Functional imaging of pericellular proteolysis in cancer cell invasion

Proteolytic interactions between cells and extracellular matrix (ECM) are involved in many physiological and pathological processes, such as embryogenesis, wound healing, immune response, and cancer. The visualization of cell-mediated proteolysis towards ECM is thus required to understand basic mechanisms of tissue formation and repair, such as the breakdown and structural remodelling of ECM, inflammatory changes of tissue integrity, and the formation of proteolytic trails by moving cells. A panel of synergistic techniques for the visualization of pericellular proteolysis in live and fixed samples allow monitoring the of proteolytic tumor cell invasion in three-dimensional (3D) fibrillar collagen matrices in vitro. These include the quantification of collagenolysis by measuring the release of collagen fragments, the detection of protease expression and local activity by dequenching of fluorogenic substrate, and the staining of cleavage-associated neoepitopes together with changes in matrix structure. In combination, these approaches allow the high-resolution mapping of pericellular proteolysis towards ECM substrata including individual focal cleavage sites and the interplay between cell dynamics and alterations in the tissue architecture.     

Distribution of diaphragmatic lymphatic lacunae.

The morphology of the submesothelial lymphatic lacunae on the pleural and peritoneal surface over the tendinous and muscular portion of the diaphragm was studied in 10 anesthetized rabbits. The lymphatic network was evidenced by injecting 1 ml of colloidal carbon solution in the pleural (n = 5) or the peritoneal (n = 5) space. After 1 h of spontaneous breathing, the animal was killed and the diaphragm was fixed in situ by injection of approximately 5 ml of fixative in pleural and peritoneal spaces. Then both cavities were opened and the diaphragm was excised and pinned to a support. According to which cavity had received the injection, the peritoneal or the pleural side of the diaphragm was scanned by sequential imaging of the whole surface by use of a video camera connected to a stereomicroscope and to a video monitor. The anatomic design appeared as a network of lacunae running either parallel or perpendicular to the major axis of the tendinous or muscular fibers. The lacunae were more densely distributed on the tendinous peritoneal area than on the pleural one. Scanty lacunae were seen on the muscular regions of both diaphragmatic sides, characterized by large areas without lacunae. The average density of lacunae on tendinous and muscular regions was 6 and 1.7/cm2 for the pleural side and 25 and 3.4/cm2 for the peritoneal side, respectively. The average width of lacunae was 137.9 +/- 1.6 and 108.8 +/- 1.7 microns on the tendinous pleural and the peritoneal side, respectively, and 163 +/- 1.8 microns on the muscular portion of the pleural and peritoneal surfaces.     

A simple method for the preparation of paraffin-embedded cell blocks from fine needle aspirates, effusions and brushings

A simple method for the preparation of paraffin-embedded cell blocks from cytologic specimens obtained by fine needle aspiration, by brushing or from effusions is described. The cells are fixed in suspension in 50% ethanol for one hour and pelleted by centrifugation in a 50-mL plastic tube. The fixative is removed, and the pellet is suspended in 3 mL of acetone for dehydration for ten minutes and thereafter repelleted. The acetone is then removed, and the cell pellet is dried at 60 degrees C for one hour. Melted paraffin is added onto the dry warmed cell mass and allowed to solidify at room temperature. A conical paraffin block with the cells in the top is obtained and can be handled as a routine tissue block.     

Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis

Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor-based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of beta 1 integrins and MT1-matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered beta 1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.     

Increases in pericellular proteolysis at developing neuromuscular junctions in culture.

To determine whether localized changes in pericellular proteolysis contribute to synapse formation, we examined the degradative actions of developing Xenopus laevis nerve and muscle cells on films of extracellular matrix proteins adsorbed to the glass surface of a tissue culture chamber. Skeletal myocytes, growing neurites, and fibroblasts all removed fluorescent fibronectin and laminin from the culture substratum at regions of close cell-surface contact. In addition, however, motor neurites also displayed a particularly enhanced rate of gelatin elimination at developing neuromuscular junctions. It has already been shown (a) that there is a similar remodeling of organized muscle basal lamina proteoglycan accumulations along the path of nerve-muscle contact and (b) that this is the earliest detectable biochemical change specific to developing neuromuscular junctions. Our observations thus suggest that the establishment of motoneuron-muscle contact leads to a further activation of pericellular proteinases along both the pre- and the postsynaptic surfaces of the developing junction. We therefore consider whether site-specific proteinase-activation cascades could contribute to the inductive signals that direct synaptic differentiation.     

