The Surface Properties of Cancer Cells: a Review

The Surface Properties of Cancer Cells: A Review

Al. ABERCROMBIEANDE. J. AMBROSE

(University College; and Chester Realty Research Institute, Institute of Cancer Research, London, England)

BIOLOGICALASPECTS 525 3. Disorganization. The malignant cells as they Local invasiveness 525 multiply fail to form tissue of the arrangement Metastasis 532 characteristic of their normal counterparts. Disorganization 532 4. Persistent growth. The malignant cell popu Growth and Mitosis 532 lation keeps up a continuous growth by mitosis. Conclusions 532 PHYSICALASPECTS 534 LOCAL INVASIVENESS Adhesivenessof Normal and Tumor Cells . . 534 Mechanisms of cellular locomotion .... 539 We must first consider what is the mechanism ITltrastructure of cell contacts 541 of movement of malignant cells when they invade their immediate surroundings. Probably the main Biochemicalproperties oftumor cellmembranes 543 mechanism is active "amoeboid" cell locomotion, In recent years attention has been increasingly which was first recorded for cancer cells nearly a turning to the cell surface as the seat of an impor century ago. Since the advent of tissue culture it tant part of the malignant transformation. This has been observed in a very wide range of meta- widening of the field of interest of cancer workers zoan cells. Until the mechanics of this style of owes its beginning largely to the work of Coman locomotion are better understood it seems prefer (34). Coman was concerned with how invasion and able to put the term "amoeboid" to one side and metastasis occur, and this crucial aspect of the to use the relatively noncommittal name "solid- cancer problemâ€”crucial in the sense that it con substrate locomotion." This emphasizes one essen cerns the difference between benign and malignant tial aspect of the mechanism : it involves adhesion, growth-â€”must still be the main preoccupation of though transient at any one point, to a solid sub a review on the cell surface in relation to cancer. strate. Only flagellated (or ciliated) cells can swim. Other aspects may, however, be linked together in The necessity for a substrate makes the nature of a speculative way, and to these we will refer brief the cell surface, and of the surrounding solid struc ly. The review is divided into two main sections: tures with which the cell surface comes into con (a) a discussion of the biological evidence for the tact, of profound importance for locomotor be existence of surface peculiarities of malignant cells havior. (M.A. has been responsible for this section) ; (6) a A number of pathologists (e.g., Willis [146], discussion of the physical evidence as to what the Young [150]) consider that the pressure developed surface peculiarities may be (E.J.A. has been re within a tumor by its growth provides the main sponsible for this section). motive power for invasion. The peripheral malig nant cells, according to this view, are forced along BIOLOGICAL ASPECTS paths of lesser resistance through the surrounding tissues, disrupting their structure and so inter The deviations from normal of malignant cells mingling with the normal cells. It may be recalled, can conveniently be discussed, from the point of however, that Wolff (147, 148) and his colleagues view of the cell surface, in the following categories. have shown that, when a malignant tumor is laid 1. Local invasion. The malignant cells move against an organ in culture, invasion of the latter outward from the focus of origin of the neoplasm, occurs. In these circumstances it seems inconceiv insinuating themselves amongst the surrounding able that the growing tumor can exert any pres normal cells. sure. We shall, therefore, concentrate on active 2. Metastasis. As a result of invasion the malig locomotion, though it is not, of course, justifiable nant cells reach moving body fluids, by which they to conclude that passive transport by means of are passively transported and become widely dis pressure in no circumstances contributes to in seminated. vasion. 525

THE NONINVASIVESTATE reaction was demonstrated by Abercrombie and The capacity to perform solid substrate locomo Heaysman (7), who investigated the interactions tion is certainly observed among the majority of of chick heart fibroblasts moving in liquid medium kinds of malignant cells when they are cultured in culture on a glass surface. Interaction was obtained vitro (see for instance Coman [35]). However, it is by placing two primary expiants about 1 mm. also found among the majority of kinds of normal apart. The movement and the distribution of the cells of a vertebrate; red cells and germ cells are cells, before and after the outgrowth from the two the most obvious exceptions. The mere existence expiants made contact with each other, were meas of an orderly pattern of cells in the body makes ured. It was found that, as soon as a complete it, however, impossible to believe that, in normal junction had been established between the two circumstances, adult cells (white blood cells are outgrowths, but not before, the cells showed a an exception) undertake extensive locomotion of an change of behavior: their speed dropped sharply, invasive kindâ€”i.e., involving penetration among their direction of movement became random in other normal cells. Direct evidence as to whether, stead of predominantly outward from the expiant, and if so in what circumstances and how far, nor and the density of population in the inter-explant mal adult cells move in vivo is of course hard to region became almost stable. The cells between the achieve, and there may be an unsuspected amount expiants, in fact, largely ceased moving, undergo of small-scale movement. Nevertheless, it seems ing apparently mere oscillations; and they re safe to suppose that the problem of invasiveness mained distributed on the glass surface, if not in is as much a question of why normal cells stay sub a perfect monolayer, at least with far fewer over stantially in place as of why malignant cells do laps than was to be expected had they been dis not. We will, therefore, consider some ideas as to posed at random without mutual interference. No why a normal adult cell (other than a white blood slowing of movement before junction of the two cell) does not move extensively among other nor outgrowths was detectable. It was, therefore, con mal cells, even though it has the power of locomo cluded that the cells were inhibiting one another tion, under two headings: (a) mutual inhibition of by mutual contact of cell surfaces, and the phe movement by contact ("contact inhibition"); (6) nomenon was termed contact inhibition. A similar other hypotheses of locomotor inactivation. quantitative analysis was applied to the interac Contact inhibition.â€”It is a well known principle tion of fibroblasts from chick embryo heart with that "epithelium will not tolerate a free edge." those from neonatal mouse skeletal muscle, and Given a suitable substrate, a free edge will advance they were found to inhibit each other in the same by movement of the epithelial sheet behind it. The way by contact (8). idea is of long standing that the movement of such The cytological details of what happens during a sheet is stopped by contact of its edge with an contact inhibition of fibroblasts were studied by other epithelial edge, because this abolishes the Abercrombie and Ambrose (4). They found that polarization of the peripheral cells between the usually, when a fibroblast made contact with a free space in front of them and their followers be second one, the ruffled membrane that forms the hind them (see Arey [21]). As early as 1913 Smith leading edge of the first fibroblast joins closely to (124) examined the interaction of different epi- the second cell, at the same time ceasing its un thelia from this point of view. Perhaps most im dulations. When the leading ruffled membrane is portant for the present review is the quantitative immobilized in this way, the cell bearing it ceases study by Lash (95) of the movement of epidermis to move in the original direction. The region of over skin wounds in amphibian larvae. By marking contact between two cells, judging from microdis individual cells he was able to show that, after section studies (94) and from the distortion pro excision of a small piece of skin, a rim of epidermis duced in the pair of cells when renewed locomotion around the wound takes part in the movement and in another direction draws them apart again until that there is a rather abrupt cessation of move they separate with a sudden recoil (6), is as a rule ment throughout this rim when the free edges meet a firm but not indissoluble adhesion between the in the middle of the wound. There remains an two plasma membranes. Some of these observa important obscurity about such epithelial move tions have been substantially confirmed by Weiss ment, as to how cells behind the free edge move (142). appropriately; but the cells at the edge seem to be The nature of the inhibitory effect of contact the pacemakers, and their movement seems to be between fibroblasts is unknown. There are various controlled by the state of their contacts with other hypotheses at present under consideration (3, 18, cells. 40), according to which a change in a contacting That fibroblasts have a similar sort of contact cell may be produced; such a change can then

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. ABERCROMBIEANDAMBROSEâ€”SurfacePropertiesof Cancer Cells 527 provide the basis for an alteration in locomotion, additional mechanisms, and how far the cell sur (a) The cell may in some way be paralyzed by the face may be involved in them. adhesive force in the region of contact (see 40). Other hypotheses of locomotor inactivation.â€”When (6) The cell surface may alter in chemical constitu expiants are placed in culture they at once become tion as a result of reaction with the surface mole surrounded by an expanse of cell-free substrate. cules of the cell it contacts, (c) The cell may be Contact inhibition no longer exists at the external unable to adhere at all to most (though obviously side of the peripheral cells of the expiant (dead not all) of the surface of the cell it contacts, so that cells apparently do not for long inhibit living ones). it cannot move across it. (d) Adhesion to the sur Different tissues, however, differ greatly in their rounding substrate may so exceed adhesion to latent periods before they take advantage of the other cells that it is impossible for a fibroblast to release from contact inhibition and in the rate at move itself from the substrate onto a neighboring which cells subsequently emerge from the expiant. cell. These four hypotheses do not seem equally Perhaps the most revealing differences are those likely on present information, but no strongly con between a normal adult tissue and the same tissue clusive evidence is available. Since they all involve which by treatment before explantation has been properties of the cell surface, they all bring contact thrown into a state of proliferation. Wounding a inhibition within the scope of this review. There tissue in vivo, or calling on any of the other mecha remains, however, a fifth hypothesis that does not nisms that induce mitosis in it, makes its cells emi necessarily involve surface properties: (e) diffusion grate earlier and in greater numbers when it is of substances out of or into the cell will be changed subsequently explanted (see 2). Now the effect where it is in contact with another cell, thus alter of this pretreatment on the propensity of the cells ing concentrations at the surface or further inside to move can hardly be via a change in their capaci the cell. Now the diffusion gradients concerned ty to move over one another's surfaces such as extend by dÃ©finitionoutside the cell boundary; yet would be produced by a change in contact inhibi no change in a ruffled membrane has been observed tion. Only a decrease in this capacity, correspond until it makes what under the light microscope ing to an increase in contact inhibition, would, by appears as actual contact with another fibroblast. encouraging movement away from one another, It seems probable, however one explains this very tend to drive cells more readily out of an expiant; short range of action of the chemotactic influence and the extremely poor initial outgrowth from un postulated by the hypothesis, that variability of treated adult tissue would then have to be inter cells and conditions would lead at least sometimes preted as due to such a lack of contact inhibition to an observed action at a distance. The hypothe that the cells were almost completely free to move sis, though it seems to us at present an unlikely one, within the expiant and hence devoid of any reason cannot, however, be dismissed out of hand. for leaving it other than random diffusive move Any mutually repulsive behavior between cells ments. This is a condition that, as already dis can immobilize them, since a cell may be so closely cussed (p. 526), is most unlikely to hold in adult surrounded by others that no space free of repul tissue. There seems to be, then, some additional sive force is presented to it. Contact inhibition is influence (or constellation of influences), inversely equivalent in effect to a repulsive force of very correlated with mitotic activity, which tends to short range, and to immobilize a cell efficiently all restrain cells. We shall refer henceforth to release the surrounding cells must be in contact with one from this influence as "mobilization." We now another, so as to block all pathways. This readily consider possible hypotheses about the nature of happens on a plane surface, since the contacts be the influence and in particular whether the cell tween fibroblasts tend to be long-lasting, so that surface may be concerned. a population forms a meshwork. Essentially the The most obvious suggestion is that mobiliza same may hold when cells are within a three- tion and its opposite are due to variations in the dimensional gel, all available solid pathways being adhesion of the cells to their surroundings. We blocked by cell processes; but the behavior of cells have already seen that contact inhibition may be in these circumstances has not been sufficiently related to adhesiveness between cells : a strong mu studied. tual adhesion is a normal consequence of contact For both epithelia and fibroblasts, therefore, re between fibroblasts, and it may indeed be the ac actions requiring contact are known which are tual cause of the failure of the cells to move over capable of immobilizing them. There seems how one another (hypothesis [a] above). We are now ever, little doubt that mutual contact inhibition is considering a different result of adhesion: an an not the sole mechanism tending to hold cells in choring down of the cells. Though both contact place in vivo. We now consider the evidence for inhibition and immobilization in this sense may

