Biochimica et Biophysica Acta, 417 (1975) 211-236 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 87020
INFLUENCE
OF GEOMETRY
ON CONTROL
OF CELL
GROWTH
J U D A H F O L K M A N and HARVEY P. G R E E N S P A N
Department of Surgery, Children's Hospital Medical Center and Harvard Medical School, 300 Longwood Avenue, Boston, Mass. and the Department of Mathematics, Massachusetts Institute of Technology, Cambridge, Mass. (U.S.A.) (Received October 15, 1975)
CONTENTS I.
Introduction
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
211
1I. Effect of geometry on cell proliferation . . . . . . . . . . . . . . . . . . . . . . A. Untransformed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. In vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. In vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Implantation of glass beads . . . . . . . . . . . . . . . . . . . . . . . b. Foreign body carcinogenesis . . . . . . . . . . . . . . . . . . . . . . B. Transformed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. In vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. In vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
212 212 212 214 214 214 216 216 222
IlL Effect of geometry on gene expression . . . . . . . . . . . . . . . . . . . . . . A. Untransformed cells (in vitro and in vivo) . . . . . . . . . . . . . . . . . . . B. Transformed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
228 228 231
IV. Theoretical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . .
231
V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
234
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
234
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
235
I. I N T R O D U C T I O N It is c l e a r t h a t cell g r o w t h is r e g u l a t e d at m a n y levels.
A few e x a m p l e s are,
s e r u m g r o w t h f a c t o r s , c e l l u l a r cyclic n u c l e o t i d e s , a n d o x y g e n gradients. O u r k n o w l e d g e o f these c o n t r o l p o i n t s is still so r u d i m e n t a r y t h a t it is p r e m a t u r e to assign p r i o r i t y , o r e v e n to d i s t i n g u i s h p r i m a r y a n d s e c o n d a r y e v e n t s in the r e g u l a t i o n o f cell growth. H o w e v e r , t h e r e are m a n y s i t u a t i o n s w h e r e t h e g e o m e t r y o f a n i n d i v i d u a l cell o r the g e o m e t r i c a l c o n f i g u r a t i o n o f a p o p u l a t i o n o f cells, s e e m to exert a p r o f o u n d influence o n g r o w t h a n d d i f f e r e n t i a t i o n .
A t t i m e s the g e o m e t r i c a l effect a p p e a r s to
212 override all other growth signals. The shape of a single diploid cell at any given time may be the final determinant of whether or not that cell will be permitted to divide, regardless of what other mitogenic factors are present. Furthermore, the geometrical arrangement of a population of tumor cells may have a profound effect on the growth rate of that population. In both the normal and the malignant state, geometry can play a significant role in proliferative capacity. We will assemble what bits and pieces of information are available in the hope that a pattern of geometrical effects on cell growth will become discernible. Some of the evidence quoted here has been around for a long time, such as the well known fact that untransformed diploid cells will not grow in suspension. Other evidence has come only recently from experiments in foreign body carcinogenesis, and also from studies of tumor angiogenesis.
II. E F F E C T OF G E O M E T R Y ON CELL PROLIFERATION
(A ) UntransJormed Cells (Diploid) (1) In Vitro When diploid cells or untransformed cells, such as mouse fibroblasts, are plated on coverslips of plastic or glass, they stick and flatten out. They proliferate until confluence is reached. By contrast when the same cells are freely suspended, either in soft agar, methyl cellulose, or in spinner culture, they fail to proliferate [1,2]. While in suspension culture, some of these cells incorporate [3H]thymidine. Most double their volume in the first 24 h, and a few may go through one cell division, but there is no further proliferation. BHK21 cells studied in suspension by Stoker [2], remained viable for up to 7 days and retained their competence for proliferation when returned to the fiat state. When these cells were returned to a plastic surface, mitotic figures appeared after 17 h and proliferation followed. All of them showed [3H]thymidine incorporation. BHK21 cells are not as normal as for example primary cultures of fibroblasts in early passage [3]. They may have passed through some genetic changes on the way to transformation. Their inability to grow in suspension can be overcome slightly with very high serum concentrations [4]. However, serum concentrations as high as 50 ~o initiated DNA synthesis in only 12 ~,, of the suspended cells, and stimulated the formation of only a few colonies. Thus, even in the presence of high serum there is a marked difference between the response of fiat cells,compared to suspended cells. The attached or flattened cells are 60 times more sensitive to the stimulating effect of serum than are suspended cells (Fig. 1). The major difference between the inhibited cells and their proliferating counterparts is their shape. Cells in suspension are rounded or spheroidal; cells attached to plastic are fiat and spread out. Inhibition of growth in the spheroidal state is characteristic of the majority of normal (untransformed) fibroblasts and epithelial cells. Cells of hematopoietic origin are exceptions and can proliferate while spheroidal and in suspension culture.
