227 - Osteoclast-Neuron Crosstalk Drives Radiation-Induced Pain

11:25am - 11:35am PT

Room 153

Presenter(s)

Sun Park, PhD - Wake Forest School of Medicine, Winston-Salem, NC

S. Park¹, A. A. Foster², J. Moore¹, K. E. Reno¹, A. Costaterryll¹, M. Farris¹, R. T. Hughes¹, M. T. Munley¹, M. Sharma³, Y. Su³, S. Singh³, G. Deep³, S. W. Almousa¹, A. P. Pluma⁴, E. A. Romero-Sandoval⁴, and J. Willey¹; ¹Department of Radiation Oncology, Wake Forest University School of Medicine, Winston-Salem, NC, ²Department of Orthopaedic Surgery, Wake Forest University School of Medicine, Winston-Salem, NC, ³Department of Internal Medicine-Gerontology and Geriatric Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, ⁴Department of Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, NC

Purpose/Objective(s):

Radiotherapy (RT)-induced chest wall pain (CWP) affects up to 40% of thoracic SBRT patients within two years, with or without rib fractures. Current treatments are only symptomatic, and the underlying mechanisms remain unclear. Our in vitro studies show that RT increases osteoclast (OC) activity, and conditioned media (CM) from irradiated OCs elevates neuronal pain markers (CGRP/SP), an effect blocked by bisphosphonates (BPs). In a recent clinical trial at our institution, BPs reduced rates of grade 2+ CWP in lung cancer patients post-SBRT. We hypothesize that irradiated OCs release effectors that activate sensory neurons, driving pain. This study aims to (1) determine if OC-derived signals enhance neuronal excitability and (2) identify key mediators of this interaction that drive RT-induced pain.

Materials/Methods:

General: For all radiation studies, OCs were differentiated from RAW264.7 cells and received one of 4 treatments: A) No RT (0Gy), B) RT (10Gy), C) BP (risedronate at 50µM), or D) RT+BP. RT exposures were performed using the Precision X-Ray SmART⁺ unit (220kVP X-Rays).

Electrophysiology: To assess whether signals from irradiated OCs enhance neuronal activity, suggesting a pain response, primary DRG neurons were cultured on a CytoView MEA plate with 15 microelectrodes per well to measure excitability in real time. Neurons were exposed to CM from differentiated OCs, and the number of active electrodes/neurons was measured every 2 hours for 24 hours.

Molecular Pathway Analysis: Small extracellular vesicles (sEVs) were isolated from all groups and analyzed via mass spectrometry and Ingenuity Pathway Analysis to identify pain inducing molecular effectors that are upregulated in irradiated OCs but suppressed by BPs. A follow-up study tested these effectors in vitro by treating neurons with CM from OCs that received No RT or RT, +/- an inhibitor for the putative molecular pain-inducing effector, then pain markers were measured via RT-qPCR.

Results:

Electrophysiological analysis showed that CM from RT-activated OCs increased neuronal activity at 20 hours, an effect blocked by BPs. Active electrode counts (out of 45) were: No RT (13), RT (33), BPs (13), and RT + BPs (14), supporting OC effectors’ role in pain. Pathway analysis identified four key pathways linking pain signaling and OC resorption, with TRPV2 (+6.8), RANK (+5.8), and TGFß receptor (R) 1 (+5.4) showing the highest RT-induced upregulation, but not with BPs. Given TGF-ß’s role in pain, a follow-up study identified blocking TGFßR1 in neurons prevented RT-induced CGRP/SP upregulation (p<0.05), confirming its role in OC-mediated neuronal activation.

Conclusion:

Osteoclast-derived signals contribute to neuronal excitability and pain after irradiation. These signals are suppressed by inhibiting OC activity with BPs. Our findings identify TGFß-signaling as a key mediator of OC-neuron crosstalk in RT-induced pain, as blocking TGFßR1 reduced RT-induced pain marker expression.