The Targeted Optic Molecular Imaging Of Tumors Mediated By Gene Recombination And In Vivo Gene-editing Systems

Objective: Luciferase(Luc) gene regulated by constitutive and tissue-specific promoter were constructed via in vitro genetic recombination, to investigate the pros and the cons of non-targeted and targeted optic molecular imaging of tumors. Furthermore, based on its precise gene-editing capacity, we aimed to utilize CRISPR/Cas9 system to targetedly activate Luc gene and thus realize bioluminescence imaging.Methods: 1st, Lipofectamine 2000, PEI and adenovirus were used as deliver vectors to transfer p CMV-Luc gene into PC-3 cells followed by bioluminescence imaging. After the treated cells were subcutaneously transplanted into nude mice, in vivo imaging was performed. In another group, Ad.p CMV-Luc was intratumorally injected into tumor-bearing animals. 2nd, normal cells, prostate cancers(PCa) and non-PCa tumor cells were treated with Ad.p DD3-Tf R-Luc which was driven by a tissue specific promoter. Then the optic imaging was performed while Tf R expression was measured by Western-blotting. 3rd, after the intratumor injection of Ad.p DD3-Tf R-Luc into tumor-bearing animals, a series of optic molecular images were carried out, with the bioluminescent range and intensity analyzed, to investigate the targeting ability of the prepared adenovirus. 4th, with synthetic genes like survivin promoter, stop sequence(SS) and its specific g RNA Sequence, a CRISPR/Cas9 expression vector p U6-g RNA(SS)-pβactin-Cas9 as well as p CMV-Luc L-SS-Luc R and p Sur-Luc L-SS-Luc R which could express the inactivated Luc gene were constructed. 5th, the plasmids p U6-g RNA(SS)-pβactin-Cas9 combined with p Sur-Luc L-SS-Luc R or p CMV-Luc L-SS-Luc R were co-transferred into PC-3 cells and 293 T cells. Then bioluminescence imaging was preformed to explore the potential of CRISPR/Cas9 system in optic molecular imaging of tumors.Result: 1st, after the treatment of the gene pCMV-Luc delivered by Lip2000、PEI and adenovirus vectors, tumor cells could emit bright bioluminescence signals, among which adenovirus vectors had the best imaging performance. Bioluminescence imaging revealed that PC-3 cells pretreated by the gene p CMV-Luc could emit fluorescence after subcutaneous transplantation, while tumor bearing mice intratumor-injected with Ad.p CMV-Luc showed bright fluorescence in tumor regions. But the bioluminescent ranges were not correlated with the actual sizes of tumors. 2nd, after the treatment of Ad.p DD3-Tf R-Luc, elevated Tf R expression and obvious fluorescent signal were detected only in prostatic cancer cells, but not in normal cells or non-prostatic tumor cells. In vivo imaging of tumor-bearing mice infected with Ad.p DD3-Tf R-Luc showed bright fluorescence signals only in tumor region while no obvious fluorescence could be observed in the surrounding area. The well-defined tumor boundary gave accurate measurements of tumor sizes, which were in consistence with the actual tumor sizes. 3rd, CRISPR/Cas9 vector p U6-g RNA(SS)-pβactin-Cas9 and the plasmids of inactivated Luc gene p CMV-Luc L- SS-Luc R and p Sur-Luc L-SS-Luc R were successfully constructed. Both normal cells and tumor cells could emit fluorescence after treated by p U6-g RNA(SS)-pβactin-Cas9 and p CMV-Luc L-SS-Luc R, and the co-transfetion of p U6-g RNA(SS)-pβactin-Cas9 and p Sur-Luc L-SS-Luc R only illuminated a small number of PC-3 cells.Conclusion: 1st, compared with cationic liposome and cationic polymer, adenovirus vectors could be more effectively to deliver reporter gene into cells. Although the Luc expression regulated by constitutive CMV promoter could be used in the optic molecular imaging, this imaging strategy showed no targeting ability, which limited their applications in in vivo molecular imaging of tumors. 2nd, in vivo and in vitro studies had verified that tissue specific promoter of DD3 could drive the specific expression of Luc gene in prostate cancer and be used in the targeted optic molecular imaging of prostate cancers, indicating that the tissue specific promoter could provide an excellent bioswitch for tumor-targeted imaging. 3rd, we successfully developed a novel optic reporter system that could be specifically activated by CRISPR/Cas9 system and realized the gene imaging of normal cells and tumor cells. Therefore, we adapted CRISPR/Cas9 system into the field of molecular imaging. Furthermore, CRISPR/Cas9 system could provide a novel, flexible and targeting strategy for molecular imaging under the combined effect of multiple targets.