213 a
I00
LIJ
J J
t !
90~ 80,
Surface
-J
70 ~
z
5O
~ 3o ~- 20 13-
•
7.
Suspension XL
--X-
- -X
O ', ' ' ' ' ' ' X ~ X ,
O
5
IO
20
q
30 50
SERUM CONCENTRATION (per cent)
Fig. 1. Response of BHK21 fibroblasts in layer culture and in Methoce! suspension culture, after exposure to increasing concentrations of serum. Cells were labelled with [aH]thymidine. From Clark et al. [4].
Diploid cells whose growth is inhibited in suspension culture, can escape this inhibition if they are permitted to stretch out on glass fibrils or flatten out on glass beads suspended in soft agar or methyl cellulose. Glass beads 75 ffm in diameter allowed ample room for cell attachment and proliferation into colonies. Silica particles averaging 5 ffm in diameter seemed to provide insufficient surface area for cell spreading and proliferation [2]. It is of interest that the mean area utilized by a normal mouse fibroblast in the flat state is approximately 2 ' 103 ffm 2 [5]. A bead of 75 # m diameter has a surface area of approximately 1.7" l04 ffm 2, enough to accommodate up to 8 cells, but a bead of 5 # m has a surface area of only 7.8 • 10 u m 2, not enough for the spreading of even one cell. Glass fibrils up to 500 # m in length also acted as rafts to which normal cells attached and proliferated while the fibrils themselves were floating in methyl cellulose. In similar studies [6], collagen fibrils have been used to support fibroblast growth in suspension culture, and Horng and McLimans [7] used sephadex beads in suspension, to support growth of pituitary cells. Stoker has applied the term "anchorage dependence" to this phenomenon, implying that an untransformed cell must anchor itself to a rigid surface before it can divide. This concept appears to be supported by Boone's recent demonstration [8], that 3T3 cells which grow well on plastic, will not grow on teflon, presumably because they cannot stick. However, Stoker's B H K cells, freely suspended, were able to go through one division without anchorage, yet could not proliferate further. This suggests a block in the phase during preparation for cell division. Thus, anchorage may be only one component of the flat state which is prerequisite for cell proliferation. Other components must be considered. Plasma membrane surface area is one possible component. When a normal cell changes from spheroidal to flat shape, its total surface area may increase approximately ten-fold. [Assume a cell of 13 ffm diameter
214 in suspension. Its surface area (as a sphere) is approximately 527 #m 2. If a mouse fibroblast when plated [5] can occupy 2600 # m 2, then its total plasma membrane surface area would be approximately 5200 #m2.] Another feature of the fiat state may be an increase in permeability of the cell membrane in comparison to the spheroidal state. Furthermore, when a cell changes from the spheroidal to the flat configuration, a variety of receptors may change position [9]. Finally, the net surface tension distributed over the plasma membrane of a spheroid cell is much greater than in a flat cell. All five phenomena may work in some cooperative fashion, or one among them may be the critical variable, or some other as yet unrecognized feature of the flat state may be important (See Theoretical Considerations).
