Fractalkine

Animals were randomly allocated into three different groups: the leptin group received a single injection of leptin, 500 ng/50 µl (Peprotech); the fractalkine group received a single injection of fractalkine, 100 ng/50 µl (Peprotech); the control group received the vehicle (0.9% NaCl/50 µl). The choice of leptin and fractalkine dose was based on previous studies and adapted to our route of administration, the subcutaneous tissue (Souza et al., 2013 (link); Tian et al., 2011 (link); Duan et al., 2007 (link)). leptin, fractalkine or saline were injected using an insulin syringe (BD Ultra-Fine^®, 29G) subcutaneously (SC) between the five callosities of the paw, the same region to which experimental mechanical and thermal stimuli were applied.

Adhesion assay protocol was adapted from Strazza et al. [28 (link)]. Non-cancer control primary NK cells were isolated from PBMC by magnetic cell sorting using human NK cell isolation kit (Stemcell, Vancouver, British Columbia, Canada) according to manufacturer’s instructions. NK cells were resuspended at a density of 1 × 10⁵/100µL of NK MACS media (Miltenyi Biotec) supplemented with 100 IU IL-2 (Peprotech) and treated with 100 ng IL-15 (Immunotools) and/or 100 ng/mL fractalkine (Peprotech) for 2 and 24 h. NK cells were also treated with OAC patient-derived TCM or ACM for 2 and 24 h with and without pre-treatment with 5 nM CX3CR1 antagonist E6130 (MedChemExpress, Monmouth Junction, NJ, USA) for 1 h prior to exposure to ACM. A 96 well plate was coated with goat-anti-human IgG (Fc specific) in PBS and incubated overnight at 4 °C. Plates were subsequently coated with 2.5 µg/mL MAdCAM-1 (R&D, Minneapolis, MN, USA) for 1 h at 37 °C. NK cells were stained with CFSE and allowed to adhere for 20 min at 37 °C. Unbound cells were washed away. Unwashed wells were used as controls. The percentage of adherent cells was determined by a fluorescent plate reader and calculated as follows: $$\frac{\mathrm{Avg} \mathrm{intensity} \mathrm{in} \mathrm{well}}{\mathrm{Avg} \mathrm{intensity} \mathrm{in} \mathrm{unwashed} \mathrm{well}} \times \frac{100}{1}$$

AZD8797 (an antagonist of CX3CR1) administration was carried out as described previously [32 (link)]. AZD8797 (HY-13848, MCE, USA) was dissolved in a DMSO solution to yield a final concentration of 2 mg/ml according to the instructions. Rats received either an intraperitoneal injection of AZD8797 (1 mg/kg) or an equal amount of PBS once per day for four days before dural IS administration. Rat fractalkine (PeproTech, USA) was dissolved in 0.9% NaCl to yield a final concentration of 1 μg/μl; bilateral intracerebral injection of fractalkine was performed stereotactically to the sp5c at the following coordinates: anteroposterior, − 14.08 mm; lateromedial, ± 2.75 mm; dorsoventral, − 8.65 mm relative to the bregma in the rat. A total of 2.5 μl was injected into each site using a 5 μl glass syringe with a fixed needle [33 (link)]. Rats in the sham groups received 2.5 μl of PBS at each site. A schematic representation of the intracerebral injection is given in Supplementary Fig. 1.