turn out to have a closely related basis in raised tion, and as usual locomotion is also activated. adhesion, as modes of behavior the two must be When (again before explantation) new axons are kept distinct. allowed to penetrate a degenerating nerve, emigra Some degree of adhesion of a cell to its substrate tion in vitro of Schwann cells but not of fibroblasts is essential for locomotion. A cell moves by a little becomes suppressed, though the proliferative state understood procedure of making and breaking ad is unimpaired (10). A wave front of this suppres hesions (19), coupled with contractions and relaxa sion can be detected passing down the nerve be tions. Its adhesions may, however, be in absolute hind the axon tips. In view of the well known close terms too high (or too low) to permit movement; association of axons and Schwann cells, it seems or its adhesion at some part of its surface may be that immobilization by adhesion has occurred. too high, relative to its adhesion to the substrate Myelination might seem the obvious form that the available to its locomotor mechanism, for it to be anchoring takes (61), which would make this case able to pull away. peculiar to nerve fibers; but there is some reason An explicit theory that cells are immobilized by to think that a similar immobilization takes place the strength of their adhesions has been proposed in unmyelinated nerves (5). by Coman (36, 37), developing a suggestion by Evidence is scanty that other types of cells, Cowdry (39). He has proposed that it is the mutual when aroused from their normal stationary state adhesion of normal epithelial cells that prevents in vivo, are released from adhesion to their sur them from invading their surroundings; insofar as roundings; this may, however, occur during heal they break free they become able to move. If nor ing in epidermis (95). The stimulation of cell emi mal epithelial cells, however, are presented in tis gration from adult expiants by trypsin treatment sue culture or in vivowith a suitable plane surface, (122) may also be adduced, though with some they move across it so closely adherent to one reservation, as suggesting the importance of adhe another as to appear like a continuous sheet. They sion to surroundings in limiting cell movement. can move through a three-dimensional gel in vitro However, even if clear evidence of the intervention also in very close association, in the form of cords of such adhesion is obtained, it is likely to be dif and strands. It is possible that the areas of mutual ficult to decide whether, when cells are mobilized, adhesion at any moment are reduced in the moving there is a primary change in their surfaces (or in state as compared with the stationary state, but the surfaces to which they are adhering) that is a the cells do not need to break free from their fel decrease in the intensity of adhesion; or whether lows in order to move (see also Clark and Clark the primary change is the activation of the mecha [32]for epithelial movement in ear chambers). Sim nism of movement, which then secondarily pro ilarly, normal fibroblasts move despite a good deal duces some degree of detachment, that is a de of mutual adhesion. It is true that the more neigh crease in the area of adhesion. boring cells a fibroblast is adherent to, the slower Summarizing these considerations about the its speed of locomotion (6) ; but there is compensa holding still of normal cells in vivo, we may say tion for this reduced speed in an improved con that contact inhibition provides one possible mech sistency of the direction of movement (owing to anism, in that when all available solid substrate contact inhibition at the points of adhesionâ€” pathways bring a cell into contact with a homolo Abercrombie and Heaysman, unpublished) so that gous cell it will usually be prevented from making net displacement over a period of time will be con headway in any direction. This behavioral mecha siderably greater for a coherent sheet of cells than nism seems to occur among fibroblasts and among for isolated cells (Abercrombie and Gitlin, unpub epithelia. The important problem of epithelio- lished) . It may be concluded, therefore, that it is by fibroblast contact relations is, however, as yet al no means certain that adhesion to homologous cells most completely unexplored. The behavior of ex- necessarily reduces the displacement of epithelial plants in culture indicates that contact inhibition, cells, as Coman has suggested. For a cell to become as it is known at present, cannot be the only mech anchored it must adhere firmly to something which anism holding normal cells still. There is evident does not itself move. ly some other influence the operation of which is An example of the anchoring effect is probably correlated with mitotic inactivity. How far the cell to be found in the relation of Schwann cells to surface is primarily involved in this second mecha axons. Schwann cells emigrate freely from expiants nism or group of mechanisms is uncertain. in vitro of peripheral nerves which have undergone a period of Wallerian degeneration, without rein- THE INVASIVESTATE nervation, before explantation (9,83). They do not Since malignant cells are not stationary in emigrate freely from expiants of intact nerves. viro, we must consider them in relation to the two Wallerian degeneration is a promoter of prolifera inhibitory mechanisms we have discussed.

In the first place, if contact inhibition stops of chick cells is infected with Rous sarcoma virus, movement in vivo, malignant cells must somehow the cells of an infected focus spread over the sur escape the influence of contact inhibition by the rounding normal cells, indicating a failure of con surrounding normal cells. Their immunity to it tact inhibition by homologous cells (see also Sachs might theoretically be because as they move they and Medina [119]). bring about the bodily removal of the normal cells Although it is clear that mutual contact inhibi from their path, so that no prolonged contact in tion exists between normal epithelial cells, as al hibition can take place. They may do this either ready mentioned, it is not known how malignant by so affecting the behavior of the normal cells and normal epithelial cells behave toward each that they move out of the way or by simply killing other. More important, however, for the actual the normal cells. Both processes may occur to some problems of invasion in vivo is, no doubt, the be extent. We have observed that a fibroblast popula havior of epithelial cells and fibroblasts to each tion in vitro may appear to move aside from an other. Though a full analysis in terms of individual encroaching outgrowth of sarcoma cells; the exact cell movements is lacking, it has been reported that behavior mechanism has not, however, been ana in cultures some normal epithelia did not invade lyzed. This may be the explanation of Leighton's a fibroblast population, whereas in the same cir observation (98) in sponge matrix culture of a cumstances the carcinomas tested did invade (58, great scarcity of fibroblasts among invading sarco 120). There is, therefore, a clear suggestion that ma cells. To be effective such a mechanism requires contact inhibition exists between nonmalignant either that the fibroblasts to be shifted can find epithelia and fibroblasts, at least in some condi some place free of other cells to move to, or that tions, but is absent when the epithelium is malig they lose their contact inhibition by other cells. It nant. Further investigation is badly needed of is well known that malignant cells may kill nearby this important relation. normal cells, either through output of substances Since contact inhibition is probably primarily a or through competitive intake of nutrients. Like reaction of cell surfaces, these results suggest that the possible repulsion of normal cells, this may to a surface change plays a key part in the malignant some extent eliminate cells from the path of moving transformation. The precise nature of the relevant cancer cells so that contact inhibition would have surface change will remain obscure until the mech no chance to operate. However, there is no doubt anism of contact inhibition is elucidated. The most that in practice malignant cells infiltrate among likely hypotheses depend on adhesiveness of the and in contact with a population of normal cells cell surface, and, as will appear, a diminution of both in vivo and in vitro. It has been observed in the general adhesive properties of malignant cells vitro that destruction of normal cells occurs only as compared with normal cells, suggested by Lud- after such infiltration, apart from the special case ford (102) and Cowdry (39), is now well authenti of penetration of an epithelium (147). It seems cated. Curtis (40) has argued that the loss of con clear that, if our hypothesis is to be sustained, con tact inhibition by sarcoma cells depends on this tact inhibition of invading malignant cells by in general loss of adhesiveness; but it still remains a vaded normal cells must be much reduced or possibility that a more specific adhesiveness be absent. tween cells of the same type is involved in contact The absence of contact inhibition of sarcoma inhibition (2). cells by normal fibroblasts has been demonstrated, According to our previous analysis, release from though only for a small number of mouse tumors. the constraint on mobility by contact inhibition is It has been shown that S-37 and S-180 cells are not necessary but not sufficient to convert normal cells inhibited by contact with normal fibroblasts (4, 7, to invasive behavior. There is also the second 8), either mouse or chick. Subsequently, this was mechanism of immobilization to be considered, shown too for transplanted fibrosarcomas (MCIM which in normal tissues is correlated with mitotic and Klein's MjC, Abercrombie and Karthauser, inactivity. Cells from expiants of a malignant tu unpublished) and for methylcholanthrene-induced mor isolated in vitro give the impression of emi primary tumors (Abercrombie and Karthauser, grating more freely than do cells from expiants of unpublished). From the point of view of invasion the corresponding normal tissue, though there in vivo, such experiments suffer from the difficulty seem as yet to be no quantitative data standard of testing contact inhibition by normal cells of the ized for the number of cells within the explant. (It kind which actually are invaded by such tumors. may be noted here that the stromal cells of a tumor The defect is to some extent mitigated by the fact are to some extent mobilizedâ€”Drew [48]). Since that very different kinds of normal fibroblast in in these conditions, where contact inhibition does hibit one another. Furthermore, Temin and Rubin not operate, the emigration of nonmalignant cells (129) have found that, when a monolayer culture is correlated with their degree of mitotic stimula-

tion, the migratory activity of the malignant cells, ary structures. Furthermore, its great importance which are mitotically active, is entirely to be ex for metastasis can hardly be doubted. pected. The nature of this mechanism of mobiliza We have discussed invasion as if the only point tion of normal cells has already been discussed, and was that the invading cells should be able to under we concluded that diminution of adhesion of the take locomotion among the invaded cells. How cells to their surroundings may play a part, though ever, the direction of locomotion must also be con there is little evidence that it does. The evidence sidered. A localized population of cells which are that lack of adhesion plays a part in the mobility undergoing random changes of direction, or as ran of malignant cells is stronger in that we at least dom as the fact that cells cannot simultaneously know that their adhesiveness is reduced. Already occupy the same position in space allows, will no in 1912 Hanes and Lambert (70) described how in doubt slowly spread by a process analogous to mo vitro individual carcinoma cells easily break free lecular diffusion reinforced by the inevitable non- from one another. Coman (34) showed that mutual random element in their movement. Such spread adhesion is much less between carcinoma cells than will probably be very slow, and it seems most im between normal epithelial cells by direct measure probable that this is how malignant invasion oc ment. There is evidence pointing the same way, curs. It is not how nonmalignant cells in tissue cul but less direct, for sarcoma cells (4, 62). Further ture extend into a cell-free medium; they have more, there are miscellaneous data suggesting a various mechanisms of orientation. A particular generalized lack of adhesiveness of malignant cells problem is raised here, since it seems that a defect to substrates of various kinds (Ludford [102] early in contact inhibition may be one of the changes remarked on this) as well as to homologous cells which makes malignant invasion possible, and yet (we have already referred to the possibility that the operation of contact inhibition is one of the such lack of adhesiveness, to normal cells, accounts main mechanisms producing the directed dispersal for the failure of contact inhibition). The relatively of a group of fibroblasts so as to occupy cell-free unflattened shape of cancer cells when cultured on space. The problems of orientation of cell move a plane surface (see, for instance, Ludford [102]; ment must therefore be briefly considered, since Gey, Shapiro, and Borysko [63]) is probably a re the cell surface is evidently involved. flection of it; and it is well known, in the case of Any mechanism by which the members of a cell ascites forms, that many tumor strains can be population repel each other, even over short dis selected for a remarkably low degree of adhesion tances, greatly increases the efficiency with which to each other and to other objects. the population spreads. Mutual repulsion may be All this suggests that if diminution of adhesion by negative chemotaxis, which requires that each to their surroundings plays a primary role in the cell is surrounded by a diffusion gradient to which mobilizing of cells, the mobilized state of cancer other cells react. It is possible that wandering cells cells is easily explained. Coman's theory, already react to each other in this way, and Twitty and mentioned in connection with the behavior of non- Niu (130) have found evidence that amphibian malignant cells, attempts to explain the whole of embryonic melanoblasts do also. There is no evi the invasiveness of malignant cells on these lines, dence yet that fibroblasts or sarcoma cells have taking, however, lack of mutual adhesion as the such a behavior mechanism. Contact inhibition key. He supposes that a malignant epithelial cell, however, produces a form of negative taxis, per easily liberated from the general mass, can for that mitting movement apart and discouraging closer reason freely move away. We have, however, al approach than the first contact, and so tending to ready pointed out that freedom from mutual ad promote movement into space where contact in hesion is not an essential condition for movement hibition does not operate. This interaction is prob and that, even if it were, it would not be sufficient ably responsible for the radial emigration of fibro for movement if contact inhibition were also pres blasts from an expiant in culture on a glass surface. ent. The theory, therefore, seems inadequate to It could be an important element in radial move account completely for invasiveness. Nevertheless, ment of sarcoma cells from a malignant focus into the lack of mutual adhesion which Coman has so surrounding tissue, if the sarcoma cells to some ingeniously demonstrated is probably a very im extent inhibit one another's movement by contact portant discovery about cancer cells. It probably but are not inhibited by surrounding normal cells. reflects a more general loss of adhesiveness, which No quantitative investigation has, however, yet could be the basis for the absence of contact in been made on the mutual interaction of sarcoma hibition exerted by the normal cells invaded, and cells. could also, as suggested above, play some part in The structure of the region under invasion is mobilization by diminishing anchorage to station likely to be an important influence on invading

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. ABERCROMBIEAND AMBROSEâ€”Surface Properties of Cancer Cells 531 cells; it is expressed in the contact guidance of Jones [85]). In part, if not wholly, it operates by Weiss (138, 139). An orientated structure of the means of diffusive substances and so is not related substrate correspondingly orientates the move to a special property of the malignant cell surface, ment of the cells upon it. In tissue culture in or except perhaps insofar as this by its leakiness pro on a gelated medium, a focus of dense population motes change in the medium around a sarcoma cell such as an expiant tends to produce a radially ori colony. entated structure in the medium around it. Under There are, of course, normal cells that invade in these conditions the radial spread of a culture, im vivo, in the sense of infiltrating by locomotion pelled by both contact guidance and contact in among other cells. Leukocytes and macrophages hibition, is highly efficient. Two expiants near to do so. Correspondingly, tested in tissue culture, gether tend to produce a pathway between them these cells show no contact inhibition by normal selves of orientated medium so that they become fibroblasts (Oldfield, unpublished). Their mutual linked by a bridge of cellsâ€”the 2-center effect of adhesiveness is low (see Levi [99], Coman [34]). Weiss (141). When one or both of the expiants is Macrophages are perhaps potentially of great in of sarcoma cells, the 2-center effect still occurs terest from our point of view, because they seem (Abercrombie and Heaysman, unpublished). The to be able to pass between an invasive state (the sarcoma cells both orientate the medium, and are ordinary migratory form) and an immobile state themselves very susceptible in their movement to (the tissue histiocyte form). The trophoblast pre the orientation. Carcinoma cells probably also sumably makes a similar transition, and indeed show contact guidance (58, 97, 98). The change in numerous orderly morphogenetic cell movements surface properties associated with malignancy has of embryogenesis and of later histogenesis need to in no way weakened this reaction. Since sarcoma be investigated from this point of view. Some of cells lack contact inhibition they are able to treat these migrations, such as the dispersal of the neural fibroblasts as a substrate; and if the fibroblasts are crest, appear to be episodes of temporary invasive- themselves orientated, this orientation is imposed ness (limited to certain substrates [41]), and a re on the sarcoma cells. In mixed cultures made on a versible and controlled counterpart of malignant glass substrate, the fibroblasts orientate each other invasiveness may be part of the repertoire of be in a radial direction by mutual contact inhibition, havior of many normal cells. Curtis (40) has con and the sarcoma cells orientate themselves to the sidered how progressive changes in invasiveness, fibroblasts by contact guidance. It is even possible initiated artificially by disaggregation, may ex that orientated sarcoma cells themselves further plain the sorting out of cells in reaggregates. orientate sarcoma cells moving on them. The tend We must now draw together these considera ency for invasion in vivo to occur along preferred tions about the role of the cell surface in the in pathways is no doubt an expression of these vari vasive state of malignant cells. We suggested that ous mechanisms of guidance, and perhaps of others for a population of normal cells to become invasive as yet undetected. two changes were necessary: it must cease to be The failure of fibroblasts to produce contact in contact-inhibited by the surrounding cells which hibition of sarcoma cells does not, in the present are to be invaded, and it must become "mobi state of our knowledge of the mechanism of contact lized," since actual movement does not necessarily inhibition, imply that sarcoma cells must fail to follow the lifting of contact inhibition. Those sar produce contact inhibition of fibroblasts. In fact, comas tested have proved to be immune to contact however, fibroblasts in vitro invade a sarcoma cell inhibition by normal fibroblasts. Carcinomas, how population (8), so that the failure of contact in ever, have not been precisely compared with nor hibition is qualitatively reciprocal. The locomotion mal epithelia in this respect. It is, nevertheless, a of fibroblasts among sarcoma cells we have tested permissible hypothesis in the present state of the is, however, quantitatively much depressed as com evidence that contact inhibition is a limitation on pared with their locomotion into cell-free space; normal fibroblast and epithelial movement which the locomotion of sarcoma cells into a colony of disappears in malignancy. This change is probably fibroblasts is unaffected or even promoted as com in the surface of the cell and may be concerned pared with their locomotion into cell-free space. with its adhesiveness. As to the second change, This effect of sarcoma cells on fibroblasts may be since "mobilization" is normally associated with complex, but it is no doubt connected with the mitotic activity it is not surprising that malignant diversion of fibroblasts from an advancing sarcoma cells have acquired this characteristic. It, too, may outgrowth already mentioned. The effect seems to conceivably be connected with a change in adhe vary considerably with different kinds of sarcoma siveness of the cell surface; but if so, since normal (see also Carrel [29], Ludford and Barlow [103], cells may be mobilized without losing contact in-