(2) In Viro Many phenomena in tissue culture have no counterpart in vivo. However, the general rule that flatness permits proliferation in a normal cell while roundness inhibits it, does have at least two analogues in experimental animals. One of these is tumor production by implantation of cells coated on glass beads, and the other is foreign body carcinogenesis. (a) Implantation of glass beads. Balb/3T3 cells proliferate as a monolayer on plastic or glass surfaces in vitro but will not grow in suspension [2]. Neither will they form tumors when injected subcutaneously into histocompatible mice. However, Boone [10] showed that tumors appeared regularly if 3T3 cells were allowed to attach to glass beads (3 m m diameter) before implantation. The inoculation of cells alone or beads alone produced no tumors. All mice receiving beads coated with cells developed tumors within 8 weeks. Cells from these tumors were capable of producing lethal tumors on subsequent transplantation in the absence of glass beads. This implies that the original 3T3 cells were not completely transformed and not tumorigenic by themselves prior to the first implantation on glass beads. However, attachment to glass beads permitted them to take the final step necessary to be a successful tumor. Again, we are unable to escape the conclusion that the flat state was an essential feature contributing to the appearance of a clone of neoplastic cells. (b) Foreign body carcinogenesis (smooth surface carcinogenesis). It has been known since 1941 [11] that implantation of a variety of smooth surfaces into mice arid rats will induce sarcomas. Glass, plastic and steel all have a similar effect. This subject has been well reviewed [12,13]. In the usual experiment, a plastic or glass coverslip is implanted subcutaneously and a sarcoma appears at the site after a long latent period, i.e., approximately 9 months. Several conclusions are convincingly established. Physical form is important; chemical composition is not. A variety of polymer sheets and films will produce tumors, but powders from these materials will not. Surface area is important. Statistical evaluation indicates a direct relationship between tumor incidence and surface area. Discs of less than 1 c m 2 rarely induce a tumor while discs of 2 cm 2 or greater, are highly carcinogenic [14]. Furthermore, tumor incidence decreases and eventually reaches zero when implants are perforated with holes in increasing number or with increasing diameter. Also,
215
SMOOTH SURFACE CARCINOGENESIS
T6
~Jr:,,
Fig. 2. Fibroblast-like cells spread out and proliferate on plastic film implanted into CBA/H-T6 mouse. When film is later transferred to a CBA/H mouse, the tumor which develops is from the T6 donor. This is our diagrammatic summary of Brand's data [16].
the fibrous capsule which forms around the implant, i.e., the foreign body reaction, is important, possibly as a source of the cells which will eventually be involved in carcinogenesis. If the fibrous capsule is removed early, along with the implant, tumors will not develop. On the other hand, tumors may still develop if only the implant is removed late in the course. Perhaps the most elegant studies in regard to etiology have been done by Brand and his co-workers [15,16]. They used two CBA mouse strains differing only by the marker gene T 6. After implantation of a plastic surface, clones of cells which later became attached to the film, began to proliferate. When the film was transplanted to the mouse of different genetic make-up, the tumor which arose was always of the donor type. (Fig. 2). Here again is a strong analogy to fiat tissue culture. Fibloblastlike cells move from the fibrous capsule and spread out on the plastic and continue to proliferate. Brand suggests that these cells are not true fibroblasts, but may derive from pericytes or endothelial cells of the microcirculation. At this writing their origin remains unknown. The pre-neoplastic cells stick to the plastic film and are carried over to the recipient animal. Burnet [17] has proposed that the continuous proliferation which occurs on the plastic promotes the selection of an intrinsic mutant which then accumulates as a clone of neoplastic cells. Recently, Buoen et al. [18] have shown that pre-neoplastic cells can be recovered from the plastic film at an early stage after implantation, and grown in tissue culture. After such a clone was expanded in vitro, cells were reimplanted into recipient animals
216 of the T6 different partner. Again, the resultant tumor had the gene marker of the donor animal. The latent period before the appearance of tumors in recipients, varied in relation to the number of passages in vitro, and also with the method of implantation. Most intriguing was the observation that cells from early culture passages, which were attached to plastic films at the time of implantation, reached neoplastic autonomy earlier than did the same cells inoculated into the animals as a suspension or pellet. This is reminiscent of the story of 3T3 cells on glass beads. So-called "pre-neoplastic" cells which have not yet acquired all the characteristics necessary to produce a tumor when inoculated as a pellet of free cells, can grow into a tumor when implanted while attached to a smooth surface. Another example of foreign body carcinogenesis is observed after exposure to asbestos. A large smooth surface is not necessary; fibers of appropriate length are sufficient. Asbestos fibers produce malignant mesotheliomas in man and in experimental animals. Maroudas et al. [19] showed that asbestos fibers from 20-320/~m in length produced mesotheliomas after injection into the pleural cavities of rats. Also BHK21 and 3T3 fibroblasts in suspension culture, attached themselves to glass fibers of this size and proliferated. Maximum growth was obtained on lengths of 200 # m or greater; no growth occurred below 20 #m. Also, fibers less than 20 #m did not cause mesotheliomas. Glass and aluminum oxide fibers similar to the long asbestos fibers, also produced mesotheliomas and supported fibroblast growth in suspension culture. An inconsistency appears to exist when plastic (or glass) film carcinogenesis is compared to carcinogenesis by asbestos or glass fibers. In the former, a large smooth area is necessary, while in the latter, only fiber length is important. This problem can be resolved by considering the large area of film as necessary for the generation of enough clones from proliferating cells, to permit one or more to reach the "preneoplastic" state during the lifetime of the animal. On this statistical basis, the number of fibers is as important as the area of film. A very low number of glass or asbestos fibers would probably not cause a tumor for the same reason that a small area of film would not be adequate. However, from the perspective of the individual cell, it appears not to matter exactly how a "flat" configuration is achieved. Whether the fibroblast is spread out in two dimensions, or stretched out in one dimension, (i.e. extended), the signal is the same: permission to proliferate. In fact, isolated fibroblasts in agar, if they are allowed to stretch in one dimension, will grow on glass fibers as thin as 0.05 # m [20]. This approaches the dimensions of collagen fibrils.
