Tumor Exosomes Mediate the Horizontal Transfer of DNA Gene Mutation

Abstract

INTRODUCTIONTumor‐derived exosomes (TEXs) are crucial in shaping the local tumor microenvironment to promote cancer progression by advancing tumor metastasis. Recently, we have demonstrated that the activation of Toll‐like receptor 4 (TLR4) boosts the immunosuppressive ability of exosomes released by tumor cells. In addition to RNA and proteins, TEXs also transport dsDNA that reflects the mutational status of their parental cells. The purpose of this study is to verify whether exosomes shed by TLR4‐activated tumor cells are able to induce a malignant transformation of normal recipient cells through the horizontal transfer of mutated DNA.METHODSSW480 tumor cells were treated with LPS (1μg/ml) and TEXs were isolated from cell supernatants by polymer precipitation‐based method. TP53R273H and KRASG12V mutations have been identify by droplet digital PCR on purified exoDNA. Normal colon epithelial cells (CCD841) were used as recipient cells and internalization of Did‐labelled vesicles was evaluated by immunofluorescence. Vitality/proliferation (Trypan Blue dye exclusion test), migration capacity (Scratch assay) and mutational status (Droplet digital PCR) of CCD841 cells were monitored after the treatment with exosomes.RESULTSSW480‐derived exosomes carried dsDNA fragments in size range of 2–10 kbp containing TP53R273H and KRASG12V mutated sequences. Exosomes were efficiently internalized by CCD841 cells within 6 hours and elicited activation of signaling pathways that induced an increase in cell proliferation and migration capacity. Copies of TP53 R273H mutated DNA and mRNA were found within recipient cells up to one month without exosomal treatment. The activation with LPS stimulated the packaging of mutated DNA into exosomes. Consequently, mutated DNA was found in greater amounts in recipient cell treated with exosomes released by TLR4‐activated cells.CONCLUSIONSThe activation of TLR4 modulates the packaging of mutated genes in exosomes, which are efficiently transferred in normal recipient cells. Although we observed that mutated TP53 gene is transcribed in recipient cells, further studies are needed to assess whether mutated gene are integrated into genome.Support or Funding InformationThis project was supported by Grant Interreg ARTE (J22F170001005)