Interaction of neoplastic cells with glass surface under flow conditions

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Experimental Cell Research 104 (1977) 335-343

INTERACTION

OF NEOPLASTIC

SURFACE

UNDER

CELLS

WITH

FLOW

CONDITIONS

J. DOROSZEWSKI, J. SKIERSKI

and L. PRZADKA

GLASS

Department of Biophyscis and Biomathematics, Medical Center of Postgraduate Education, Warsaw, Poland

SUMMARY The adhesion to glass of L 1210cells flowing in transparent parallel plate microchannel was studied by a cinematographic method. Most cells settle on the surface when their velocity immediately preceding attachment does not exceed approx. 100 pmlsec, the greatest adhesion rate accompanying relatively small velocities. The arrest of cells on the glass surface is either permanent or temporary and in a certain range of fluid velocities numerous cells are arrested several times consecutively for brief periods. Two types of surface attachment may be distinguished: cells are either totally immobilized on the surface (firm adhesion) or are able to petform under the influence of the fluid impulses some movements around the attachment site (loose adhesion). When the adherent cells are subjected to the shearing force of rapidly flowing fluid, they detach from the surface, the tearing away being frequently preceded by an accelerating gliding movement. The influence of hydrodynamic forces on the cell-surface interaction and adhesion processes is discussed, as well as some problems concerning possible mechanisms of the cell binding to the surface under dynamic conditions.

The arrest of malignant cells on endothelial surfaces constitutes an important aspect of the neoplastic dissemination process. Under natural conditions, i.e., inside the vascular network, cells interacting with endothelium flow in the stream of blood or lymph and, therefore, the influence of hydrodynamic factors on cell adhesion should not be neglected. However, in view of the great complexity of conditions in natural environment and because of considerable methodological difficulties involved in the study of dynamic cell adhesion, relatively simple physical models are useful for numerous research purposes. The aim of the present work is to investigate some basic phenomena connected

with the interaction of neoplastic cells with the surface of the channel in which they flow, suspended in the fluid stream. This work was centered on the following subjects: (1) the velocity of all movement immediately before adhesion and the influence of cell velocity on the adhesion process; (2) the behaviour of the adhered cells and general features of adhesive cell binding; (3) the fate of surface-fixed cells and some aspects of their detachment. MATERIAL

AND METHODS

The experiments were carried out on the L 1210 cells grown as peritoneal ascites on the DBA-2 mice. The cells were collected 5-7 days after inoculation. As medium, phosphate-buffered saline (PBS) was used in Exp

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IO4 (1977)

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Doroszewski, Skierski and Przqdka 2

1

3

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Fig. 1. Flow channel. (Top) View from above (natural

scale); (botrom) lateral view (dimension h, x20). 1, Parafilm layer; 2, coverslip;J, channel;l, inlet;5, outlet;6, glass slide. all experiments; in some cases (for comparative purposes) autologous serum was added to the medium in concentrations up to 1%. The cell viability estimated with the aid of standard trypan blue exclusion test was equal to about 95%. In most experiments the suspension concentration was about IO6cells/ml. The cell suspension flows through a microchannel composed of parallel glass plates (microscope slide and a coverslip) separated by a layer of parafilm (fig. 1). The width of the slit between the two plates is equal to 100 pm, the two other dimensions of the channel being 3 cm and 3 mm. The cell suspension or fluid enters the channel by the inlet tube and leaves it by the outlet conduit. The fluid was set in motion by the aid of a gas pressure device regulated by a precise needle valve; thanks to this method the flow rate may be maintained with satisfactory stabilization at a very low level of approx. 1 pl/min and may be raised to relatively high values of several mllmin. In these conditions the flow is laminar, the Reynolds numbers being below unity even for the highest maximal velocities. The cells interacted with a clean glass surface of the lower wall of the channel. The glass slides were prepared in a standardized manner by washing with a detergent solution and subsequent rinsing with distilled water during 24 h and with the aid of ethyl alcohol and ether. All experiments were performed at room temperature (ca 22°C). The parallel plate flow chamber is connected with the microscope “Ergaval” C. Zeiss-Jena (phase contrast) or “MPI-3” (PZO Warsaw) microscope (Nomarski interference contrast). The cell suspension flow and cell interactions are recorded on a 16 mm film by the aid of the “Krasnogorsk” (USSR) camera. Typical filming speed was 16 frames/set.

