Faculty Bibliography

Pain is rated by oral cancer patients as the worst symptom and significantly impairs a patient's ability to eat, talk, and drink. Mediators, secreted from oral cancer microenvironment, excite primary afferent neurons, which in turn generate pain. Oral cancer cells release TNFalpha which induces acute inflammation and nociception in mice. We hypothesize that TNFalpha activates Schwann cells to amplify pain signals. First, we confirmed the involvement of TNFalpha in oral cancer pain in patients and animal models. We found that oral cancer tissues collected from patients have higher TNFalpha concentration compared to anatomically matched normal tissues. Differences in TNFalpha concentration between the tumor and anatomically matched normal tissues correlate positively with total pain scores. In a Nitroquinoline 1-oxide (4NQO) mouse oral cancer model we demonstrated reduced mechanical hypersensitivity (P<0.05, N=8) with the dolognawmeter gnawing assay when TNFalpha was neutralized with C-87. Using a non-contact co-culture model, we found that HSC-3 cells induced a more activated human primary Schwann cells phenotype with increased proliferation (P<0.05) and migration (P<0.05); introduction of C-87 in the co-culture reduced Schwann cell proliferation (P<0.05) and migration (P<0.05) induced by HSC-3 cells. After removal of the co-cultured cancer cells, cancer-activated Schwann cells secrete greater amounts of TNFalpha and nerve growth factor (NGF), another known nociceptive mediator in the oral cancer microenvironment, compared to Schwann cells initially co-cultured with DOK (P<0.05) or naive Schwann cells (P<0.05). To determine whether activated Schwann cells mediate oral cancer pain, we cultured Schwann cells in hypoxic conditions - a known cancer stimulus that induces robust Schwann cell activation. Schwann cell supernatant was then collected and injected into the mouse cheek. Supernatant from hypoxia-activated Schwann cells induced greater facial allodynia (measured with von Frey filaments) in mice (P<0.05, N=7), compared to supernatant from Schwann cells cultured in normoxic conditions (N=5). C-87 significantly reduced facial allodynia caused by hypoxiaactivated Schwann cells (P<0.05, N=5). We infer from our results that TNFalpha plays a role in the activation of Schwann cells and that cancer-activated Schwann cells are a source of nociceptive mediators in the cancer microenvironment. Inhibition of Schwann cell activation might be clinically useful for alleviating oral cancer pain

Chronic inflammation of the salivary glands from pathologic conditions such as Sjögren's syndrome can result in glandular destruction and hyposalivation. To understand which molecular factors may play a role in clinical cases of salivary gland hypofunction, we developed an aquaporin 5 (AQP5) Cre mouse line to produce genetic recombination predominantly within the acinar cells of the glands. We then bred these mice with the TNF-αglo transgenic line to develop a mouse model with salivary gland-specific overexpression of TNF-α; which replicates conditions seen in sialadenitis, an inflammation of the salivary glands resulting from infection or autoimmune disorders such as Sjögren's syndrome. The resulting AQP5-Cre/TNF-αglo mice display severe inflammation in the salivary glands with acinar cell atrophy, fibrosis, and dilation of the ducts. AQP5 expression was reduced in the salivary glands, while tight junction integrity appeared to be disrupted. The immune dysregulation in the salivary gland of these mice led to hyposalivation and masticatory dysfunction.

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.

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.

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.

Oral cancer patients 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 utilized two oral cancer mouse models: a cell line supernatant injection model and the 4-nitroquinoline-1-oxide (4NQO) chemical carcinogenesis model. We used the two 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. While 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 (TNFa), was identified in high concentration in oral cancer cell line supernatant and in the tongue tissue of 4NQO-treated mice with oSCC. Inhibition of TNFa signaling abolished oral cancer cell line supernatant-evoked functional allodynia and disrupted T cell infiltration. With these data, we identified TNFa as a prominent mediator in oral cancer-induced nociception and inflammation highlighting the need for further investigation in neural-immune communication in cancer pain.

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.

We propose a new mechanism of sensory modulation through cutaneous dopaminergic signalling. We hypothesize that dopaminergic signalling contributes to differential cutaneous sensitivity in darker versus lighter pigmented humans and mouse strains. We show that thermal and mechanical cutaneous sensitivity is pigmentation dependent. Meta-analyses in humans and mice, along with our own mouse behavioural studies, reveal higher thermal sensitivity in pigmented skin relative to less-pigmented or albino skin. We show that dopamine from melanocytes activates the D1-like dopamine receptor on primary sensory neurons. Dopaminergic activation increases expression of the heat-sensitive TRPV1 ion channel and reduces expression of the mechanically-sensitive Piezo2 channel; thermal threshold is lower and mechanical threshold is higher in pigmented skin.

Virus-mediated gene delivery shows promise for the treatment of chronic pain. However, viral vectors have cytotoxicity. To avoid toxicities and limitations of virus-mediated gene delivery, we developed a novel nonviral hybrid vector: HIV-1 Tat peptide sequence modified with histidine and cysteine residues combined with a cationic lipid. The vector has high transfection efficiency with little cytotoxicity in cancer cell lines including HSC-3 (human tongue squamous cell carcinoma) and exhibits differential expression in HSC-3 ( approximately 45-fold) relative to HGF-1 (human gingival fibroblasts) cells. We used the nonviral vector to transfect cancer with OPRM1, the mu-opioid receptor gene, as a novel method for treating cancer-induced pain. After HSC-3 cells were transfected with OPRM1, a cancer mouse model was created by inoculating the transfected HSC-3 cells into the hind paw or tongue of athymic mice to determine the analgesic potential of OPRM1 transfection. Mice with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared with control mice. The effect was reversible with local naloxone administration. We quantified beta-endorphin secretion from HSC-3 cells and showed that HSC-3 cells transfected with OPRM1 secreted significantly more beta-endorphin than control HSC-3 cells. These findings indicate that nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment has an analgesic effect in a preclinical cancer model, and nonviral gene delivery is a potential treatment for cancer pain.

Tumor necrosis factor-alpha (TNF-alpha) is a proalgesic cytokine that is commonly expressed following tissue injury. TNF-alpha expression not only promotes inflammation but can also lead to pain hypersensitivity in nociceptors. With the established link between TNF-alpha and inflammatory pain, we identified its increased expression in the teeth of patients affected with caries and pulpitis. We generated a transgenic mouse model (TNF-alphaglo) that could be used to conditionally overexpress TNF-alpha. These mice were bred with a dentin matrix protein 1 (DMP1)-Cre line for overexpression of TNF-alpha in both the tooth pulp and bone to study oral pain that would result from subsequent development of pulpitis and bone loss. The resulting DMP1/TNF-alphaglo mice show inflammation in the tooth pulp that resembles pulpitis while also displaying periodontal bone loss. Inflammatory infiltrates and enlarged blood vessels were observed in the tooth pulp. Pulpitis and osteitis affected the nociceptive neurons innervating the orofacial region by causing increased expression of inflammatory cytokines within the trigeminal ganglia. With this new mouse model morphologically mimicking pulpitis and osteitis, we tested it for signs of oral pain with an oral function assay (dolognawmeter). This assay/device records the time required by a mouse to complete a discrete gnawing task. The duration of gnawing required by the DMP1/TNF-alphaglo mice to complete the task was greater than that for the controls; extended gnaw time in a dolognawmeter indicates reduced orofacial function. With the DMP1/TNF-alphaglo mice, we have shown that TNF-alpha expression alone can produce inflammation similar to pulpitis and osteitis and that this mouse model can be used to study dental inflammatory pain.