Widespread pain and anxiety are commonly reported in cancer patients. We hypothesize that cancer is accompanied by attenuation of endogenous opioid-mediated inhibition, which subsequently causes widespread pain and anxiety. To test this hypothesis we used a mouse model of oral squamous cell carcinoma (SCC) in the tongue. We found that mice with tongue SCC exhibited widespread nociceptive behaviors in addition to behaviors associated with local nociception that we reported previously. Tongue SCC mice exhibited a pattern of reduced opioid receptor expression in the spinal cord; intrathecal administration of respective mu (MOR), delta (DOR), and kappa (KOR) opioid receptor agonists reduced widespread nociception in mice, except for the fail flick assay following administration of the MOR agonist. We infer from these findings that opioid receptors contribute to widespread nociception in oral cancer mice. Despite significant nociception, mice with tongue SCC did not differ from sham mice in anxiety-like behaviors as measured by the open field assay and elevated maze. No significant differences in c-Fos staining were found in anxiety-associated brain regions in cancer relative to control mice. No correlation was found between nociceptive and anxiety-like behaviors. Moreover, opioid receptor agonists did not yield a statistically significant effect on behaviors measured in the open field and elevated maze in cancer mice. Lastly, we used an acute cancer pain model (injection of cancer supernatant into the mouse tongue) to test whether adaptation to chronic pain is responsible for the absence of greater anxiety-like behavior in cancer mice. No changes in anxiety-like behavior were observed in mice with acute cancer pain.

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 PAR2, which can sensitize the TRPV1 channel. We show co-expression of TRPV1 and PAR2 on tongue afferents and using a conditioned place aversion assay, demonstrate that PAR2 mediates oral cancer-induced, TRPV1-evoked sensitivity in an oral cancer mouse model. The findings provide insight into oral cancer-mediated chemosensitivity.

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.

Neutrophil elastase is a serine protease that has been implicated in the pathogenesis of inflammatory bowel disease. Due to post-translational control of its activation and high expression of its inhibitors in the gut, measurements of total expression poorly reflect the pool of active, functional neutrophil elastase. Fluorogenic substrate probes have been used to measure neutrophil elastase activity, though these tools lack specificity and traceability. PK105 is a recently described fluorescent activity-based probe, which binds to neutrophil elastase in an activity-dependent manner. The irreversible nature of this probe allows for accurate identification of its targets in complex protein mixtures. We describe the reactivity profile of PK105b, a new analogue of PK105, against recombinant serine proteases and in tissue extracts from healthy mice and from models of inflammation induced by oral cancer and Legionella pneumophila infection. We apply PK105b to measure neutrophil elastase activation in an acute model of experimental colitis. Neutrophil elastase activity is detected in inflamed, but not healthy, colons. We corroborate this finding in mucosal biopsies from patients with ulcerative colitis. Thus, PK105b facilitates detection of neutrophil elastase activity in tissue lysates, and we have applied it to demonstrate that this protease is unequivocally activated during colitis.

Cancer pain creates a poor quality of life and decreases survival. The basic neurobiology of cancer pain is poorly understood. Adenosine triphosphate (ATP) and the ATP ionotropic receptor subunits, P2X2 and P2X3, mediate cancer pain in animal models; however, it is unknown whether this mechanism operates in human, and if so, what the relative contribution of P2X2- and P2X3-containing trimeric channels to cancer pain is. Here, we studied head and neck squamous cell carcinoma (HNSCC), which causes the highest level of function-induced pain relative to other types of cancer.

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.

Individual bacteria and shifts in the composition of the microbiome have been associated with human diseases including cancer. To investigate changes in the microbiome associated with oral cancers, we profiled cancers and anatomically matched contralateral normal tissue from the same patient by sequencing 16S rDNA hypervariable region amplicons. In cancer samples from both a discovery and a subsequent confirmation cohort, abundance of Firmicutes (especially Streptococcus) and Actinobacteria (especially Rothia) was significantly decreased relative to contralateral normal samples from the same patient. Significant decreases in abundance of these phyla were observed for pre-cancers, but not when comparing samples from contralateral sites (tongue and floor of mouth) from healthy individuals. Weighted UniFrac principal coordinates analysis based on 12 taxa separated most cancers from other samples with greatest separation of node positive cases. These studies begin to develop a framework for exploiting the oral microbiome for monitoring oral cancer development, progression and recurrence.

