Review of functional in vitro models of the blood-cerebrospinal fluid barrier in leukaemia research.

Acute lymphoblastic leukaemia represents the most common paediatric malignancy. Although survival rates approach up to 90% in children, investigation of leukaemic infiltration into the central nervous system (CNS) is essential due to the presence of ongoing fatal complications. Recent in vitro studies mostly employed models of the blood-brain barrier (BBB), as endothelial cells of the microvasculature represent the largest surface between the blood stream and the brain parenchyma. However, crossing the blood-cerebrospinal fluid barrier (BCSFB) within the choroid plexus (CP) has been shown to be a general capability of leukaemic blasts. Hence, in vitro models of the BCSFB to study leukaemic transmigration may be of major importance to understand the development of CNS leukaemia. This review will summarise available in vitro models of the BCSFB employed to study the cellular interactions with leukaemic blasts during cancer cell transmigration into the brain compartment across primary or immortal/immortalised BCSFB cells. It will also provide an outlook on prospective improvements in BCSFB in vitro models by developing barrier-on-a-chip models and brain organoids.

Structure and biomechanics of the endothelial transcellular circumferential invasion array in tumor invasion.

Cancer cells breach the endothelium not only through cell-cell junctions but also via individual endothelial cells (ECs), or transcellular invasion. The underlying EC forms a circular structure around the transcellular invasion pore that is dependent on myosin light chain kinase (MLCK) and myosin II regulatory light chain (RLC) phosphorylation. Here we offer mechanistic insights into transcellular invasive array formation amid persistent tensile force from activated EC myosin. Fluorescence recovery after photobleaching (FRAP) experiments, sarcomeric distance measurements using super-resolution microscopy and electron microscopy provide details about the nature of the myosin II invasion array. To probe the relationship between biomechanical forces and the tension required to maintain the curvature of contractile filaments, we targeted individual actin-myosin fibers at the invasion site for photoablation. We showed that adjacent filaments rapidly replace the ablat11ed structures. We propose that the transcellular circumferential invasion array (TCIA) provides the necessary constraint within the EC to blunt the radial compression from the invading cancer cell.

ß2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier.

In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of ß2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, ß2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

Programming membrane fusion and subsequent apoptosis into mammalian cells.

By the delivery of specific natural or engineered proteins, mammalian cells can be programmed to perform increasingly sophisticated and useful functions. Here, we introduce a set of proteins that has potential value in cell-based therapies by programming a cell to target tumor cells. First, the delivery of VSV-G (vesicular stomatitis virus glycoprotein) allowed the cell to undergo membrane fusion with adjacent cells to form syncytia (i.e., a multinucleated cell) in conditions of low pH typically occurring at a tumor site. The formation of syncytia caused the clustering of nuclei along with an integration of the microtubule network and ER. Interestingly, the formation of syncytia between cells that are dynamically blebbing, a mode of migration preferred during tumor metastasis, resulted in the loss of these morphology changes. Lastly, the codelivery of VSV-G with L57R (an engineered photoactivated caspase-7) allowed cells to undergo low pH-dependent membrane fusion followed by blue light-dependent apoptosis. In cell-based therapies, the clearance of syncytia between tumor cells might further trigger an immune response against the tumor.

Endothelial endoglin is involved in inflammation: role in leukocyte adhesion and transmigration.

Human endoglin is an RGD-containing transmembrane glycoprotein identified in vascular endothelial cells. Although endoglin is essential for angiogenesis and its expression is up-regulated in inflammation and at sites of leukocyte extravasation, its role in leukocyte trafficking is unknown. This function was tested in endoglin heterozygous mice (Eng(+/-)) and their wild-type siblings Eng(+/+) treated with carrageenan or LPS as inflammatory agents. Both stimuli showed that inflammation-induced leukocyte transendothelial migration to peritoneum or lungs was significantly lower in Eng(+/-) than in Eng(+/+) mice. Leukocyte transmigration through cell monolayers of endoglin transfectants was clearly enhanced in the presence of endoglin. Coating transwells with the RGD-containing extracellular domain of endoglin, enhanced leukocyte transmigration, and this increased motility was inhibited by soluble endoglin. Leukocytes stimulated with CXCL12, a chemokine involved in inflammation, strongly adhered to endoglin-coated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin α5ß1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin α5ß1 via its RGD motif, and this adhesion process is stimulated by the inflammatory chemokine CXCL12, suggesting a regulatory role for endoglin in transendothelial leukocyte trafficking.

Ultrastructural descriptions of heterotypic aggregation between eosinophils and tumor cells in human gastric carcinomas.

A histological variant of gastric adenocarcinoma, characterized by an intense tumor-associated tissue eosinophilia (TATE), has been occasionally reported in the literature. The purpose of this ultrastructural study was to determine the interactions between frequently occurring eosinophils and tumor cells in gastric carcinoma characterized by TATE. Fresh tumor tissue of 92 gastric carcinomas was processed for both light and electron microscopic examination. Intense TATE was found in 7 out of 92 (7.6%) gastric carcinomas (6 of intestinal-type and 1 of diffuse-type). Electron microscopy, selectively performed in 7 cases with intense TATE, revealed eosinophils, singly or in groups, in contact with damaged or necrotic tumor cells. Activated eosinophils showing piecemeal degranulation were also found in intimate contact with viable tumor cells, characterized by plasma membrane caveolar invaginations. The authors regard this close morphological relationship as in vivo evidence for possible cross-talk between eosinophil and viable tumor cell, a conclusion that has already been drawn from experimental studies, but until now inadequately supported by ultrastructural observations in a human tumor.

