Tumor necrosis factor alpha secreted from oral squamous cell carcinoma contributes to cancer pain and associated inflammation

Abstract

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-Mediated Endogenous Analgesia Contributes to Sex Differences in Oral Cancer Pain

“…Human oral cancer cell line, HSC-3, and non-tumorigenic keratinocyte cell line, HaCaT, were utilized to produce the acute supernatant cancer pain and control models, respectively. Cells were maintained and culture supernatant was collected as previously described (Scheff et al, 2017 ). All cell lines were cultured in 10 cm 2 cell culture dishes at 37°C with 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum and penicillin/streptomycin (50 U/mL).…”

“…We developed an acute oral cancer pain model by injecting cell culture supernatant into the tongue (Scheff et al, 2017 ). Mice received 50 μl injections (under isoflurane general anesthesia) of either HSC-3 or HaCaT cell culture supernatant over a 5 s period, into the left lateral tongue for three consecutive days.…”

“…To study the development of oral nociception with carcinogenesis, female and male mice were first trained in the dolognawmeter over 5–7 sessions (Dolan et al, 2010 ). Subsequently these mice were offered carcinogen 4-nitroquinoline-1-oxide (4NQO; 100 μg/mL; Sigma Aldrich, St. Louis, MO, USA) in their drinking water or the equivalent dilution of the vehicle, propylene glycol for 16 weeks (Scheff et al, 2017 ). The mice were then monitored weekly under light anesthesia for tumor incidence, location and size for an additional 12 weeks.…”

“…Tongue tissue was dissected into cold DMEM containing antibiotics (penicillin/streptomycin, 10 U/mL) and 20 mM HEPES and was dissociated as previously described (Scheff et al, 2017 ). Tongue tissue was dissected and minced in DMEM with antibiotics, collagenase-H (0.5 mg/mL; Sigma Aldrich, 34 Units/mg), DNase (0.5 mg/mL) and 20 mM HEPES, and then incubated at 37°C for 1 h. The tissue was then mechanically dissociated using a fire-polished pipette, washed twice with fresh DMEM containing antibiotics and HEPES, and resuspended in Ca 2+ /Mg 2+ free phosphate buffered saline (Sigma Aldrich) containing 3% fetal bovine serum, 1 mM EDTA, and 0.02% sodium azide and filtered through a 40 μm cell strainer (Falcon brand, Fisher Scientific, Waltham, MA, USA).…”

“…Microglia, fundamental to neuropathic pain signaling in males, are not involved in female pain processing; female mice achieve similar levels of pain hypersensitivity using adaptive immune cells, likely T lymphocytes (Sorge et al, 2015 ). We described a role for peripheral infiltrating T cells in oral cancer pain in female mice (Scheff et al, 2017 ). The role of infiltrating immune cells in oral cancer pain in males, however, has not been investigated.…”