The secretion of enzymes into the pericellular environment.

Connective tissue cells are capable of both synthesizing and degrading the macromolecular components of the extracellular matrix. The degradation of proteoglycan and collagen has been shown to be associated with the extracellular release of proteolytic enzymes, some of which are of lysosomal origin. The identity in carilage of two previously unrecognized proteases, capable of proteoglycan breakdown (CPGases), has recently been achieved by the use of a new assay for proteoglycan degradation. These enzymes have been shown to be synthesized and released in response to vitamin A. The third proteoglycan degrading enzyme of connective tissue cells, cathepsin D, has been located in the pericellular environment by trapping with specific antibody and the pattern of release studied in organ culture, experimental arthritis and in human rheumatoid tissues. The secretion of this enzyme and possibly also of the other CPGases is thought to be of importance in the local (pericellular) turnover of matrix macromolecules and, in association with collagenase, to be the cause of the excessive degradation in the pannus erosion of articular cartilage in rheumatoid arthritis.     

Additional techniques in serous effusions.

Cytological examination is a valuable diagnostic tool in case of a serous effusion. The first manifestation of malignancy may be an effusion of the pleural, pericardial, or peritoneal cavity, especially in carcinoma of the ovary, or lung, and malignant mesothelioma. In other malignancies effusions may occur in the course of the disease. The contribution by Mother by et al. in this issue of ACP focuses on the contribution of image and flow cytometry to establish the presence or absence of malignancy in serous effusions. They point out that the sensitivity of DNA image cytometry in equivocal effusions may be as high as 87.5%, and that for the detection of malignancy, DNA image cytometry is superior to flow cytometry.     

Structural and histochemical aspects of the pericellular environment in cartilage.

Mature cartilage contains pericellular regions of matrix of fine texture, consisting of filamentous material and granules containing proteoglycan. Intercellular matrix contains collagen fibres with structural elements resembling those of the pericellular regions in the spaces between the fibres. Membrane bound bodies may be present at the margin of the pericellular region. Histochemically, chondroitin sulphate is found in the pericellular region in all zones but keratan sulphate is similarly stainable only in the deep zones of ageing cartilage.     

Stress-concentrating effect of resorption lacunae in trabecular bone

Analyses of the distributions of stress and strain within individual bone trabeculae have not yet been reported. In this study, four trabeculae were imaged and finite elements models were generated in an attempt to quantify the variability of stress/strain in real trabeculae. In three of these trabeculae, cavities were identified with depths comparable to values reported for resorption lacunae ( approximately 50 microm)-although we cannot be certain, it is most probable that they are indeed resorption lacunae. A tensile load was applied to each trabeculum to simulate physiological loading and to ensure that bending was minimized. The force carried by each trabecula was calculated from this value using the average cross sectional area of each trabecula. The analyses predict that very high stresses (>100 MPa) existed within bone trabecular tissue. Stress and strain distributions were highly heterogeneous in all cases, more so in trabeculae with the presumptive resorption lacunae where at least 30% of the tissue had a strain greater than 4000 micoepsilon in all cases. Stresses were elevated at the pit of the lacunae, and peak stress concentrations were located in the longitudinal direction ahead of the lacunae. Given these high strains, we suggest that microdamage is inevitable around resorption lacunae in trabecular bone, and may cause the bone multicellular unit to proceed to resorb a packet of bone in the trabeculum rather than just resorb whatever localized area was initially targeted.     

Gordonia lacunae sp. nov., isolated from an estuary

An actinomycete, strain BS2^T, was isolated from a sand sample collected from an estuary in Plettenberg Bay, Western Cape Province, South Africa. Based on 16S rRNA gene sequence similarities and chemotaxonomy, strain BS2^T was identified as a member of the genus Gordonia. It exhibited weak antibiosis against Mycobacterium aurum A+ and Mycobacterium smegmatis LR222. Phenotypic and phylogenetic results allowed for the differentiation of strain BS2^T from other species within the genus Gordonia. DNA–DNA hybridization further differentiated strain BS2^T from its nearest phylogenetic neighbour, Gordonia terrae NRRL B-16283^T (57.5±4.4% DNA relatedness). Strain BS2^T is therefore a novel species within the genus Gordonia, for which the name Gordonia lacunae is proposed, with the type strain being BS2^T (=DSM 45085^T=NRRL B-24551^T).     

