Open AccessIn Vivo Visualized Tracking of Tumor-Derived Extracellular Vesicles Using CRISPR-Cas9 System
Author(s) -
Yangyang Ye,
Qian Shi,
Ting Yang,
Fei Xie,
Xiang Zhang,
Bin Xu,
Jingwen Fang,
Jiangning Chen,
Yujing Zhang,
Jing Li
Publication year - 2022
Publication title -
technology in cancer research and treatment
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.754
H-Index - 63
eISSN - 1533-0346
pISSN - 1533-0338
DOI - 10.1177/15330338221085370
Subject(s) - in vivo , cas9 , microbiology and biotechnology , crispr , microvesicles , in vitro , reporter gene , green fluorescent protein , biology , melanoma , cancer cell , guide rna , cancer research , chemistry , gene expression , gene , cancer , biochemistry , genetics , microrna
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.