(B) Transformed cells (1) In vitro Transformed cells have in a sense escaped the restriction that geometry imposes on the growth ofuntransformed cells in suspension. Transformed cells can proliferate equally well in the flat state or the spheroidal state; i.e., while plated on a culture dish
217 or suspended in spinner culture or soft agar. Of all the characteristics acquired during malignant transformation, this may be of the most fundamental importance to understanding neoplasia. At the moment we have only a dim perception of how this change might come about, and no explanation at all at the molecular level. For the purpose of this paper, transformation is important from another perspective. Transformed cells, having escaped one level of geometrical growth control, form colonies in suspension culture. However, these colonies do not grow indefinitely; they are subject to another form of growth control by geometry. This is the control of cell populations. Cell populations may be considered as either two-dimensional or three-dimensional, depending upon the configuration of their growth. When transformed cells are grown in flat tissue culture, growth continues only in two dimensions [21,22]. The third dimension, or thickness of the cell population, becomes constant. It is a thin layer, rarely more than 6-8 cells thick [5]. Transformed cells in this fiat configuration will increase their number indefinitely, provided two requirements are met: (1) there must always be sufficient open space, i.e., the dish must be continually enlarged or the population periodically subdivided and transferred to a new dish; (2) there must be sufficient fresh nutrient, i.e., a continuous or frequent change of media. Under these conditions, two-dimensional growth is not self-limited. Cultures have been passaged for many years with continuous increase in cell number. Depending on the space available, such a cell culture may exceed 108 cells in a few weeks. When the same cells are grown in suspension culture (soft agar or methocel), the population expands in all three dimensions. However, expansion does not continue indefinitely despite the provision of sufficient open space and frequent exchange of nutrient. The total population does not increase beyond l 0 6 cells even after months of continuous incubation, although the cells remain viable [21]. Therefore, growth in three dimensions is self-limited, in contrast to two-dimensional growth which is not! The study of three-dimensional growth was initiated by suspending transformed cells in soft agar [21]. As soon as a colony became visible it was removed to a separate flask. The diameter of each spheroid was measured every other day by projection of its image. Each spheroid was transferred every 2-3 days with a wide-bore pipette to a new flask containing 10 ml fresh agar. This procedure prevented any significant changes in pH. Approximately 10000 measurements were made over a period of 1 year. All spheroids enlarged along an approximately linear growth curve for 5-23 weeks before reaching a critical diameter beyond which there was no further expansion. This was termed the stage of"population dormancy". For L-5178Y (mouse leukemia) cells the diameter of the dormant phase was 3.8 mm ± 0.5 mm at approximately 25 days (Fig. 3). For V-79 (hamster lung) cells, the dormant diameter was 4.0 mm -k 0.8 mm at 175 days (Fig. 4). For B-16 (mouse melanoma) cells it was 2.4 mm :k 0.4 mm reached at approximately 100 days. These are the maximum diameters which can be reached by any single spheroid of a given cell line. When multiple spheroids
218 SPHEROIDS OF LSI78y CELLS
4 E ~
3
7.
~ 2 I
'
2o
40
8o
so
IO0
DAYS
F i g . 3. D i a m e t e r a n d s t a n d a r d d e v i a t i o n o f 50 i s o l a t e d s p h e r o i d s o f L - 5 1 7 8 Y cells. E a c h s p h e r o i d w a s m a i n t a i n e d in 10 ml. soft a g a r a n d t r a n s f e r r e d e v e r y 2 - 3 d a y s to a n e w flask. F r o m F o l k m a n a n d H o c h b e r g [21].
