STING suppresses bone cancer pain via immune and neuronal modulation

Abstract

Patients with advanced stage cancers frequently suffer from severe pain as a result of bone metastasis and bone destruction, for which there is no efficacious treatment. Here, using multiple mouse models of bone cancer, we report that agonists of the immune regulator STING (stimulator of interferon genes) confer remarkable protection against cancer pain, bone destruction, and local tumor burden. Repeated systemic administration of STING agonists robustly attenuates bone cancer-induced pain and improves locomotor function. Interestingly, STING agonists produce acute pain relief through direct neuronal modulation. Additionally, STING agonists protect against local bone destruction and reduce local tumor burden through modulation of osteoclast and immune cell function in the tumor microenvironment, providing long-term cancer pain relief. Finally, these in vivo effects are dependent on host-intrinsic STING and IFN-I signaling. Overall, STING activation provides unique advantages in controlling bone cancer pain through distinct and synergistic actions on nociceptors, immune cells, and osteoclasts.

Fig. 1. STING agonists reduce bone cancer-induced pain and functional impairment.

a Experimental design to test the antinociceptive effects of DMXAA in the LLC model of bone cancer. b Von Frey testing to determine cancer-induced mechanical allodynia, as assessed by withdrawal threshold (left) or withdrawal frequency (0.16 g stimulus; right) in mice treated with vehicle or DMXAA (20 mg/kg, i.p.) (n = 11 vehicle-treated mice and n = 9 DMXAA-treated mice) ***P < 0.001. c Assessment of cancer-induced cold allodynia after LLC inoculation in mice treated with vehicle or DMXAA (n = 11 vehicle-treated mice and n = 9 DMXAA-treated mice). d Comparison of spontaneous pain as indicated by flinching behaviors (left) or guarding behaviors (right) in vehicle and DMXAA-treated mice on d14 after tumor implantation (n = 11 vehicle-treated mice and n = 8 DMXAA-treated mice). e Measurement of body weight in vehicle or DMXAA-treated mice at the indicated timepoints (n = 11 vehicle-treated mice and n = 9 DMXAA-treated mice). f Open field testing at d14 post-inoculation to determine distance traveled (m) and mean speed (cm/s) over a 30 min duration in vehicle or DMXAA treated mice (n = 8 mice/group). Left: representative traces. Right: quantification. g Von Frey testing to measure cancer-induced mechanical allodynia in mice treated with vehicle, ADU-S100 (20 mg/kg, i.p.) or ZA (zoledronic acid; 100 µg/kg, i.p.), n = 8 vehicle-treated mice and n = 7 ADU-S100-treated mice, and n = 8 ZA-treated mice, ***P < 0.001. h Measurement of cancer-induced cold allodynia in mice with indicated treatment on day 7, 10 and 14 after LLC inoculation (n = 8 vehicle-treated mice and n = 7 ADU-S100-treated mice, and n = 8 ZA-treated mice). i Spontaneous pain as determined by flinching behaviors (left) or guarding behaviors (right) over a 2-min interval on d14 post inoculation (n = 8 vehicle-treated mice and n = 7 ADU-S100-treated mice, and n = 8 ZA-treated mice). j Body weight measurements in mice with the indicated treatments (n = 8 vehicle-treated mice and n = 7 ADU-S100-treated mice, and n = 8 ZA-treated mice). k Open field testing, measuring distance traveled (m) and mean speed (cm/s) over a 30 min duration in vehicle, ADU-S100, and ZA-treated mice at d14 after tumor inoculation (n = 6 mice/group). Left: representative traces; right: quantification. All data displayed represent the mean ± SEM, repeated-measures two-way ANOVA with Bonferroni’s post-hoc test (b, c, e, g, h, j); two-tailed Student’s t-test (d, f); one-way ANOVA with Bonferroni’s post-hoc test (i, k). Source data are provided as a Source Data file.

Fig. 2. STING agonists attenuate cancer-induced bone destruction.

