The mechanisms by which tumors are able to evade cellular immune responses are still largely unknown. It is likely, however, that the initial recruitment of lymphocytes to tumor vessels is limited by cell retention in normal tissue, which results in a low flux of these cells into the tumor vasculature. We grew MCaIV. (20mouse mammary carcinoma) tumors in the leg of SCID mice and injected 111In-oxine-labeled, primed T lymphocytes directed against the tumor intravenously. The systemic distribution of cells in normal organs was similar between mice injected with primed and control lymphocyte populations, except for a delayed clearance of primed lymphocytes from the lungs. Kinetics of lymphocyte localization to the tumor were identical between the primed and control lymphocyte populations. Splenectomy before the injection of primed lymphocytes increased delivery of cells to the lungs and liver after 1 hour with no significant improvement in tumor localization. Within 24 to 168 hours after injection, localization of cells in the liver of splenectomized mice was higher than in the control group. However, no significant difference in tumor localization was observed between groups. A physiologically based compartmental model of lymphocyte distribution predicted the compartmental sequestration and identified model parameters critical for experimental planning and therapeutic optimization.

Interactions between endothelial cell receptors and the extracellular matrix (ECM) play a critical, yet poorly understood role in angiogenesis. Based on the anti-adhesive role of decorin, we hypothesized that decorin binding to ECM molecules such as thrombospondin-1 (TSP-1) plays a regulatory role in endothelial tube-like structure (TLS) formation. To test this hypothesis, endothelial cells were plated on TSP-1, decorin, or mixed substrates of TSP-1 plus decorin. TLS formation was induced by applying type I collagen on the confluent endothelial monolayer. Cartilage decorin inhibited the formation of TLSs in a concentration-dependent manner. On substrates of high decorin concentrations (2.5 and 5.0 μg/cm2) the reduction in TLSs was due either to a reduction in the number of adhering cells or to decreased cell migration. At low decorin concentrations (0.05 and 0.25 μg/cm2) the reduction in TLSs was independent of the number of attached cells. Time-lapse video microscopy revealed that decorin substrates facilitated homotypic aggregation and isolated cord formation at the expense of endothelial migration and TLS formation. Consistent with the reduced migration, endothelial cells formed fewer vinculin-positive focal adhesions and actin-stress fibers on decorin substrates. Endothelial migration and TLS formation were also significantly inhibited by skin decorin and the protein core of cartilage decorin. The inhibition of TLS formation by the protein core of cartilage decorin was potentiated by TSP-1. These findings suggest that decorin alone or in combination with TSP-1 interferes with the activation of endothelial cell receptors by ECM molecules, thus blocking intracellular signals that induce cytoskeletal reorganization, migration, and TLS formation.

Physical interactions between circulating cells and the vascular wall play a central role in inflammation, metastasis, atherosclerosis, and therapeutic cell delivery. Unfortunately, traditional in vitro flow assays cannot be used to visualize the details of cell-surface interactions in blood flow because of inappropriate geometry and the poor penetration of light in erythrocyte solutions. To overcome these obstacles, we have developed an agarose-cast cylindrical vessel system to examine the profiles of cells interacting with surfaces under flow conditions. This design allows observation and quantification of cell deformation as cells adhere to surfaces under dynamic flow conditions without modifying the microscope or optical path. Furthermore, our flow system is uniquely suited for monitoring the profiles of adherent leukocytes deforming in response to erythrocyte suspension flow. We have used this flow system to study the role of erythrocytes in leukocyte-substrate interactions. Our results show that the cell deformation index (the ratio of the cell length to cell height) is higher in erythrocyte solutions compared to erythrocyte-free saline. This novel lateral view flow system provides a powerful technique for visualizing and quantifying the morphological changes of cells in contact with substrates exposed to shear stress.

The delivery of cells to specific regions of the vasculature is a critical step in many therapeutic strategies. These include the packaging of DNA or RNA in cell "vehicles" for delivery to tissues, the reconstitution of differentiated cells to an organ using embryonic stem cells, and the enhancement of the immune response using effector lymphocytes. In most cases, these cells must be injected systemically. Unfortunately, ex vivo manipulation or activation can affect cell visco-elastic properties, making it difficult for the injected cells to traverse capillary beds. Compounding the problem is the fact that common agents used in the laboratory for increasing cell deformability generally have adverse side effects on the therapeutic potential of the cells. Using micropipet aspiration techniques, cytotoxicity assays and in vivo trafficking studies we show that: (1) the rigidity of injected effector cells directly affects resistance to passage through tissue; (2) modulation of cytoskeletal organization can be used to decrease cell rigidity, but can also compromise therapeutic efficacy; and (3) thioglycollate, an agent which does not influence effector lymphocyte cytotoxic activity, reduces cell rigidity and entrapment in the lungs.

