Confocal Laser Endoscopy For Imaging Mesenchymal Stem Cell Target Tocolon Cancer

Backgroud and aims:Colon cancer is the world’s fourth leading cause of death due to cancer in males and the third leading cause in females.Despite considerable advances in anti-tumor therapies in recent years,colorectal carcinoma remains one of the most challenging diseases.In particular,it has a highly invasive nature,which precludes surgical excision and resists a number of antitumor agents.Mesenchymal stem cells(MSCs)are pluripotent stem cells which have self-renewing properties and a strong capacity to migrate into inflamed tissues and active tumors.Mesenchymal stem cells(MSCs)can serve as vehicles for therapeutic genes.However,little is known about MSC behavior in vivo.Currently,there are several methods to track the fluorescence signals of MSCs in vivo.However,clinical possible methods to individually document the MSC homing to tumor surroundings is lacking.Conventional radiology imaging have been applied as non-invasive methods to track MSC migration and monitor therapeutic efficacy in tumor models.However,they cannot effectively discriminate between the debris of dead MSCs and live cells.What’s more,they are unable to evaluate the MSC homing efficiency to the tumor site.Probe-based confocal laser endomicroscopy(pCLE),a newly invented diagnostic tool,provides real-time optical section images with a cellular resolution similar to that of histology.It can provide non-invasive inspection in histology with*1000 magnification.The endomicroscopy system uses a blue laser that delivers an excitation wavelength of 465 nm and light emission at 505-585 nm,which is compatible with the fluorescence feature of enhanced green fluorescent protein(EGFP).Here,we demonstrated that probe-based confocal laser endomicroscopy(pCLE)can be used to track MSCs in ivivo and individually monitor tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL)gene expression within carcinomas.Tumor necrosis factor(TNF)-related apoptosis inducing ligand(TRAIL)is expressed as a type II transmembrane protein and is a member of the TNF superfamily.TRAIL is an appealing anticancer molecule because it induces cancer cell death without affecting healthy cells.Recent reports have shown that generating stable modified MSCs to obtain cellular vehicles leads to targeted and consistent TRAIL delivery,suggesting that synergistic antitumor effects may be achieved using combination therapies.However,determining the most appropriate clinical application of TRAIL-expressing MSCs is currently hampered by a lack of knowledge of how these cells behave in vivo.The aims of our current work were as follows:(1)Extraction and identification of bone marrow mesenchymal stem cells.Bone marrow mesenchymal stem cells(MSCs)were used as carriers.Tumor necrosis factor related apoptosis ligand(TRAIL)gene was used as therapeutic gene.Constructing anti-tumor target MSC(TRAIL-MSC).(2)To investigate the feasibility of using pCLE to visualize the homing of TRAIL-MSCs to tumors in vivo.To monitor these MSCs homing to the tumor in vivo,in order to the effects exerted on migration-related gene expression by MSCs as delivery vehicles in tumor xenografts in mice.Methods:Part 1:Preparation,Characterization and Adenoviral infection of MSC1.Preparation and characterization of MSCMSCs were obtained and cultured from the bone marrow of 3-to 4-week-old female BALB/c nu/nu mice.Cultured MSCs were tested for their ability to differentiate into adipogenic,chondrogenic and osteogenic cell lineages.The flow cytometric analysis of surface markers showed that the isolated MSCs were positive for CD29.CD44 and CD90 but negative for CD34 and CD 45,which agreed with the characteristics of MSCs.2.Adenoviral infection of MSCIsolated BALB/c nu/nu mice MSCs(MSCs)were characterized and engineered to co-express the TRAIL and enhanced green fluorescent protein(EGFP)genes.3.Tumor-bearing mouse modelHT29 cells were maintained in Dulbecco’s Modified Eagle’s Medium(DMEM)with 10%fetal bovine serum(FBS).100 U/ml penicillin,and 100 mg/ml streptomycin.Four-week-old BALB/c nu/nu mice weighing 15-20g were