a Representative radiographs of tumor-bearing femora from vehicle or DMXAA treated mice. Bone destruction score is indicated in each image and arrows show bone lesions with scores over 3 while arrowhead shows the detached patella. b Quantification for (a) (n = 11 vehicle-treated mice and n = 9 DMXAA-treated mice), ***P < 0.001. c Micro-CT images showing trabecular and cortical bone destruction in the distal part of tumor-bearing femora on d11 after LLC inoculation. Scale bars, 1 mm. d Morphometric quantification of micro-CT images with analysis of trabecular bone (Conn.D; upper) or cortical bone (BV/TV) in tumor-free femora from naïve mice or LLC inoculated femora from vehicle or DMXAA-treated mice (n = 5 naive mice, n = 5 vehicle-treated tumor-bearing mice, and n = 7 DMXAA-treated tumor-bearing mice). e, f Radiographical analysis of bone destruction in mice administered vehicle, ADU-S100, or ZA at the indicated timepoints after tumor inoculation. e Representative X-ray images. Bone destruction score is labeled on the bottom of each photo and arrow indicates bone destruction score more than 3. f Quantification of images in e (n = 8 vehicle-treated mice and n = 7 ADU-S100-treated mice, and n = 8 ZA-treated mice), ***P < 0.001. g Images of femurs and quantification of the proportion with bone fracture from tumor bearing femora taken from vehicle or DMXAA-treated mice on d17 after LLC inoculation. Arrows indicate the disconnection and absence of the distal part of the femora (n = 8 mice/group). h Images of tumor bearing femora with indicated treatment harvested on d17 after LLC inoculation (left) and quantification of the proportion with bone fracture (right). Arrows indicate lesion and loss of the distal aspect of the femora (n = 8 vehicle-treated mice and n = 7 ADU-S100-treated mice, and n = 8 ZA-treated mice). All data indicate the mean ± SEM, repeated-measures two-way ANOVA with Bonferroni’s post-hoc test (b, f); two-tailed Student’s t-test (d); two-sided Fisher’s exact test (g, h). Source data are provided as a Source Data file.

Fig. 3. The protective effects of STING agonists are mediated by host IFN-I signaling.

a Analysis of serum IFN-α (left) and IFN-β (right) levels at BL and again 4 h or 24 h after DMXAA or ADU-S100 treatment on d3 after tumor implantation (n = 7 vehicle-treated mice, n = 5 DMXAA-treated mice, and n = 5 ADU-S100-treated mice), ***P < 0.001. b IFN-α (left) and IFN-β (right) levels in bone marrow (BM) lysates from mice treated with vehicle, DMXAA or ADU-S100 (n = 8 vehicle-treated mice, n = 5 DMXAA-treated mice, and n = 5 ADU-S100-treated mice), ***P < 0.001. c von Frey testing to determine withdrawal threshold (left) and frequency (right) from Ifnar1^+/+ or Ifnar1^−/− mice treated with vehicle or DMXAA (2 x 20 mg/kg, i.p.). d Analysis of cold allodynia in vehicle or DMXAA-treated Ifnar1^+/+ mice and Ifnar1^−/− mice. e–f Bone destruction scores from radiographs of tumor bearing femora in Ifnar1^+/+ and Ifnar1^−/− mice with the indicated treatment on d0, d8, d11 and d15 after LLC injection. Arrows show bone lesions with destruction scores over 3. e Representative X-ray images. f Quantification for e. g Body weight measurement after vehicle or DMXAA treatment. Sample sizes for c–g were as follows: n = 7 vehicle-treated Ifnar1^+/+ mice, n = 7 DMXAA-treated Ifnar1^+/+ mice, n = 6 vehicle-treated Ifnar1^−/− mice, n = 6 DMXAA-treated Ifnar1^−/− mice. All data indicate the mean ± SEM, repeated-measures two-way ANOVA with Bonferroni’s post-hoc test (a, c, d, f, g); one-way ANOVA with Bonferroni’s post-hoc test (b). Source data are provided as a Source Data file.

Fig. 4. Ex vivo STING activation inhibits neuronal hyperexcitability of DRGs from cancer-bearing mice.

a Schematic of whole-mount DRG preparation, drug treatment, and bright field image with a recording micropipette sealed on a small-diameter neuron (nociceptor). b Representative traces of rheobases: current clamp recordings of the membrane potential from small diameter DRG neurons of naïve mice or bone cancer-bearing animals with or without DMXAA (30 µM, 2 h). Current injection for action potential induction starts from 0 pA and increases 10 pA per step for 30 ms. c The averages of rheobases from naïve mice, vehicle or DMXAA-treated group (naïve: n = 8 neurons/4 mice; vehicle: n = 9 neurons/4 mice; DMXAA: n = 10 neurons/4 mice). d Left: injected current steps for the induction of action potentials starts from 0 pA and increases 10 pA per step for 300 ms. Right: representative traces showing the response to a 110 pA current injection (red line in left) from small diameter DRG neurons of naïve mice or bone cancer mice with or without DMXAA treatment. e Action potentials in response to increasing current amplitude from naïve mice or bone cancer mice with or without DMXAA (naïve: n = 8 neurons/4 mice; vehicle: n = 9 neurons/4 mice; DMXAA: n = 10 neurons/4 mice), ***P < 0.001. All data indicate the mean ± SEM, one-way ANOVA with Bonferroni’s post hoc test (c); repeated-measures one-way ANOVA with Bonferroni’s post-hoc test (e). Source data are provided as a Source Data file.