RESULTS Flow of cells before adhesion Cells flowing in the fluid stream were filmed Exp Cell Res 104 (1977)

and movie pictures were studied frame by frame; analysis of cell positions on successive film frames permits to estimate their velocity. Images of slowly flowing cells are clearly delineated and cells in rapid movement are visible as blurred shadows (fig. 2). The velocity of cells may be calculated by measuring the distances between consecutive cell images or the length of cell traces. If the velocity of cell suspension is great, i.e., more than 200-300 pmlsec (blurred shadows), adhesion of cells constitutes a rare phenomenon. When cells are flowing more slowly, the adhesion may be observed frequently (fig. 3) and the number of adhering cells is-as a first approximationinversely proportional to the velocity of the medium flow. The speed of numerous (though not of all) cells flowing through the camera field of observation is decreasing. The adhesion rate is greatest if the suspension velocity is small: numerous cells flowing with speed below 100 pm/set settle on the glass surface during the time of observation. More detailed analysis of the speed of cells undergoing adhesion was performed for the last part of their travel in medium stream (fig. 4). It appears that in the last fraction of a second (& or + set) most cells are arrested after a short braking. Before stopping, the cell movement is sometimes monotonously retarded; there are, however, numerous cells which move with approximately constant speed before adhesion to the surface or even slightly accelerate. The arrest of cells is not always definitive and irreversible. Numerous cells are arrested only for a relatively short period of time once or several times during their passage through the field of observation. Such a phenomenon may be called ‘temporary adhesion’. Fig. 5 represents a graph of velocity of a cell which is temporarily arrested

Interaction of neoplastic cells with glass surface

337

Fig. 2. Adhesion of cells in the fluid stream. Microphotograms magnified from 16 mm film frames; microscope optical system, ~22. Photograms taken from one film sequence, separated by l/2 set intervals. Cell b and c have adhered to the surface and occupy the same position on all photograms. Cell a flows in the

fluid stream and is seen in different positions on photograms 1,2,3; it adheres to the glass (photogram 3) and remains attached in the site of contact (photogram 4) in spite of continuing movement of fluid. Flowing cells are either sharply delineated or are visible as blurred shadows.

on the glass surface; its speed between the third and fourth second is equal to zero. Before arrest the velocity of cell is decreasing, and after having detached it evidently accelerates and reaches approximately the same speed as before adhesion. In the experiments in which the medium was supplemented with serum, the rate of adhesion was smaller, but as concerns general features the phenomena of cellsurface interaction were similar to those occurring in the absence of this factor. The influence of serum on the L 1210 cell dynamic adhesion was, however, not examined in a quantitative manner in this study.

Behaviour offixed cells The cells which have permanently settled on the surface are for the most part firmly attached to the glass and do not change their positions. Some cells, however, when subjected to pulsatile or oscillatory movements of fluid may move within a certain range around the site of adhesion as if they were attached in a loose and unstable manner. The mobility of some of adherent cells is so evident that it seems appropriate to coin the term ‘loose adhesion’ (fig. 6) in contradistinction to ‘firm’ or ‘close’ adhesion. Under the influence of the fluid waves a cell attached to the surface by the loose adExp Cell Res LO4 (1977)

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Fig. 3. Abscissa: time (set); ordinate: velocity (pm/ set). Mean values in 1 set intervals. Velocity of cells flowing relatively slowly before adhesion; four cells are arrested on the surface, one cell is not attached. L

aks

0.5

ilk

LO

hesion mechanism performs rather complex 4. Abscissn: time (set); ordinate: velocity (wm/ and irregular movements, driving away Fig. set) . Mean values in the fraction of a second intervals. from the place of its initial settling and apVelocity of cells in the last second before adhesion; proaching it again (fig. 7). examples c, d, e, f are more typical than a and 6. The frequency of appearance of the loose adhesion in comparison with firm cell attachment is difficult to assess exactly be- Detachment of settled cells cause it varies in a rather large range. In The fate of cells which have settled on the some experiments the proportion of loosely surface, i.e. phenomena appearing when bound cells was as great as 20-30 %, in adhesive bonds have already been formed, other cases loose adhesion constituted only constitute an important problem. In this work general features of the process of cell a few per cent. The influence of time factor on the mo- detachment have been studied. bility of cells attached to the substratum has The rapid stream of fluid may detach not been studied in detail; however, some from the surface cells which are firmly atpreliminary experiments concerning this problem were performed. Observation of individual cells subjected to the action of gentle oscillations of the medium reveals that numerous cells immediately after the first contact and arrest on the glass surface are able to perform movements of relatively great amplitude (ten or more cell diameters); in a few minutes-if cell does not flow away-its movements become more and more restricted. On the other hand, in 5. Abscissa: time (set); ordinnte: velocity (pm/ all experiments, numerous cells firmly at- Fig. set). tached and immobilized immediately after Mean values in 1 set intervals. Temporary adhesion: velocity of cell a drops to zero for a period of 3 set, the first contact with the surface, without then increases to previous level. Cell b flows without any transitory phase, were also observed. contact with surface. Exp CellRes

104 (1977)

