The study was approved by the Institutional Review Board of New York University College of Dentistry and University of California San Francisco (UCSF). All patients provided written informed consent in accordance with the Declaration of Helsinki. Patients were enrolled with the following inclusion criteria: 1) biopsy-proven HNSCC, and 2) no history of prior surgical, chemotherapeutic, or radiation treatment for oral SCC. The validated UCSF Oral Cancer Pain Questionnaire [26] was administered to enrolled patients. The questionnaire consisted of eight questions on spontaneous and functional pain, which were rated on a visual analog scale (0 to 100 mm). None of the patients were taking analgesics or were receiving cancer treatment at the time of questionnaire completion. Demographic information was collected for each patient including age, sex, ethnicity, HNSCC location (tongue, floor of mouth, buccal mucosa, gingiva, palate), tumor size (greatest dimension based on clinical examination), and evidence of metastasis.
Animals
Female athymic, immunocompromised (BALB/c nu/nu) mice and BALB/c mice were purchased from Charles River Laboratories. All experiments were performed according to the policies of the International Association for the Study of Pain and approved by the New York University Institution Animal Care and Use Committee.
HPLC analysis
HNSCC and anatomically matched, contralateral normal oral epithelium from 13 oral cancer patients were surgically removed, immediately snap frozen in liquid nitrogen and stored at −80°C. ATP was quantified with HPLC coupled to UV detection. Each tissue sample was weighed and ground in 10% Trichloroacetic acid buffer (MP Biomedical). Samples were incubated on ice for 1 hour, and were periodically vortexed for 3 seconds. After incubation, samples were centrifuged for 15 min at 10000 rpm in 4°C. The supernatant was collected into pre-cooled centrifuge tubes and neutralized with 0.2 M K2HPO4. The sample was kept on ice for an additional 15 minutes to precipitate the insoluble salts and centrifuged again for 10 minutes at 10000 rpm at 4°C. The resulting supernatant was decanted into a collecting tube, drawn into a 1 ml syringe and passed through a 0.22 μM filter (Fisher Scientific). The samples were then transferred to vials and injected at a volume of 10 μl into the HPLC system equipped with a Waters 2795 Separations Module Microsampler (Waters Corporation). The mobile phase was pumped at 1 ml/min and consisted of 0.1 M KH2PO4, pH 5.0. Samples were passed through a 100 × 4.1 mm C18 column (Waters Corporation). A Waters 2487 Absorbance Detector was set at 260 nm (Waters Corporation). Identification and quantification of ATP in samples were accomplished using retention time and area under the curve produced by injecting an ATP standard (Sigma-Aldrich) into the HPLC system under identical conditions. ATP standard calibration concentration ranged from 0 to 500 μM. A good linear relationship was observed between ATP concentrations against area under the curve (R2 = 0.999) or peak height (R2 = 0.998). ATP concentration was calculated based on area under the curve and was normalized against the weight of each extracted tissue.
Cell culture
Cancer cells
The human head and neck cancer cell line, HSC-3 (ATCC) derived from a human tongue SCC, was cultivated in Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5 g/L glucose, l-glutamine and sodium pyruvate, supplemented with 10% fetal bovine serum (FBS), 25 μg/mL fungizone, 100 μg/mL streptomycin sulfate, and 100 U/mL penicillin G and cultivated at 37°C in 5% CO2.
Neurons
Mouse TG neurons were harvested and cultured as previously described [27]. Briefly, BALB/c mice were euthanized with isoflurane. Trigeminal ganglia were removed, transferred into HBSS and enzyme-digested by incubation with papain (Worthington), collagenase type II (CLS2) (Worthington), and dispase type II (MB). Dissociated neurons were plated on glass coverslips coated with poly-d-lysine and laminin and maintained for approximately 2 hr at 37°C at 5% CO2/95% air in F12 media (Gibco BRL) supplemented with 10% FBS.
Co-culture
Coverslips containing TG neurons were transferred into culture dishes containing HSC-3 cells in fresh F12 medium supplemented with 10% FBS and cultured for 1 day before co-culture experiments. Anti-NGF antibody (R&D Systems) was used at 50 ng/ml and added directly into culture medium.
