The Mechanism Of Microvascular Perfused Tumor Cell Adhesion To Microvessel Wall In Vivo

Tumor distant metastasis consists of a series of discrete processes that tumor cell need to escape from the primary sites, intravasate into the circulation, as well as survive and prosper at distant sites with undetected and selectivity. Tumor hematogenous metastasis processes is a critical issue of tumor metastasis. Tumor cell adhesion to the microvascular wall of host organ and successful extravasations of microvessel is aimportant step in tumor distant organs metastasis.Many studies have observed the interactions between circulating tumor cells and the microvasculature in vivo, ex vivo and in vitro. Regardless of these attempts, to date few accurate in vivo model systems have been studied about the mechanism governing tumor cell adhesion without loss of their physiological and dynamic microenvironment. Less attention has been given to identifying the critical process, fluideffect, adhesion molecular, or other factor contributing to the microvascular permeability increase by tumor cell adhesion. The mechanism of the tumor cell adhesion and extravastion of the endothelial barrier is poorly understood.Our work is performed on an individually perfused microvessel of Sprague-Dawley (SD) rat. We developed a novel individually microvessel perfused tumor cell in vivo model, in which experimental conditions can be well defined. We mainly study the effect of adhesion molecule, fluid shear stress and the permeability of microvessel on cancer adhesion to microvessel wall in vivo.We also extended previous approach to the study of tumor cell adhesion to normal microvascular endothelium and adhesion-associated changes in microvessel structure in well controlled flow conditions in vivo. Adhesion rates of the human malignant breast cancer cell in a single postcapillary venule of rat mesentery in vivo were measured by intravital video microscopy. Then we quantified the tumor cell adhesion increasing the microvessel hydraulic conductivity Lp and solute permeability P to FITC-albumin under the similar condition of tumor cell perfusion. We applied a mathematical model for the interendothelial cleft revised from our previous study to calculate the changes in Lp and P to albumin by changing the structural components of the microvessel wall, e.g., degrading the surface glycocalyx, increasing the gap between endothelial cells. We compared the measured Lp and P to albumin with those calculated to predict the most likely structural changes induced by tumor cell adhesion. The adhesion-induced glycocalyx degradation was thus examined by applying in vivo fluorescence microscopy and immuno-labeling of the heparan sulfate (HS) in the EGL of the microvessels.Understanding these mechanisms can provid a new way to prevent tumor hematogenous metastasis with target therapy by stoping tumor adhesion related contribute factor such as adhesion molecules, blood flow, vascular permeability and EGL of the microvessel. Chemotherapy and radiotherapy cancer should be not only to kill tumor cells, but also consider the reduction and elimination of tumor adhesion factor, such as inhibitor VEGF and protection of microvascular EGL. Therapeutic result will be greatly improved and provid a future of molecular targeted therapy. Our study has mainly three parts as following:Part1Tumor Cell Adhesion to Microvessel Wall:Single Vessel Perfusion in vivo ModelObjective:To develope a novel tumor cell single microvessel perfusion of rat in vivo model, in which the conditions of experiment can be well defined. Adhesion of tumor cell at individually postcapillary venule of rat mesentery in vivo was observed by dynamic intravital video microscopy. Part2and part3would base on this model.Method:We evaluated Cellular Viability Rates by the effects of perfusion on different differences in pipette size, water pressure and time period. Then we choose the properly perfusion pipette. Tumor cells were perfused at mesenteric microvessel of rat by single vessel perfusion technique. The perfusion flow velocity was determined by the movement a fluorescently labeled tumor cell when the vessel was cannulated and perfused at a known driving pressure measured from water manometer. The cell out number was counted under certain velocity and concentration. We measure different type tumor cell adhesion rate by CCD camera record.Result: 1) Effect of pipette diameter, drive pressure and time on cellular viability ratesThe results demonstrate that higher water pressures, a smaller pipette diameter, and longer time exposure result in a lower viability rate in some of the three experimental cell types, to varying degrees. There is no significantly different viability rate between before perfusion and after perfusion60minutes. So we use the pipette tip withdiameter30μm.2) Cell perfusion velocityWe tested three perfusion velocities in our study, a reduced velocity of～200μm/s when the driving