In Vivo Visual Imaging Of Bladder Urothelial Cancer Cells With Quantum Dot-based Fluorescent Probes

BackgroundThe typical biological behavior characteristics of BUC includes recurrence and progression.Improving the pre-judgment of BUC patients with high risk of recurrence and progression,which can effectively enhance the level of diagnosis and prognosis.The biology behavior analysis of tumor cells in vivo can be used to assess the risk of recurrence and progression of the tumor,and its detection depends on the development of fluorescence image technology,however,there are technical difficulties in the implementation process.Quantum dots(QDs)are a kind of semiconductor nanocrystal,which has been widely used in the field of biomedicine because of their unique optical properties,which provides an effective research strategy for the biology behavior of tumor cells in vivo.In this paper,the feasibility of noninvasive visualization of tumor-targeted imaging in bladder urothelial carcinoma was analyzed by using the QD-based probes at the level of cells and animals.ObjectiveOn one hand,to investigate the effect of quantum dots(QDs)on survival rate,proliferation,migration and invasion of the human bladder cancer cell line EJ.On the other hand,to prepare the fluorescent probes of quantum dot(QD)conjugated with prostate stem cell antigen(PSCA)monoclonal antibody(QD-PSCA),study their specific labeling imaging of bladder cancer cells,analyze the feasible of non-invasive tumor-targeted imaging in vivo,and provide the experimental basis for non-invasive tumor-targeted imaging in vivo.In the end,to investigate the in visual targeted imaging of fluorescent probes of QD-PSCA and QD-TAT on the bladder urothelial cancer(BUC)cells subcutaneous transplanted tumors in nude mice,in order to provide effective basis for specific recognition,early diagnosis and medication of tumor.MethodsThe cell counting kit-8(CCK-8)test was used to measure the survival rate of EJ cells following incubation with QDs in different concentrations.The live cells were marked by QD-based nonspecific labeling.The labeling rate was detected by flow cytometry.Additionally,the effect of the QDs on tumor cell migration and invasion was evaluated by the scratch test and transwell chamber assay,respectively,and cell proliferation rate was assessed using a hemocytometer.The method of chemical covalent coupling was used to prepare QD605-PSCA fluorescent probes,then,the optical properties of the probes coupled and uncoupled with PSCA were measured and assessed by an ultraviolet spectrophotometer and a fluorescence spectrophotometer.Direct immune-fluorescence labeling was utilized to detect and analyze the targeted imaging of the probes for bladder cancer EJ cells and BIU-87 cells,and observe the duration of the fluorescence after labeling.QDs with an emission wavelength of 605 nm(QD605)were conjugated with PSCA and TAT respectively to prepare of QD605-PSCA and QD605-TAT fluorescent probes.In vitro,QD605-PSCA and QD605-TAT were co-cultured with BIU-87 cells,respectively,observed the labeling imaging with a fluorescence microscope;The BIU-87 cells labeled with QD605-TAT were injected subcutaneously in nude mice,in vivo visualization imaging on animals was observed at different time points by using a Maestro in-vivo imaging system;The BIU-87 BUC subcutaneous xenografted tumor models in nude mice were established successfully,through the tail vein injection of QD605-PSCA and QD605-TAT probes,respectively,then,the in vivo visual imaging and the in vivo distribution of QD605-PSCA and QD605-TAT probes were observed and analyzed at different time points with the Maestro in-vivo imaging system.Results1.Detection of the toxicity of QDs on bladder cancer living cells The survival rate of cells was decreased with the increasing of QDs concentrations after incubated with QDs for 24 hours using cell counting kit-8(CCK-8)test,however,in the common concentration(5nM,lOnM and 20nM),no statistical difference in survival rate was observed between experimental and control groups(P>0.05);in the high concentration(40 nM,80nM),there were obvious difference between experimental and control groups(P<0.05).2.Influence of QDs on cell growth,migration and invasive The labeling rate of EJ cells labeled with QD605 after 2 h was 98.5%.Different concentrations of QD605 had no obvious effect on proliferation of EJ cells.In the scratch test and transwell chamber assay,the ability of migration and invasion was no significantly difference between EJ cells labeled with QD605 and EJ cells unlabeled with QD605(P>0.05).3.Optical properties of QD605-PSCA probes and fluorescent labeling of QD605-PSCA probes on bladder cancer cells The absorption spectrum and emission spectrum of the QD605 and QD605-PSCA probes were determined using the ultraviolet spectrophotometer and fluorescence spectrophotometer,respectively.TheQD605-PSCA probes retained the fluorescent properties of QD605 and the immunogenicity of the PSCA protein.After QD605 coupled with PSCA,the absorption spectrum of the QD605-PSCA probes was wide and continuous,and the absorbance of QD605-PSCA was higher compared with that of QD605(P<0.05).The emission spectrum of QD605-PSCA was narrow and symmetric,and there was an obvious fluorescence emission peak in the vicinity of a wavelength of 608 nm.The fluorescence intensity of QD605-PSCA was no obviously difference compared with that of QD605(P>0.05).The probes could specifically recognize PSCA protein in bladder cancer cells,the specific fluorescence was mainly expressed in the cell membrane of living cells,the fluorescence was stable and had a longer duration.4.Fluorescent labeling of QD605-PSCA and QD605-TAT probes on bladder cancer living cells In vitro experiments showed that QD605-PSCA probes could be bound specifically to EJ cells and BIU-87 cells,the QD605-TAT could be delivered into tumor cells through the non-specific penetrating transmembrane ability of peptides.5.In vivo visual imaging of BUC xenografted tumors in mouse models In vivo imaging showed that the fluorescence signals of the tumor site were gradually weakend with the passage of time during one week.The BIU-87 BUC subcutaneous xenografted tumor models in nude mice were established successfully,the visualization targeting fluorescence imaging of the transplanted tumor could be acquired after tail vein injection of QD605-PSCA probes,however,in BUC transplantation tumor site,there had no seen obvious fluorescence imaging after tail vein injection of QD605-TAT probes;In histopathological examinations suggested that the fluorescence signals of probes can be detected in the parts of transplanted tumor and liver,there was no obvious fluorescence signals could be observed in other organs,and non-specific fluorescence signals of QD605-TAT probes in the parts of xenografted tumor site and other organs were not be observed.Conclusion1.The quantum dots had a certain degree of influence on the activity of bladder cancer cell line EJ at high concentrations.However,at concentrations at which QDs are conventionally used,they had little impact on the survival of EJ cells.Moreover,the ability of proliferation,migration and invasion of EJ cells were not affected by QDs.2.The QD-PSCA fluorescent probes could have the ability of specific targeted labeling and imaging with bladder cancer cells,and the probes possess good optical stability.This is contributed to the research development of bladder cancer-targeted non-invasive imaging in vivo,tumor early diagnosis and targeted therapy.3.Quantum dot-peptide fluorescent probes have great application value in the imaging and dynamic tracking of living cells,and in the research development of tumor in vitro.Quantum dot-antibody fluorescent probes could be used to identify the specific targeted imaging of tumor,and have considerable development prospects in non-invasive tumor lesion recognition,dynamic observation and drugs targeting treatment in vivo.