Mouse models
Six to eight week-old female athymic, immunocompromised (BALB/c nu/nu) mice and BALB/c mice were purchased (Charles River Laboratories). Mice were housed in a temperature-controlled room on a 12:12 light:dark cycle (0600–1800 h light), with ad libitum access to food and water. All experiments were performed according to the policies and recommendations of the International Association for the Study of Pain and approved by the New York University Institution Animal Care and Use Committee.
SCC supernatant model
HSC-3 cells were grown in 10 cm cell culture dishes to 90% confluency; the medium was changed to serum-free DMEM (2 mL volume), and incubated for 48 hours. Culture supernatant was then collected on the day of injection. 50 μl of SCC supernatant was injected into the right hind paw of BALB/c mice anesthetized with isoflurane. Control mice received the same volume of serum-free DMEM in the right hind paw. 3 mg/kg or 10 mg/kg A-317491 was directly dissolved into 50 μl SCC supernatant. AF-353 was first dissolved in DMSO (Sigma-Aldrich) and the solution was then added into 50 μl of SCC supernatant. Both A-317491 and AF-353 are strong antagonists to P2X3 and P2X2/3 receptors, and are weak antagonists to P2X2 receptors. AF-353 is a more potent antagonist, with high oral bioavailability and CNS penetration [14, 28].
Paw SCC model
A paw cancer pain model was created by inoculating 106 HSC-3 cells, suspended in vehicle consisting of 50 μl volume DMEM and Matrigel™ (Becton Dickinson & Co), into the right hind paw of athymic BALB/c nu/nu mice. Subcutaneous injection of 3 mg/kg A-317491 or 3 mg/kg AF-353 was performed daily from day 10 through day 28 post-inoculation. Mouse hind paw volume was measured by using a plethysmometer (IITC Life Science) (Additional file 1: Figure S1C).
Tongue SCC model
The tongue SCC model was produced in athymic BALB/c nu/nu mice as previously described [22]. The anatomic and functional features of this mouse cancer model parallel those found in HNSCC patients [22]. After baseline Dolognawmeter gnaw times were established, BALB/c nude mice were inoculated with 50 μl total volume (of 106) HSC-3 cells in DMEM and Matrigel™ into the tongue through a transoral approach. Control mice received 50 μl of vehicle injection. 20 μl of an ATP-hydrolyzing enzyme apyrase (100 μM, Sigma-Aldrich), A-317491 (3 mg/kg, Sigma-Aldrich), AF-353 (3 mg/kg, Afferent Pharmaceuticals) or normal saline solution was injected into the tongue of mice on post-inoculation day 14.
Behavioral assessment
Paw withdrawal assay
Testing was performed by an observer blinded to the experimental groups. The paw withdrawal threshold was measured by an electronic von Frey anesthesiometer (IITC Life Sciences). Paw withdrawal threshold was defined as the force (g) sufficient to elicit a distinct paw withdrawal flinch upon application of the probe tip. A mean of eight withdrawal thresholds was calculated.
Orofacial function measurement
Behavioral testing with the Dolognawmeter was performed as previously described [29]. Briefly, each animal was placed in a tube in which access to escape was obstructed by a series of two polymer dowels. The animal voluntarily gnaws through the two dowels to escape from confinement within the tube. Each polymer dowel is connected to a digital timer. When a bar is severed by the mouse, a timer dedicated to the respective dowel is stopped. Animals were trained for 10 gnawing trials and then a baseline gnaw-time was established for each animal. Gnawing behavior was measured 15 minutes after drug treatment on post-inoculation 14. Data were analyzed as percent change of gnaw-time from baseline for each mouse.
Immunofluorescence
Coverslips with plated neurons were fixed with 4% PFA and blocked with superblock (Thermo Fisher Scientific) for 30 min. Human HNSCC tissues were resected and fixed with 4% PFA, dehydrated, embedded in paraffin, and cut into 8-μm sections. Sections were then deparaffinized and blocked with superblock. H&E staining was performed to confirm cancer lesions. For immunofluorescence labeling, neurons or tissue sections were then incubated for 24 h at 4°C in rabbit anti-P2X3 (1:500, Alomone Labs) and goat anti-P2X2 (1:500, Santa Cruz Biotechnology). The sections were then washed in phosphate-buffered saline (PBS) with Triton X-100 and incubated in secondary antibody chicken anti-rabbit Alexa-594 (1:1000, Invitrogen) and donkey anti-goat Alexa-488 (1:1000, Invitrogen) in a dark chamber for 2 hours at room temperature. Control experiments were performed by incubation in secondary antibody alone and by applying P2X2 blocking peptides (Santa Cruz Biotechnology), and P2X3 blocking peptides (Alomone Labs). The coverslips or sections were washed and visualized with images acquired using a Nikon Ti Eclipse microscope (Nikon).
