Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets. By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography. We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients. Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. .

We performed whole transcriptome analysis of osteosarcoma bone samples. Initially, we sequenced total RNA from 36 fresh-frozen samples (18 tumoral bone samples and 18 non-tumoral paired samples) matching in pairs for each osteosarcoma patient. We also performed independent gene expression analysis of formalin-fixed paraffin-embedded samples to verify the RNAseq results. Formalin-fixed paraffin-embedded samples allowed us to analyze the effect of chemotherapy. Data were analyzed with DESeq2, edgeR and Reactome packages of R. We found 5365 genes expressed differentially between the normal bone and osteosarcoma tissues with an FDR below 0.05, of which 3399 genes were upregulated and 1966 were downregulated. Among those genes, BTNL9, MMP14, ABCA10, ACACB, COL11A1, and PKM2 were expressed differentially with the highest significance between tumor and normal bone. Functional annotation with the reactome identified significant changes in the pathways related to the extracellular matrix degradation and collagen biosynthesis. It was suggested that chemotherapy may induce the modification of ECM with important collagen biosynthesis. Taken together, our results indicate that changes in the degradation of extracellular matrix seem to be an important mechanism of osteosarcoma and efficient chemotherapy induces the genes related to bone formation. Impact statement Osteosarcoma is a rare disease but it is of interest to many scientists all over the world because the current standard treatment still has poor results. We sequenced total RNA from 36 fresh-frozen paired samples (18 tumoral bone samples and 18 non-tumoral paired samples) from osteosarcoma patients. We found that differences in the gene expressions between the normal and affected bones reflected the changes in the regulation of the degradation of collagen and extracellular matrix. We believe that these findings contribute to the understanding of OS and suggest ideas for further studies.

Surrounded by speakers of Indo-European, Dravidian and Tibeto-Burman languages, around 11 million Munda (a branch of Austroasiatic language family) speakers live in the densely populated and genetically diverse South Asia. Their genetic makeup holds components characteristic of South Asians as well as Southeast Asians. The admixture time between these components has been previously estimated on the basis of archaeology, linguistics and uniparental markers. Using genome-wide genotype data of 102 Munda speakers and contextual data from South and Southeast Asia, we retrieved admixture dates between 2000–3800 years ago for different populations of Munda. The best modern proxies for the source populations for the admixture with proportions 0.29/0.71 are Lao people from Laos and Dravidian speakers from Kerala in India. The South Asian population(s), with whom the incoming Southeast Asians intermixed, had a smaller proportion of West Eurasian genetic component than contemporary proxies. Somewhat surprisingly Malaysian Peninsular tribes rather than the geographically closer Austroasiatic languages speakers like Vietnamese and Cambodians show highest sharing of IBD segments with the Munda. In addition, we affirmed that the grouping of the Munda speakers into North and South Munda based on linguistics is in concordance with genome-wide data.