hibition, this change in the cell surface is different through the lung circulation, whereas others, equal at least in degree from that which we suppose elim in size or larger, do not (152). Large ascites tumor inates contact inhibition. There are, however, no cells, likely to be very nonadhesive, may circulate strong grounds at present for thinking that mobili for a long time after intra vascular injection (20). zation is primarily due to a change in the cell It may be that there are also qualitative surface. differences in adhesiveness of tumor cells which METASTASIS match qualitative differences in the endothelia The passive spread of cancer cells via flowing of different organs (such as may occur in the body fluids, and the consequent establishment of normal development of primordial germ cells new foci of growth and invasion, probably involve of birds [123]). These might somehow account the properties of the malignant cell surface. It can for the different patterns of metastatic deposit hardly be doubted that adhesiveness plays a major found with different tumors administered to the circulation by the same route (see Zeidman part in determining the initial detachment of the cancer cells, and it may also play a part in their [151]). The properties of tumor cell surfaces may also produce varying ability to invade the different arrest in other parts of the body. It is no doubt usually through the invasion dis organs in which an embolus comes to rest (86, 147, 148). It is obvious, however, that the establish cussed in the previous section that the malignant ment of a metastasis involves several unknowns, cells make contact with a flowing body fluid. In and the whole concept of qualitatively specific ad order to be swept away they must already be sepa rated or be readily separable into single cells or hesion between cells is at present in a somewhat uncertain state (40), so such speculation is prema into small groups. This implies that their mutual ture. It should be added that for these problems adhesiveness must be low. It may also require that of the arrest of tumor emboli the plasticity of cell their adhesiveness to the endothelium bounding shape must also be considered (151). the body fluid should at least be below a certain threshold. (A similar suggestion has been made DISORGANIZATION about the liberation of red cells from the marrow It is well known that malignant tumors often into the circulation [91]). We have already dis lack the typical pattern of corresponding normal cussed the strong evidence for diminished adhe tissues. This is an expression partly of the dedif- siveness of sarcoma cells, mutual and to other ferentiation of intracellular specializations, partly structures, in the previous section and need not of a simplification of cell shape, partly of a disor repeat it here. ganization of cell arrangement. This section of the To form a successful metastasis a tumor em- review does no more than call attention to the bolus must settle down in a place favorable for probability that the simplication of shape and the growth. Where it settles will depend partly on the disorganization of arrangement may be, at least in size of the embolus; and, insofar as the embolus is part, an expression of surface abnormalities of the multicellular, its size is likely to reflect mutual ad cell. The importance of the cell surface for embry hesiveness. Coman (35) has, for instance, shown onic development of the form and association of that large clumps in the vascular system may tend cells has been reviewed by Holtfreter (78) (see to stick in arterioles where conditions for growth also Birbeck and Mercer [23]). are unsuitable. Since the settling of a tumor em- That the form characteristic of many kinds of bolus may involve adhesion to endothelium (149), cells in situ is not maintained by internal structural where it settles may also be partly determined by rigidity, but rather by cell surface relations, is the degree of its adhesiveness in this respect. Willis shown by the manner in which these cells round (146) as early as 1952 suggested that it might de up into a sphere when suspended freely in liquid. pend on surface charge of the tumor cell. It has, The potent influence of adhesion to surfaces on however, already been suggested that too great an shape is also well known to the tissue culturist who affinity for the endothelium may hinder embolism. compares the effects of different patterns of solid This would imply an optimal range of adhesive substrate, such as meshworks of fibrin with strands ness reminiscent of that required of ascites cells of different caliber (143). On the whole the malig (Klein [93]) which need to be relatively nonadhe- nant cell, observed in relatively standardized con sive to occupy their fluid habitat, and somewhat ditions such as on a plane surface, shows a tenden adhesive to evoke ascitic fluid from the peritone cy towards a reduced surface/volume ratio (see for um. Quantitative differences in adhesiveness to instance Ludford [102], Gey, Shapiro, and Borysko vascular endothelium may perhaps explain such [63]). This suggests a general diminution in its ad observations as that some tumor cells pass readily hesiveness to substrate which, along with its dimi-

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. ABERCROMBIEANDAMBROSEâ€”SurfacePropertiesof Cancer Cells 533 nution of mutual adhesion (34), could contribute lular substrate (which we have called its mobiliza to the simplification of shape actually found. tion) do not distinguish it from a population of The cellular arrangement of a complex tissue, normal cells in a state of proliferation. The malig composed of different types of cell, will naturally nant change, as far as these properties are con be disorganized if only one of the cell types grows cerned, is simply to put the population into a state in population and invades the others. The disor of permanent proliferation. However, a second set ganization extends, however, to the mutual ar of properties appears in most malignant cells which rangement of the malignant cells, all of the same is unusual among proliferating normal cells: the type. Normal cells of a given type, at any rate capacity to migrate with other cells serving as sub epithelia, have a mutual arrangement which is strateâ€”i.e., to invade (which we refer to a lack of capable of reasserting itself after disturbanceâ€”for contact inhibition) ; the liability to passive dissem instance after dissociation, as first shown by Holt- ination; and the disorganization of structure. freter (77). The mutual arrangement of epithelial The two properties in the first group, mitosis cells is plausibly interpreted as an outcome of mu and mobilization, are certainly closely linked to tual contact reactions. It seems at present that gether in normal cells (though the correlation is not they must involve a pattern on each cell of surfaces perfect [2]). That they are linked in malignant cells favorable and unfavorable for mutual adhesion. is, therefore, not surprising. It has been tentatively With the surface changes, manifested by the gen suggested in this review that their onset in a popu eral diminution of adhesion among malignant cells, lation of cells, when it is stimulated to proliferate there may go a partial or complete loss of the pow or become malignant, may be related to some er of mutual arrangement. The partial retention of change in cell surfaces; but the connection is specu this power is presumably what Leighton (96) is lative, and if it exists it is not clear which is cause concerned with in his "aggregate replication" of and which is effect. On the other hand, there is carcinomas, the increase in numbers of nests or good reason for supposing that the appearance in aggregates of malignant cells during the growth of a population of the three characteristics of the sec the tumor. ond groupâ€”lack of contact inhibition, dissemina tion, disorganizationâ€”are largely an expression of GROWTH AND MITOSIS a surface change. The possibility has frequently The last main characteristic of malignant tu emerged in this discussion that lowered adhesion mors to be mentioned is the persistent replicative is the fundamental aspect of this surface change. activity of a proportion of the malignant cell popu Passive dissemination seems to require lowered ad lation, accompanied by the rhythm of repeated hesiveness for the critical process of disintegration mitosis which usually goes with such growth. Can from the main mass; diminution of adhesion seems this characteristic be linked to a change in the cell capable of accounting for much of the disorganiza surface? If it is assumed that growth is normally tion of cancers; and though the nature of contact limited by intake of nutrients, it is clear that it inhibition is still uncertain, diminished adhesive may (125), particularly if pinocytosis is important ness is at least the basis of some plausible hypothe for such intake (62, 90). Increased pinocytosis in ses about it. malignant cells has been reported (62, 100). It has On the evidence presented, then, a generalized been found that the pinocytosis of normal cells diminution of adhesiveness seems to be an impor was stopped where they form the adhesions asso tant part of the malignant transformation; but ciated with contact inhibition; and these mutual this hypothesis must not be pushed too far. It is adhesions are much less in evidence with malignant evident that invasion, dissemination, and disor cells (4). There is, therefore, some possibility that ganization may vary among different tumors in an the growth activity of malignant cells may be independent way (59). Even in the absence of linked with persistent surface activity associated quantitative methods for the assay of these charac with diminished adhesiveness. teristics, it seems that a theory based simply on a nonspecific loss of adhesiveness cannot account for CONCLUSIONS their distribution in different tumors. A more so We now attempt to draw together what has been phisticated theory is probably required. said about the biological evidence that changes in If it is difficult to unify these three characteris the cell surface may be an important element in tics by making them exclusively the result of a the differences between normal and malignant single kind of surface change, it is clearly more cells. difficult still to add in the persistent mitosis and The mitotic activity of a population of malig mobilization of malignant cells as well. However, nant cells, and its capacity to migrate on a noncel- even though this may mean that qualitatively dis-

tinct changes are involved in the successive steps PHYSICAL ASPECTS of carcinogenesis, it becomes tempting to think of the cell surface as at least one of the primary sites INTRODUCTION of carcinogenic action. Two of the more mysterious In the biological summary the importance of forms of carcinogenesis, that produced by implan the cancer cell surface in relation to the invasive tation of sheets of material and that produced by behavior and adhesive properties of the cells was long culture in vitro, might fit with such a concep emphasized. We shall now attempt to examine the tion, since they may involve presenting cells with physical and chemical aspects of these changes un prolonged abnormal contacts (which may, how der three headings : (a) the adhesiveness of normal ever, act selectively rather than adaptively). At and tumor cells, (6) mechanisms of cellular loco least one chemical method may act initially on the motion, and (c) the ultrastructure of cell contacts. cell surface (60). Malignancy is, however, usually This part of the review will be concluded with a a lasting change which becomes independent of summary (d) of the more general biochemical as pects of the structure of the tumor cell membrane with particular reference to possible future lines of research. The primary function of the plasma or cell mem CL brane is to provide a barrier of low permeability CL which will isolate and protect the internal structure of the cell. It might therefore be expected that Å’ lipides, which are largely impermeable to hydro- CL philic molecules, would play an important part in CL such structures. This was originally suggested by CL Overton in 1895 (114), who found that fatty sub stances readily penetrate cells, whereas substances that are insoluble in fats penetrate very slowly, if at all. But measurements of the surface tension of L< cells by Harvey (71) indicated that the tension was much lower (0.2-0.08 dynes/cm) than would be expected for a pure lipide layer. Danielli and Har vey (43) and Danielli (42) were able to show that lipides readily adsorb protein at the lipide-water interface; protein can reduce the surface tension to rr the magnitude observed in living cells. CL I The model for the structure of the cell mem brane as proposed by Danielli is, therefore, as CHART1.â€”Lipoprotein structure of the cell membrane ac shown in Chart 1. It consists of a double layer of cording to Danieli!. L=lipide chains; O=polar groups of lipide in which the polar regions lie toward the lipides; Pi = tangentially arranged protein chains; PS = aqueous phase while the hydrocarbon regions lie globular proteins. perpendicular to the surface and are packed in a manner similar to that which is known to occur in crystals of long-chain fatty acids. The protein whatever induces it; it must, therefore, involve the mechanisms of replication. It is conceivable chains are much longer than the fatty acid chains and may be expected to lie parallel to the surface that the cell surface may be a component in one of the membrane. Although modified in certain re or more replication cycles, so that it takes part in spects by more recent work, the Danielli model replicating itself and hence is a possible site for a has proved satisfactory in accounting for most of primary carcinogenic action. But what the changes the general properties of cell membranes. There is in replication are that bring about lasting trans much experimental evidence in favor of a double formations like differentiation and malignancy is lipide layer. The most direct evidence concerning really still obscure, and it is wiser for the present the chemical nature of the lipides comprising the merely to say that the cell surface, at whatever cell membrane has been obtained by the analysis remove from the primary transformation it is al of erythrocytes. The total lipide content of these tered, seems to play an important part in the mani cells is just sufficient to provide a bimolecular layer festation of malignancy. over the surface, which strongly suggests that the

