1. The Effect Of Rabbit Mesenchymal Stem Cells On The VX2 Bladder Tumor Growth And Associated Stroma Remodelling In Vivo 2. Safety And Efficacy Of Holmium Laser Resection For Primary Non-Invasive Bladder Cancer Versus Transurethral Resection

BACKGROUDBone marrow-derived mesenchymal stem cells(MSCs) come from bone marrow non-hematopoietic tissue,which are a kind of pluripotent adult stem cells,have high degree of plasticity,and are capable of developing into diverse functional cell lineages, including bone,cartilage,myoblasts,fat and neural cells cells.Instead of embryonic stem cells,MSCs avoid the ethical,moral and legal constraints,and have been considered to represent ideal cell sources of regenerative medicine and tissue engineering currently.As a consequence,MSCs has aroused widespread interest. Moreover,MSCs can be readily transfected,allowing for easy ex vivo modification., have excellent migratory ability,the tumor tropism and low immunogenicity of the characteristics,which make them ideal vector cells in tumor gene therapy and targeted therapy.However,with the increasing apprehension of physiological characteristics regarding MSCs and the deepening of tumor research in recent years.Some scholars have found that MSCs themselves might be associated with tumorigenesis closely, suggesting that long-term biosafety of MSCs for clinical applications deserves further assessment.In previous work,We have initially observed that cultured autologous MSCs transfusion into VX2 bladder tumor tissue could promote tumor growth,and found that MSCs could be differentiated into myofibroblast in vitro and in vivo tumor micro-environment,which is regarded as an important cancer-associated fibroblast in tumor stroma.Therefore,MSCs derived myofibroblast or other stroma cells may be involved in tumor stroma remodeling.And activated tumor micro-environment or stroma remodeling plays a key role in tumor growth and infiltration.In this section, we intend to amplify of MSCs in vitro first,then establish tumor model through bladder submucosal innoculation of different proportions of autologous MSCs and VX2 tumor cells,observing the effect of different numbers of MSCs on VX2 bladder tumor occurrence and development in New Zealand rabbit.In this way,we try to study the relationship accurately between MSCs and VX2 bladder tumor occurrence and development;Meanwhile,by monitoring the tumor tissue growth factors and some stromal remodeling enzyme,we try to disclose the concrete signaling mechanism of MSCs induced stroma remodeling and the interaction between tumor cells and stroma cells.OBJECTIVETo investigate the effect of MSCs on the VX2 bladder tumor occurrence and development,to explore tumor tissue growth factor and stromal remodeling enzyme expression change induced by MSCs and whether MSCs are involved in the tumor stroma remodeling.METHODS1.Isolation,culture and identification of rabbit MSCs in vitro:A total of 30 male New Zealand rabbits were used.Marrow aspirates were obtained from the proximal tibia of each animal,and density gradient centrifugation followed by repeated adherence in plastic bottle were applied in MSCs isolation and amplification.Morphology and flow cytometry detection of surface antigen were used to identify and confirmation cultured MSCs.2.Rabbit VX2 bladder tumor model establishment and monitoring:After successful MSCs cultivation,VX2 tumor cell suspension was prepared and mixed with DAPI labeled autologous MSCs in a total volume for 300μl PBS.The mixed cell suspension was transplanted by bladder submucosal innoculation.Based on the ratio of two different cells,30 New Zealand rabbits were randomly divided into 3 groups with 10 in each group,as follows:Group A(10⁶ VX2),Group B [(VX2/MSCs=1:1):10⁶ VX2 + 10⁶ MSCs]and Group C[(VX2/MSC=1:10): 10⁶ VX2 + 10⁷ MSCs].Multisection ultrasound scanning was conducted 3,4,5,6, 7,14,21,28d respectively after cell innoculation to monitor tumor occurrence and growth.The tumor growth curve was set up and inguinal lymph nodes metastasis was observed to compare tumor progression in 3 groups.All animals were sacrificed at 4 weeks point.Bladder tumor specimens were obtained to make frozen and paraffin section,and distant organ metastasis was recorded.3.Immunohistochemistry(IHC) assay of tumor growth factors and remodeling enzymes:growth factors(TGFβ1,bFGF,HGF) and matrix remodeling proteases (MMP2,MMP9) expression was detected in developed tumor,and Image Pro Plus 5.0 Software was used to analyze IHC intensity semi-quantitatively.Integral optical density(IOD) value represents the level of protein expression and was applied to compare the difference in 3 groups.4.Real-time quantitative PCR assay of the above-mentioned growth factors and matrix remodeling protease mRNA:SYBR Green I method was used technically, and data analysis using 2^{ΔCtΔCt} method was conducted.RESULTS1.Cultured MSCs in vitro present uniform morphology and its growth curve showed strong amplification ability,and were identified as rabbit MSCs,rather than hematopoietic cells.2.The effect of MSCs on tumor development:on 5^th day after cell inoculation,2 (2/10),2(2/10),6(6/10) animals in Group A,B,C respectively could be found by ultrasound in the bladder injection site,and 6(6/10),7(7/10),8(8/10) animals could be monitored in Group A,B,C respectively on 6^th day.At the 1^st,2^nd and 3^rd week time point,tumor volume in group A＜group B＜group C,and the difference has statistical significance(P all＜0.001).Tumor growth curve showed that MSCs could promote VX2 bladder tumor growth.The