The rising incidence and persistent thrombosis in multiple cancers including those that are immunosuppressive highlight the need for understanding the tumor coagulome system and its role beyond hemostatic complications. Immunotherapy has shown significant benefits in solid organ tumors but has been disappointing in the treatment of hypercoagulable cancers, such as glioblastoma and pancreatic ductal adenocarcinomas. Thus, targeting thrombosis to prevent immunosuppression seems a clinically viable approach in cancer treatment. Hypercoagulable tumors often develop fibrin clots within the tumor microenvironment (TME) that dictates the biophysical characteristics of the tumor tissue. The application of systems biology and single-cell approaches highlight the potential role of coagulome or thrombocytosis in shaping the tumor immune microenvironment (TIME). In-depth knowledge of the tumor coagulome would provide unprecedented opportunities to better predict the hemostatic complications, explore how thrombotic stroma modulates tumor immunity, reexamine the significance of clinical biomarkers, and enable steering the stromal versus systemic immune response for boosting the effectiveness of immune checkpoint inhibitors in cancer treatment. We focus on the role of coagulation factors in priming a suppressive TIME and the huge potential of existing anticoagulant drugs in the clinical settings of cancer immunotherapy.

When a leukocyte enters a blood vessel, it may continue to move with flowing blood, collide with the vessel wall, adhere transiently or stably, and finally extravasate (1). These interactions are governed by both local hydrodynamic and adhesive forces. The former are determined by the vessel diameter, fluid velocity, viscosity, and hematocrit, and the latter by the number, strength and kinetics of bond formation between adhesion molecules, and by surface area of contact (1-6). Cellular deformability affects both types of forces (7-9). Two families of cell adhesion molecules (CAMs) are involved in leukocyte rolling and stable adhesion. In general, the selectins (P, L, and E) mediate rolling, while the IgG superfamily members (ICAM-1 and VCAM-1) on endothelial cells, with their cognate receptors (β(2) and β(1) integrin receptors) on the leukocytes, mediate firm adhesion, with some overlap in these functions (10-12). The expression of CAMs on the endothelial cells and leukocytes can be modulated by cytokines secreted by a variety of cells (e.g., cancer cells, fibroblasts, macrophages) (13,14). Cellular deformability can be modulated by altering the cytoskeleton, membrane, or cytoplasm, with the cytoskeleton playing the dominant role (7,15,16). In this chapter, we describe methods to quantitate cellular deformability in vitro, CAM expression in vitro, leukocyte-endothelial interaction (LEI) in vitro, and LEI in vivo.

BACKGROUND:Angiogenesis is characteristic of chronic inflammatory reactions. The process of angiogenesis is reported to be proinflammatory in part due to enhanced adhesion events and in part due to increased perfusion and permeability to sites of inflammation. However, little is known about the association between angiogenesis and rejection.METHODS:Severe combined immune deficient mice are permissive for the growth of human skin allografts and human peripheral blood mononuclear cells (PBMC). Human PBMC were injected into mice by intravenous or intraperitoneal injection. The infiltration of cells and the associated angiogenesis reactions in the skin allografts were analyzed temporally by videomicroscopy and spatially by immunohistochemistry.RESULTS:Human alloreactive mononuclear cells migrated to human skin but not mouse skin within hours after the intravenous infusion of PBMC. Within 3 days, areas of angiogenesis were observed in the skin grafts at the sites of infiltrates. The vessel densities in skin grafts were 24+/-6 vessels per calibrated grid at baseline on the day of the infusion and increased to 55+/-16 vessels per calibrated field by day 10. Skin grafts harvested from humanized severe combined immune deficient mice 7-14 days after the intraperitoneal infusion of human PBMC showed a similar increased density of vessels that were spatially associated with mononuclear cell infiltrates.CONCLUSIONS:A significant angiogenesis response was associated with the cell infiltrates in the human skin allografts. The onset of angiogenesis appeared after the initial development of localized infiltrates and preceded the development of microvascular destruction. These findings suggest that alloreactive T cells and/or monocytes mediate the angiogenesis response in skin allografts.

The goal of this study was to compare growth characteristics of cells shed from a tumour with the native tumour cells. The human colon adenocarcinoma LS174T and its highly metastatic subline LS LiM 6 were grown as tissue-isolated tumours in nude mice and perfused to collect shed cells. The tumours were then excised and prepared into single-cell suspensions. Clonogenicity in 0.3–0.9% agarose, apoptotic fraction, and in vivo tumorigenicity were determined for each population. In both tumour lines, shed cells were less clonogenic, more apoptotic and less tumorigenic than cells isolated directly from their native tissue. These findings suggest that shed cells have a low metastatic potential compared to native tumour cells, most likely because they represent an apoptotic population.

