Identification and Functional Analysis of EPOR(+) Tumor-Associated Macrophages in Human Osteosarcoma Lung Metastasis

BACKGROUND: Tissue-resident macrophages can be educated to tumor-associated macrophages (TAMs) by the tumor microenvironment and many types of macrophages express erythropoietic receptor (EPOR); However, little is known about the expression of EPOR on TAMs and the identity of EPOR(+) TAMs in osteosarcoma lung metastasis has thus far remained elusive. METHODS: EPOR-eGFPcre mice were used to determine the expression of EPOR on lung tissue-resident macrophages. Flow cytometry, RT-PCR, and Western blot were examined to define the identity of EPOR(+) TAMs in 106 osteosarcoma lung metastasis specimens. Moreover, the clinicopathologic factors and prognosis of patients with CD163(+)EPOR(+) macrophages were compared. RESULTS: We found that a subpopulation of mouse lung tissue-resident macrophages express EPOR and EPO enhances the proliferation of EPOR(+) macrophages in mouse lung. A subpopulation of CD163(+) macrophages expresses EPOR in human osteosarcoma lung metastasis specimens. CD163(+)EPOR(+)TAMs increase 2.5 times in human osteosarcoma lung metastasis tissues; CD206, CD163, and PD1, which are known to have a significant role in TAM function had high expression in CD163(+)EPOR(+) TAMs compared with CD163(+)EPOR(−) TAMs. Furthermore, CD163(+)EPOR(+) TAMs had higher M2 marker and cytokine expression in osteosarcoma tissues compared with para-osteosarcoma tissues. EPO enhanced the expression of M2 cytokines in primary CD163(+)EPOR(+) TAMs. Importantly, the percentage of CD163(+)EPOR(+) TAMs had a positive linear association with malignant phenotypes as well as poor disease-free survival and overall survival time. CONCLUSIONS: We have characterized TAMs expressing EPOR and CD163(+)EPOR(+) macrophages as TAMs in osteosarcoma lung metastasis patients, which are highly associated with tumor aggressiveness.