Contractile injection systems (CISs) are cell-puncturing nanodevices that share ancestry with contractile tail bacteriophages. Photorhabdus virulence cassette (PVC) represents one group of extracellular CISs that are present in both bacteria and archaea. Here, we report the cryo-EM structure of an intact PVC from P. asymbiotica. This over 10-MDa device resembles a simplified T4 phage tail, containing a hexagonal baseplate complex with six fibers and a capped 117-nanometer sheath-tube trunk. One distinct feature of the PVC is the presence of three variants for both tube and sheath proteins, indicating a functional specialization of them during evolution. The terminal hexameric cap docks onto the topmost layer of the inner tube and locks the outer sheath in pre-contraction state with six stretching arms. Our results on the PVC provide a framework for understanding the general mechanism of widespread CISs and pave the way for using them as delivery tools in biological or therapeutic applications.

Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO) production in post-capillary venules and arterioles of rat mesentery under reduced (low) and normal (high) flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS) of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA). Rats (SD, 250–300g) were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for ~10 min under a low flow (~300 μm/s) and for ~60 min under a high flow (~1000 μm/s). In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.

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.

The glycocalyx on the surface of endothelium lining blood vessel walls modulates vascular barrier function, cell adhesion and also serves as a mechano-sensor for blood flow. Reduction of glycocalyx has been reported in many diseases including atherosclerosis, inflammation, myocardial edema, and diabetes. The surface glycocalyx layer (SGL) is composed of proteoglycans and glycosaminoglycans, of which heparan sulfate is one of the most abundant. To quantify the SGL thickness on the microvessels of rat mesentery and mouse cremaster muscle in situ, we applied a single vessel cannulation and perfusion technique to directly inject FITC-anti-heparan sulfate into a group of microvessels for immuno-labeling the SGL. We also used anti-heparan sulfate for immuno-labeling the SGL on rat and mouse aortas ex vivo. High resolution confocal microscopy revealed that the thickness of the SGL on rat mesenteric capillaries and post-capillary venules is 0.9 ± 0.1 μm and 1.2 ± 0.3 μm, respectively; while the thickness of the SGL on mouse cremaster muscle capillaries and post-capillary venules is 1.5 ± 0.1 μm and 1.5 ± 0.2 μm, respectively. Surprisingly, there was no detectable SGL in either rat mesenteric or mouse cremaster muscle arterioles. The SGL thickness is 2.5 ± 0.1 μm and 2.1 ± 0.2 μm respectively, on rat and mouse aorta. In addition, we observed that the SGL is continuously and evenly distributed on the aorta wall but not on the microvessel wall.

Prior studies have indicated that the β4 integrin promotes mammary tumor invasion and metastasis by combining with ErbB2 and amplifying its signaling capacity. However, the effector pathways and cellular functions by which the β4 integrin exerts these effects are incompletely understood. To examine if β4 signaling plays a role during mammary tumor cell adhesion to microvascular endothelium, we have examined ErbB2-transformed mammary tumor cells expressing either a wild-type (WT) or a signaling-defective form of β4 (1355T). We report that WT cells adhere to brain microvascular endothelium in vitro to a significantly larger extent as compared to 1355T cells. Interestingly, integrin β4 signaling does not exert a direct effect on adhesion to the endothelium or the underlying basement membrane. Rather, it enhances ErbB2-dependent expression of VEGF by tumor cells. VEGF in turn disrupts the tight and adherens junctions of endothelial monolayers, enabling the exposure of underlying basement membrane and increasing the adhesion of tumor cells to the intercellular junctions of endothelium. Inhibition of ErbB2 on tumor cells or the VEGFR-2 on endothelial cells suppresses mammary tumor cell adhesion to microvascular endothelium. Our results indicate that β4 signaling regulates VEGF expression by the mammary tumor cells thereby enhancing their adhesion to microvascular endothelium.