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. ABERCROMBIEANDAMBROSEâ€”SurfacePropertiesof Cancer Cells 535 lipide analyzed is membrane material. This ghost rather similar to keratin but with an extremely low material has the composition: 70 per cent phos- content of cystine (0.7 per cent) (145). The arginine pholipide, 25 per cent cholesterol, 5 per cent cho content is relatively high. It is, therefore, likely lesterol ester, 0.4 per cent cerebroside (55) ; in hu to be combined with the phospholipide by van der man erythrocytes the phospholipide contains: 30 Waals' attraction between side chains and by elec per cent lecithin, 15 per cent phosphatidyl ethanol- trostatic linkages between carboxyl, phosphate, amine, 13 per cent phosphatidylserine, 19 per cent and choline; primary chemical bonds are unlikely sphingomyelin, 10 per cent other lipide (45). The to be involved. chemical constitution of the more commonly oc curring lipides is shown in Chart 2. It will be seen ADHESIVENESS OF NORMAL that the phospholipides can readily be fitted into AND TUMOR CELLS a model of the Danielli type, with the long fatty The adhesiveness of surfaces depends upon the acid chains providing the hydrocarbon portion relative magnitudes of the attractive and repulsive while the phosphate and amino groups provide the polar regions. In general, phospholipides display a number of the physicochemical properties of cell membranes, as has been mentioned by Holtfreter (78). For example, lecithin when placed in water CO CO co co spreads out to produce the familiar threads of mye- IO OCHj-CH-HjCO OCHj-CH-HjCO linic form. Optical studies have indicated that the OP-O9 OP-O molecules in these myelinic forms are arranged in I a series of double layers with a radial orientation. O The myelinic threads are hollow tubes with the polar ends of the lipide molecules at the lipide- H3N water interfaces. Myelinic forms occur even more readily with mixtures of lecithin and cholesterol, which suggests that both phospholipide and steroid may produce a particularly stable double-layered structure. X-ray diffraction studies of phospholip co ides and of the sheath of myelinated nerve, which CO CO I IO NH resembles a myelinic form in many respects, indi OCH2-CH-H2CO CHCH-CH-CH2O cate that the spacing between layers has a charac 1 e I l Â« OP-O OH OP-O teristic value for individual lipides but reaches a i I stable and independent value for mixtures of lip- O O S Ã‡Â»i ides. The spacing obtained for nerve myelin is 171 M A for two double layers. A method of packing phos HC-CO pholipide and steroid into a double-layered struc e ture has been suggested by Fincan (57). The choline portions are turned upward toward the polar ends of the cholesterol; the phosphate groups lie at the surface. When extracted from whole tissues, phospholip ides are generally tightly bound to protein, as might be extected from Danielli's observations. CO I They can only be separated from protein by com NH CH-CH-CH- paratively polar solvents, such as methyl alcohol. CH20 The neutral lipides, on the other hand, may be Â¿H removed by comparatively nonpolar solvents such HÃ‡foH) I as ether. The general pattern of the lipoprotein in (HO)CH O myelin consists of double lipide layers which are R (HO)Â¿H HC- 52 A thick, whereas the protein layers are 28 A thick. Comparatively little is known about the CH20H chemical and physical properties of the protein CHARTi.â€”Lipide components of the erythrocyte mem brane. L = lecithin; K = phosphatidyl ethanolainine; C = fraction of cell membranes. Stromatin as isolated steroid; S = phosphatidyl serine; M = sphingomyelin; R = from red cell ghosts has an amino acid content cerebroside.

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. Cancer Research Vol. 22, June 1962 forces between them. In general terms, the re presence of a reaction rather similar to antigen- pulsive forces can be understood simply as arising antibody interactions. Specific surface antigens are from the electrostatic repulsion between charged well known, as in the agglutination tests for blood surfaces. These repulsive forces operate over con group substances on erythrocytes. Reactions of siderable distances and in the case of Coulomb this type, however, appear to be more characteris forces fall off inversely as the square of the dis tic of the individual than of the organ. For example tance between the charges. The attractive forces Kebus, Gerner, and Coombs (89) have detected the in general decrease more rapidly with increas H-blood group substance on Hela carcinoma cells ing distance. The attractive forces are also more after 8 years of continuous tissue culture. This complex; for example, they may involve van blood antigen is associated with the 'O' group of der Waals' and related forces and electrostatic at the original donor of the carcinoma cells. It would tractions between acidic and basic groups; these certainly not be expected that rat and mouse liver attractive forces can operate over considerable cells would associate preferentially according to distances. In addition, if the surfaces come into accepted views of antigen-antibody reactions. It is close contact, hydrogen bonds can form between not likely, therefore, that blood group substances the > NH and > C = 0 groups of proteins, â€”OH provide the main component for organ specificity. groups of steroids, amide linkages in phospholip- Intercellular adhesions differ from antigen-anti ide, etc. (These bonds involve energies of 2-5 K body reactions in another important respect. They cal/mole, whereas covalent bonds require 20-50 K occur between the surfaces of identical cells and cal/mole to separate the combining atoms.) Link would not, therefore, be expected to possess the ages between negatively charged groups such as complimentary character proposed for antigens and carboxyl by bivalent ions such as calcium may also antibodies. In this connection the sensitivity of take place. All the cell-substrate adhesions brought embryonic tissues to calcium may be important. about in this way are readily reversible and can be Identical surfaces of specific structure could clearly broken moderately easily at the temperature of be bound by bivalent cations as already men the body. tioned. An alternative possibility would be the se Intercellular adhesions exhibit more complex be cretion of a cementing material which is deposited havior. Cells such as erythrocytes, which are com between the cells. As will be mentioned later, elec paratively nonadhesive both to substrate and to tron microscopy has shown clearly that in certain each other, carry a high negative surface charge tissues a dense secretion of layers takes place be (17). This is consistent with the principles already tween individual cells. Recent work by Easty and mentioned which indicate that the main repulsive Mutolo (53) suggests that in adult tissues a protein forces between surfaces are due to electrostatic re cement may be considerably more important than pulsion between like charges. Insofar as the attrac calcium in maintaining intercellular adhesions. In tive forces between cells are concerned, calcium agreement with Essner, Sato, and Belkin (56) they ions play a predominant role, at least in the early have found that proteolytic enzymes (trypsin, stages of embryonic development. Amphibian em pepsin, and chymotrypsin) are effective in break bryos at the blÃ¡stula stage may be disaggregated ing up islands of hepatoma cells and liver cells. by low concentrations of EDTA, which removes Diisopropyl fluorophosphate-inactivated trypsin, calcium by chelation. If the calcium in the medium on the other hand, is without effect, indicating is replaced, the cells will reaggregate and eventu that proteolytic hydrolysis is involved in the sepa ally sort themselves out to produce normal struc ration. Erythrocytes which have been agglutinated tures. In addition to forming linkages between car with antiserum (^-immune globulin) are not dis boxyl groups (â€”COOCa++ OOC â€”)calcium ions persed by trypsin, unlike the intact tissues. This will reduce the net negative charge on the cell sur result suggests that the protein cement is not of face. Both these factors may be of importance in the same type as antibody protein. It is therefore the establishment of intercellular adhesions. In possible that in embryonic tissues and tissue cul mixed cultures of mammalian cells it is also found tures an early stage of intercellular adhesion occurs that the cells eventually associate with cells of a which is sensitive to calcium in the medium. This similar type (109, 140). Selective affinities are also adhesion probably takes place directly between the observed in organ culture (147). The tissue specific cell membranes. In adult tissues this reaction ap ity does in fact appear to be more organ-specific pears to be at least partially replaced by a definite than species-specific. Rat liver cells will associate secretion of a cementing protein. L. Weiss (136, with mouse liver cells, for example, rather than 137) has demonstrated the importance of protein with other tissue cells of the rat. The presence of in determining the adhesiveness of both normal specific reactions of this type possibly suggests the and tumor tissues. He has found that trypsin-

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. ABERCROMBIEANDAMBROSEâ€”SurfacePropertiesof Cancer Cells 537 treated cells require a higher concentration of se experimentally an oil-water-oil system and has rum in the medium to make them adhere to glass found that the two surfaces are stabilized at a dis than is required by untreated cells. The constitu tance of 100-200 A. This result is in good agree ent of whole serum which appears to be mainly re ment with the Verwey and Overbeek theory, which sponsible for the adhesive properties is the /3.2lipo- predicts such a distance for balance between the protein-rich serum fraction. attractive and repulsive forces. According to Curtis A more physical approach to the problem of cel it is therefore possible to account for the separa lular adhesiveness has been given by Curtis (40), tion of the parallel membranes at ~150 A, as seen who has developed a theory to account for the in the electron micrographs (Chart 5). More recent properties of the cell membrane based on the methods of fixation have shown that a deposit does known rheological properties or flow character in fact lie between the membranes. But Curtis has istics of protein monolayers and on the Verwey pointed out that this may occur at a later stage after the membranes have been initially stabilized

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CHART3.â€”(a) Cross section through a moving Amoeba. L = leading pseudopodium; T = tail; S =â€¢solidsubstrate. (6) Undulating contacts between a filamentous alga and the solid substrate. C = contacts between membrane and substrate. (c) Mechanism of cellular locomotion involving undulating contacts between membrane and substrate. C, C1 = moving contact; T = transverse undulations of membrane; M = compressional wave traveling in cytoplasm. and Overbeek (133) theory of adhesions between surfaces, which throws some light on the mecha nism leading to cellular adhesions. Protein films are known to exhibit viscosity changes with shear, the viscosity decreasing with increasing shear at low concentrations giving the phenomenon of thix- otropy (tendency to become fluid at high rates of shear). A rise in viscosity occurs with shear at high concentrations giving rise to rheopexy (tend CHART4.â€”Morphologyof cell membranes and cell contacts ency to become rigid at high rates of shear) ; it is to be expected that the cell membrane will also as shown in the electron microscope. exhibit effects of this kind. According to the Ver a) Intercellular contact with separation of ~150 A between densely staining membranes. wey and Overbeek (133) theory of adhesion, sur V) Less continuous contacts seen between cells in region of faces of similar constitution should attract one active cell proliferation in the hair root. another by van der Waals' forces and repel one fc') Higher power picture of contact showing each membrane another if they differ considerably; the electrical resolved into two layers ~70 A apart. c) Region of intercellular contacts showing dense deposits (P) charges of the two surfaces will drive them apart in cytoplasm. (Desmosomes.) if of similar sign and draw them together if of op d) Multilayered deposit between cells seen in the upper part of posite sign. Van den Teupel (131) has examined the hair root and some other differentiated tissues.

at a distance of ~150 A by the balance between mor cells from kidney cortex and on normal liver the attractive and repulsive forces. By combining cells and hepatoma cells. The hamster kidney tu the rheological and the adhesion model, Curtis has mors were originally induced with stilbestrol. Both been able to account for a number of the properties the normal and tumor cells have been grown in cul of cell membranes. An expansion of the surface of ture (15) and their contacts examined. The normal the cell may be expected to increase its adhesive kidney epithelium grows as discrete islands of cells ness because the van der Waals-London forces will with closely adhering contacts between the mem be unaffected, whereas the charge density may be branes. The tumor cells grow much more inde expected to decrease. This effect can explain the pendently with fewer stable adhesions between increased adhesiveness of spreading membranes their membranes. The culture studies show that and can account for the changes in contacts which the membranes of these tumor cells are less adhe occur continuously during locomotion by means of sive than the normal cells from which they are undulations between the moving cell and the solid derived. According to the electrophoretic studies surface, to be described later. When two moving the tumor cells also have almost twice the mobility

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cells meet in opposition the effective shear will be of the normal cells from which they are derived greatly increased, the surface viscosity will fall, (Chart 5). This increased mobility can be ex and the membranes will expand; this will lead to plained by an increase in the net negative charge increased adhesiveness, and an adhesion will form per unit area carried by the tumor cell membrane. between the two cells. Contact inhibition can be An increased negative charge was also found when understood in these terms. hepatoma cells were compared with normal liver How are we to explain the reduced adhesiveness cells. This method of measurement is expected to of the tumor cell surface in such terms? Since the demonstrate surface effectsâ€”e.g.,Moyer (110) has repulsive force between the cells is mainly due to shown that collodion particles when coated with repulsion between like charges, a method of exam protein display the same mobility as the protein ining the charge on the surface of tissue cells may molecules in solution. With tumors containing cells throw some light on the adhesive process. Cell elec- of varying size, the mobility is found to be inde trophoresis can be used for this investigation. The pendent of size, which also supports this view. In tissues are gently dispersed and suspended in a addition, the known electrical resistance of the cell medium in which they are known to remain viable membrane (44) ensures even more effectively that for many hours. The velocity of individual cells in the cell surface is practically isolated from the in an electric field is then determined in a chamber terior in electrical studies of this type. The increase mounted under the microscope. Experiments of in negative surface charge can be correlated direct this kind have been carried out on normal and tu- ly with a reduced mutual adhesiveness of the cells