Interaction of neoplastic cells with glass surface

Fig. 6. Loose adhesion. The arrow-marked cell is seen

in different positions when fluid flows from left to right (I-3) and from right to left (between 3 and 4). Other cells are attached motionless to the surface

tached as well as loosely adhering ones. Thus, loose adhesion is not a prerequisite for the detachment of cells. Usually, however, the detachment of a cell is preceded by its slow movement resembling crawling or creeping; cells move in this way for a distance of several diameters and then either stop (if the fluid velocity remains relatively small and constant) or accelerate and disappear. The rapidity of a pre-detachment movement is greater or smaller depending on the fluid velocity; it is either uniform, slowly accelerated or variable and the motion terminates in an abrupt acceleration and disappearance of the cell (figs 8, 9). The velocity of detached cell being relatively great, it is visible only on a single film-frame and appears as a blurred image. In some cases,

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(firm adhesion). Microphotograms magnified from 16 mm film frames; microscope optical system, X25; Nomarski contrast.

if a cell detaches under the influence of a relatively slow fluid stream, it can be traced on numerous frames during its post-detachment travel among other settled cells.

t

YOpm ’

I

Fig. 7. Scheme of movements performed by a cell loosely attached to the surface (loose adhesion) based

on the frame-by-frame analysis. Points correspond to the positions of the cell on consecutive frames. Exp Cell Res 104 (1977)

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Several types of phenomena connected with cell-surface interaction in flow conditions may be distinguished. Dynamic cell adhesion may be either permanent or temporary and the attachment of cells to the surface is either loose or firm. As concerns the process of detachment of cells settled on the surface, it may be either abrupt or gradual. Various combinations of these types of cell surface interFig. 8. Abscissa: time (set); ordinate: distance (pm). actions have been observed and the quesCell detachment: displacement of the cell under the influence of the fluid stream-accelerating movement tion whether these phenomena are linked ending in abrupt outflow of the cell. with different mechanisms and processes Fig. 9. Abscissa: time (set); ordinate: distance (km). Cell detachment: displacement and tearing away of or rather correspond to various temporal cell takes place in a fraction of a second. phases of the same process can at present not be answered. Most cells settle on the glass surface The minimal fluid velocity necessary for when their velocity immediately preceding the detachment of L 1210 cells is equal to attachment is in the range 10-100 pmlsec. 100-200 pm/set. When fluid of sufficient Very few cells settle with final velocity velocity flows above the surface on which above 100 pm/set and almost all cells numerous cells have settled, they detach whose velocity at the end of their travel is one after another. However, even when less than 20 pm/set are attached to the fluid velocity is relatively great (mean linear glass. velocity in the channel of several mmlsec), The process of the adhesion of cells unthere always remain at least a few cells der dynamic conditions may be described which do not detach. briefly as follows. As a result of the interplay of various DISCUSSION forces, the flowing cell approaches the wall. L 1210 leukaemia constitutes one of experi- When the distance between the cell memmental systems widely used in cancer re- brane and the surface becomes sufficiently search and is recommended for the screen- small, adhesive bonding arises; its strength ing of chemotherapeutic agents [lo]. Ad- may be greater or smaller, depending on hesion of neoplastic cells to artificial sur- various factors (e.g. on the area of confaces has been studied by numerous authors tacting surfaces). If the adhesion bonds are either under static conditions or in dynamic very weak, the cell is almost immediately perfusion systems. In the study on de- detached and flows further (such an event formability and adhesiveness of erythro- cannot be observed directly under the cytes and adhesion of platelets, various microscope). If the bonds appearing at the types of flow channel have been used [9, point when the cell-surface contact is es13, 151 and the micro-cinematographic tablished are stronger, the cell may be armethod has proved a valuable tool in study- rested for a short period during which it is ing cell motions and interactions in flowing immobilized (temporary adhesion). The systems (e.g. [ 111). momentary retaining of a cell may be due to Exp Cell Res 104 (1977)

Interaction of neoplastic cells with glass surface the fact that the adhesive bonds break only after a certain time, necessary to cause sufficient strain. If the bonding strength is relatively great, the cell adheres to the surface in a stable manner and does not leave the site in which it came into contact with the surface (firm or loose permanent adhesion) . When a cell which flows above the channel wall in the fluid stream becomes attached to the surface by adhesive bonds, its velocity relative to that of the fluid becomes smaller. The differences in the relative cell-and-fluid velocities give rise to a hydrodynamic force whose action results in a tendency to maintain or to set the adhering cell in motion. The hydrodynamic shearing force is proportional, although in a rather complex way, to the velocity of the fluid and this is the reason (or, at least, one of the most important reasons) why the number of cells settling on the surface is small or equal to zero if the fluid velocity is great. In addition to the direct action of hydrodynamic shearing force on cells settling in a gravitational field, several other phenomena and mechanisms may be expected to play a certain role in the process of cell adhesion in flow conditions. The most important of these are the occurrence of a boundary skimming layer in which the concentration of cells is greatly reduced [2, 7, 11, 141and the radial migration or displacement of cells [ 17, 201. The deceleration of cells before adhesion is probably attributable to two different reasons. On the one hand, under the influence of gravitational force, cells move downward and leave the region of relatively great fluid velocity which is situated near the centre of the channel. Alternatively, in the last part of the cell path, during a very short time a direct interaction of cells with the surface becomes probable. It

