# Three-Dimensional Analysis of the Effect of Osteosarcoma on Sensory Nerves Innervating the Femur in a Murine Model of Osteosarcoma-Induced Bone Pain

## TL;DR

This study shows how osteosarcoma cancer changes sensory nerves in mouse bones, which could help develop better pain treatments for bone cancer.

## Contribution

The study reveals that osteosarcoma differentially impacts subtypes of sensory nerves in bone using 3D analysis.

## Key findings

- Osteosarcoma reduces myelinated nerve density in bone marrow but not peptidergic nerves.
- Periosteal peptidergic nerve density increases in osteosarcoma-affected bones.
- Altered nerve distribution correlates with pain behavior in mice with osteosarcoma.

## Abstract

This study aimed to determine how cancer affects sensory nerve density and distribution, using 3D analysis and mapping tools applied to a murine model of osteosarcoma-induced bone pain. Pain-like behavior, nerve density and distribution were assessed in osteosarcoma bearing and control mice. Comparisons between osteosarcoma bearing and control mice revealed that osteosarcoma differentially affects the density and distribution of different subtypes of peripheral sensory nerves in bone. Understanding how osteosarcomas affect different populations of sensory nerves could lead to more targeted mechanism-based treatments for bone cancer-induced pain.

Background: The ways in which peripheral sensory nerves interact with osteosarcomas are important to understand because it could lead to development of new approaches to treat bone cancer pain. This study aimed to determine how cancer affects sensory nerve density and distribution in a murine model of osteosarcoma-induced bone pain. Methods: The femoral marrow cavities of male C3H/HeNHsd mice were injected with either NCTC 2472 primary osteosarcoma (cancer) cells or phosphate buffered saline (control). Pain behavior was assessed using limb use score and static weight bearing assays. At the experimental endpoint, femurs were collected, decalcified, immunolabeled, cleared and imaged using light sheet microscopy (Ultramicroscope Blaze, Miltenyi Biotec). The distribution of sensory nerves was traced through the marrow cavity of the proximal femur and the periosteum overlying the third trochanter (Imaris, Bitplane). Results: Weight bearing on the injected limb was decreased in osteosarcoma-injected but not saline-injected mice. Filament tracing revealed a reduced density of neurofilament 200 kDa-positive (NF200+; myelinated nerve marker) but not calcitonin gene-related peptide-positive (CGRP+; peptidergic nerve marker) sensory nerves in the marrow cavity of osteosarcoma-injected relative to saline-injected mice. There was increased density of CGRP+ but not NF200+ nerves in the periosteum of osteosarcoma-injected relative to saline-injected mice. Conclusions: Osteosarcoma differentially affects the density and distribution of different subtypes of peripheral sensory nerves in bone. Understanding how osteosarcomas affect different populations of sensory nerves could lead to more targeted mechanism-based treatments for bone cancer-induced pain.