Extended Data Fig. 2: Antinociceptive effects of STING agonists in naive mice and in mouse models of neuropathic and cancer pain.

a, Naive mice were administered vehicle or the STING agonist DMXAA via i.t. injection (red arrows), followed by Von Frey testing to determine mechanical thresholds at 4 h following the first (day 1, D1) or second (D2) injection. STING agonists induced a dose-dependent increase in paw withdrawal thresholds, which was further amplified by multiple injections. 10 μg (35 nmol) exhibited the largest effect, and therefore, was used throughout the rest of the study. BL, baseline. b, Naive mice were administered vehicle or ADU-S100, a STING agonist with cross-species activity, via i.t. injection (red arrows) and tested as in a. 25 μg (35 nmol) exhibited the largest increase in paw withdrawal thresholds and this dose was used throughout the rest of the study. c, Systemic administration of DMXAA and ADU-S100 increased paw withdrawal threshold in naive mice for up to 24 h. d, In the CIPN model, i.p. DMXAA and ADU-S100 suppressed mechanical allodynia for up to 48 h. Some toxicity was observed with systemic administration in the CIPN model, as 3 mice in the DMXAA group died 24 h after the second injection. No mice died in the vehicle or ADU-S100 groups. e, A chemotherapy-induced peripheral neuropathy (CIPN) model of neuropathic pain was established with paclitaxel. f, g, In the CIPN model, i.t. DMXAA and ADU-S100 suppressed mechanical allodynia (f) and cold allodynia (g), as determined by response duration (in seconds) to acetone. h, Intrathecal administration with the natural STING ligand 3′,3′-cGAMP also reduced CIPN-induced mechanical allodynia. i, STING agonists were also tested in the sciatic nerve chronic constriction injury (CCI) model of neuropathic pain. j, Intrathecal treatment with DMXAA and ADU-S100 led to prolonged inhibition of mechanical allodynia (as determined by withdrawal threshold). k, l, Administration of DMXAA and ADU-S100 also suppressed cold allodynia (k) in the BCP model. These effects were not secondary to direct antitumour effects, as tumour burden was unaffected by STING agonist treatment and treatment groups still show significant tumour growth (l). m–o, Effects of naloxone on morphine-, DMXAA-, and ADU-S100-induced antinociception. Naloxone (10 mg/kg, i.p.) reversed morphine (2 nmol i.t.)-induced antinociception (m) but had no effect on the antinociceptive effects of DMXAA (35 nmol, i.t.) (n) or ADU-S100 (35 nmol, i.t.) (o). p, Repeated administration of DMXAA (35 nmol, i.t.) did not induce tolerance in naive mice. q–t, Pain and glial reaction after spared nerve injury (SNI). q, Paradigm of experimental design. Pain behaviours and spinal dorsal horn glial cell reaction were assessed following repeated administration with vehicle or DMXAA (red arrows) in mice receiving SNI. r, s, Mechanical allodynia (r) and cold allodynia (s) were significantly reduced in DMXAA-treated mice at later time points (starting at D12). t, Quantification of GFAP⁺ (astrocytes), Iba1⁺ (microglia), or DAPI (all cells) in the spinal dorsal horn (SDH) of mice at D21 after sustained vehicle or DMXAA treatment as indicated in q. SNI increased astrocyte activation (GFAP) in vehicle- but not DMXAA-treated mice. Values represent the ipsilateral (injured) SDH normalized to the contralateral (uninjured) SDH. All data are expressed as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001. Statistical comparisons were conducted with two-way ANOVA with Dunnett’s (a–d, f, g, k), Sidak’s (h, j, l, s), Bonferroni’s (m–p, r, t), or Tukey’s post hoc test (m–o). See Supplementary Information for complete sample sizes, sex, and statistical information.

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