Quantification of the Release of NGF from Osteoblasts and Osteocytes in Response to Mechanical Load

Prostate cancer (PCa) is the second most common type of cancer in men with an estimated 161,360 new cases to be diagnosed in 2017 according to the American Cancer Society. Approximately 80% of prostate cancer patients experience metastases to bone. Within the bone environment, prostate cancer metastases form osteosclerotic lesions that cause a reduction in bone strength and an increase in mechanical strains in the bone. This new environment leads to the release of signaling molecules and growth factors from bone cells that heighten cancer invasion and proliferation. Nerve Growth Factor (NGF) is a signaling molecule released from osteoblasts that we have previously shown can increase proliferation in bone‐derived PCa cells. From these previous studies, I hypothesized that mechanical load increases the amount of NGF released from osteoblasts and osteocytes. To determine the effects of mechanical load on bone cells, osteoblasts and osteocytes were subjected to different magnitudes (5, 12, and 20 dynes/cm 2 ) of oscillatory fluid shear. At each magnitude, cells in culture were sheared for 2 hours. Media was collected and cells were lysed. NGF concentrations were quantified from the conditioned media using an ELISA assay. Protein concentrations were determined from the cell lysate using a BCA assay. Results were measured in NGF concentration per protein concentration to account for differences in cell density. Osteocytes released significantly more NGF during fluid shear than static cells, although NGF release was variable. The static osteocytes released approximately 0.022 pg/mg NGF while the maximum concentration of NGF released in sheared cells was 0.054 pg/mg at 5 dynes/cm 2 . However, osteoblastic MC3T3 cells had no significant response to shear in terms of NGF release. This indicates that while both types of cells exhibited a basal release of NGF, only the osteocytes increased this release in response to fluid shear. Additionally, these findings support the concept that the osteocytes are more sensitive than osteoblasts to mechanical load. Osteocytes are located in lacuna‐canalicular system and are exposed to comparable forces of fluids flowing through these small channels. Since osteocytes make up the majority of bone cells, their NGF release could have a significant impact on the bone microenvironment. We can then predict that the changes in load caused by ostesclerotic lesions could increase prostate cancer cell invasion and proliferation through upregulation of NGF release. Future studies include the treatment of prostate cancer cells with conditioned media from sheared osteocytes and observing any changes in proliferation. These data will further our understanding of the cellular mechanisms involved in prostate cancer proliferation and help to identify potential therapeutic targets for the treatment of metastatic prostate cancer in bone. Support or Funding Information This study was supported by the Milton H. Stetson Undergraduate Research Fellowship, a grant from the National Institutes for Health (DC014461) and a private donation from Dr. Jeremie Axe at First State Orthopedics. We also thank Nick Trompeter and Lindsey Stevens for their help and advice. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .