Background & Aims

Schwann cells (SC), which ensheath peripheral nerve endings, contribute to oral cancer-induced mechanical hyperalgesia in mice(1). SC can respond to mechanical stimuli and convey these mechanical signals to nociceptive neurons to drive pain(2). The receptors on SC required for signal transduction of mechanical stimulation have not been identified.
The TRPV4 receptor responds to mechanical stimuli and is a candidate in SC-mediated mechanical pain. TRPV4 is also activated by shear stress, hypotonic solution, heat and various agonists. We hypothesize that TRPV4 on SC is sensitized in oral cancer patients, leading to mechanical hyperalgesia. A better understanding of how TRPV4 activity is modulated on SC will inform the relevance of SC TRPV4 to oral cancer pain.
The aims of this study are to: i) validate functional expression of TRPV4 on SC using different modes of activating TRPV4 ii) determine how SC TRPV4 is sensitized and iii) if this sensitization occurs in oral cancer pain models.

Methods

TRPV4 mRNA from human and mouse SC was quantified with RT-PCR (Primers Hs01099348_m1, Mm00499025_m1, Applied Biosystems). Human SC (Neuromics, HMP303) and mouse SC (ABM, T0769) were seeded to collagen coated (100 ?g/mL) 96-well plates at 20,000 cells/well. After 48 hours, cells were loaded with the calcium indicator Fura-2 AM for 30 minutes, washed, and left to settle at 37°C in a Flexstation®3 Multi-Mode Microplate Reader. At 5-second intervals, fluorescence at 538 nm following alternate excitation at 340 and 380 nm was measured. A fluorescence ratio (F340/F380) was calculated. After a baseline period, graded concentrations of the TRPV4 agonist GSK1016790A (GSK101) were applied and F340/F380 measured for 5 minutes. Another group was administered the TRPV4 antagonist HC-067047 30 minutes prior to receiving GSK101.
In a separate experiment, human SC were treated with an isotonic (300 mOsm/L) or hypertonic (335 mOsm/L) solution for 10 minutes prior to the addition of GSK101 (5 nM).

Results

Human and mouse SC both expressed TRPV4 mRNA transcripts, with ?Ct of 12.1±0.6 and 7.7±0.1 respectively, normalised to human/mouse GAPDH. Cytosolic calcium levels (F340/F380) increased in both human and mouse SC following application of GSK101. This increase was dose-dependent and was reduced by pretreatment with a TRPV4 antagonist.
Pretreatment of human SC with an isotonic (300 mOsm/L) solution doubled the GSK101 (5 nM) – dependent calcium influx compared to pretreatment with a hypertonic (335 mOsm/L) solution, as measured by total area under the curve (107.0±7.0 vs 47.3±7.0, p = 0.006, 1-way ANOVA). Pretreatment with an isotonic solution also doubled peak cytosolic calcium levels (peak F340/F380) after GSK101 application, compared with the group that received an isotonic solution (0.58±0.05 vs 0.26±0.04, p = 0.040, 1-way ANOVA). Baseline cytosolic calcium levels were similar across the hypertonic and isotonic groups (p=0.99, 1-way ANOVA). Data shown as Mean ± SEM.

Conclusions

TRPV4 is functionally expressed by human and mouse SC and TRPV4 activity in human SC is increased by cell swelling when going from a hypertonic environment to an isotonic one. Given that SC swelling, which deforms the cell membrane, sensitizes TRPV4 to its specific agonist GSK101, we plan to study how stiffness of the extracellular matrix can influence TRPV4 activity on SCs, since oral cancers exhibit increased tissue stiffness (3,4) which also causes deformation of the cell. We will explore whether modulation of extracellular matrix stiffness contributes to oral cancer pain through SC TRPV4. Additional studies will examine i) whether chemical mediators in the oral cancer microenvironment modulate TRPV4 activity, using a co-culture model of an oral cancer cell line (HSC-3) and SC and ii) the effect of SC TRPV4 activation on neuronal excitability.

References

1. Salvo, E., Saraithong, P., Curtin, J.G., Janal, M.N. and Ye, Y., 2019. Reciprocal interactions between cancer and Schwann cells contribute to oral cancer progression and pain. Heliyon, 5(2).
2. Abdo, H., Calvo-Enrique, L., Lopez, J.M., Song, J., Zhang, M.D., Usoskin, D., El Manira, A., Adameyko, I., Hjerling-Leffler, J. and Ernfors, P., 2019. Specialized cutaneous Schwann cells initiate pain sensation. Science, 365(6454), pp.695-699.
3. Zhang, J.Y., Zhu, W.W., Wang, M.Y., Zhai, R.D., Wang, Q., Shen, W.L. and Liu, L.K., 2021. Cancer-associated fibroblasts promote oral squamous cell carcinoma progression through LOX-mediated matrix stiffness. Journal of Translational Medicine, 19(1), pp.1-16.
4. Hasegawa, K., Fujii, S., Matsumoto, S., Tajiri, Y., Kikuchi, A. and Kiyoshima, T., 2021. YAP signaling induces PIEZO1 to promote oral squamous cell carcinoma cell proliferation. The Journal of Pathology, 253(1), pp.80-93.

Presenting Author

Sam Nicholson

Poster Authors

Sam Nicholson

BSc

New York University

Lead Author

E-mail

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Orofacial Pain