Background & Aims
Considered one of the most painful cancers, oral cancer pain poses a significant clinical challenge, and understanding the molecular mechanisms involved is crucial for the development of effective therapeutic interventions.The most severe oral cancer pain is induced by stretch.Therefore, the focus of this study is to measure functional expression of transient receptor potential vanilloid 4 (TRPV4), a stretch-activated ion channel, on non-neuronal cells in the oral cancer microenvironment using a calcium mobilization assay.Studies to date have not demonstrated TRPV4 expression on trigeminal ganglion neurons, unlike epithelial (1,2) and human schwann cells (SCs)(3).TRPV4 antagonists are currently being tested in clinical trials, making TRPV4 an attractive oral cancer pain target.The cells studied include SCs, human dysplastic oral keratinocytes (DOK; dysplasia patients do not report pain while those with oral cancer report severe pain (4)), and human oral squamous carcinoma cells(HSC-3).
Methods
Functional expression of TRPV4 was studied using a calcium mobilization assay. SCs (Neuromics Inc.,HMP303), HSC-3 (JCRB,0623) and DOK (ECACC, 94122104) were plated in a 96-well plate at 80% confluency. Wells were treated with 1µM Fura2-AM at 37ºC/5% CO2 for 30 minutes before incubating in calcium buffer for 30 minutes. A selective TRPV4 agonist (GSK1016790A, 0,10,30,100,300nM) was used to compare responses between cell types. A TRPV4 antagonist (HC067, 1µM) was used as a control to confirm TRPV4-dependent responses. Control wells were treated with the antagonist 30 minutes prior to imaging. All wells received the agonist and TRPV4-dependent calcium influx was quantified with a FlexStation3®(Molecular Devices). Measurements were taken at 5 second intervals for 6 minutes following application. This was followed by ionomycin (1µM) to confirm cellular responses. Statistical analysis was performed in GraphPad Prism10.
Results
Significance was determined using the AUC of the F340/F380 response for 6 minutes following the application of the agonist. For all concentrations above 10nM GSK101 the response of SCs to the agonist was at least 50% greater than DOK and HSC-3, which did not differ in response (p<0.01, 1-way ANOVA, n = 8 wells). Following pretreatment of the antagonist, TRPV4 responses to 10,30,100nM GSK101 were significantly decreased for all cell types (p<0.01, vehicle and agonist vs. antagonist and agonist; 1-way ANOVA, n=8 wells). There was no significant difference in the response to ionomycin between any treatments.
Conclusions
TRPV4 is expressed on SC and oral cancer cells found within the cancer microenvironment. The responses of SCs, DOK, and HSC-3 to the TRPV4 agonist were robust and dose dependent. The TRPV4 response on SC is greater than DOK and HSC-3. Future studies are designed to test trigeminal neurons, human oral keratinocytes, an additional human oral cancer cell line (HSC-4), and macrophages in order to complete the picture of the TRPV4-responsive cells in the oral cancer microenvironment. Cell-specific genetic deletion will be used to test the contribution of TRPV4 on different cells within the oral cancer microenvironment to oral cancer pain.
References
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4. Bhattacharya, A., Janal, M.N., Veeramachaneni, R. et al. Oncogenes overexpressed in metastatic oral cancers from patients with pain: potential pain mediators released in exosomes. Sci Rep 10, 14724 (2020). https://doi.org/10.1038/s41598-020-71298-y
Presenting Author
Grace Harden
Poster Authors
Topics
- Specific Pain Conditions/Pain in Specific Populations: Cancer Pain & Palliative Care