341

appears that there are some mechanisms of cell-surface interaction which may operate only when the cell movement is slow, but for which the total immobilization of a cell prior to adhesion is not indispensable. The velocities of cell suspension flow in the microchannel in our experiments are comparable as concerns the order of magnitude, to those of blood flow in capillaries, although physiological data in this domain are not very precise. For example: Dintenfass [7] estimates that the flow velocity in capillaries is equal approximately to 0.04 cmlsec, McDonald [8] gives a value of 0.05 cmlsec for the velocity in capillaries. According to Atherton & Born [l] the mean velocity of the blood flow in venules of the hamster cheek pouch is between 200 and 900 pmlsec; they found, however, that rolling granulocytes moved much more slowly; their mean velocity was about 20 pm/set in cheek pouch venules and approx. 10 pm/set in venules of mouse mesentery. Some problems concerning the influence of cell velocity on their adhesion to the endothelial surface are considered by Weiss [21]; he calculates minimal velocities of cells normal to the intima which are required to give the cells enough energy to overcome the potential energy barrier to contact; it appears that for erythrocytes a velocity of at least 0.72 cmlsec of the movement perpendicular to the surface is required. This value cannot be compared to our results which concern the velocities of the fluid flowing parallel with the channel wall. The interpretation of loose adhesion is not entirely obvious. Its appearance is not regular, the degree of cell mobility may vary and it is not always in time. Direct observation in the microscope or of filmed sequences of loose adhesion suggests that the cells are retained on the glass Exp Cell Rcs 104 (1977)

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by some invisible elastic threads or tethers. hesion and it seems reasonable to suppose In this study the magnification did not ex- that the pre-detachment gliding of cells on ceed 100 times and it is evident that to visu- the surface is connected with the elongaalize these structures greater microscope tion of the cell membrane structures in the power and resolution would be necessary. region of the attachment site. Thus, the In the light of the theoretical analysis and process of elongation of a cell membrane experimental observations it appears prob- structure consists either in pulling out of an able that various extensions and projections already formed extension, in stretching the cell membrane itself, or in distorting extraof the cell surface, i.e. microvilli, filopodial protrusions, “cell-probes” etc., play an im- cellular bridging material. Formation of portant role in cell-substratum and cell-cell tethers binding erythrocytes to glass and adhesion. Protoplasmatic filaments binding their elongation and disruption in the procells to the surface have been described by cess of shearing force detachment is well several authors [3,18,19,24] whose investi- known [2, 131. As described in the preceding section, it gations, however, concerned the adhesion appears that many cells were detached of cells in static conditions and were not related to the influence of the medium flow. without destruction. However, methods It was found by micromanipulation tech- employed in this investigation were not nique, by interference reflection micro- appropriate to studying in detail rapidly scopy and by other methods [4, 12, 191and flowing cells; for this reason it is not posothers, that under cell culture conditions sible to decide whether all the cells or only the adhesion of cell to a solid substratum a fraction of them were detached intact and was frequently restricted to small points undamaged. The general problem connected with the situated in narrow areas near the cell margins and to the pseudopods. interpretation of the results of this work is Another possible explanation of the phe- the extent to which cell adhesion to glass in nomenon of loose adhesion consists in ad- a flow channel may be considered a valid mitting the existence of an extracellular working model of the interaction between material binding the outer surface of the cell neoplastic cells and the vascular surface. It membrane with another surface. Subjected is obvious that the similarity of such a to a strain, such material may be able to model situation to the biological system is stretch away, thus permitting an attached restricted to only a few general features of cell to conserve some freedom of move- the phenomena under study. It is probable ment. The possible role of such binding sub- that the mechanism of cell attachment to stances, in form of adhesive “glues”, “mic- glass is not identical with that of cell adroexudates” or other binding layers is sug- hesion to the endothelial surface and to gested by several authors [ 16,22,23]. Other other cells; the problem of differences and authors, however, were unable to find con- similarities between these two phenomena clusive evidence in favour of the hypothesis remains, however, open to discussion [9of an extracellular binding material (for a 121. This work is directed chiefly to the inreview of problems related to morphology vestigation of some basic facts of the dyof cell contacts, see Curtis [5, 61). namic adhesiveness of neoplastic cells and The cell detachment process resembles in to the elaboration of appropriate methods. many aspects the phenomenon of loose adExp Cell Res 104 (1977)

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Exp Cc// Res 104 (1977)