Visualized Tracking of Tumor-Derived Extracellular Vesicles Using CRISPR-Cas9 System
| Autor: | Yangyang Ye PhD, Qian Shi MA, Ting Yang MA, Fei Xie PhD, Xiang Zhang PhD, Bin Xu PhD, Jingwen Fang MA, Jiangning Chen PhD, Yujing Zhang PhD, Jing Li PhD |
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| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: | |
| Zdroj: | Technology in Cancer Research & Treatment, Vol 21 (2022) |
| Druh dokumentu: | article |
| ISSN: | 1533-0338 15330338 |
| DOI: | 10.1177/15330338221085370 |
| Popis: | Introduction: Tumor extracellular vesicles (EVs) and their relevance to various processes of tumor growth have been vigorously investigated over the past decade. However, obtaining direct evidence of spontaneous EV transfer in vivo remains challenging. In our previous study, a single-guide RNA (sgRNA): Cas9 ribonucleoprotein complex, which can efficiently delete target genes, was delivered into recipient cells using an engineered EV. Aim: Applying this newly discovered exosomal bio-cargo to track the uptake and distribution of tumor EVs. Methods: Tumor cells of interest were engineered to express and release the sgRNA:Cas9 complex, and a reporter cell/system containing STOP-fluorescent protein (FP) elements was also generated. EV-delivered Cas9 proteins from donor cells were programmed by a pair of sgRNAs to completely delete a blockade sequence and, in turn, recuperated the expression of FP in recipient reporter cells. Thus, fluorescently illuminated cells indicate the uptake of EVs. To improve the efficiency and sensitivity of this tracking system in vivo , we optimized the sgRNA design, which could more efficiently trigger the expression of reporter proteins. Results: We demonstrated the EV-mediated crosstalk between tumor cells, and between tumor cells and normal cells in vitro . In vivo , we showed that intravenously administered EVs can be taken up by the liver. Moreover, we showed that EVs derived from melanoma xenografts in vivo preferentially target the brain and liver. This distribution resembles the manifestation of organotrophic metastasis of melanoma. Conclusion: This study provides an alternative tool to study the distribution and uptake of tumor EVs. |
| Databáze: | Directory of Open Access Journals |
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