135 - Nociceptive Nerves Promote Disease Progression and Metastasis of Osteosarcoma

Publication Number: 2130.135

Anthony C. Restaino, UPMC Childrens Hospital of Pittsburgh, Pittsburgh, PA, United States; Paola D.. Vermeer, University of South Dakota, Sanford School of Medicine, Sioux Falls, SD, United States; Caitlin S. Williamson, Sanford Children's Hospital, Sioux Falls, SD, United States; Ethan B. Lamfers, Sanford Research, Sioux Falls, SD, United States; Samuel Milanovich, Sanford Roger Maris Cancer Center, Fargo, ND, United States

Background: Cancer neuroscience has become more of a focus in peripheral solid cancers. Previous studies by our lab and others have shown that peripheral sensory and sympathetic nerves mediate cancer progression through various mechanisms, including direct signaling, immune dysregulation, and microenvironment changes. However, these studies have not been conducted in pediatric diseases, including osteosarcoma.

Objective: Investigate the presence and composition of neural structures, the mechanism of nerve recruitment, and the function of nerves in osteosarcoma progression and metastasis.

Design/Methods: Patient osteosarcoma samples were IHC and IF stained with nerve markers to identify presence and type of intra-tumoral nerves. In vitro studies utilized SAOS-2 and K7M2 cell lines. Proliferation studies were conducted with MTT assays. Migration studies were conducted by modified scratch assay. Microelectroarray were used to measure impact of cancer on nerve electrical activity. Dorsal root ganglia were collected from Balb/c mice and exposed to condition media from OS cell lines, stained for BIII-tubulin and measured with Sholl analysis to determine nerve outgrowth. In vivo studies utilized K7M2 orthotopically injected into mice pretreated with placebo or Resiniferatoxin to ablate sensory nerves and measured every seven days. Nerve tracing experiments were conducted using wheat germ agglutinin. At terminal growth size, primary bone lesions and lungs were collected, and stained for BIII-tubulin to identify intra-tumoral nerves.

Results: IHC staining of human osteosarcoma samples demonstrated TRPV1+ intra-tumoral nerves. In vitro studies demonstrated that co-culture of OS cells with DRG increased cell proliferation, migration, and neurite extension. ELISA studies showed increased VEGF and PDGF secretion from OS cells in the presence of DRG, and proliferation, migration, and neurite extension was suppressed with co-treatment of Sorafenib. In vivo studies demonstrated that chemical ablation of TRPV1+ nerves significantly decreased cancer progression and metastasis, and increased survival.

Conclusion(s): Here we demonstrate that osteosarcoma cells are capable of recruiting nerves into the tumor microenvironment through the release of VEGF, and co-culture of nerves and OS cells results in increased cell proliferation and migration, and further studies showed OS cells increased electrophysiological activity. In vivo experiments demonstrate that loss of TRPV1+ nerves significantly reduces tumor growth and metastasis. These studies advocate for new potential therapies targeting the tumor microenvironment in osteosarcoma patients.