Fig. 5. Direct antinociceptive effect of STING agonist is dependent on STING and Ifnar1 in sensory neurons.

a Von Frey testing to determine cancer-induced mechanical allodynia, as assessed by withdrawal frequency in mice treated with vehicle (n = 12 mice), DMXAA (20 mg/kg, i.p., n = 6 mice) or ADU-S100 (20 mg/kg, n = 6 mice), ***P < 0.001. Measurement of mechanical allodynia as indicated by paw withdrawal frequency (b) or cold allodynia (c) 1 h, 4 h or 24 h after a single i.p. injection of vehicle (n = 8 mice), DMXAA (20 mg/kg, n = 9 mice), or morphine (10 mg/kg, n = 8 mice) on d11 after LLC inoculation, ***P < 0.001. d-e Measurement of mechanical allodynia by von Frey testing (d) and cold allodynia from acetone response (e) after DMXAA i.p. injection in WT, STING^gt/gt or Ifnar1^−/− mice (n = 7 mice/group). Measurement of mechanical allodynia by von Frey testing (f) and cold allodynia by acetone response duration (g) after vehicle/DMXAA i.p. injection in Ifnar1^fx/fx; Na_v1.8-Cre (Ifnar1-cKO) mice (n = 6 mice for vehicle group and n = 9 mice for DMXAA group) or in Ifnar1^fx/fx (WT) mice (n = 5 mice for vehicle group and n = 7 mice for DMXAA group), ^#P = 0.0027 (Ifnar1^fx/fx + vehicle vs. Ifnar1^fx/fx; Nav1.8-Cre + vehicle); P = 0.0011 (Ifnar1^fx/fx + vehicle vs. Ifnar1^fx/fx; Nav1.8-Cre + DMXAA); P = 0.0020 (Ifnar1^fx/fx + DMXAA vs. Ifnar1^fx/fx; Nav1.8-Cre + vehicle); P = 0.0006 (Ifnar1^fx/fx + DMXAA vs. Ifnar1^fx/fx; Nav1.8-Cre + DMXAA); ***P < 0.001. h–i Measurement of mechanical allodynia by von Frey testing after DMXAA and ADU-S100 treatment (at d3 and d7, i.p.) in STING^fx/fx; Na_v1.8-Cre (STING-cKO) mice or STING^fx/fx (WT) mice. h Schematic of experimental design. i Paw withdrawal frequency. n = 6 mice for STING^fx/fx; Na_v1.8-Cre (Vehicle) group and n = 7 mice for the rest groups, ***P < 0.001. Notably, DMXAA and ADU-S100 attenuated mechanical allodynia at later time points (d10, d14) in STING-cKO mice. All data indicate the mean ± SEM, repeated-measures two-way ANOVA with Bonferroni’s post-hoc test (a–i). Source data are provided as a Source Data file.

Fig. 6. Systemic STING agonists reduce local tumor burden in the bone cancer tumor microenvironment.