To investigate whether vascular endothelial growth factor (VEGF) enhances cancer cell adhesion to normal microvessels, we used in vivo video microscopy to measure adhesion rates of MDA-MB-435s human breast cancer cells and ErbB2-transformed mouse mammary carcinomas in the postcapillary venules of rat mesentery. An individual postcapillary venule in the mesentery was injected via a glass micropipette with cancer cells either in a perfusate of mammalian Ringer solution containing 1% bovine serum albumin as a control, or with the addition of 1 nm VEGF for test measurements. Cell adhesion was measured as either the number of adherent cells or the fluorescence intensity of adherent cells in a vessel segment for ∼60 min. Our results showed that during both control and VEGF treatments, the number of adherent cells increased almost linearly with time over 60 min. The VEGF treatment increased the adhesion rates of human tumour cells and mouse carcinomas 1.9-fold and 1.8-fold, respectively, over those in control conditions. We also measured cancer cell adhesion after pretreatment of cells with an antibody blocking VEGF or an antibody blocking α6 integrin, and pretreatment of the microvessel with VEGF receptor (KDR/Flk-1) inhibitor, SU1498, or anti-integrin extracellular matrix ligand antibody, anti-laminin-5. All antibodies and inhibitor significantly reduced adhesion, with anti-VEGF and SU1498 reducing it the most. Our results indicate that VEGF enhances cancer cell adhesion to the normal microvessel wall, and further suggest that VEGF and its receptor, KDR/Flk-1, as well as integrins of tumour cells and their ligands at the endothelium, contribute to mammary cancer cell adhesion to vascular endothelium in vivo.

To investigate the mechanical mechanisms behind tumor cell arrest in the microvasculature, we injected fluorescently labeled human breast carcinoma cells or similarly sized rigid beads into the systemic circulation of a rat. Their arrest patterns in the microvasculature of mesentery were recorded and quantified. We found that 93% of rigid beads were arrested either at arteriole–capillary intersections or in capillaries. Only 3% were at the capillary–postcapillary venule intersections and in postcapillary venules. In contrast, most of the flexible tumor cells were either entrapped in capillaries or arrested at capillary or postcapillary venule–postcapillary venule intersections and in postcapillary venules. Only 12% of tumor cells were arrested at the arteriole–capillary intersections. The differential arrest and adhesion of tumor cells and microbeads in the microvasculature was confirmed by a χ2 test (p < 0.001). These results demonstrate that mechanical trapping was responsible for almost all the arrest of beads and half the arrest of tumor cells. Based on the measured geometry and blood flow velocities at the intersections, we also performed a numerical simulation using commercial software (ANSYS CFX 12.01) to depict the detailed distribution profiles of the velocity, shear rate, and vorticity at the intersections where tumor cells preferred to arrest and adhere. Simulation results reveal the presence of localized vorticity and shear rate regions at the turning points of the microvessel intersections, implying that hemodynamic factors play an important role in tumor cell arrest in the microcirculation. Our study helps elucidate long-debated issues related to the dominant factors in early-stage tumor hematogenous metastasis.

Tumor cell adhesion to vessel walls in the microcirculation is one critical step in cancer metastasis. In this paper, the hypothesis that tumor cells prefer to adhere at the microvessels with localized shear stresses and their gradients, such as in the curved microvessels, was examined both experimentally and computationally. Our in vivo experiments were performed on the microvessels (post-capillary venules, 30–50 μm diameter) of rat mesentery. A straight or curved microvessel was cannulated and perfused with tumor cells by a glass micropipette at a velocity of ~1mm/s. At less than 10 min after perfusion, there was a significant difference in cell adhesion to the straight and curved vessel walls. In 60 min, the averaged adhesion rate in the curved vessels (n = 14) was ~1.5-fold of that in the straight vessels (n = 19). In 51 curved segments, 45% of cell adhesion was initiated at the inner side, 25% at outer side, and 30% at both sides of the curved vessels. To investigate the mechanical mechanism by which tumor cells prefer adhering at curved sites, we performed a computational study, in which the fluid dynamics was carried out by the lattice Boltzmann method, and the tumor cell dynamics was governed by the Newton’s law of translation and rotation. A modified adhesive dynamics model that included the influence of wall shear stress/gradient on the association/dissociation rates of tumor celladhesion was proposed, in which the positive wall shear stress/gradient jump would enhance tumor cell adhesion while the negative wall shear stress/gradient jump would weaken tumor cell adhesion. It was found that the wall shear stress/gradient, over a threshold, had significant contribution to tumor cell adhesion by activating or inactivating cell adhesion molecules. Our results elucidated why the tumor cell adhesion prefers to occur at the positive curvature of curved microvessels with very low Reynolds number (in the order of 10−2) laminar flow.