Study of the interactions between CD99, immunologic microenvironment and microRNAs and their prognostic significance in human osteosarcoma

Osteosarcoma is the most frequent primary tumor of the bone, characterized by an aggressive and metastatic potential. Few improvements were achieved in term of therapies in the last two decades. Understanding mechanisms underlying chemosensitivity of tumor cells and the discovery of new prognostic factors could improve patients’ outcome. In this study, expression of CD99 was evaluated by IHC in 100 OS biopsies, discovering the potential of CD99 as a predictive biomarker of ifosfamide response. The importance of CD99 in ifosfamide sensitivity was also studied in osteosarcoma cell lines. CD99 is associated with an increased sensitivity in OS cell lines, corresponding to a greater apoptosis rate. For the same OS specimens, expression of immune infiltrate markers and their correlation with CD99 was studied and CD99 tumoral expression correlates to an enrichment in CD8+ and CD68+ cells in OS samples. Taken together these data confirm a role of CD99 in ifosfamide sensitivity in osteosarcoma, thus CD99 is able to increase both the cytotoxic and the immunomodulatory effects of ifosfamide, making osteosarcoma cells more sensitive. For understanding the regulation of CD99 in osteosarcoma, miRNA mediated post-transcriptional regulation was examined, identifying hsa-miR-330-3p as a modulator of CD99 expression. Moreover miR-330-3p affects downstream effector of CD99, like ROCK2, leading to a modulation of migration capabilities. Finally, a small RNA sequencing pilot study has been conducted on 14 OS samples, with the aim to identify a miRNome able to distinguish at diagnosis patients with high probability to relapse. This 3 miRNAs signature (hsa-miR-99b-3p, hsa-miR-130b-5p and hsa-miR-139-5p) was validated by qPCR in a larger OS cohort. Enrichment pathways analyses show the involvement of these miRNAs in immunological processes and cellular metabolism, underlining the importance of these mechanisms in the pathogenesis and response to therapy in OS.