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. ABERCROMBIEANDAMBROSEâ€”SurfacePropertiesof Cancer Cells 539 displayed in biological experiments. For example, spleen cells. It was found that several of the poly various sublines of a single tumor have been ex mers showed selective effects. One polymer, poly amined by this method (MCIM sarcoma). A num ethylene Â¡mine,agglutinated the tumor cells selec ber of sublines of this tumor have been obtained tively, having less affinity both for spleen cells and by Klein and Klein (93) using selective transplan erythrocytes. Erythrocytes carry a high negative tation. The MSSstrain is a solid subcutaneous tu surface charge like tumor cells, but these results mor which does not produce ascites cells but grows suggest that, at least in this transplanted tumor, again as a solid tumor when implanted in the peri the distribution of charges on the cell surface dif toneum. It does not metastasize readily. MÃ©tas fers from that of the erythrocytes. tases in the lung are found as discrete emboli which It may be concluded that the adhesions between do not infiltrate into the lung tissue. MTLFis an cell surfaces are probably due to the protein or intermediate form of solid subcutaneous tumor which gives rise partly to ascites cells and partly to solid tumor in the peritoneum. MAsis a solid sub MAA cutaneous tumor which produces an ascites tumor 20 in the peritoneum at the first implant. It metas- 10 tasizes readily, with a high degree of infiltration MAS73 into the lung tissue. MAAis the actual ascites tu 20 mor, transplanted continuously in the peritoneum. 10 In Chart 6 it can be seen that there is a progressive increase in the net negative charge carried by the MTLF 20 cells as one passes from the MSS to the MAA 10 strains. Lack of adhesiveness between the tumor cells can therefore be most readily explained by a MSS high electrical charge resulting in electrostatic re 20 pulsion between the surfaces. How could such an 10 increase in surface charge be brought about? The 1-0 1-5 2-0 simplest explanation would be that the membranes MOBILITY ji/Â«c/Â»oU/cm. have a reduced capacity to bind positive ions, no tably calcium. A reduced calcium content of tumor CHART6.â€”Electrical mobilities for sublines of the MCIM sarcoma. tissue as compared with normal tissue has been Mss = Solid tumor which does not produce disseminating found by Beebe (24), Brunsching, Dunham and mÃ©tastases;MTLF= Intermediate form; MASTS= Solid tumor Nichols (28), Clowes and Frisbie (33), de Long, producing disseminating mÃ©tastases;MAA= Ascites form. Coman, and Zeidman (47), Dunham, Nichols, and Brunsching (49); de Long, Coman, and Zeidman mucoprotein component of the membrane. An in have suggested that the decreased calcium content crease in the net negative charge on the cell surface may be related to the decreased adhesiveness of the reduces adhesiveness, and this change probably tumor cells. They have pointed out that in normal explains in part the reduced adhesiveness of tumor growth conditions, as in embryonic development, cells. changes in the sodium/potassium ratio occur, but no decrease in calcium content is found (47). Al MECHANISMS OF CELLULAR ternatively, there may be some alteration in the LOCOMOTION quantity and distribution of the actual charged The cell surface plays a fundamental role in all chemical groupings themselves on the surface of types of cellular locomotion. We shall now examine the tumor cell. One way of investigating this dis some physical theories of such locomotion with tribution is to examine the way in which long-chain particular reference to the invasiveness of tumor polymers carrying positive charges are absorded on cells. Unicellular organisms and almost all tissue the surface. For example, Katchalsky and his col cells of higher animals are capable of migrating on laborators (112) have found that polylysine readily solid surfaces under suitable conditions. Most of combines with the erythrocyte surface, neutraliz the earlier observations of these movements were ing the negative surface charge of the cell and lead carried out on protozoa, particularly the large ing to rapid cellular agglutination. Ambrose, Easty, amoeba, Amoeba proteus. Goldacre (67) proposed and Jones (16) studied the agglutination of sus the mechanism for locomotion illustrated in Chart pensions of Walker carcinoma cells by various 3, a. Contraction of the proteins in the tail of the positively charged polyelectrolytes of this type. organism causes the cytoplasm to flow forward Comparisons were made with erythrocytes and toward the leading pseudopodium. Allen has

proposed that the driving mechanism for proto 3, c). Undulations of the membranes are also seen plasmic streaming is located in the leading pseudo- when migrating tissue cells are examined by time- podium. But whichever view of the location of the lapse filming with the interference microscope (4). driving forces is accepted, evidently the membrane These undulations can be seen in phase contrast of the leading pseudopodium expands and flows by focusing carefully on the upper surfaceâ€”i.e., forward in the direction in which the cell is mov the cell-liquid interface. Observations of similar ing. Meanwhile, the membrane in the tail is re cellsunder the surface contact microscope, however, tracted and flows into the cytoplasm. In other immediately show that these undulations are also words oÃdmembrane is withdrawn from the tail, is occurring at the contacts between the cell and the carried forward by protoplasmic streaming, and surface of the solid substrate (18). The undulating new membrane is formed continuously at the lead contacts are particularly conspicuous in the ad ing edge. This mechanism of locomotion is usually vancing regions of epithelial sheets. When groups called amoeboid movement. It has been observed of fibroblasts streaming from an explant are exam in a number of organisms, including mammalian ined, they are all seen to exhibit rapid undulations white blood cells. It implies the presence of a labile of the membranes and contacts. Migrations of tis membrane into and from which new molecules or sue cells by this process can explain a number of micelles can be introduced and readily withdrawn. otherwise unrelated phenomena. Fibroblasts and The model illustrated in Chart 3, a, is satisfactory epithelium rarely exhibit streaming of the cyto from this point of view. It is by no means true, plasm in the direction of migration, as is the case however, that cells with labile membranes are with Amoebae. Intermittent streaming of the cyto unique in being able to migrate across solid sur plasm takes place away from the leading edge of faces. An extreme case of organisms with coherent the cell, as can be seen when droplets are enclosed membranes which can still migrate are the blue- by pinocytosis and carried toward the nucleus. green filamentous algae. They consist of cells line When groups of closely adhering fibroblasts and arly arranged with a moderately rigid membrane, epithelial cells in culture are examined, they are as commonly occurs in plant cells. The continuous often seen to stream uniformly across the sub flow of cytoplasm associated with amoeboid move strate in the direction of a free border. In inter ment is excluded in these organisms, yet a number ference contrast and under the surface contact of them migrate rapidly on surfaces. By the use of microscope the free borders of sheets are seen to a new type of microscope (12, 13) it has now be exhibit extremely active undulations. These undu come possible to observe the locomotor mechanism lations travel backward from the leading edge of directly. This microscope depends upon the reflec the cell. The leading edge therefore acquires domi tion of light at the surface of a glass prism and nance in guiding the general movements of the enables those regions of the cell which are in con group of adhering cells. In the biological section tact with the surface to be examined individually ; mention was made of possible blocking mecha the interior of the cell is not illuminated. In this nisms which could play a part in the phenomenon microscope, it is seen that the locomotion of Oscil of contact inhibition. Physical or chemical factors latoria and other algae is accompanied by waves affecting the intermittent contacts between cell of contact between the membrane and substrate and substrate of the type shown in Chart 3, c, could which travel continuously along the length of the possibly operate in this way. filament. These waves can readily provide a mech Migration of cells by amoeboid movement or by anism of locomotion as illustrated in Chart 3, b. If membrane undulations requires continuous supply these waves are accompanied by a compressional of energy by the cell. Goldacre (67) has proposed wave, also traveling along the length of the organ that contractile proteins are stimulated in the tail ism as indicated in Chart 3, c, the membrane of of the Amoeba and that these proteins are carried the organism will make intermittent contact with forward to the leading psuedopodium, where they the surface, each contact occurring at a different unfold. An ATP enzyme system similar to acto- point on the membrane, if the compressional wave myosin has in fact been isolated from Amoebae. is out of phase with the transverse wave. In this Little is known at present about the source of en way the organism is able to move along the sur ergy of membrane undulations, but there are a face, rather like a Gastropod. few clues. Weber (134) has shown that glycerol- An examination of mammalian tissue cells such extracted fibroblasts contract in the presence of as fibroblasts and epithelial sheets indicates that ATP. Ambrose and Curtis (13) have found indica a similar mechanism is involved in their migration. tions by surface-contact microscopy that the am Undulating contacts between the membrane and plitude of membrane undulations in living fibro the substrate surface are again involved (Chart blasts is increased in the presence of ATP in the

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. ABERCROMBIEANDAMBROSEâ€”SurfacePropertiesof Cancer Cells 541 medium. It is possible that contractile proteins of peared, do independent cells break away from the the actomyosin type are present on or near the tumor mass and give rise to mÃ©tastases. membranes of mammalian tissue cells. The mechanism of locomotion in tumor cells has ULTRASTRUCTURE OF CELL been studied by interference microscopy (Aber- CONTACTS crombie and Ambrose [4]) and by surface contact Electron microscopy has been used for the study microscopy (Ambrose, unpublished). As with nor of the morphology of cell membranes and cell con mal tissue cells, undulations of the membrane in tacts, both with normal and tumor tissues. The contact with substrate are generated with both preparation of specimens for electron microscopy sarcoma and carcinoma cells. The main locomotor involves both fixation and drying. With a delicate mechanism in tumor cells is evidently by the same colloidal system such as the lipoprotein complex of intermittent contacts (Chart 4, c) between cell and cell membranes, it might be expected that satis substrate, as in the case of normal tissue cells. factory fixation would be difficult to achieve. Men Qualitatively, however, the type of movement in tion has already been made, however, of the tumor cells differs from that of normal cells. The studies of nerve myelin by EnstrÃ¶m and Finean movements of tumor cell membranes tend to be (54, 57) using x-ray diffraction and electron mi less coordinated than those of their normal coun croscopy. They have also examined the effect of terparts. Normal fibroblasts tend to develop one successive stages of fixation and drying on the x-ray expansive fanlike pseudopodium which leads and diffraction pattern of myelin and have compared directs the cell. Many sarcoma cells tend to devel the results with direct observation in the electron op a number of less expansive pseudopodia and in microscope. Although some qualitative changes in the anaplastic, more rounded types the membrane spacing occur, the basic structure appears to be may be divided into large numbers of small and preserved after fixation. It has been tempting to independently moving villi. This is probably just suggest that the plasma membrane of other cells another manifestation of the general degeneration might correspond to a single layer of the type ob of control mechanisms observed with so many as served in the multiple structure of the myelinic pects of tumor cell behavior. In the biological sec sheath. Evidence in support of this view has been tion it was pointed out that complete independence obtained by Mercer from studies of Amoeba pro- of cells so that they were able to move like proto teus, using fixation with osmium tetroxide. There zoa was not necessarily the sine qua non of migra are two outer layers of densely staining material tion. Normal epithelial cells in tissue culture or in with a central more transparent region. These vivo will move on a suitable plane surface while so three layers could correspond to the two outer lay closely adherent to each other as to appear like a ers of protein with a lipide layer between as en continuous sheet; they can move through a three- visaged by Danielli (43) (Chart 1). With other dimensional gel in vitro in very close association in types of cell the membrane is usually seen as a the form of cords or strands. Locomotion by mem single layer, but it may be resolved into a three- brane undulations giving rise to intermittent con layered structure similar to Amoeba proteus, par tacts with the substrate can account for such ticularly if potassium permanganate fixation is movements. It is only necessary that a free border used (Robertson [116]; Mercer, private communi be present to generate the leading membrane cation). Contacts between neighboring cells also movements, when the whole group of cells will show interesting effects in the electron microscope. migrate in the direction of the free border, each This has been clearly illustrated by Birbeck and Mercer's systematic study of the developing hair cell being coordinated in its movements by the leading cell. Similarly, in the early stages of tumor follicle (23). This is a particularly interesting sys invasiveness, it is not necessary to postulate com tem in which to examine cellular differentiation. In plete loss of contact inhibition between all tumor the hair root, where cells are actively dividing, the cells. Some local loss of contact inhibitionâ€”e.g., membranes show convoluted forms, and continu between tumor cells and the surrounding normal ous close contacts between cells are not observed tissuesâ€”may be all that is required. The tumor (Chart 4, 6). In those regions where contacts do cell mass may then be able to migrate by mem occur the membranes of adjoining cells lie side by brane undulations on the normal tissue as sub side, at a distance apart of 150-200 A. With os strate, while maintaining close contacts with neigh mium tetroxide fixation this space appears to be boring tumor cells. There are suggestions, from transparent, but with other methods, for example histological sections, that the early stages of inva after staining with lead salts (Mercer, private com siveness can occur in this way. Only later, when munication), the space between the membranes is tumor cells of much reduced adhesiveness have ap at least as dense as the general cytoplasmic back-

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. 542 Cancer Research Vol. 22, June 1962 ground in the interior of the cell. Further up the the type just described may have more general hair follicle, as differentiation of the cells takes effects in the actual organism. For example, prob place, the areas of contact progressively increase lems of embryonic development are concerned (Chart 4, a). In these regions of parallel contacts, with mechanisms of communication between cells. increased density may be observed within the cyto In the past, emphasis has been placed on organizer plasm in the region in the immediate neighborhood substances, chemical messengers which are se of the contact (Chart 4, c). These are the so-called creted by one cell and act upon a receptor in a tlesmosomes. Occasionally in these regions of thick neighboring cell. Emphasis has turned in recent ened cytoplasm adjoining a contact, tonofibrils years, however, toward a study of direct reactions may be seen. These are fibers which lie at right between cells (77, 78, 140). Control by simple angles to the cell surface. They are also conspicu physical contact is clearly illustrated in the phe ous in the region of the contact between muscle nomenon of contact inhibition (6, 7) described in cells. They are thought to provide attachments the biological section. The adhesions between cell between the cell contact and the interior of the membranes which give rise to the contact inhibi cytoplasm, so that considerable tension can be tion effects described in the biological section, af maintained between adjoining cells. Further up the fect the movements of cells. But they may also hair cuticle (Chart 4, Â¿),in the region where kera- modify the cells in other respects. As already men tinization is taking place, a definite secretion of a tioned, electron microscopy indicates that, in layered structure between the cells can be de the region of an adhesion between membranes, a tected. Similar results have been obtained more thickening may occur, just within the cyto recently by Rogers (117). plasm. Evidently an increased secretion of some The presence of multi-layered structures in nerve kind has been stimulated by the contact. myelin has also been demonstrated in a most in In a few cases actual passage of vacuoles from teresting way by Robertson (116). The Schwann one cell to another has been observed. Mela- cell is considered to have wound itself round the nocytes secrete melanin into cells by this process nerve axon to produce a spiral structure. The outer (106). Cell-to-cell interactions, arising from con surfaces of the Schwann cell membrane in the un- tacts of an electrical nature, may also arise. This swollen condition are separated from one another has been extensively investigated with growing by a distance of ~150 A, as in the case of tissue plants by Lund and his collaborators (104). They cells as previously described. It is interesting to have shown that differences in electric potential of note that on the cytoplasmic side the inner sur up to 60 millivolts can be detected between the faces of the membranes come into intimate con apex and proximal region of the cotylon of growing tact. The individual membranes have also been seeds. These potentials, which occur in many types resolved by Robertson into two layers ~20 A of cell, are considered to arise from differences in thick, with a lighter zone <~35 A thick lying be the ionic permeability of the membrane in various tweenâ€”i.e., the individual membranes have the regions of the cell. In mammalian nerve cells, the same general appearance as the plasma membranes transfer of the action potential is known to be ac illustrated in Chart 4, 6. Apart from the keratin- companied by a reversible change in the permea ized regions of the hair follicle and the myelin bility of the cell membrane to potassium and sodi sheath, multilayers are not generally observed at um ions (73, 82). Goldacre (66) has demonstrated cell contacts. Contacts between differentiated tis with electronic models that a small number of elec sue cells generally take the form shown in Chart 4, trical interactions between units can give rise to a. The membranes lie parallel to one another and quite complex patterns of behavior. The study of are separated by ~150 A. A more sensitive method bioelectric potentials and currents in relation to of studying the cell surface by electron microscopy cell-to-cell interactions generally may provide an is to use some shadowing procedure to show up the interesting field for future research, particularly surface contours instead of sectioning the tissue. their possible failure and lack of coordination in For example, Hillier and Hoffman (72) prepared cancer tissue. erythrocyte ghosts and shadowed the surface with When tumor tissues are examined by electron evaporated metal in a high vacuum. When this microscopy the morphology of the membrane in sec procedure was carried out after careful washing, an tion is similar to that of normal cells. The plasma interesting plaque formation was observed. These membrane is observed as a continuous layer ~70 plaques were considered to correspond to the pro A thick. However, an altered appearance of the tein portion of the membrane, because they were contacts with neighboring cells can generally be insoluble in lipide solvents. observed in anaplastic tumors. In these tumors, Intercellular adhesions between normal cells of the contacts are similar to those observed in the