weight of bladder tumor at 4^th week was 4.0±1.2 g,11.3±3.5 g,12.8±3.9g in Group A,B,C respectively.Animals in Group A did not present inguinal lymph node metastasis(ILNM) in first 3 weeks,ILNM appeared in 3(3/10) rabbits at the 4^th week point.Animals in Group B did not present ILNM in first 2 weeks,and 3(3/10) rabbits showed ILNM at 3^rd week point,and a total of 7(7/10) at 4^th week point.5(5/10) rabbits in Group C appeared ILNM at 3^rd week point and a total of 8(8/10) at 4^th week point.Bladder tumor could be found in all animals when they were sacrificed at 4^th week point,and generated tumor approximately were regular and most are round in shape.No obvious satellite focus was found.HE staining showed that tumor pathology was in line with the general characteristics of transplanted VX2 tumor.3.Tumor tissue growth factors and stromal remodeling protease IHC results:growth factors:Stroma cells(fibroblasts,endothelial cells and inflammatory cells,etc.) and tumor cells(nucleus,cytoplasm) have positive expression.TGFβ1,bFGF and HGF IOD value in Group B(15396.10±3230.57,9280.66±3754.99 and 13203.17±3990.86,respectvely) were significantly higher than Group A (8948.70±2462.91,5632.70±1943.18 and 7824.43±4077.94,respectvely),P all＜0.05;TGFβ1,bFGF and HGF IOD value in Group C were 20875.40±6737.11,13811.14±4311.62 and 14523.69±3865.41,respectively,which was higher than in Group A,P all＜0.05,and TGFβ1,bFGF IOD value is higher than Group B,the difference has statistical significance(P＜0.05);Matrix metalloproteinases:The positive expression was mainly located in stromal cells (fibroblasts,endothelial cells,inflammatory cells,etc.) and tumor cell membrane, cytoplasm,particularly in active tumor infiltrating area.MMP2 IOD values in Group A,B,C were 25337.20±8151.51,51507.50±17679.55 and 61535.66±19449.11,respectvely.Levels in Group B,C was higher than in Group A,and the difference has statistical significance(P＜0.05);MMP9 IOD values in group A,B, C were 34172.91±5243.75,60582.28±22975.45 and 90287.45±31362.21, respectvely.Group B,C was higher than Group A(P＜0.05),Group C was higher than Group B(P = 0.024).4.Tissue tumor growth factor and stromal remodeling protease mRNA levels: growth factors:TGFβ1,bFGF and HGF mRNA levels in Group B(3.250±0.587, 2.970±0.490 and 2.101±0.527,respectvely.) was significantly higher than Group A(1.087±0.194,1.134±0.165 and1.185±0.247,respectvely)(both P＜0.05);TGFβ1,bFGF and HGF mRNA levels in Group C were 3.620±0.670, 3.651±0.744 and 3.052±0.438,respectively,higher than the Group A(all P＜0.05),and bFGF,HGF mRNA levels were higher than Group B(P＜0.05).Matrix MetaUoproteinases:MMP2 mRNA levels in Group A,B,C were 1.134±0.243, 3.67±0.945 and 3.845±1.159,and levels in Group B,C is higher than group A, (both P＜0.05);MMP9 mRNA levels in Group A,B,C were 0.955±0.166,3.360±0.628 and 4.045±1.084,respectively,and differences among all groups were statistically significant(all P＜0.05).CONCLUSIONS1.VX2 tumor cell suspension submucosal injection is a homogeneous and stable method to establish bladder tumor model,and can better reflect the pathogenesis of bladder tumor.2.Bone marrow derived MSCs can promote the bladder VX2 tumor growth and development in vivo.3.MSCs can induce high expression of certain growth factors in tumor micro-environment,which presumebly is the basic mechanism to promote tumor growth.4.MSCs can stimulate matrix protease(MMPs) expression,which is probably one of the pathway to promote stromal remodeling and tumor invasion and metastasis. OBJECTIVESTo assess the safety and efficacy of holmium laser resection for primary clinically non-muscle invasive bladder cancer(HOLRBT) compared with standard transurethral resection(TURBT).METHODSData of a total of 212 patients with primary non-muscle invasive bladder cancer (NMIBC) was collected in this study.These patients treated by holmium laser resection(HOLRBT group) or transurethral electroresection(TURBT group) were divided into 2 groups.Patients in each group were stratified into 3 risk subgroups (low,intermediate and high risk) according to prognostic factors for recurrence,based on EAU guideline.Patient demographics and tumor characteristics regarding each group were summarized and compared.Then,HOLRBT and TURBT groups were compared,concerning intraoperative complications and postoperative characteristics, including obturator nerve reflex and bladder perforation incidence,the proportion needing postoperative bladder irrigation,as well as postoperative catheter drainage period and hospital stay.Efficacy indicated by recurrence-free survival in overall group and stratified subgroup was meanwhile analyzed and compared by Kaplan-Meier technique,as well as log-rank test. RESULTSPatient demographics and tumor characteristics in the 2 groups were comparable, and no statistical significance was found between the 2 groups.HOLRBT is superior to TURBT in terms of intraoperative complications such as obturator nerve reflex and postoperative catheterization time and hospital stay(P＜0.001).The mean follow-up after surgey was 34 months.Recurrence-free survival after HOLRBT treatment is similar with that of TURBT(P = 0.283).P values in low,intermediate and high risk subgroups were 0.816,0.352,and 0.592,respectively.CONCLUSIONSOur results have indicated that HOLRBT is a feasible,safe and effective alternative for the management of primary clinically NMIBC when compared to TURBT,with similar recurrence-free survival and less perioperative complications, meanwhile providing sufficient material for pathology evaluation.