Semliki forest virus-based virotherapy coupled with the inhibition of galectin-3 empowers antitumor response against pediatric osteosarcoma

Resumen

Osteosarcoma (OS) is the most common bone cancer arising during childhood. These tumors are characterized for being very aggressive and highly metastatic, being these metastases normally found in the lungs of the patients. Moreover, standard treatment including chemotherapy, radiotherapy, target therapy, as well as the most promising immunotherapy strategies, have shown to be ineffective in metastatic and non-responder osteosarcoma patients. This therapeutic failure is due to the presence of a highly immunosuppressive environment inside these tumors, which is mainly formed by myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), pro-tumoral mesenchymal stem cells (MSCs) and cancer-associated fibroblast (CAFs). The interaction of these populations with OS cells favor this immunosuppressive environment, in which numerous inhibitory cytokines and immunosuppressive molecules are secreted leading to a reduction in the infiltration and activity of effector CD8 T cells, NK cells or CD4 helper T cells. Among these immunosuppressor molecules, and in the context of OS tumors, galectin-3 (Gal3) is seen as a promising target since it was described to actively participate in different pro-tumoral activities including tumor progression, T cell immunosuppression and the generation of metastases. On the other hand, due to the limited effect of standard treatments in these chemo- and radio-resistant OS cells, the use of virotherapy has been presented as a good strategy to induce the initial apoptosis of tumor cells, which leads to the secretion of tumor antigens, boosting the antigen recognition, and promoting T cell infiltration. Considering all the previous information, in this work we focused on the use of a Semliki Forest virus (SFV) that express truncated-based Gal3 inhibitors for the treatment of pediatric osteosarcoma. Based on this idea, we generated four different SFV vectors containing: the carboxy-terminal domain of Gal3 (C); the amino-terminal domain of Gal3 (N); the Gal3 inhibitor peptide C-12; and the amino-terminal domain of Gal3 fused to the inhibitor peptide C-12 (N-C12). Whereas the inhibitors N and N-C12 showed to efficiently block the binding of Gal3 to the surface of CD8 and CD4 T cells in vitro, only the treatment with the vector SFV expressing the N-C12 (SFV-Gal3-N-C12) showed to have a potent anti-tumor and anti-metastatic therapeutic effect in two orthotopic OS mouse models. On one hand, the treatment with SFV-Gal3-N-C12 reduced, in primary OS tumors, the expression of a set of genes that are described to be involved in the generation of lung metastases in OS patients. On the other hand, treatment with SFV-Gal3-N-C12 resulted in an increase in the infiltration of CD8 T cells in both primary tumors and lung metastases, which were able to generate tumor-specific responses, and presented a slower progression into exhausted phenotypes, in primary tumors. Apart from this modulation of the immune system, SFV-Gal3-N-C12 also showed to reduce the presence of two pro-metastatic immune populations, CD4+PD1+ and CD4+CD25+ T cells, in both primary tumors and lung metastases. Moreover, transcriptional analyses exhibited the potential of SFV-Gal3-N-C12 to decrease the expression of immunosuppressive molecules inside the primary tumors. Overall, this work demonstrated that SFV-Gal3-N-C12 could constitute a potential therapeutic agent for osteosarcoma patients expressing Gal3.