Blockade of the Immune Checkpoint B7-H3 Sensitizes Rhabdomyosarcoma to Anti-Tumor Immune Response
Roxane R. Lavoie, R.A., Fabrice Lucien, PhD, Haidong Dong, M.D., PhD, Patricio C. Gargollo, M.D., Candace F. Granberg, M.D..
Mayo Clinic, Rochester, MN, USA.
BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children and up to 20% of children may present with aggressive and/or metastatic disease. Multimodal therapies including chemotherapy and radiotherapy have improved patient survival in the last few decades, yet the 5-year overall survival rate is less than 40%. Additionally, the therapeutic regimen for RMS patients is extremely toxic, resulting multiple deleterious long-term effects in surviving patients. This highlights the urgent need to develop novel therapeutic approaches for pediatric RMS (pedRMS). Since immunotherapy has been introduced to the armamentarium of oncologists, unprecedented clinical responses have been observed in adult cancers. In contrast, immunotherapy is at its infancy in pedRMS, and significant efforts are required to characterize the tumor immune microenvironment and assess the clinical utility of immunotherapy. Immune checkpoint molecules such as B7-H1 (PD-L1) are highly expressed by cancer cells to evade the host's immune system, and blockade of immune checkpoint molecules with neutralizing antibodies have demonstrated positive clinical responses in several cancers. Our group sought to assess the therapeutic potential of immune checkpoint blockade in pedRMS.
METHODS: Expression profiles of immune checkpoint molecules in pedRMS patient-derived cell lines were analyzed by qPCR, western-blot and flow cytometry and confirmed by proteomic profiling. Immunohistochemistry was employed to validate expression of immune checkpoint molecules in patient-derived tumor sections. We developed an in vitro T-cell killing assay to determine the functional impact of immune checkpoint molecule expression on the anti-tumor immune response. Finally, we performed in vivo experiments with two animal models to evaluate the efficacy of immune checkpoint blockade to enhance T-cell mediated antitumor immune response and therapeutic response in pedRMS.
RESULTS: We determined expression profiles of B7 immune checkpoint molecules by flow cytometry of patient-derived RMS cells and observed no expression of B7-H6 and B7-H7, weak/moderate expression of B7-H1 (PD-L1) or PD-L2 and high expression of B7-H3 (Fig 1A). B7-H3 expression was confirmed by cell-surface proteomic profiling and immunohistochemistry of patient-derived xerograph (Fig 1B) and patient-derived fresh tumor sections. Knockdown of B7-H3 was associated with decreased pedRMS tumor cell viability when co-cultured with T-lymphocytes (Fig 1C). In animals bearing pedRMS tumors, B7-H3 knockdown resulted in greater tumor infiltration of immune cells and reduction of tumor burden.
CONCLUSIONS: B7-H3 is constitutively expressed in pedRMS and participates to immunosuppression and disease progression. Blockade of B7-H3 restores anti-tumor immune response in vitro and in vivo. This work highlights the therapeutic potential of targeting B7-H3 and supports the implementation of immunotherapy for the treatment of pediatric rhabdomyosarcoma.
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