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) reduces survival. In this study we hypothesized that methylation of key genes mediates cisplatin resistance. We determined whether a demethylating drug, decitabine, could augment the anti-proliferative and apoptotic effects of cisplatin on SCC-25/CP, a cisplatin-resistant tongue SCC cell line. We showed that decitabine treatment restored cisplatin sensitivity in SCC-25/CP and significantly reduced the cisplatin dose required to induce apoptosis. We then created a xenograft model with SCC-25/CP and determined that decitabine and cisplatin combination treatment resulted in significantly reduced tumor growth and mechanical allodynia compared to control. To establish a gene classifier we quantified methylation in cancer tissue of cisplatin-sensitive and cisplatin-resistant HNSCC patients. Cisplatin-sensitive and cisplatin-resistant patient tumors had distinct methylation profiles. When we quantified methylation and expression of genes in the classifier in HNSCC cells in vitro, we showed that decitabine treatment of cisplatin-resistant HNSCC cells reversed methylation and gene expression toward a cisplatin-sensitive profile. The study provides direct evidence that decitabine restores cisplatin sensitivity in in vitro and in vivo models of HNSCC. Combination treatment of cisplatin and decitabine significantly reduces HNSCC growth and HNSCC pain. Furthermore, gene methylation could be used as a biomarker of cisplatin-resistance.

Oral cancer pain is attributed to the release from cancers of mediators that sensitize and activate sensory neurons. Intraplantar injection of conditioned media (CM) from human tongue cancer cell line HSC-3 or OSC-20 evokes nociceptive behavior. By contrast, CM from noncancer cell lines, DOK, and HaCaT are non-nociceptive. Pain mediators are carried by extracellular vesicles (EVs) released from cancer cells. Depletion of EVs from cancer cell line CM reverses mechanical allodynia and thermal hyperalgesia. CM from non-nociceptive cell lines become nociceptive when reconstituted with HSC-3 EVs. Two miRNAs (hsa-miR-21-5p and hsa-miR-221-3p) are identified that are present in increased abundance in EVs from HSC-3 and OSC-20 CM compared to HaCaT CM. The miRNA target genes suggest potential involvement in oral cancer pain of the toll like receptor 7 (TLR7) and 8 (TLR8) pathways, as well as signaling through interleukin 6 cytokine family signal transducer receptor (gp130, encoded by IL6ST) and colony stimulating factor receptor (G-CSFR, encoded by CSF3R), Janus kinase and signal transducer and activator of transcription 3 (JAK/STAT3). These studies confirm the recent discovery of the role of cancer EVs in pain and add to the repertoire of algesic and analgesic cancer pain mediators and pathways that contribute to oral cancer pain.

The incidence of oral cancer in the United States is increasing, especially in young people and women. Patients with oral cancer report severe functional pain. Using a patient cohort accrued through the New York University Oral Cancer Center and immune-competent mouse models, we identify a sex difference in the prevalence and severity of oral cancer pain. A neutrophil-mediated endogenous analgesic mechanism is present in male mice with oral cancer. Local naloxone treatment potentiates cancer mediator-induced orofacial nociceptive behavior in male mice only. Tongues from male mice with oral cancer have significantly more infiltrating neutrophils compared to female mice with oral cancer. Neutrophils isolated from the cancer-induced inflammatory microenvironment express beta-endorphin and met-enkephalin. Furthermore, neutrophil depletion results in nociceptive behavior in male mice. These data suggest a role for sex-specific, immune cell-mediated endogenous analgesia in the treatment of oral cancer pain.