Bendicht U. Pauli

Tumor invasion and host extracellular matrix

SummaryIn this review some of the major mechanistic pathways by which tumor cells are thought to invade host tissues are discussed. Tumor invasion has been conceived to be the result of pathological, close-range interactions between malignant cells and host stroma. The sequence of events that characterize invasion can be summarized as follows: (a) Tumor cell clusters break from the confinement of the primary tumor. Loss of intercellular junctions (desmosomes), alterations in the chemical composition and physical properties of the cell surface coat (loss of fibronectin and heparan sulfate; excessive amounts of hyaluronate), and loosening of cell-substrate interactions (loss of hemidesmosomes, fibronectin, and heparan sulfate), are among the most frequently listed causes of tumor cell shedding. (b) Increased proteolytic activities at the invasion front cause focal alterations in the surrounding extracellular matrix, thereby changing its physical properties. Collagenases and cathepsins, as well as elastase and other neutral proteinases are the enzymes most frequently associated with matrix destruction and invasion. In some tissues this process is effectively regulated by inhibitors of matrix-degrading, proteolytic enzymes. (c) Tumor cells migrate into the altered matrix, possibly moving as aggregates along guidance tracks provided by host structures (blood vessels, lymphatics, nerves) or matrix macromolecules (collagen and fibronectin tracks). Migration seems to be preceded by increased swelling of glycosaminoglycan (i.e., hyaluronate) in the matrix, ahead of the migrating cell population. Various host cell types (mast cells, fibroblasts, endothelial cells, macrophages, etc.) may participate in these events.

THE MURINE CALCIUM-SENSITIVE CHLORIDE CHANNEL (MCACC) IS WIDELY EXPRESSED IN SECRETORY EPITHELIA AND IN OTHER SELECT TISSUES

Abstract The tissue and cellular expression pattern of a recently cloned murine calcium-sensitive chloride channel (mCaCC) was determined. In situ hybridization was performed on formalin-fixed, paraffin-embedded murine tissues using digoxigenin-labeled, single-stranded RNA probes. The data were substantiated with northern blot and reverse transcriptase–polymerase chain reaction analyses. All three assays consistently indicated strong expression in tissues with secretory or ion regulatory functions, such as mammary gland, respiratory and intestinal epithelia, gall bladder, pancreas, kidney, uterus, and epididymis. Additional mCaCC expression was observed in germinal centers of lymphatic tissues, in spermatids, and in keratinocytes of the skin, esophagus, and cornea. The results are in accordance with previous electrophysiological reports on calcium-activated chloride conductances in various murine exocrine secretory epithelia and suggest a role of mCaCC in transepithelial ion transport. However, expression in other than secretory tissues indicates a more complex function.

Correlation between numbers of desmosomes and the aggressiveness of transitional cell carcinoma in human urinary bladder.

Quantitative electron microscopy has been used to examine the correlation between numbers of desmosomes and the histopathological grade and stage of papillary transitional cell carcinomas in human urinary bladder. Numbers of desmosomes (desmosomal density) per 100 μm of cell perimeter were quantitated in 6 examples of normal epithelium, 11 noninvasive papillary transitional cell carcinomas, 8 invasive transitional cell carcinomas arising from papillary lesions, 3 invasive transitional cell carcinomas which had prominent foci of glandular and squamous differentiation, and 1 squamous cell carcinoma. Desmosomal densities were increased in noninvasive transitional cell carcinomas, as compared with normal epithelium, but decreased in invasive transitional cell carcinomas. However, in areas of glandular or squamous differentiation in invasive tumors, desmosomal densities were increased, possibly reflecting the changes in cell phenotype. The decrease in numbers of desmosomes in invasive transitional cell carcinomas may contribute to reductions in cell adhesiveness.

Isoantigens A, B and H in urinary bladder carcinomas following radiotherapy

ABH tissue isoantigens were measured by the Specific Red Cell Adherence (SRCA) test in 66 surgical specimens of urinary bladder, including 53 transitional cell carcinomas, 2 squamous cell carcinomas and 11 controls. The SRCA test was strongly positive in 10 of 11 controls. ABH isoantigens were absent or equivocally present in 68 percent of noninvasive carcinomas (stage 0) and in 65 percent of invasive carcinomas. Clinical histories revealed that all patients with invasive carcinoma who had strongly positive SRCA test results had received prior radiotherapy to the bladder region. None of the patients with invasive bladder carcinoma with negative or weakly positive SRCA tests had been radiated. Histopathology of tumors in both groups was similar. Results of this retrospective study support the hypothesis that radiation may induce differentiation in tumors, possibly through an enhancement of Golgi apparatus function. The SRCA test should not be used as a predictor of the biological behavior of future recurrences in patients with bladder carcinoma who have received therapeutic radiation since radiation may produce “false positive” SRCA test results.

Rotary replication of lens gap junctions

We have further characterized the structure of chick lens gap junctions by rotary-shadowed freeze-etch electron microscopy. The crystalline gap junctions, seen in lens epithelial cells and differentiating cells, had connexons arranged in hexagonal clusters separated by particle-free aisles. These connexons measured 7–8 nm in diameter and had center-to-center spacing of 8.5 to 9.5 nm. The noncrystalline gap junctions seen in lens fibers had connexons that were nonordered. Connexons of these gap junctions measured 8–9 nm in diameter and had center-to-center spacing of 8 to 15 nm. Two distinct populations of connexons were seen in these two types of lens gap junctions. One population displayed a central, electron-luscent zone surrounded by six electrondense globules that represented the metal caps of rotary-shadowed subunits of connexons. The second population had a similar ultrastructure but, in addition, contained a central electron-dense core measuring 1.5–2.0 nm that corresponded to the metal cap deposited over the rotary-shadowed central pore of the connexon. This ultrastructure closely resembled that seen previously in rotary-shadowed gap junctions of other epithelial cells and was consistent with the Unwin and Zampighi (1980) model of liver gap junction structure. These results demonstrate that lens gap junctions have the ultrastructure that clearly characterizes gap junctions in other tissues.