Microdialysis
Mice were anesthetized with ketamine/xylazine (9:1, vol/vol). A microdialysis probe (CMA30, CMA-Microdialysis) was inserted through a guide cannula into the tongue of mice with tongue SCC or normal mice. Tongues were perfused with PBS at a constant flow rate of 1.0 μl/min using a CMA-402 microsyringe pump. After a 30 minute equilibration period, samples were collected for 90 minutes and kept at 4°C. Six mice were used in each group.
ATP Luminescence assay
ATP concentration in the mouse tongue microdialysate samples and HNSCC supernatant were determined using ENLITEN ATP assay kit (Promega). Luminescence intensity was determined using a luminometer (GloMax-Multi Detection System, Promega). Calibration curves were obtained using standard ATP samples with subtraction of background luminescence of PBS. For SCC supernatant, 5×104, 105, 2×105 cells were seeded onto separate culture plates and incubated in 3 ml of serum-free medium. Media was collected after 12 hours of incubation and ATP quantification was performed immediately.
Calcium imaging
Cultured TG neurons were loaded with 1 μM of the cell permeable calcium sensitive dye, Fura 2 AM (Molecular Probes) for 30 min and washed with HBSS before use. Coverslips containing neurons were placed in a chamber with constant infusion of phenol-red free DMEM at room temperature. Fluorescence was detected by a Nikon Eclipse TI microscope (Nikon) fitted with a 20x fluor/NA 0.75 objective lens. Fluorescence images of 340 and 380 excitation wavelengths were collected and analyzed with the TI Element Software (Nikon). For drug treatments, neurons were pre-incubated with either AF-353 (1 μM), or A-317491 (1 μM) for 20 min prior to SCC supernatant application. Cells were counted as SCC supernatant responsive if the 340/380 ratio is ≥0.2 from baseline.
Electrophysiology
Coverslips with neurons were transferred to a recording chamber and perfused continuously with external solution containing the following (in mM): 140 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 glucose and 10 HEPES (pH 7.3 adjusted with NaOH, 320 mOsm/kg with sucrose), at room temperature. Patch pipettes were double-pulled (P-2000, Sutter, CA) from quartz glass capillaries (Q100-50-10, Sutter). They were adjusted to 2–8 MΩ when filled with a pipette solution (in mM): KCl 145, MgCl2 3, CaCl2 2.25, EGTA 1, HEPES 10 (pH 7.3 adjusted with KOH, 310 mOsm). After establishing the whole-cell configuration, the voltage was clamped at −60 mV using Axopatch 200B amplifier (Axon Instrument) and controlled by Clampex software (pClamp 10.2; Axon Instrument). DMEM, SCC supernatant, and drugs were applied using a fast-step SF-77B perfusion system (Warner Instrument) with three-barreled pipette placed near the cell. Current amplitudes were measured at the peak of the inward component.
qRT-PCR
Human oral SCC and anatomically matched, contralateral normal oral epithelium from 10 oral cancer patients were surgically removed and immediately snap frozen in liquid nitrogen and stored at −80°C. Fresh TG neurons from mice with tongue SCC or normal mice were collected and stored at −80°C (n = 8 in each group). Tissues were homogenized and total RNA isolation of each sample was conducted with a Qiagen AllPrep DNA/RNA Micro Kit (Qiagen Inc.). Reverse transcription was carried out with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc.) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with the Taqman Gene Expression Assay Kit (Applied Biosystems Inc.). Primers were purchased from Life Technologies (Mouse P2X3: Mn00523699_m1; Mouse P2X2: Mn00462952_m1). The housekeeping gene β-actin was used as the internal control gene. Relative quantification analysis of gene expression data was calculated using the 2 − ΔΔCt method.
Statistical analysis
SigmaPlot 11.0 for Windows was used to perform the statistical analysis. Student’s t-test, paired t-test, one-way Analysis of Variance (ANOVA) with a Tukey multiple comparisons post-hoc test, two-way ANOVA, and regression analysis were used where appropriate. Significance level was set at *P < 0.05, **P < 0.01, ***P < 0.001. Results were presented as mean ± SEM.