Background: Mesenchymal stem cells (MSCs) play a key role in the progression of osteosarcoma (OS). In response to tumor-associated signals, including tumor-secreted extracellular vesicles (EVs), MSCs increase the expression of inflammatory cytokines promoting tumor cell aggressiveness. Previous data has demonstrated that IL-6 and IL-8 signaling mediate metastasis in OS. The aim of this study was to evaluate tumor EV-induced alterations of MSCs and identify combination therapies that can counteract MSC-induced resistance to antimetastatic drugs. Methods: Tumor EV-induced alterations of the MSC transcriptome were analyzed by RNA-seq. Gene set enrichment analysis (GSEA) was applied to discriminate TGFβ-dependent and independent pathways. EV RNA-induced expression changes were identified by transfecting purified EV-RNA in MSCs and by using a selective dsRNA antagonist. We selected candidate targets to block MSC-induced drug resistance and evaluated their effect in an orthotopic xenograft model of OS. Ladarixin (an allosteric inhibitor of the CXCL8/IL-8 receptors CXCR1 and CXCR2; 30 mg/kg 6x per week i.p.) and tocilizumab (anti-IL6 receptor antibody; 100 µg/mouse every other day i.p.) were administered starting from day one until the experimental endpoint. Metastasis were quantified by histological examination. This study was funded by the Dutch Cancer Society (KWF), POR FESR Campania 2014‐2020 and Dompé Farmaceutici SpA. Results: EVs from aggressive cancer cell lines induced an inflammatory MSC (iMSC) phenotype, characterized by increased expression of chemokines, including IL-8 as the most upregulated. Apart from IL-6, these alterations were mostly independent from TGFβ signaling and related to pattern recognition receptor (PRR) activation. We demonstrated that tumor EV-associated non-coding RNAs trigger TLR3 signaling in MSCs activating an innate immune response leading to high induction of IL-8 and other chemokines. Ladarixin and tocilizumab combination significantly reduced metastasis formation in a spontaneous metastasis model and overcame iMSC-induced resistance observed with single antimetastatic treatments. No effect was observed on primary tumor growth. Conclusions: EV-associated TGFβ together with EV-RNA induce iMSCs development in OS. Ladarixin in combination with tocilizumab reduced metastasis formation in a xenograft mouse model of OS, and, importantly, may prevent the occurrence of iMSC-induced tumor resistance to antimetastatic drugs.