pressure was controlled at<0.5cmH2O,-1000μm/s, which was a mean blood flow velocity in this type of micro vessel when the driving pressure was2cmH2O, and-2500μm/s with5cmH2O or more.3) Perfusion cell number in different velocityWe got the tumor cell perfusion number after60minute under different velocity,5770±158.75under velocity of2500μm/s,2913±443.3under velocity of～1000μm/s and471±33.89under<250μm/s and.4) The adhesion rate in different type cellWe measured the normal epithelial cell and malignant mammary breast cancer cell, MDA-MB-231, MDA-MB-435s, WT and1335T cell adhesion rate on normal microvessel. There was almost no cell adhesion at5000um2vessel segment. However cancer cell adhesion rate was more than normal epithelial cell. The adhesion rate of MDA-MB-231, WT and1355T as5.65±0.7,5.56± 0.65,and5.87±0.31respectively were more than that MDA-MB-435s2.90±0.51at5000um2vessel segment.Conclusion:We successfully developed a novel individually microvessel perfusion tumor cell in vivo model, in which experimental conditions can be well control by perfusion velocity, concentration, and vessel permeability.Part2The Effect of VEGF and Shear Stress on Tumor Cell Adhesion to Microvascular Endothelium in vivoObjective:To investigate whether vascular endothelial growth factor (VEGF) and shear stress affects on cancer cell adhesion to normal microvesselsMethod:1) Test VEGF effect on tumor cell adhsionA single straight post-capillary venule of female SD rats was cannulated with a micropipette filled with1%BSA-Ringer solution containing MDA-MB-435s with4million/ml. The venule was perfused with a driving pressure to maintain a normal flow velocity of～1000μm/s. To test the effect of VEGF on cell adhesion to the microvessel wall injected simultaneously with1nM VEGF in1%BSA-Ringer into a single vessel in the same way as control. To test the effect of anti-VEGF and SU-1498on cell adhesion, cells were treated with20μg/ml anti-VEGF and50μM SU-1498at least incubated one hour at- 4℃in1%BSA-Ringer solution. We perfused the microvessel with50μM SU-1498for～45minute before injected the cell.2) The flow effect on tumor cell adhesionTo investigate the flow effect on tumor cell adhesion, we measure the tumor cell adhesion under low flow velocity (～200μm/s), normal velocity (～1000μm/s) and high velocity (-2500μm/s)3) VEGF effect on microvascular permeability measurementTo test the effect of1nM VEGF on permeability(P) of0.1mg/ml sodium fluorescence solutes, for each test solute, after making several control measurements (10min) with a θ pipette, we replaced with a new θ pipette of measure solution P with Su-1498or BSA30minutes. And then θ pipette was replaced again, both washout and dye solutions also contained with VEGF or Su-1498&VEGF.4) Sliver staining of boundearies of endothelial cells forming microvessel wallTo identify the locations of adherent tumor cell, silver stain was applied to individually perfused microvessels to illustrate the junctions of endothelial cells forming microvessel walls. We have calculated the location of adhesion cell on the vessel wall and gotten the rate of the adhesion location on the endothelial cell place.Result:1) VEGF increases the tumor cell adhesion to microvessel In conditions of normal vessel permeability, there was a monotonic statistically significant increase of cell adhesion from its basal value about1.5fold.2) Anti-VEGF and SU-1498decrease tumor cell adhesionWe observed anti-VEGF and SU-1498almost completely abolished cancer cell adhesion to the However, VEGF failed to significantly increase the adhesion of cancer cell to vessel wall as compare with untreated up to60min. With pretreatment Su-1498, VEGF failed to significantly increase the adhesion of cancer cell to vessel wall as compare with untreated up to60min,3) VEGF increase microvascular permeabilityWe paired measurements of P in individual rat mesenteric microvessels indicate that P for sodium fluorescence solutes showed a transient increase during treatment with VEGF, peaked at～30s and returned to baseline values in-2min.4) The effect of shear stress on tumor cell adhesionReducing shear rate induced tumor cell adhesion more than higher shear stress.5) Preferential Location of tumor cell adhesion in intact venular microvesselsWe have calculated the location of adhesion cell on the vessel wall. There are328tumor cells in total, and301cells were located at endothelial borders. The rate was191.86%cell at the endothelial cell boundary. We found that over92%of the adherent leukocytes had a definite position at the junctions of endothelial cells, and the remaining8%were at uncertain positions due to their location close to the side of the vessel.Conclusion:In summary, our well-controlled single-vessel perfusion in vivo study showed that VEGF increased microvessel permeability and enhanced human breast cancer cell adhesion in the post-capillary venule of a rat mesentery. Anti-VEGF and an inhibitor of VEGFR2almost completely