Intercellular Junctions In Fanft-Induced Carcinomas Of Rat Urinary Bladder In Tissue Culture: In Situ Thin-Section, Freeze-Fracture, And Scanning Electron Microscopy Studies

This paper describes a set of simple methods for comparative light and electron microscopy studies on tissue cultured tumour cells derived from both noninvasive and invasive carcinogen‐induced rat urinary bladder carcinomas. Cells are grown on Thermanox plastic coverslips and fixed in situ. Each plastic coverslip is then divided with scissors into four parts: the first is processed for light microscopy, the second for thin‐section electron microscopy, the third for freeze‐fracture electron microscopy, and the fourth for scanning electron microscopy. In some experiments, portions of the culture which have first been examined by light microscopy are subsequently prepared for electron microscopy. In this way, the culture conditions are kept constant and a comparison of structural features (i.e. intercellular junctions) by several preparative techniques is possible.

The isolation and characterization in vitro of normal epithelial cells, endothelial cells and fibroblasts from rat urinary bladder

Epithelial cells, microvascular endothelial cells, and fibroblasts have been isolated in culture from normal urinary bladders of Fischer rats. Normal epithelial cells were cultured most efficiently when transitional epithelial sheets were plated on to collagen-coated roller flasks. The epithelial sheets were obtained by two micro-dissection techniques. In the first method, the epithelium was peeled as a large coherent sheet from the submucosal connective tissue following subepithelial injection of a collagenase solution, and after incubation of the bladders in the same enzyme solution. Epithelial sheets with intact basal cell layers were essential for culture success. On collagenous matrices, epithelial differentiation was similar to that in vivo. The in vitro transitional epithelium was composed of three cell layers, namely superficial, intermediate, and basal cells. Basal cells were attached to newly synthesized basal lamina by means of hemidesmosomes. Superficial cells were sealed at their apical lateral membranes by a junctional complex, i.e. a terminal bar. Asymmetric luminal membrane plaques were not apparent. In the second method, the epithelium was separated from the underlying connective tissue after collagenase--trypsin digestion of everted urinary bladders. Although the digest consisted mainly of epithelial cells, these rarely survived the first passage when plated on conventional plastic growth surfaces. After the third culture week, epithelial cells usually died and slowly growing colonies of fibroblasts or large flattened epitheloid cells became apparent. Epitheloid cells were identified by their typical ultrastructure as endothelial cells, showing Weibel--Palade bodies and pinocytotic caveolae. These cells were reactive with antiserum against factor VIII. The free surface of monolayer cultures was non-thrombogenic when incubated in the presence of platelets. Fibroblasts were isolated from heavily contaminated epithelial cell cultures after differential trypsinization. These three cells types represent the normal control cells of an in vitro tumor model for the study of invasiveness. All three cell types are involved in the formation and functional maintenance of the epithelial--stromal junction. The study of cell--cell and cell--matrix interactions may provide important clues for the understanding of tumor invasiveness, a process that starts at the epithelial--stromal junction and proceeds with its destruction.

Alterations of intercellular junctions in acinic cell carcinoma of the canine pancreas.

SummaryIntercellular junctions in spontaneous canine pancreatic acinic cell adenocarcinomas were compared to those in control canine pancreas. The neoplastic cells displayed proliferation and fragmentation of tight junctions and reduction in size and number of gap junctions. Marked decrease in desmosomal density was observed only in the poorly differentiated carcinoma. In the well differentiated carcinomas a few of the desmosomes were characteristic of those found in squamous cells. No quantitative or qualitative differences in cell junctions were noted between primary and metastatic tumor.

Structure of gap junctions in cultures of normal and neoplastic bladder epithelial cells.

Normal rat urinary bladder epithelial cells contain small subunit (PF-1) and large subunit (PF-2) gap junctions, whereas carcinoma cells only contain PF-1 gap junctions. The absence of PF-2 gap junctions, which are composed of larger connexons with slightly larger ionic channels, may contribute to altered metabolic coupling between urinary bladder carcinoma cells.

Endocytosis: A Property of the Glomerular Visceral Epithelial Cell

The sequential intravenous injections of protamine and heparin into rats results in the deposition of protamine-heparin aggregates along the lamina rara externa of the glomerular basement membrane. Morphologic studies indicate that these foreign aggregates are removed from the glomerular basement membrane by virtue of phagocytic activity by the glomerular visceral epithelial cells. We have studied the disappearance rate of protamine-heparin aggregates from the glomerular basement membrane in animals given a single dose of these compounds and in animals receiving chronic administration. The disappearance rate was measured utilizing a computerized morphometric technique to determine the numerical density of protamine-heparin aggregates per 100 micrometer glomerular basement membrane in sequential biopsy specimens taken from 15 to 600 min post-injection. The disappearance rate described a linear function in both acute and chronic animals, with a half-disappearance time of approximately 120 min. Disappearance of the aggregates appeared to be due to epithelial cell phagocytosis. This model provides a method of studying this function in the intact glomerulus in both normal and disease states.