Characterization of Macrophages and Osteoclasts in the Osteosarcoma Tumor Microenvironment at Diagnosis: New Perspective for Osteosarcoma Treatment?

SIMPLE SUMMARY: Due to the great genetic instability of osteosarcoma (OS), a recurrent molecular therapeutic target has not been identified to date. Therefore, characterization of the OS tumor microenvironment (TME) might offer new therapeutic perspectives. The OS2006 trial, originally designed to evaluate the impact of zoledronic acid (ZA, osteoclast-inhibitor) addition to conventional OS-therapies, was ended preliminary due to a negative impact on patient survival. Through retrospective biomarker analysis of the unique biological samples collected during the trial, we demonstrate here that ZA not only acts on harmful osteoclasts but also on protective macrophages, clarifying its detrimental effect. By multiplex immunohistochemistry, applied on additional OS biopsies, an important bipotent macrophage-population (CD168+/CD163+), homogenously distributed throughout OS tumor areas, was identified. These bipotent cells might play a determining role in the evolution of OS and offer a novel therapeutic approach. A clear definition of the macrophage populations present at diagnosis could re-enforce therapeutic decisions. ABSTRACT: Biological and histopathological techniques identified osteoclasts and macrophages as targets of zoledronic acid (ZA), a therapeutic agent that was detrimental for patients in the French OS2006 trial. Conventional and multiplex immunohistochemistry of microenvironmental and OS cells were performed on biopsies of 124 OS2006 patients and 17 surgical (“OSNew”) biopsies respectively. CSF-1R (common osteoclast/macrophage progenitor) and TRAP (osteoclast activity) levels in serum of 108 patients were correlated to response to chemotherapy and to prognosis. TRAP levels at surgery and at the end of the protocol were significantly lower in ZA+ than ZA− patients (p(adj) = 0.0011; 0.0132). For ZA+-patients, an increase in the CSF-1R level between diagnosis and surgery and a high TRAP level in the serum at biopsy were associated with a better response to chemotherapy (p = 0.0091; p = 0.0251). At diagnosis, high CD163+ was associated with good prognosis, while low TRAP activity was associated with better overall survival in ZA− patients only. Multiplex immunohistochemistry demonstrated remarkable bipotent CD68+/CD163+ macrophages, homogeneously distributed throughout OS regions, aside osteoclasts (CD68+/CD163−) mostly residing in osteolytic territories and osteoid-matrix-associated CD68−/CD163+ macrophages. We demonstrate that ZA not only acts on harmful osteoclasts but also on protective macrophages, and hypothesize that the bipotent CD68+/CD163+ macrophages might present novel therapeutic targets.