Transcellular migration of neutrophil granulocytes through the blood-cerebrospinal fluid barrier after infection with Streptococcus suis.

BACKGROUND: A critical point during the course of bacterial meningitis is the excessive influx of polymorphnuclear neutrophils (PMNs) from the blood into the brain. Both paracellular and transcellular routes of leukocyte transmigration through the blood-brain barrier have been described in CNS diseases so far. Thus, we investigated the mechanism of PMN transmigration through the blood-CSF barrier under inflammatory conditions. METHODS: In an "inverted" Transwell culture model of the blood-CSF barrier, the zoonotic agent Streptococcus suis (S. suis) was used to stimulate porcine choroid plexus epithelial cells (PCPECs) specifically from the physiologically relevant basolateral side. Barrier function was analyzed by measuring TEER and TR-dextran-flux, and tight junction morphology was investigated by immunofluorescence. Route and mechanism of PMN transmigration were determined by immunofluorescence, electron microscopy and FACS analysis. Quantitative real time-PCR was used to determine expression levels of ICAM-1 and VCAM-1. RESULTS: Here, we show that the transmigration of PMNs through PCPECs was significantly higher after stimulation with TNFα or infection with S. suis strain 10 compared to its non-encapsulated mutant. Barrier function was not significantly affected by PMN migration alone, but in combination with S. suis infection. Tight junction and cytoskeletal actin reorganisation were also observed after stimulation with S. suis or TNFα. Most strikingly, PMNs preferentially migrated across PCPECs via the transcellular route. Extensive sequential analyses of the PMN transmigration process with Apotome(®)-imaging and electron microscopy revealed that paracellular migrating PMNs stop just before tight junctions. Interestingly, PMNs subsequently appeared to proceed by transcellular migration via funnel-like structures developing from the apical membrane. It is noteworthy that some PMNs contained bacteria during the transmigration process. Flow cytometric and transmigration inhibition studies with integrin-specific antibodies showed that PMN traversal is dependent on CD11b/CD18. Analysis of cell adhesion molecules in PCPECs revealed a significant increase of ICAM-1 and VCAM-1 expression after TNFα and S. suis stimulation. CONCLUSION: Our data underline the relevance of the blood-CSF barrier as a gate for leukocyte entry into the CNS and suggest a novel transcellular migration step during the pathogenesis of bacterial meningitis.

Radial glial cells and the lamination of the cerebellar cortex.

Radial glial cells are stem cells that play an important role in neuronal migration and proliferation in the developing brain. However, how radial glial cells contribute to the lamination of the cerebellar cortex is not well understood. We therefore used immunohistochemistry and BrdU labeling to follow radial glial cell differentiation, cell migration and cerebellar cortex development in mice from embryonic day 8 to postnatal day 180. We report that radial glial cells represent the stem cell population of the neuroepithelium of the neural tube, and act as progenitors for both neurons and neuroglia. In addition, radial glial cells not only give rise to the principal cells of the cerebellar cortex, the Purkinje and granule cells, but they also provide a scaffold for the migration of these cells. We conclude that radial glial cells play a pivotal role in establishing the laminar structure of the cerebellar cortex.

Transplacental traffic after in utero mesenchymal stem cell transplantation.

Transplacental traffic of fetal progenitor and differentiated cells is a well-known phenomenon in pregnancies. We hypothesize that intrauterine stem cell transplantation leads to microchimerism in the dams and that this is gestational age-dependent. EGFP+ fetal liver-derived mesenchymal stem cell (MSC) (10(5) per fetus) were injected intraperitoneally into congeneic and allogeneic recipient fetuses at E12 versus E13.5 of murine pregnancy (56 dams). Engraftment in maternal organs was evaluated using TaqMan quantitative polymerase chain reaction (PCR) and fluorescence microscopy during pregnancy (1, 3, and 7 days after in utero transplantation [IUT]) and after delivery (1 and 4 weeks after delivery). One day after IUT donor cells were mainly found in the placenta (E12: 9/10 dams vs. E13.5: 4/8 dams) and laparotomy site (E12: 5/10 dams vs. E13.5: 4/8 dams). Three days after IUT these probabilities decreased significantly in the placenta to 3/8 and 1/3, respectively, whereas it was increased within the surgical wound to 8/8 and 2/4. One week after IUT donor cells could be detected in other single maternal organs, such as bone marrow or spleen. The surgical wound was chimeric in all dams. One week after delivery the surgical wound was still a major site of engraftment in both groups. E12 IUT resulted in detectable donor cell microchimerism in the maternal bone marrow (3/4), liver (2/4), lungs (1/4), spleen (1/4), and thymus (1/4), whereas engraftment probabilities were lower following E13.5 IUT (BM: 1/4, liver: 2/4, lungs: 1/4, spleen: 1/4, thymus: 0/4). At 4 weeks after delivery persistent microchimerism was found only after E12 IUT in various maternal organs (BM: 1/4, spleen: 1/4, lungs: 1/4) and within newly created surgical wounds (3/4), but completely not in the E13.5 group. Allogeneic IUT did also not result in any detectable long-term fetal microchimerism. An earlier IUT might lead to a higher transplacental traffic of donor MSC and persistent microchimerism within maternal tissues. Even 4 weeks after delivery, these cells are present in surgical wounds.