Lymphocyte adhesion and trafficking is difficult to observe in vivo over time. We used magnetic resonance imaging (MRI) to identify magnetically labeled lymphocytes in phantom experiments and in tissue. A method of lymphocyte labeling was developed that is based on fluid-phase endocytosis of nanometer-sized biocompatible superparamagnetic particles. The maximum cell uptake in culture was 0.11 ng Fe/cell corresponding to 5 × 106 particles/lymphocyte. Cells stably retained the label and were fully viable for at least 3 days. Labeled lymphocytes showed adhesion to human endothelial cells similar to unlabeled cells, indicating no effect of labeling on cell surface expression of adhesion proteins. No particle-mediated cytotoxicity could be observed. The detection threshold of MRI for detecting labeled lymphocytes in the current study was 2.5 × 106 cells/30 μL sampling volume. Following intravenous injection of labeled lymphocytes into rats, cells accumulated in spleen, lymph nodes and liver with a similar bio-distribution as unlabeled cells. Lymphocyte accumulation in the spleen resulted in MRI signal intensity changes readily detectable by MRI. These findings suggest that intracellular lymphocyte labeling with superparamagnetic particles is feasible, does not alter the viability or tissue distribution of labeled cells and allows the detection of labeled lymphocytes by MRI.

ABSTRACTObjective: Previous work indicated that E‐selectin mediates transient interactions between leukocytes and cytokine‐activated endothelium in vitro. Here we examine the role of E‐selectin in blood leukocyte interactions with microvascular endothelium in vivo.Methods: E‐selectin‐deficient (E−/− mice were produced by gene targeting. The effect of this null mutation on leukocyte‐endothelial interactions was determined by intravital microscopy before and 4 to 5 hours after local administration of the proinflammatory cytokine tumor necrosis factor α (TNFα) in dermal microvessels with low blood flow (dorsal skin‐fold chambers, intact ear skin), and after endotoxin activation in exteriorized mesenteric microvessels with higher blood flow.Results: E−/− mice were viable, fertile with normal circulating leukocyte and platelet profiles. Approximately 60% of circulating leukocytes rolled in dermal microvessels of both normal (E+/+) and E−/− mice without inflammatory stimulation. After local administration of TNFα, rolling increased modestly and equivalently in both genotypes. The main effect of TNFα was a dramatic increase in leukocyte stable adhesion and, unlike rolling, this manifestation of endothelial activation was significantly reduced in E−/− animals. This reflected fewer dermal microvessels supporting higher adhesion densities in E−/− mice, and a similar trend was observed in mesenteric microvessels.Conclusions: E‐selectin plays a previously unappreciated role in facilitating and/or mediating stable adhesion of leukocytes to inflamed microvascular endothelium.

In this review we discuss (1) the role of the endothelial cell in the initiation of acute and chronic rejection; (2) studies that demonstrate that endothelial cells have the ability to selectively costimulate T cell activation: and (3) the bi-directionality of T cell-endothelial cell interactions in rejection.

Localization of activated natural killer (A-NK) cells in the microvasculature of growing tumors is the result of recognition of the intracellular and vascular cell-adhesion molecules ICAM-1 and VCAM-1 on the tumor endothelium, mediated by lymphocyte function-associated protein LFA-1 and vascular lymphocyte function-associated protein VLA-4. In vitro and in vivo studies of A-NK cell adhesion to endothelial cells showed that vascular endothelial growth factor (VEGF) promotes adhesion, whereas basic fibroblast growth factor (bFGF) inhibits adhesion through the regulation of these molecules on tumor vasculature. Thus, some angiogenic factors may facilitate lymphocyte recognition of angiogenic vessels, whereas others may provide such vessels with a mechanism that protects them from cytotoxic lymphocytes.

The binding of circulating cells to the vascular wall is a central process in inflammation, metastasis, and therapeutic cell delivery. Previous in vitro studies have identified the adhesion molecules on various circulating cells and the endothelium that govern the process under static conditions. Other studies have attempted to simulate in vivo conditions by subjecting adherent cells to shear stress as they interact with the endothelial cells in vitro. These experiments are generally performed with the cells suspended in Newtonian solutions. However, in vivo conditions are more complex because of the non-Newtonian flow of blood, which is a suspension consisting of 20-40% erythrocytes by volume. The forces imparted by the erythrocytes in the flow can contribute to the process of cell adhesion. A number of experimental and theoretical studies have suggested that the rheology of blood can influence the binding of circulating leukocytes by increasing the normal and axial forces on leukocytes or the frequency of their collision with the vessel wall, but there have been no systematic investigations of these phenomena to date. The present study quantifies the contribution of red blood cells (RBCs) in cell capture and adhesion to endothelial monolayers using a combination of mathematical modeling and in vitro studies. Mathematical modeling of the flow experiments suggested a physical mechanism involving RBC-induced leukocyte dispersion and/or increased normal adhesive contact. Flow chamber studies performed with and without RBCs in the suspending medium showed increases in wall collision and binding frequencies, and a decrease in rolling velocity in the presence of erythrocytes. Increased fluid viscosity alone did not influence the binding frequency, and the differences could not be attributed to large near-wall excesses of the lymphocytes. The results indicate that RBCs aid in the transport and initial engagement of lymphocytes to the vascular wall, modifying the existing paradigm for immune cell surveillance of the vascular endothelium by adding the erythrocyte as an essential contributor to this process.