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. ABERCROMBIEANDAMBROSEâ€”SurfacePropertiesof Cancer Cells 543 actively dividing region of the hair root, as indi 4, a. The adjacent membranes are separated by cated in Chart 4, b. The membranes exhibit small about 100-150 A. Kidney tumor cells, on the other areas of contact with neighboring cells, the re hand, exhibit a number of longprocesses or pseudo- maining regions showing a highly convoluted form. podia (J-l p diameter) around the cell borders, Where contacts do occur they appear to be mor with very few parallel contacts with neighboring phologically similar to those of normal cells. The cells. The decrease in the mutual adhesiveness of membranes are separated by a distance of ~150 A, the membranes in cancer cells is clearly demon and the cytoplasm in the region adjoining the con strated by the shadowing procedure. Nowell and tact tends to produce a desmosome structure Berwick (113) have also compared normal and leu- (Mercer, private communication). These observa kemic lymphocytes by the shadowing method. tions support the earlier studies of tissue cul Here again, no difference between the texture of tures filmed under the interference microscope and the normal and tumor cells could be detected in the micromanipulation experiments which indicate the range from 50 to 300 A. On biochemical grounds that anaplastic tumor cells move considerably it is hardly to be expected that differences between more independently and are less adhesive than the the lipoprotein structure of normal and tumor cell homologous normal cells. membranes would show up in the 100 A range. As in the case of normal cells, the most sensitive They would be expected to occur in the range from method of studying the cell surface by electron 10 to 100 A. The changes observed by Coman microscopy is to shadow the surface layers. Several and Anderson and by Berwick, with cells separated investigators have compared normal and tumor from tissues, may perhaps correspond more to cells by this method. The most favored procedure changes in the elastic or other physical properties has been to prepare carbon replicas. The surface of the plasma membrane of cancer cells (see also of the cell is coated with carbon in high vacuum. Catalano, Nowell, Berwick, and Klein [30]), rather The surface contours of the carbon are accentuated than to permanent morphological changes. by shadowing with gold or chromium. The cellular It may be concluded that on the whole the material is then dissolved away by enzymatic di studies of ultra-structure provide additional evi gestion to leave the carbon replica for examination dence for altered surface properties associated with in the electron microscope. This method was first less coherent intercellular contacts, at least in ana- used for comparisons between rabbit Vx2 epider- plastic tumors. moid cells and epithelial cells separated mechani cally from the rabbit's ear by Coman and Ander BIOCHEMICAL PROPERTIES OF son (37). The normal epithelial cells exhibited a TUMOR CELL MEMBRANES number of large projections or prickles, but the In the three previous sections of this part of general texture of the membrane surface was com the review we have tried to learn something of the paratively smooth over the range from 30 to 300 A. physical factors underlying the changes in biologi The carcinoma cells, on the other hand, did not cal behavior of the cell surface during carcinogene- exhibit prickles but showed many irregularities in sis. We shall now consider the biochemistry of the the range from 30 to 300 A. More recently, Ber complete plasma or cell membrane in more general wick (26) has carried out similar observations us terms. ing tryptic digestion to separate the cells. He has That the internal structure of the membrane as also found that the surface of the Vx2 carcinoma opposed to the cell surface may be changed during cells is rougher in the range from 50 to 300 A. In carcinogenesis is suggested by observations of cell the case of a benign tumor (Shope papilloma), the permeability. Particularly striking have been the texture more closely resembled that of the normal observations indicating that a considerable leakage epithelial cells. However, Easty and Mercer (52) of enzymes takes place from the cytoplasm in have shown that, when cells which have not been growing tumors. For example, MalmgrÃ©n,SylvÃ©n, subject to mechanical or enzymatic treatment are and RÃ©vÃ©sz(105)found that ascites tumor cells examined after fixation in tissue culture (53), the release a considerable number of proteolytic en texture in the range from 50 to 300 A is similar both zymes into the ascitic fluid. By careful tests it was with normal and tumor cells. The contacts between established that this release took place from the neighboring cells, however, show distinct differ living cells and was not due to the presence of dead ences. In the case of normal kidney epithelium, the or dying cells in the suspension. In 1957, Libenson shadowing indicates that the membranes between and Jena (101) prepared samples of interstitial adjoining cells of the sheets are in close contact fluid from transplanted human tumors. From elec- with uniform and parallel membranes running trophoretic studies on fluids incubated for from 24 around the circumference of the cells, as in Chart to 96 hours at pH 4, they concluded that the deg-

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. 544 Cancer Research Vol. 22, June 1962 radation of added albumin was more marked in was found. This ratio has a value of approximately the cell-free tumor fluid than in the subcutaneous lf:l for normal liver and approximately 1:2 for tissue fluid distant from tumor sites, providing evi the hepatoma. This ratio was found when the phos- dence for the presence of proteolytic enzymes in tu phatides were extracted from various regions of the mor fluid. SylvÃ©n(126)has carried out an extreme cell, suggesting that the phosphatides may have a ly detailed and thorough analysis of this problem constant proportion of fatty acids irrespective of by a microanalytical method. A fine capillary was their position in the cell. The proportions of fatty used to withdraw interstitial fluid from unicentric acids differed only when cells from different tissues mouse transplants, from the surrounding normal or different species were compared. It is, therefore, tissue, and from the peritoneum. In all cases, care possible that van Deenen and his collaborators was taken to ensure that regions containing only have detected an alteration in fatty acid composi living and actively metabolizing cells were tested. tion of the phosphatides of tumor cell membranes. The cell-free tumor fluid had a protein content Earlier evidence for a change in total tumor phos about 100 per cent higher than that of the peri pholipide was obtained by Rapport, Skipski, and toneal fluid, but the 'over-all' dipeptidase activity Alonzo (115), who isolated a glyosphingolipide was increased 40-100 times; the arginase and glu- which they could not detect in normal tissues. Ka- tathione reducÃaseactivities increased 5-20 times; saki, Ikoda, Katani, Nakawa, and Saka (87) iso the catheptic activity at pH 4.5 increased 3 times lated a characteristic phospholipide from several over the corresponding normal plasma. The amino human tumors which they could not detect in nor peptidase and LDH activity were also found to be mal tissues. There is also some more indirect evi increased dramatically (SylvÃ©n,personal commu dence from enzyme studies. Easty, Easty, and Am nication) . The interesting point here is that no mi- brose (51) examined the effect of sixteen enzymes tochondrial enzymes are releasedâ€”i.e., only those on the adhesiveness of viable ascites tumor cells to enzymes which are in a soluble state in the cyto clear glass. They found that only four of these had plasm are released. These increases cannot be due a marked effect on the adhesiveness; these were to a general retention of protein in the tumor com wheat germ lipase, acid phosphatase, elastase, and partment, and it appears that the local increase in trypsin. When the effect of enzymes on growing enzyme concentration is due mainly to leakage cultures of the stilbestrol-induced kidney tumor from the tumor cells. This is supported by other and normal kidney epithelium already described in vitro work by SylvÃ©nand Bois (127). Evidence were compared (15), it was found that the lipase for the leakage of other types of enzyme from tu showed the most strikingly selective action on the mor cells has been obtained by Bosch (27). He tumor cultures. The lipase inhibited the growth of found that ribose-5-phosphate, glucose, and fruc the tumor cells at a lower concentration than that tose were metabolized by enzymes liberated into required for the normal cells. Cormack, Easty, and the extracellular medium by ascites tumor cells. Ambrose (38) showed by the use of fluorescent The release of enzymes into the interstitial fluid labels that there was a characteristic difference may play an important role in tumor invasiveness, between the reaction of the lipase with the normal particularly the catheptic and peptidase activity and tumor-cell membranes. which may break down the normal stroma. Concerning the protein or muco-protein com According to the studies of de Grier and van ponent of the tumor cell membrane little is known Deenan (46) there is a direct correlation between at present. Taylor (128) has shown that the pres permeability and phospholipide composition when ence of serum or other proteins in the medium de erythrocytes from various species are compared. creases the adhesiveness of tumor cells to clean A correlation was found between permeability to glass. It is therefore probable that the adhesive glycol and percentage of lecithin in a range of material on the surface is secreted by the cells and erythrocytes. This result suggests that the altered is not adsorbed from the medium. Bryn Jones (84) permeability of tumor cells may be related to has made use of a counterstaining procedure for changes in phospholipide composition. Veerkamp, proteins and has demonstrated a characteristic col Mulder, and van Deenen (132) have in fact carried or reaction with tumors, which he considers may out a detailed analysis of the lipides of normal rat be due to an alteration in the state of tanning of liver and a primary hepatoma. No striking differ the tyrosine residues in the surface protein. ences could be observed in the proportions of the Serological methods have provided some evi various phospholipides. In addition, however, the dence for altered surface antigens in tumor tissue. constituent fatty acids of the phospholipides were Weiler (135) demonstrated several years ago that compared by use of gas chromatography. A con there was an antigenic loss in stilbestrol-induced siderable change in the ratio of stearic to oleic acid kidney tumors of the hamster when compared with