maintained under specific pathogen-free conditions and used in accordance with institutional guidelines under the approved protocols in this animal study.Solitary tumors were administered of with 2×106 HT29 tumor cells as suspensions in 100 ml of PBS at a site above the right flank of the mice.Part 2:In vivo cellular imaging the movement of MSC and the MSC can exert anti-tumor effects1.Cell injection and Macroscopic fluorescence imagingAs the tumor size reached 5-10mm,TRAIL-MSCs were injected from nails at 5×106 as suspensions in 100 ml of PBS subcutaneously into the colon cancer-bearing mice.The macroscopic fluorescence imaging analysis was used to monitor MSC migration in vivo during 18 days.2.The pCLE imaging in vivoThe number of MSCs co-expressing EGFP and TRAIL(TRAIL-MSCs)at tumor sites was quantified with pCLE in vivo,while their presence was confirmed using immunofluorescence(IF)and quantitative polymerase chain reaction(qPCR).3.Antitumor effect of TRAIL-MSCOn day 18,these animals were sacrificed.The therapeutic effects of TRAIL-MSCs were evaluated by measuring the volumes and weights of subcutaneous HT29-derived xenograft tumors.Results:Part 1:Preparation,Characterization and Adenoviral infection of MSC1.Preparation and characterization of MSCMSCs were extracted from the bone marrow of female BALB/c nu/nu mice and characterized.The immunophenotyping results and the differential capacity of isolated MSCs.The flow cytometric analysis of surface markers showed that the isolated MSCs were positive for CD29,CD44 and CD90 but negative for CD34 and CD 45,which agreed with the characteristics of MSCs.MSCs differentiate into adipogenic,chondrogenic and osteogenic cell lineages.2.Adenoviral infection of MSCThe flow cytometric analysis indicated that the infection efficiency of the CMV-EGFP-EF-la-TRAlL lentiviral vector into the MSCs was 81.6%± 3.1(P<0.05.n=3).Furthermore,84.6%± 1.0(P<0.05,n=3)of the MSCs transduced with the lentiviral vector containing EGFP were GFP-positive.3.Tumor-bearing mouse modelApproximately 2-4 weeks after injection of HT29 cells,colon xonagraft mice had the tumor size reached 5-10mm.Part 2:In vivo cellular imaging the movement of MSC and the MSC can exert anti-tumor effects.In a first study of macroscopic fluorescence imaging in tumor-bearing mice,a significant change in signal movement was observed in mice treated with TRAIL-MSCs compared with mice treated with inontransduced MSCs.The findings from the pCLE images were consistent with the IF and qPCR results.After an intravenous injection of TRAIL-MSCs.fluorescent cellular signals could be visualized at the surface of the tumors.The signals detected inside the tumor sites were weaker than those at the surface(P<0.05.n=5).Intravital imaging of the subcutaneous xenograft tumors revealed that BALB/c mice treated with TRAIL-MSCs exhibited specific cellular signals,whereas no specific signals were observed in the control mice.The weights and volumes of the tumors derived from mice injected with TRAIL-MSCs were significantly reduced relative to control mice(P<0.05,n=5).Conclusion:Our study demonstrates,for the first time,that in vivo endomicroscopic cellular imaging of TRAIL-MSCs in xenografts with colorectal neoplasia is possible using pCLE.Furthermore,we have extended our experiments to infer the value of endomicroscopy for assessing the adequacy of TRAIL-MSCs for use as a cell-based anti-tumor therapy.Our data revealed that the endomicroscopic tracking of EGFP-labeled TRAIL-MSCs may be of critical importance for monitoring MSC homing effects and establishing individualized stem cell therapies,,as well as maximizing the benefits and preventing the shortcomings of the therapeutic use of MSCs.Significance:The pCLE results indicated that endomicroscopy could effectively quantify injected MSCs that homed to subcutaneous xenograft tumor sites in vivo and correlated well with the therapeutic effects of the TRAIL gene.By applying pCLE for the in vivo monitoring of cellular trafficking,stem cell-based anticancer gene therapeutic approaches might be feasible and attractive options for individualized clinical treatments.