Osteocyte lacunae tissue strain in cortical bone

Current theories suggest that bone modeling and remodeling are controlled at the cellular level through signals mediated by osteocytes. However, the specific signals to which bone cells respond are still unknown. Two primary theories are: (1) osteocytes are stimulated via the mechanical deformation of the perilacunar bone matrix and (2) osteocytes are stimulated via fluid flow generated shear stresses acting on osteocyte cell processes within canaliculi. Recently, much focus has been placed on fluid flow theories since in vitro experiments have shown that bone cells are more responsive to analytically estimated levels of fluid shear stress than to direct mechanical stretching using macroscopic strain levels measured on bone in vivo. However, due to the complex microstructural organization of bone, local perilacunar bone tissue strains potentially acting on osteocytes cannot be reliably estimated from macroscopic bone strain measurements. Thus, the objective of this study was to quantify local perilacunar bone matrix strains due to macroscopically applied bone strains similar in magnitude to those that occur in vivo. Using a digital image correlation strain measurement technique, experimentally measured bone matrix strains around osteocyte lacunae resulting from macroscopic strains of approximately 2000 microstrain are significantly greater than macroscopic strain on average and can reach peak levels of over 30,000 microstrain locally. Average strain concentration factors ranged from 1.1 to 3.8, which is consistent with analytical and numerical estimates. This information should lead to a better understanding of how bone cells are affected by whole bone functional loading.     

LRP-1 silencing prevents malignant cell invasion despite increased pericellular proteolytic activities

The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.     

A novel cell ablation strategy blocks tobacco anther dehiscence.

We utilized a new cell ablation strategy to ablate specific anther cell types involved in the dehiscence process. The tobacco TA56 gene promoter is active within the circular cell cluster, stomium, and connective regions of the anther at different developmental stages. We introduced a cytotoxic TA56/barnase gene into tobacco plants together with three different anticytotoxic barstar genes. The anticytotoxic barstar genes were used to protect subsets of anther cell types from the cytotoxic effects of the TA56/barnase gene. The chimeric barstar genes were fused with (1) the tobacco TP12 gene promoter that is active at high levels in most anther cell types; (2) the soybean lectin gene promoter that is active earlier in the connective, and at lower levels in the circular cell cluster and stomium, than is the TA56 promoter; and (3) the tobacco TA20 gene promoter that is active at high levels in most anther cell types but has a different developmental profile than does the TP12 promoter. Normal anther development and dehiscence occurred in plants containing the TA56/barnase and TP12/barstar genes, indicating that barstar protects diverse anther cell types from the cytotoxic effects of barnase. Anthers containing the TA56/barnase and lectin/barstar genes also developed normally but failed to dehisce because of extensive ablation of the circular cell cluster, stomium, and contiguous connective regions. Anthers containing the TA56/barnase and TA20/barstar genes failed to dehisce as well. However, only the stomium region was ablated in these anthers. The connective, circular cell cluster, and adjacent wall regions were protected from ablation by the formation of barnase/barstar complexes. We conclude that anther dehiscence at flower opening depends on the presence of a functional stomium region and that chimeric barnase and barstar genes containing promoters that are active in several overlapping cell types can be used for targeted cell ablation experiments.     

Measurement of pH and ionic composition of pericellular sites.

The development of ion selective microelectrodes has made it possible to measure the normal steady state in the pericellular environment together with ion fluxes in response to physiological or pathological disturbances. Combined intracellular and extracellular measurements indicate that there is a considerable range of ability between various types of cells in the efficiency with which they can tolerate changes in pericellular conditions. Macrophages are extremely tolerant while cells of the cerebral cortex require a very finely controlled local environment. Combination of ion selective probes with microelectrodes which measure substrate and oxygen availability extend the information which can be obtained about ionic composition of cellular environment and the factors which are important in its homostasis.