normal kidney tissue, using antibodies with fluo ing Unmyelinated Nerves. J. Anat., London, 93:9-14, rescent labels. Hughes, Louis, Duneen, and Spec- 1959. 6. ABEKCROMBIE.,M.,and HEAYSMAN,J.E. M. Social Be tor (80) have shown that such effects can be pro havior of Cells in Tissue Culture. I. Speed of Movement duced by nonspecific reactions, but correspond to of Chick Heart Fibroblasts in Relation to Their Mutual some biochemical change in the composition of the Contacts. Exp. Cell Research, 5:111-31, 1953. 7. . Social Behaviour of Cells in Tissue Culture. II. tumor cells. More recently, Nairns, Richmond, Monolayering of Fibroblasts. Ibid., 6:293-306, 1954. McEntegard, and Fothergill (111) have detected 8. ABERCROMBIE,M.; HEAYSMAN,J. E. M.; and KAR- staining of the cell membranes in normal tissues THAU8ER,H.M. Social Behaviour of Cells in Tissue Cul using antibodies with fluorescent labels. This ture. III. Mutual Influence of Sarcoma Cells and Fibro staining did not occur with homologous induced blasts. Exp. Cell Research, 13:276-92, 1967. tumor tissue. Easty and Ambrose (50) obtained 9. ABERCROMBIE,M.,and JOHNSON,M.L. The Outwander- ing of Cells in Cultures of Nerves Undergoing Wallerian evidence with an anti-serum prepared against Degeneration. J. Exp. Biol., 19:266-83, 1942. transplanted ascites tumors and adsorbed against 10. ABERCROMBIE,M.; JOHNSON,M. L. ; and THOMAS,G.A. normal tissue, for the presence of a specific reac The Influence of Nerve Fibres on Schwann Cell Migration Investigated in Tissue Culture. Proc. Roy. Soc., s.B, tion which appeared to take place initially with the 136:448-60, 1949. plasma membrane of the tumor cells. Kay (88) has 11. ABRAMSON,H.A.; MOVER,L. S.; and GORIN,H. The demonstrated the loss of a surface antigen in some Electrophoresis of Proteins. Reinhold Pubi. Co., Vol. 44: human tumors, while Green (68) has proposed on 1942. the basis of his immunological studies that car- 12. AMBROSE,E. J. A Surface Contact Microscope for the cinogenesis is due to the loss of a tissue-specific Study of Cell Movements. Nature, London, 198:1194, 1956. lipoprotein antigen, present in the endoplasmic 13. . The Movements of Fibrocytes. Exp. Cell Re reticulum and possibly in the plasma membrane. search, Suppl. 8, pp. 54-73, 1961. The biochemical characterization of the tumor 14. AMBROSE,E. J.; EASTY,D. M.; and ANDREWS,R. D. cell membrane and surface must await the devel Inhibition of Tumor Growth by Lipases and Lipids in Tissue Culture. Exp. Cell Research (in press). opment of more refined physicochemical and bio 15. AMBROSE,E. J.; EASTY, D. M.; EASTY, G. C.; and chemical technics. When isolated and uncontami- DUDGEON,A.The Effects of Enzymes on Cell Growth in nated plasma membranes have been separated Tissue Culture. Exp. Cell Research (in press). from a number of tumor cells and their normal 16. AMBHOSE,E. J.; EASTY,D. M.; and JONES, P. C. T. Specific Reactions of Polyelectrolytes with the Surfaces counterparts and subjected to biochemical analy of Normal and Tumour Cells. Brit. J. Cancer, 12:439-47, sis, it may then become possible to relate the 1958. altered biological properties of the tumor cell sur 17. AMBROSE,E. J.; JAMES,A. M.; and LOWICK,J. H. B. face to actual changes in metabolic pathways with Differences between the Electrical Charge Carried by Normal and Homologous Tumour Cells. Nature, London, in the cell. 177:576-77, 1956. ACKNOWLEDGMENTS 18. AMBROSE,E. J., and JONES, P. C. T. Studies of Cell \Ve should like to thank Professor A. Haddow, F.R.S., for Movements Using the Surface Contact Microscope. Sur face Contact Microscopy: Studies in Cell Movements. his interest in this work. One of us (M.A.) wishes to acknowl Med. & Biol. 111.,11:104-10, 1961. edge the support of a Research Grant (C-4847) from the Na tional Cancer Institute, Public Health Service of the United 19. AMBROSE,E. J.; JONES, P. C. T.; and LEVENE,A. L. States. One of us (E.J.A.) has been supported by grants to the Interference Microscopy of Normal and Tumour Cells in Chester Beatty Research Institute (Institute of Cancer Re Tissue Culture. CinÃ©FilmShown at Vllth Int. Cancer Congress London, 1958. search: Royal Cancer Hospital) from the Medical Research Council, the British Empire Cancer Campaign, the Anna 20. AMBRUS,J. L.; AMBRUS,L. M.; BYRON,J. W.; GOLD Fuller Fund, and the National Cancer Institute of the National BERG,M. E. ; and HARRISON,J.W. E. Study of Metastasis with Aid of Labelled Ascites Tumour Cells. Ann. N.Y. Institutes of Health, U.S. Public Health Service. Acad. Sci., 63:938-61, 1956. 21. AREY,L. B. Wound Healing. Physiol. Rev., 16:327-406, REFERENCES 1936. 1. ABEKCHOMBIE,M.Localised Formation of New Tissue in 22. BANGHAM,A.D.; PETHICA,B. A.; and SEAMAN,G.V. F. an Adult Mammal. Symp. Soc. Exp. Biol., 11:235-54, Charged Groups at the Interface of Some Blood Cells. 1957. Biochem. J., 69:12-19, 1958. 2. . Exchanges between Cells. A Symposium on the 23. BIRBECK,M. S. C., and MERCER,E. H. Cell Membranes Chemical Bases of Development. Baltimore: Johns Hop and Morphogenesis. Nature, London, 178:985-86, 1956. kins Press, 1958. 24. BEEBE, S. P. The Chemistry of Malignant Growths, II, 8. . The Bases of the Locomotory Behaviour of Fibro The Inorganic Ion Constituents of Tumours. Ann. J. blasta. Exp. Cell Research, Suppl. 8, pp. 188-98, 1961. Physiol., 12:167-72, 1904-5. 4. ABERCROMBIE,M., and AMBROSE,E. J. Interference 25. BENNETT,L. R., and CANNON,F. E. Effects of Lytic Microscope Studies of Cell Contacts in Tissue Culture. Agents on Plasma Membranes of Ehrlich Ascites Tumour Exp. Cell Research, 15:332-45, 1958. Cells and Mouse Erythrocytes. J. Nat'l. Cancer Inst., 5. ABERCROMBIE,M.;EVANS,D. H. L.; and MURRAY,J. G. 19:999-1011, 1957. Nuclear Multiplication and Cell Migration in Degenerat 26. BERWICK,K.L. A Comparison of Surface Ultra-structure

of Normal Papillomas and Carcinomatous Epidermal sium and Calcium Content of Carcinomas and Papillomas Cells. Cancer Research, 19:858-55, 1959. of Colon. Cancer Research, 6:233-34, 1946. 27. BOSCH,L.The Release of Enzymes from Ascites-Tumour 50. EASTY,G. C., and AMBROSE,E.J. The Antigenic Com Cells into the Extra Cellular Medium. Biochim. et Bio- position of Mouse Ascites Tumour Cells Using in Vitro phys. Acta, 30:444-45, 1958. and Gel Diffusion Techniques. Brit. J. Cancer, 11:287-95, 28. BRUNCHWIG,A.;DUNHAM,L.J.; and NICHOLS,S.Potas 1957. sium and Calcium Content of Gastric Carcinoma. Cancer 51. EASTY,G. C.; EASTY,D. M.; and AMBROSE,E.J. Studies Research, 6:296-97, 1946. of Cellular Adhesiveness. Exp. Cell Research, 19:539-47, 29. CARREL,A.,and EBELING,A.H. The Fundamental Prop 1960. erties of Fibroblasts and Macrophages. III. The Malig- 52. EASTY,G. C., and MERCER,E. H. An Electron Micro nant Fibroblast of Sarcoma 10of the Crocker Foundation. scope Study of the Surface of Normal and Malignant J. Exp. Med., 48:105-23, 1928. Cells in Culture. Cancer Research, 20:1608-13, 1960. 80. CATALANO,P.;NOWELL,P.; BERWICK,L.; and KLEIN,G. 53. EASTY,G. C., and MUTOLO,V.The Nature of the Inter Surface Ultrastructure of Tumour Sublines Differing in cellular Material of Adult Mammalian Tissues. Exp. Cell Adhesiveness. Exp. Cell Research, 20:634-35, 1960. Research, 21:374-85, 1960. 31. CHRISTENSEN,L.K. Trypsin Splitting and Denaturation 54. ENGSTRÃ–M,A.,and FINEAN,J. B. Biological Ultra-struc of 0-Lactoglobulin. Nature, London, 163:1003-4, 1949. ture. New York: Academic Press, Inc., 1958. 32. CLARK,E. R., and CLARK,E. L. Growth and Behaviour 55. EHICKSON,B. N.; WILLIAMS,H. H.; BERNSTEIN,S. S.; of Epidermis as Observed Microscopically in Observation ARVIN,I.; JONES,R. L.; and MACY,I. C. The Lipid Dis Chambers Inserted in the Ears of Rabbits. Am. J. Anat., tribution of Posthemolytic Residue or Stroma of Erythro- 93:171-219, 1953. cytes. J. Biol. Chem., 122:515-28, 1938. 33. CLOWES,G. H. A., and FHISBIE,W. S. On the Relation- 56. ESSNER,E.; SATO,H.; and BELKIN,M. Experiments on ship between the Rate of Growth, Age and Potassium and an Ascites Hepatoma. I. Alkaline Degradation of the Calcium Content of Mouse Tumours (Adenocarcinoma, Cementing Substance and Digestion and Separation of Jensen). Am. J. Physiol., 14:173-92, 1905. Cells in Tumour. Exp. Cell Research, 7:430-37, 1954. 34. COMAN,D. R. Decreased Mutual Adhesiveness, a Prop- 57. FINEAN,J. B. Further Observations on the Structure of erty of Cells from Squamous Cell Carcinomas. Cancer Myelin. Exp. Cell Research, 6:202-15, 1953. Research, 4:625-29, 1944. 58. FISHER,A.; LASER,H.; and MEYER, H. WechselbezieÂ« 35. . Mechanisms Responsible for the Origin and Dis hungen zwischen normalen und bÃ¶sartigen Gewebe. tribution of Blood-borne Tumour MÃ©tastases:A Review. Ztschr. Krebsforsch., 29:270-301, 1929. Ibid., 13:397-404, 1953. 59. FOULDS,L.The Experimental Study of Tumour Progres 36. . Reduction in Cellular Adhesiveness upon Contact sion: A Review. Cancer Research, 14:327-39, 1954. with a Carcinogen. Ibid., 20:1202-4, 1960. 60. GALLACHER,C.H.; GUPTA,D. N.; JUDAH,J. D.; and 37. COMAN,D. R., and ANDERSON,T. F. A Structural Dif REES, K. R. Biochemical Changes in Liver in Acute ference between the Surfaces of Normal and of Car Thioacetamide Intoxication. J. Pathol. Bact., 72:193- cinomatous Epidermal Cells. Cancer Research, 16:541- 201, 1956. 43, 1955. 61. GEREN,B. B. The Formation from the Schwann Cell 38. CORMACK,D.H.; EASTT,G. C.; and AMBROSE,E.J. In Surface of Myelin in the Peripheral Nerves of Chick Em teraction of Enzymes with Normal and Tumour Cells. bryos. Exp. Cell Research, 7:558-62, 1959. Nature, 190:1207-8, 1961. 62. GEY, G. D. Some Aspects of the Constitution and Be 39. COWDRY,E.V. Properties of Cancer Cells. Arch. Pathol., haviour of Normal and Malignant Cells Maintained in 30:1245-74, 1940. Continuous Culture. Harvey Lecture, 1954-55, pp. 154- 40. CURTIS,A.S. G. Cell Contacts, Some Physical Considera 229, 1956. tions. Am. Naturalist, 94:37-56, 1960. 63. GEY,G. D. ; SHAPIRO,P.; and BORYSKO,E.Activities and 41. DALTON,H. C. Inhibition of Chromoblast Migration as a Responses of Living Cells and Their Components as Factor in the Development of Genetic Differences in Pig Recorded by Cinephase Microscopy and Electron Mi mentation in White and Black Axolotls. J. Exp. Zool., croscopy. Ann. N.Y. Acad. Sci., 68:108-9, 1954. 115:151-74, 1950. 64. GOLDACRE,R.J. The Action of Several Anaesthetics and 42. DANIELLI,J. Protein Films at the Oil-Water Interface. the Mechanisms of the Response of Touch. Symp. Soc. Cold Spring Harbor Symp., 6:190-95, 1938. Exp. Biol., 6:128, 1952. 43. DANIELLI,J., and HARVEY,E. H. The Microscope Cen- 65. GOLDACBE,R. J. Properties of Biological Membranes. trifuge and Some of Its Applications. J. Franklin Inst., (General Discussion.) Faraday Soc. Dis. No. 21, p. 275, 214:1,1932. 1956. 44. DAVSON,H., and DANIELLI,J. F. The Permeability of 66. GOLDACRE,R.J., and BEAN,A. D. A Model for Morpho Natural Membranes. Cambridge: University Press, 1943. genesis. Nature, London, 186:294-95, 1960. 45. DAWSON,R.M. C.; REMINGTON,N.;and LINDSAY,D.B. 67. GOLDACRE,R.J., and LORCH,I. J. Folding and Unfolding The Phospliolipids of the Erythroeyte 'Ghosts' of Various of Protein Molecules in Rektion to Cytoplasmic Stream Species. Biochem. J., 77:226-30, 1960. ing, Amoeboid Movement, and Osmotic Work. Nature, 46. DE GRIER, J., and VANDEENEN, L.L.M. Some Lipid London, 166:497-500, 1950. Characteristics of Red Cell Membranes of Various Ani- 68. GREEN, H. N. The Immunologies! Theory of Cancer. mal Species. Biochim. et Biophys. Acta, 49:286-96, 1961. Acta UniÃ³.Internat, contra cancrum, 17:215-33, 1961. 47. DELONG,R. P.; COMAN,D.R.; and ZEIDMAN,I.The Sig- 69. HAKIM,A. A. Crystallisation of Antigenic Phospholipid nificance of Low Calcium and High Potassium Content in from Human Tumours. Naturwissenschaften, 46:84-85, Neoplastic Tissues. Cancer Research, 3:718-21, 1950. 1959. 48. DREW,A. H. A Comparative Study of Normal and Ma- 70. HANES,F. M., and Lambert, R. A. AmÃ¶boideBewegung lignant Tissues Grown in Artificial Culture. Brit. J. Exp. en von Krebszellen als ein Faktor des invasiven und Pathol., 3:20-27, 1922. metatastischen Wachstums maligner Tumoren. Virch. 49. DUNHAM,L.J.; NICHOLS,S.;and BRONSCHWIG,A.Potas Arch, path Anat. Physiol., 209:12-21, 1912.