a In vivo bioluminescence of flux emitted by LL/2-Luc2 carcinoma (LLC) cells in tumor bearing femur after vehicle or DMXAA treatment (2 × 20 mg/kg, i.p.) measured at d8, d11, and d15 post tumor inoculation (n = 10 vehicle-treated mice, n = 11 DMXAA-treated mice). Images (left) were obtained at 15 min after i.p. injection of D-luciferin (30 mg/kg). Right, experimental scheme and quantification of a. b Ratio of maximum thigh circumference reflecting local tumor burden in mice with each indicated treatment on d17 after LLC implantation. Left, vehicle or DMXAA treatment (2 × 20 mg/kg, i.p.; n = 11 vehicle-treated mice and n = 9 DMXAA-treated mice). Right, vehicle, ADU-S100 (2 × 20 mg/kg, i.p.) or ZA (zoledronic acid; 2 × 100 µg/kg, i.p.) treatment (n = 8 vehicle-treated mice and n = 7 ADU-S100-treated mice, and n = 8 ZA-treated mice), ***P < 0.001. c Ratio of maximum thigh circumference in mice administered with vehicle, DMXAA or ADU-S100 (2 × 100 µg/kg, i.p.) on d17 after implantation of E0771 breast cancer cells (n = 7 mice/group), ***P < 0.001. d Images of lung tumor nodules in mice with each indicated treatment on d17 after LLC inoculation. Left, representative dorsal and ventral murine lung image, with arrows showing metastatic tumor nodules. Right, H&E staining for sections from lung samples in Left. Arrows indicates the areas with tumor cells, scale bar, 2 mm. 1 and 2 are enlarged images showing tumor tissue and peritumoral areas, respectively. Scale bar, 50 µm. Note that tumor cells have large and irregular nuclei with loss of the normal alveolar structure. e Quantification of panel d (n = 8 vehicle-treated mice, n = 7 ADU-S100-treated mice, and n = 8 ZA-treated mice). f-g FACS analysis of CD4⁺ and CD8⁺ T cells (f) or Treg cells (g) within the bone marrow tumor microenvironment in mice treated with vehicle or DMXAA (2 × 20 mg/kg, i.p.) on d8 post-LLC inoculation (n = 5 mice/group). The gating strategy for this figure is provided in Supplementary Fig. 7. h Local tumor burden as determined by the ratio of maximum thigh circumference after vehicle or DMXAA treatment in WT (n = 10 mice for vehicle or DMXAA group) and Rag1^−/− mice (n = 13 mice for vehicle or DMXAA group) on d17 after LLC implantation, ***P < 0.001. i Local tumor burden as determined by the ratio of maximum thigh circumference in Batf3^+/+ and Batf3^−/− mice with indicated treatment measured on d17 after LLC implantation (n = 8 mice/group), ***P < 0.001. Data indicate the mean ± SEM, repeated-measures two-way ANOVA with Bonferroni’s post hoc test (a, b, c, h, i); one-way ANOVA with Bonferroni’s post hoc test (e); two-tailed Student’s t-test (f, g). Source data are provided as a Source Data file.

Fig. 7. STING agonists attenuate cancer-induced osteoclastogenesis.

a Representative images (left) and quantification (right) of TRAP staining to reveal osteoclast numbers in the tumor-bearing distal femora from mice treated with vehicle or DMXAA (2 × 20 mg/kg, i.p.) measured on d11 after LLC inoculation (n = 3 mice for naïve group and n = 5 mice for vehicle or DMXAA group). Scale bar, 500 µm. b Images (left) and quantification (right) of ALP staining to reveal osteoblasts in the tumor-bearing distal femora at d11 from mice with the indicated treatments (n = 3 mice for naïve group and n = 5 mice for vehicle or DMXAA group). Scale bar, 500 µM. c Measurement of serum CTX-I and PINP levels by ELISA at BL or d17 in vehicle or DMXAA (2 × 20 mg/kg, i.p.) treated mice (n = 8 vehicle-treated mice and n = 7 DMXAA-treated mice). d TRAP staining revealing osteoclast numbers after differentiation from RAW264.7 cells stimulated with 35 ng/ml RANKL, in the presence of increasing concentrations of DMXAA. Arrows indicate TRAP⁺ multinucleated osteoclasts. Left, representative TRAP-stained images. Right, quantification (n = 3 biologically independent experimental replicates), ***P < 0.001. Scale bar, 200 µm. e ELISA quantification of IFN-α and IFN-β levels in the culture medium of BMDM cells 24 h after DMXAA (30 µM) or ADU-S100 (30 µM) co-incubation. RANKL: 35 ng/ml, MCSF: 20 ng/ml (n = 3 biologically independent experimental replicates). f, g TRAP staining for osteoclasts differentiated from BMDM cells from WT mice, STING^gt/gt mice or Ifnar1^−/− mice, each treated with vehicle, DMXAA (30 µM) or ADU-S100 (30 µM). RANKL: 35 ng/ml, MCSF: 20 ng/ml. f Representative images of TRAP staining. Arrows indicate TRAP⁺ multinucleated osteoclasts. Scale bar, 100 µm. g Quantification for (f), ***P < 0.001. Sample sizes for f-g refer to independent cultures taken from individual mice and are as follows: WT/vehicle: n = 6, WT/DMXAA: n = 6, WT/vehicle: n = 6, STING^gt/gt/vehicle: n = 3, STING^gt/gt/DMXAA: n = 3, STING^gt/gt/ADU-S100: n = 3, Ifnar1^−/−/vehicle: n = 3, Ifnar1^−/−/DMXAA: n = 3, Ifnar1^−/−/ADU-S100: n = 3. Data indicate the mean ± SEM, one-way ANOVA with Bonferroni’s post hoc test (a, b, d, e, g); repeated-measures two-way ANOVA with Bonferroni’s post-hoc test (c). Source data are provided as a Source Data file.

