Adhesion of malignant mammary tumor cells MDA-MB-231 to microvessel wall increases microvascular permeability via degradation of endothelial surface glycocalyx

Abstract

To investigate the effect of tumor cell adhesion on microvascular permeability (P) in intact microvessels, we measured the adhesion rate of human mammary carcinoma MDA-MB-231, the hydraulic conductivity (L(p)), the P, and reflection coefficient (σ) to albumin of the microvessels at the initial tumor cell adhesion and after ∼45 min cell perfusion in the postcapillary venules of rat mesentery in vivo. Rats (Sprague-Dawley, 250-300 g) were anesthetized with pentobarbital sodium given subcutaneously. A midline incision was made in the abdominal wall, and the mesentery was gently taken out and arranged on the surface of a glass coverslip for the measurement. An individual postcapillary venule was perfused with cells at a rate of ∼1 mm/s, which is the mean blood flow velocity in this type of microvessels. At the initial tumor cell adhesion, which was defined as one adherent cell in ∼100- to 145-μm vessel segment, L(p) was 1.5-fold and P was 2.3-fold of their controls, and σ decreased from 0.92 to 0.64; after ∼45-min perfusion, the adhesion increased to ∼5 adherent cells in ∼100- to 145-μm vessel segment, while L(p) increased to 2.8-fold, P to 5.7-fold of their controls, and σ decreased from 0.92 to 0.42. Combining these measured data with the predictions from a mathematical model for the interendothelial transport suggests that tumor cell adhesion to the microvessel wall degrades the endothelial surface glycocalyx (ESG) layer. This suggestion was confirmed by immunostaining of heparan sulfate of the ESG on the microvessel wall. Preserving of the ESG by a plasma glycoprotein orosomucoid decreased the P to albumin and reduced the tumor cell adhesion.

Direct Tracking of Particles and Quantification of Margination in Blood Flow

“…A pressure-driven pump system with a flow controller (Fluigent, Villejuif, France) was used to pump each solution through the microfluidic device at a physiologically relevant average velocity of 1 mm/s (44)(45)(46)(47). This velocity corresponds to a volumetric flow rate of 0.24 mL/min and an apparent wall shear rate of 61 s À1 for our channel dimensions.…”

Automatic Classification and Quantification of Cell Adhesion Locations on the Endothelium

“…For each microvessel, 4 photomicrographs were collected [4] [2], generating a total of 20 photomicrographs. Fig.…”

“…To determine whether tumor cells prefer to adhere at the endothelial junctions rather than randomly adhere on the vessel surface, using the quantification approach as devised in [14] and [2], we first measured the length of endothelial junctions per unit area for the microvessel shown in Fig. 4, the averaged value is 107.9 ± 14.4 mm/mm 2 .…”

“…The tumor cell adhesion image in a single perfused microvessel was obtained using the same method as described in [2]. To identify the locations of adherent tumor cells, silver staining was applied to the individually perfused microvessel to illustrate the junctions of endothelial cells forming the microvessel wall [14].…”

“…Another equally important question concerns the preferential pathway for circulating leukocytes to cross the microvessel wall to perform their normal immune functions in the infected tissues. Previously, human operators classify and quantify cell adhesion locations via eyeballing and tape measuring [4] [2]. However, to obtain significant results, many photomicrographs need to be analyzed with tens to hundreds of adherent cells in each photomicrograph.…”