current treatments for cigarette smoking and opioid addiction do aid in successful abstinence from the individual's vice, there is great need and room for improvement. The best of smok- ing cessation therapies reports a mere 19% success after one year, while only 50% of individuals who enter clinics for opioid addiction remain for the duration of the treatment, of which 65% return to using within 4 weeks. 3,5 Clearly, a therapy that better controls cravings or abates withdrawal symptoms to decrease the likelihood of relapse is needed. Carbon nanotube (CNT) membranes present the opportu- nity to create a transdermal patch that can vary its rate of delivery throughout its application to the skin to attain therapeutic plasma levels and plasma profi les of a specifi c drug. The purpose of this report is to discuss the use of carbon nanotube membranes in a transdermal system to aid in abstinence from cigarettes and withdrawal from opioids.

Although the advent of marketed transdermal drug-delivery products is nearing three decades, considerable strides in this noninvasive route of administration have been slow compared with its intravenous and oral counterparts, primarily due to the extreme barrier capabilities of the skin. The development of prodrug and codrug systems provides opportunity to enhance drug flux through the skin and thereby create highly desirable and efficacious treatment. The pharmacologically inactive prodrug is synthesized to increase or maximize its transdermal diffusion capabilities and then metabolized in vivo to the active compound. Similarly, codrugs are mutual prodrugs that are linked together in order to improve the drug-delivery properties of one or both drugs. This article reviews the current advances in prodrug and codrug transdermal therapy and their respective mechanisms for improvement in percutaneous absorption.

Recent attention has been given to gender differences in neurotrauma, and the anecdotal suggestion is that females have better outcomes than males, suggesting that circulating levels of estrogen (E(2)) may be neuroprotective. In order to address this issue, both young adult male and ovariectomized female rats were subjected to a T10 spinal cord injury (SCI), and E2 levels were maintained at chronic, constant circulating levels. Animals were clinically evaluated for locomotor changes using the Basso-Beattie-Bresnahan (BBB) scoring system. Morphologic differences were evaluated with unbiased stereology. Data analysis failed to reveal any significant benefit for the E2 therapy in either males or females. We did find a non-estrogen-dependent difference between male and female rats in length of injury, and percent of spared tissue, with female outcomes more favorable. These results suggest that E(2) does not provide a viable therapy following SCI.

Persistent inflammation results in an increase in the magnitude and duration of high K(+)-evoked Ca(2+) transients in putative nociceptive cutaneous dorsal root ganglion (DRG) neurons. The purpose of the present study was to determine whether recruitment of Ca(2+)-induced Ca(2+) release (CICR) contributes to these inflammation-induced changes. Acutely dissociated, retrogradely labeled cutaneous DRG neurons from naïve and complete Freund's adjuvant inflamed adult male Sprague-Dawley rats were studied with ratiometric microfluorimetry. Ryanodine only attenuated the duration but not magnitude of the high K(+)-evoked Ca(2+) transient in neurons from inflamed rats. However, there was no significant impact of inflammation on the potency or efficacy of ryanodine-induced block of the caffeine-evoked Ca(2+) transient, or the impact of sarco-endoplasmic reticulum ATPase (SERCA) inhibition on the high K(+)-evoked Ca(2+) transient. Furthermore, while there was no change in the magnitude, an inflammation-induced increase in the duration of the caffeine-evoked Ca(2+) transient was only observed with a prolonged caffeine application. In contrast to the high K(+)-evoked Ca(2+) transient, there was no evidence of direct mitochondrial involvement or that of the Ca(2+) extrusion mechanism, the Na(+)/Ca(2+) exchanger, on the caffeine-evoked Ca(2+) transient, and block of SERCA only increased the duration of this transient. These results indicate the presence of Ca(2+) regulatory domains in cutaneous nociceptive DRG neurons within which cytosolic Ca(2+) increased via influx and release are highly segregated. Furthermore, our results suggest that changes in neither CICR machinery nor the coupling between Ca(2+) influx and CICR are primarily responsible for the inflammation-induced changes in the evoked Ca(2+) transient.

Aim To test the hypothesis that the clinical efficacy of triptans reflects convergent modulation of ion channels also involved in inflammatory mediator (IM)-induced sensitization of dural afferents. Methods Acutely dissociated retrogradely labeled rat dural afferents were studied with whole cell and perforated patch techniques in the absence and presence of sumatriptan and/or IM (prostaglandin E2, bradykinin, and histamine). Results Sumatriptan dose-dependently suppressed voltage-gated Ca2+ currents. Acute (2 min) sumatriptan application increased dural afferent excitability and occluded further IM-induced sensitization. In contrast, pre-incubation (30 min) with sumatriptan had no influence on dural afferent excitability and partially prevented IM-induced sensitization of dural afferents. The sumatriptan-induced suppression of voltage-gated Ca2+ currents and acute sensitization and pre-incubation-induced block of IM-induced sensitization were blocked by the 5-HT1D antagonist BRL 15572. Pre-incubation with sumatriptan failed to suppress the IM-induced decrease in action potential threshold and overshoot (which results from modulation of voltage-gated Na+ currents) and activation of Cl− current, and had no influence on the Cl− reversal potential. However, pre-incubation with sumatriptan caused a dramatic hyperpolarizing shift in the voltage dependence of K+ current activation. Discussion These results indicate that although the actions of sumatriptan on dural afferents are complex, at least two distinct mechanisms underlie the antinociceptive actions of this compound. One of these mechanisms, the shift in the voltage dependence of K+ channel activation, may suggest a novel strategy for future development of anti-migraine agents.

The biochemical sequelae that follow traumatic brain injury (TBI) are numerous and affect many different brain functions at different points of time as the secondary cascades progress. The complexity of the resulting pathophysiology is such that a singular therapeutic intervention may not provide adequate benefit and a combination of drugs targeting different pathways may be needed. Two of the most widely studied injury mechanisms are oxidative stress and inflammation. Numerous studies have suggested that pharmacological agents targeting either of these pathways may produce an improvement in histological and functional outcome measures. We hypothesized that combining melatonin, a potent antioxidant, with minocycline, a bacteriostatic agent that also inhibit microglia, would provide better neuroprotection than either agent used alone. To test this hypothesis, we subjected anesthetized adult male rats to a 1.5mm controlled cortical impact and administered melatonin or vehicle in the acute post-injury period followed by daily minocycline or vehicle injections beginning the following day in a 2×2 study design. The animals were allowed to recover for 5 days before undergoing Morris water maze (MWM) testing to assess cognitive functioning following injury. There was no significant difference in MWM performance between the vehicle, melatonin, minocycline, or combination treatments. Following sacrifice and histological examination for neuroprotection, we did not observe a significant difference between the groups in the amount of cortical tissue that was spared nor was there a significant difference in [(3)H]-PK11195 binding, a marker for activated microglia. These results suggest that neither drug has therapeutic efficacy, however dosing and/or administration issues may have played a role.