Abstract 3630: TrkA+ sensory neurons modulate macrophage phenotype in osteosarcoma
Authors: Qin Qizhi , Ramesh Sowmya Ramesh , Clemens Thomas L. , James Aaron W. 
Published: June 2023

Abstract
Introduction: Osteosarcoma (OS) is the most common primary malignancy of the skeleton, and inordinately affects young adults. Despite improved outcomes with current multimodal care, the prognosis for patients with metastatic disease is grim. Peripheral neural regulation of cancer growth has long been suggested by clinical observations in pancreatic and prostate cancers. Prior reports suggest peripheral nerve sprouting in osteosarcoma, but the functional roles of sensory nerves in osteosarcoma are unknown. Here, the role of TrkA+ sensory nerves in OS disease progression was assayed.
Methods: All experiments were conducted under IACUC approval at Johns Hopkins University. TrkAF592A mice which are homozygous for a phenylalanine-to-alanine point mutation in exon 12 of Ntrk1 gene were crossbred with NOD scid mice (TrkAF592A/Scid). TrkA inhibition was performed by 1NMPP1 treatment. In vivo tumor growth was examined using a periosteal xenograft model. 1 million human 143B-luc OS cells were injected. Analysis was performed using serial measurements of tumor size and IVIS imaging. Nerve distribution and macrophage polarization within the tumor microenvironment was documented using immunostaining for Beta III Tubulin (TUBB3), CD86, CD206 and F4/80.
Results: Inhibition of TrkA led to a significant reduction in Tubb3+ nerves (88.4%) within and around the OS xenografts. In addition, tumors within TrkAF592A/Scid animals demonstrated slower growth, including a 51.5% reduction in tumor size and 60.0% reduction in IVIS luciferase activity at 4 wks. Histologic examination showed a reduction in macrophage infiltration and reprogramming of macrophages from an M2-like to M1-like phenotype in tumors with chemical denervation. Remarkably, this was associated with a reduction in Ki67 proliferative index (80.1%) and CD31 immunofluorescent staining (60.8%) within the tumors. Overall survival of mice with TrkA inhibition was significantly prolonged (p=0.0003). Retrograde tracing identified the sensory nerve fibers innervating the periosteum originated from L3-L5 lumbar DRGs and RNA seq identified OS-derived neurotrophins and axon guidance molecules.
Discussion: Disruption of TrkA+ nerves significantly mitigates OS disease progression in a mouse xenograft model, including a significant improved overall survival. These findings are associated with a reduction of macrophage infiltration and macrophage re-polarization. Ongoing studies are examining the molecular mechanisms that mediate nerve-to-macrophage interaction in peripheral nerves and DRG neurons using a combination of sequencing approaches.
Significance/Clinical Relevance: Sensory neurons positively regulate osteosarcoma growth and disease progression. Use of chemical or microsurgical approaches to denervate tumors may represent a future adjunctive therapy to both improve pain and prognosis in high grade bone sarcomas.
Citation Format: Qizhi Qin, Sowmya Ramesh Ramesh, Thomas L. Clemens, Aaron W. James. TrkA+ sensory neurons modulate macrophage phenotype in osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3630.