abolished this adhesion. Reducing shear rate induced tumor cell adhesion more than higher shear stress. These results quantitatively demonstrate that adherent tumor cell preferentially attach to the junctions of endothelial cells forming microvessel walls.Part3Adhesions of Tumor Cells to the Microvessel Wall Increases Its Permeability by Degrading the Endothelial Surface GlycocalyxObjective:To investigated the effect of tumor cell adhesion on microvessel permeability and the mechanical and chemical change in intact microvessels by MDA-M-231Method:1) The measurement of the tumor cell adhesion rate, Lp, P to albumin of the microvesselsWe investigated the effect of tumor cell adhesion on microvessel permeability in intact icrovessels by measuring the adhesion rate of human mammary carcinoma MDA-MB-231. Then we measured three groups to observe the effect of tumor cell adhesion on Lp/P, which were the control, the initial adhesion, and after perfusion45minute.First control group, Lp/P was first measured baseline Lp of a vessel with Ringer-1%BSA10minute, then Lp was measured as shame control lasted10min after continuously perfused Ringer-1%BSA about30minute.For the initial adhesion and after perfusion45min, each group measurement was accomplished with two steps. The first step is to perfuse tumor cell a certain time. The initial adhesion defined tumor cell adhesion number was about1per5000μm(about)100μm length segment. The second step includes the removal of the cannulation pipette and then recannulated to apply Lp measurement with10minute.2) Mathematic predicationWe predicted the most likely change in the structural components of the endothelium induced by tumor cell adhesion by compromising the integrity of inter-endothelial cleft. We adapted the mathematical with P and Lp result.3) Anti-HS immunostaining of microvascular EGLAfter perfusing with a blocking solution, FITC conjugated anti-HS was perfused into the microvessel by a cannulating micropipette for2hr and the free dye was washed away. We investigated three groups to observe the effect of tumor cell adhesion on EGL, which were the control, the initial adhesion, and after perfusion45minute.4) Investigate the effect of ORM on tumor cell adhesion, P, and EGL We then measured the tumor cell adhesion rate for60min with control and orosomucoid treatment. We measured P to albumin by using0pipette, the fluorescence intensity of anti-HS immunostaining of micro vascular EGL at pretreatment with orosomucoid.Result:1) MDA-MBA-231adhesion to the microvessel wallThe time for initial adhesion was7.0±2.8(SD, n=14) min. The initial time was defined as one adherent cell in100μm vessel segment. Figure1shows the number of adherent cells as a function of time. The averaged5adherent cells in5000μm2vessel segment time were39.4±3.64minutes.2) MDA-MB-231adhesion increases in microvascular LpAt the initiation of cell adhesion, Lp increased from a mean control of1.01±0.04×10-7cm/s·cmH2O-1(n=5, SE)to1.84±0.08×10-7cm/s-cmH2O-1(n=13), a1.7±0.1-fold increase. After-45min tumor cell perfusion, Lp further increased to2.94±0.16×10-7cm/s-cmH2O-1(n=12), a2.75±0.14fold mcrease.4) Tumor cell adhesion increases in microvascular permeabilityDiffusion permeability to albumin increased from a mean baseline value of7.08±0.42to15.6±2.1×10-7cm/s, a2.2±0.3fold increases at the initial adhesion. After-45min tumor adhesion, P increased to39.9±4.4×10"7cm/s, a5.6±0.6fold increase.5) Model prediction Comparing these measured Lp and P data with the model predictions for Lp and P changes when degrading the endothelial surface glycocalyx.We could find that if normal Lf=400nm, the initial tumor adhesion and after perfusion45min would degrade the glycocalyx layer by30-45%and91-95%, respectively.6) Tumor cell adhesion degrade the EGLIn vivo fluorescence microscopy revealed that the amount of heparan sulfate in the EGL was reduced by60.25%(±7%, SE, n=15) of the initial adhesion,81.7%(±3%, SE n=14) after-45min tumor cell adhesion, respectively. This observation confirms the prediction from the mathematical modeling.7) Orosomucoid decrease tumor cell adhesion and P by maintain EGL The cell adhesion rate under the orosomucoid treatment decreased from5.58±0.50under control to3.23±0.32at5000μm2segment during60min period. At orosomucoid pretreatment, P was0.547(±0.08SE, n=7) compare with controls; the amount of heparan sulfate in the EGL fluorescence was increased1.38(±0.11SE, n=18)-fold of their control.Conclusion:Combining the predictions model and immunolabeling of the HS in the EGL of the microvessels suggests that tumor cell adhesion to the microvessel wall degrade EGL to increase microvascular permeability. Meaning while maintain mechanical and chemical stability of the endothelial surface layer can decrease tumor cell adhesion and the permeability of microvessel wall.Therapeutic strategies targeting the endothelial glycocalyx is great potential to prevent tumor cell adhesion and metastasis.