71. HAHVEY,E. H. A Determination of the Tension at the 95. LASH,J. W. Studies on Wound Closure in Urodeles. J. Surface of Eggs of the Sea Urchin. Biol. Bull., 60:67; The Exp. Zool., 128:13-28, 1955. Tension of the Surface of Marine Eggs Especially of the 96. LEIGHTON,J. Aggregate Replication, a Factor in the Sea Urchin Arbacia. Ibid., 61:273, 1932. Growth of Cancer. Science, 129:466-67, 1959. 72. i IM.I.Iii: J., and HOFFMANN,J.F. On the Ultrastructure 97. LEIGHTON,J.; KALLA,R. L.; KLEIN, I.; and BELKIN,M. of the Plasma Membrane Determined by the Electron Pathogenesis of Tumour Invasion. I. Interaction between Microscope. J. Cell & Comp. Physiol., 42:203-48, 1953. Normal Tissues and Transformed Cells in Tissue Culture. 73. HODGKIN,A.L.; HUXLEY,A.F.; and KATZ,B. Ionic Cur Cancer Research, 19:23-27, 1959. rents Underlying Activity in the Giant Nerve Axon of the 98. LEIGHTON,J.; KLINE, I.; BELKIN,M.; and TETENBAUM, Squid. Arch, des Sci. Physiol., 3:129-50, 1949. Z. Studies on Human Cancer Using Sponge-Matrix Tissue 74. HÃ–GMAN,C.F. Blood Group Antigens of Human Cells in Culture. III. The Invasion Properties of a Carcinoma Tissue Culture. Exp. Cell Research, 21:137-43, 1960. (Strain HÃ©la)asInfluenced by Temperature Variations, 75. HOLTFRETER,J. A Study of the Mechanics of Gastrula- by Conditioned Media, and in Contact with Rapidly tion. R. I. J. Exp. Zool., 94:261-318, 1943. Growing Chick Embryonic Tissue. J. Nat'l. Cancer Inst., 76. . A Study of the Mechanics of Gastrulation. Pt. II. 16:1353-65, 1956. Ibid., 96:171-212, 1944. 99. LEVI,G. Conservazione e perdita deirindependenza delle 77. . Experimental Studies on the Development of cellule dei tessuti. Arch. exp. Zellforsch., 1:1-57, 1925. Pronephros. Rev. CaÃ±ad.Biol., 3:220-50, 1944. 100. LEWIS,W. H. Normal and Malignant Cells. Science, 81: 78. . Significance of the Cell Membrane in Embryonic 545-53, 1935. Processes. Ann. N.Y. Acad. Sci., 49:709-60, 1948. 101. LIBENSON,L.,and JENA,M. Catheptic Activity of Blood 79. HOLTZER,H., and HOLTZER,S. The in Vitro Uptake of Serum in Cancer. Cancer, 10:1004-7, 1957. Fluorescein Labelled Plasma Proteins; I. Mature Cells. 102. LUDFORD,R. J. Differences in the Growth of Trans- Compt. Rend. Trav. Lab. Carlsberg, 31:373-402, 1960. pljintable Tumours in Plasma and Serum Culture Media. 80. HUGHES,P. E.; Louis, C. J.; DUNEEN,J. K.; and SPEC- Proc. Roy. Soc., B, 112:250-63, 1932. TOR,W. G. Role of Organ Specific Antigens during 4- 103. LUDFOHD,R.J., and BARLOW,H.The Influence of Malig Methylaminoazobenzene Carcinogenesis in the Rat Liver. nant Cells upon the Growth of Fibroblasts in Vitro. Can Nature, 180:289-90, 1957. cer Research, 4:694-703, 1944. 81. HUXLEY,J. Cancer Biology, Viral and Epigenetic. Biol. 104. LUND,E. J. Bioelectric Fields and Growth. Austin: Uni Rev. (Cambridge), 32:1-87, 1957. versity of Texas Press, 1947. 82. HUXLEY,J. S., and DEBEER,G. R. The Elements of Ex 105. MALMGRÃ‰N,H.;SYLVÃ‰N,B.;and RÃ‰VESZ,L.Catheptic perimental Embryology. Cambridge: University Press, and Dipeptidase Activities of Ascites Tumour Cells. Brit. 1934. J. Cancer, 9:473-79, 1955. 83. INGEBR:GTSEN,R.A Contribution to the Biology of Pe 106. MERCER,E. H. Reported in Biology of Cancer, E. J. ripheral Xerves in Transplantation. II. Life of Peripheral AMBROSE(ed.), British Association for Advancement of Nerves of Mammals in Plasma. J. Exp. Med., 23:251-64, Science, 57:473-79, 1958. 1916. 107. MITCHISON,M.,and SWANN,M.,The Mechanical Proper 84. JONES,BRYNM. Some Studies Relating to the Problem ties of the Cell Surface. I. The Cell Elastimeter. J. Exp. of Cellular Adhesion. Proc. Roy. Phys. Soc. Editi., 28: Biol., 31:443-60, 1954. 85-42, 1960. 108. . The Mechanical Properties of the Cell Surface. 85. JONES, L. O. In Vitro Studies on the Effect of Spleen, II. The Unfertilised Sea Urchin Egg. Ibid., 31:461-72, Striated Muscle and Kidney upon the Growth of Sarcoma 1954. 180 and Mammary Carcinoma of Mice. Cancer Research, 9:27-34, 1949. 109. MOSCONA,A.The Development in vitro of Chimeric Ag gregates of Dissociated Embryonic Chick and Mouse 86. KAUTZ,J. Differential Invasion of Embryonic Chick Tis Cells. Proc. Nat. Acad. Sci., 43:184-93, 1957. sues by Mouse Sarcoma 180 and 37. Cancer Research, 12:180-87, 1952. 110. MOYER,L. S. Electrokinetic Aspects of Surface Chem istry. I. The Electrophoresis of Adronbed Albumin. J. 87. KASAKI,T.; IKODA,T.; KATANI,Y.; NAKAGAWA,S.;and Phys. Chem., 42:71-83, 1938. SAKA,T. A New Phospholipid, Malignolipin, in Human Malignant Tumours. Science, 127:1176-77, 1958. 111. NAIRN,R. C.; RICHARD,H. G.; MCENTEGARD,M.G.; 88. KAY,H. E. M. A and B Antigens of Normal and Malig and FOTHERGILL,J. E. Immunological Ditferences be nant Cells. Brit. J. Cancer, 11:409-14, 1957. tween Normal and Malignant Cells. Brit. M. J., 2:1335- 89. KEBUS,K.; GERNER,B. \V.; and COOMBS,R.R. A. Blood 40, 1960. Group Antigens on Hela Cells by Mixed Agglutination. 112. NEVE,A.; DEVRIES,A.; and KATCHALSKY,A.Interaction Immunology, 2:262-67, 1959. of the Basic Polyamino Acids with the Red Blood Cells. I. 90. KENT,H. H., and GEY,G. O. Changes in Serum Proteins Combination of Polylysine with Single Cells. Biochim. et during Growth of Malignant Cells in Vitro, Proc. Soc. Biophys. Acta, 17:538, 1955. Exp. Biol. & Med., 94:205-8, 1957. 113. NOWELL,P.C., and BERWICK,L.The Surface Ultrastruc 01. KEY, J. A. Studies on Erythrocytes with Special Refer ture of Normal and Leukemic Rat Lymphocytes. Cancer ence to Reticulum, Polychrornatophilia, and Mitochon Research, 18:1067-69, 1958. dria. Arch. Int. Med., 28:511-49, 1921. 114. OVERTON,E. Ãœberdie osmatischen Eigenschaften der 92. KLEIN, G. The Nature of Mammalian Lymphosarcoma lebenden Pflanzen und Tierzellen. Vjschr. Naturf. Ges. Transformation by Isolated Chromatid Fractions. Cancer ZÃ¼rich,40:159,1895. Research, 12:589, 1952. 115. RAPPORT,M. L.; GRAF,L.; SKIPSKI,V. P.; and ALONZO, 93. KLEIN, G., ., i KLEIN, E. Conversion of Solid Neo N. F. Immunological Studies of Organ and Tumor Lipids. plasms into Ascites Tumours. Ann. N.Y. Acad. Sci., 63: Iv. Isolation and Properties of Cytolipin H. Cancer, 12: 640-65, 1956. 438-45, 1959. 94. KREDEL,F. The Physical Relations of Cells in Tissue Cul 116. ROBERTSON,J.D. Experimentally Induced Variations in tures. Bull. Johns Hopkins Hosp., 40:216-27, 1927. the Contact Relationships of Axon and Schwann Cell

Membranes Compared with Similar Variations Occurring Stability of Lyophobic Colloids. Amsterdam: Elsevier, during Growth. J. Physiol., 142:9-11, 1958. 1948. 117. ROGERS,G. E. Electron Microscope Studies of Hair and 134. WEBEB,H. H. The Motility of Muscle and Cells. Cam Wool. Ann. H. V. Acad. Sci., 83:378-99, 1959. bridge: Harvard Univ. Press, 1958. 118. ROTRSTEIN,A. Enzymology of the Cell Surface. Proto- 135. WEILER,E. Antigenie Differences between Normal Ham plasmatalogia, 2:E4. Vienna: Springes, 1954. ster Kidney Carcinoma and Studies of Kidney Carcinoma 119. SACHS,L., and MEDINA,D. In Vitro Transformation of Complement Fixation Reactions with Cytoplasmic Par ticles. Brit. J. Cancer, 10:553-59, 1956. Normal Cells by Polyoma Virus. Nature, London, 189: 457-58, 1961. 136. WEISS,L. Studies of Cellular Adhesion in Tissue Culture. The Effect of Serum. Exp. Cell Research, 17:499-507, 120. SANTESSON,L.Characteristics of Epithelial Mouse Tu mour Cells in Vitro and Tumour Strains in Vivo. Acta 1959. Pathol. Microbiol. Scandinav. (Suppl.), 24:1-257, 1935. 137. -â€”â€¢â€”.Studiesof Cellular Adhesion in Tissue Culture. 121. SCHNEIDER,N. Sur les possibilitÃ©sdepropagation d'un II. The Adhesion of Cells to Gel Surfaces. Ibid., 17^OS- IS, 1959. sarcome de souris sur des organes embryonaires de poulet 138. WEISS,P. The Problem of Specificity in Growth and De ÃdiffÃ©rentsstatesdu dÃ©veloppement.Arch.Anat. microsc. velopment. Yale J. Biol. & Med., 19:235-78, 1947. et Morphol. expÃ©r.,47:573-60-1,1958. 139. . Nerve Patterns: the Mechanics of Nerve Growth. 122. SIMMS.H. S., and STILLMAN,N.P. Substances Affecting Growth (Suppl.), 5:163-203, 1941. Adult Tissues in Vitro. I. The Stimulating Action of Tryp- 140. . Some Introductory Remarks on the Cellular As sin on Fresh Adult Tissue. J. Gen. Physiol., 20:603, 1937. pects of Differentiation. J. Emb. Exp. Morph., 1:181-211, 123. SIMON,D. Contribution Ã l'Ã©tudedela circulation et du 1953. transport des gonocytes primaires dans les blastodermes 141. . "Attraction Fields" between Growing Tissue d'oiseau cultures in vitro. Arch. Anat. microsc. et Mor Cultures. Science, 116:293-96, 1952. phol. expÃ©r.,49:93-176, 1960. 142. . Cell Contact. Internat. Rev. Cytol., 7:391-423, 124. SMITH, G. M. Morphological Changes in Tissue with 1958. Changes in Environment; Replacement of Surface Epi 143. WEISS, P., and GARBER,B. Shape and Movement of thelium of Grafted Tissues by Adjacent Epithelium. J. Mesenchyme Cells as Functions of the Physical Structure Med. Research, 28:423-39, 1913. of the Medium. Contributions to a Quantitative Morphol 125. SWANN,M. M. The Control of Cell Division, A Review. ogy. Proc. Nat. Acad. Sci., 38:264-80, 1952. II. Special Mechanisms. Cancer Research, 18:1118-60, 144. WESTROPP,J.W., and GREEN,H. N. Binding of Hepato- 1958. carcinogen to a Structural Lipoprotein of Rat Liver; Its 126. SYLVÃ‰N,B.Protein Content and Enzymatic Assays of Bearing on the ImmunologieÂ«]Theory of Cancer. Nature, Interstitial Fluid from Normal Tissues and Transplanted London, 186:350, 1960. Mouse Tumours. Cancer Research (in press). 145. WILLIAMS,H. H.; EHICKSON,B. N.; and MACY,I. G. 127. SYLVÃ‰N,B.,and Bois, I. Protein Content and Enzymatic Chemical Structure of Red Blood Cells. Quart. Rev. Assays of Interstitial Fluid from Some Normal Tissues Biol., 16:80, 1941. and Transplanted Mouse Tumours. Cancer Research, 146. WILLIS, R. A. The Spread of Tumours in the Human 20:831-36, 1960. Body. London: Butterworth & Co., 1952. 128. TAYLOR,A.C. Attachment and Spreading of Cells in Cul 147. WOLFF,E., and SCHNEIDER,N.La culture d'un sarcome ture. Exp. Cell Res. (Suppl.), 8:154-73, 1961. de souris sur des organes de poulet explantÃ©esin vitro. 129. TEMIN,H. M., and RUBIN,H. Characteristics of an Assay Arch. Anat. micr. Morph. expÃ©r.,46:173-97, 1957. for linn- Sarcoma Virus and Rous Sarcoma Cells in Tissue 148. WOLFF,E., and WOLFF,E. Les rÃ©sultatsd'une nouvelles Culture. Virology, 6:669-88, 1958. mÃ©thodedeculture de cellules cancÃ©reusesin vitro. Rev. 130. TWITTY,V. C., and Niu, M. C. The Motivation of Cell Franc. Ã‰tudesClin,et Biol., 3:945-51, 1958. Migration Studied by Isolation of Embryonic Pigment 149. WOOD, S. Pathogenesis of Metastasis Formation Ob Cells Singly and in Small Groups in Vitro. 3. Exp. Zool., served in Vitro in the Rabbit Ear Chamber. Arch. Pathol., 126:541-74, 1954. 66:550-68, 1958. 131. VANDENTEMPEL,M. Distance between Emulsified Oil 150. YOUNG,J. S. The Invasive Growth of Malignant Tu Globules upon Coalescence. J. Coll. Sci., 13:125-33, 1958. mours; an Experimental Interpretation Based on Elastic 132. VEERKAMP,J. H.; MULDER, I.; and VAN DEENEN, Jelly Models. J. Path. Bact., 77:321-39, 1959. L. L. M. Comparative Studies on the Phosphatides of 151. ZEIDMAN,I. Metastasis; a Review of Recent Advances. Normal Rat Liver and Primary Hepatoma. Ztschr. Cancer Research, 17:157-62, 1957. Krebsforsch., 64:137^8, 1961. 152. ZEIDMAN,L,and Buss, J. M. Transpuhnonary Passage of 133. VERWEY,E.J. W., and OVERBEEK,J.T. G. Theory of the Tumour Cell Emboli. Cancer Research, 12:731-33, 1952.

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