Fig. 8. The analgesic and bone-protective effects of STING agonists largely retain in T cell-deficient Rag1^−/− mice.

a Mechanical allodynia from von Frey test in WT or Rag1^−/− mice treated with vehicle or DMXAA (2 × 20 mg/kg, i.p.) on baseline (BL), day 7, 10 and 14 after tumor inoculation. Left, withdrawal threshold. Right, withdrawal frequency, ***P < 0.001. b Cold allodynia from acetone test in WT or Rag1^−/− mice with indicated therapy, ***P < 0.001. c, d Radiographical analysis of bone destruction in WT or Rag1^−/− mice administered vehicle or DMXAA, measured at BL, d8, d11 and d15 post LLC inoculation. c Representative X-ray images. Bone destruction score is labeled on the bottom of each photo and arrows indicate bone destruction scores of more than 3. d Quantification for (c). e Quantification of the proportion of mice with distal bone fractures, harvested and analyzed at d17 post-inoculation and with the indicated genotypes and treatment groups. Sample sizes for a–e were as follows: n = 10 vehicle-treated WT mice, n = 10 DMXAA-treated WT mice, n = 13 vehicle-treated Rag1^−/− mice, and n = 13 DMXAA-treated Rag1^−/− mice (pooled from two independent experiments). Data are Mean ± SEM, repeated-measures two-way ANOVA with Bonferroni’s post hoc test (a, b, d); two-sided Fisher’s exact test (e). Source data are provided as a Source Data file.

Fig. 9. STING agonists reduce fracture-induced bone pain in tumor-free mice.

a Schematic of mouse model of fracture-induced bone pain. b Experimental diagram indicating single treatment of vehicle, DMXAA, or ADU-S100 and behavioral testing. c, d Mechanical allodynia from von Frey test (c) and cold allodynia from acetone test (d) in mice treated with vehicle, DMXAA, or ADU-S100 (20 mg/kg i.p.) on d3 after bone fracture, ***P < 0.001. e Experimental diagram of repeated administration of vehicle, DMXAA, or ADU-S100 and behavioral testing for the measurement of long-term effects. f Von Frey test to determine mechanical allodynia, as assessed by withdrawal threshold (left) or withdrawal frequency (right) in mice treated with vehicle, DMXAA or ADU-S100 (3 × 20 mg/kg, i.p.) at d14 and d42 after bone fracture, ***P < 0.001. g Locomotor function of mice treated with vehicle, DMXAA or ADU-S100 (3 × 20 mg/kg, i.p.) at d14 and d42 after injury. Sample sizes for c, d, f, and g were as follows: n = 6 vehicle-treated mice, n = 7 DMXAA-treated mice, n = 7 ADU-S100-treated mice. Data are mean ± SEM, repeated-measures two-way ANOVA with Bonferroni’s post hoc test (c, d, f, g). Source data are provided as a Source Data file.

Fig. 10. Schematic of mechanisms by which STING agonists directly and indirectly attenuate bone cancer-induced pain.

a In untreated metastatic bone cancer, the pathophysiology driving cancer pain is multifaceted. Metastatic cancer cells present within the bone marrow tumor microenvironment (TME) produce pro-osteoclastogenic signals, driving cancer-induced osteoclastogenesis and osteoclast overactivation. Both cancer cells and osteoclasts produce nociceptive mediators, which directly activate peripheral nociceptive afferents present in the TME to produce pain through a direct mechanism. Additionally, cancer-induced osteoclast overactivation also leads to increased bone resorption, leading to increased bone destruction and bony fractures, which also produce pain through nociceptor activation. b STING agonists dramatically attenuate metastatic bone cancer-associated pain through multiple mechanisms, each mediated by host-intrinsic type-I interferon signaling. First, STING-mediated IFN-I signaling directly suppresses excitability of peripheral nociceptors (neuromodulation), leading to acute suppression of pain for the duration of the IFN-I response. In addition, STING-mediated IFN-I signaling promotes CD8⁺ T cell migration into the bone marrow TME, augmenting antitumor immunity and reducing tumor burden. In addition, IFN-I drives suppression of cancer-induced osteoclastogenesis. These two non-neuronal, immunomodulatory effects lead to sustained inhibition of pain by (1) reducing cancer cell- and osteoclast-derived pro-nociceptive mediators and (2) reducing osteoclast-mediated bone resorption, thereby attenuating subsequent bone destruction and bony fractures that frequently evoke pain and skeletal-related events (SRE). Thus, the immunomodulatory and neuromodulatory effects of STING agonists each individually suppress pain through actions on different cell types, and also synergistically suppress cancer pain through a convergence of shared downstream actions. Pre-OCs pre-osteoclasts, TILs tumor infiltrating lymphocytes.