V-79
SPHEROIDS
"~ 4 E
,.=, 3
INFLUENCE
OF GEOMETRY
ON CONTROL
OF CELL
GROWTH
J U D A H F O L K M A N and HARVEY P. G R E E N S P A N
Department of Surgery, Children's Hospital Medical Center and Harvard Medical School, 300 Longwood Avenue, Boston, Mass. and the Department of Mathematics, Massachusetts Institute of Technology, Cambridge, Mass. (U.S.A.) (Received October 15, 1975)
CONTENTS I.
Introduction
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
211
1I. Effect of geometry on cell proliferation . . . . . . . . . . . . . . . . . . . . . . A. Untransformed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. In vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. In vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Implantation of glass beads . . . . . . . . . . . . . . . . . . . . . . . b. Foreign body carcinogenesis . . . . . . . . . . . . . . . . . . . . . . B. Transformed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. In vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. In vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
212 212 212 214 214 214 216 216 222
IlL Effect of geometry on gene expression . . . . . . . . . . . . . . . . . . . . . . A. Untransformed cells (in vitro and in vivo) . . . . . . . . . . . . . . . . . . . B. Transformed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
228 228 231
IV. Theoretical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . .
231
V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
234
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
234
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
235
I. I N T R O D U C T I O N It is c l e a r t h a t cell g r o w t h is r e g u l a t e d at m a n y levels.
A few e x a m p l e s are,
s e r u m g r o w t h f a c t o r s , c e l l u l a r cyclic n u c l e o t i d e s , a n d o x y g e n gradients. O u r k n o w l e d g e o f these c o n t r o l p o i n t s is still so r u d i m e n t a r y t h a t it is p r e m a t u r e to assign p r i o r i t y , o r e v e n to d i s t i n g u i s h p r i m a r y a n d s e c o n d a r y e v e n t s in the r e g u l a t i o n o f cell growth. H o w e v e r , t h e r e are m a n y s i t u a t i o n s w h e r e t h e g e o m e t r y o f a n i n d i v i d u a l cell o r the g e o m e t r i c a l c o n f i g u r a t i o n o f a p o p u l a t i o n o f cells, s e e m to exert a p r o f o u n d influence o n g r o w t h a n d d i f f e r e n t i a t i o n .
A t t i m e s the g e o m e t r i c a l effect a p p e a r s to
212 override all other growth signals. The shape of a single diploid cell at any given time may be the final determinant of whether or not that cell will be permitted to divide, regardless of what other mitogenic factors are present. Furthermore, the geometrical arrangement of a population of tumor cells may have a profound effect on the growth rate of that population. In both the normal and the malignant state, geometry can play a significant role in proliferative capacity. We will assemble what bits and pieces of information are available in the hope that a pattern of geometrical effects on cell growth will become discernible. Some of the evidence quoted here has been around for a long time, such as the well known fact that untransformed diploid cells will not grow in suspension. Other evidence has come only recently from experiments in foreign body carcinogenesis, and also from studies of tumor angiogenesis.
II. E F F E C T OF G E O M E T R Y ON CELL PROLIFERATION
(A ) UntransJormed Cells (Diploid) (1) In Vitro When diploid cells or untransformed cells, such as mouse fibroblasts, are plated on coverslips of plastic or glass, they stick and flatten out. They proliferate until confluence is reached. By contrast when the same cells are freely suspended, either in soft agar, methyl cellulose, or in spinner culture, they fail to proliferate [1,2]. While in suspension culture, some of these cells incorporate [3H]thymidine. Most double their volume in the first 24 h, and a few may go through one cell division, but there is no further proliferation. BHK21 cells studied in suspension by Stoker [2], remained viable for up to 7 days and retained their competence for proliferation when returned to the fiat state. When these cells were returned to a plastic surface, mitotic figures appeared after 17 h and proliferation followed. All of them showed [3H]thymidine incorporation. BHK21 cells are not as normal as for example primary cultures of fibroblasts in early passage [3]. They may have passed through some genetic changes on the way to transformation. Their inability to grow in suspension can be overcome slightly with very high serum concentrations [4]. However, serum concentrations as high as 50 ~o initiated DNA synthesis in only 12 ~,, of the suspended cells, and stimulated the formation of only a few colonies. Thus, even in the presence of high serum there is a marked difference between the response of fiat cells,compared to suspended cells. The attached or flattened cells are 60 times more sensitive to the stimulating effect of serum than are suspended cells (Fig. 1). The major difference between the inhibited cells and their proliferating counterparts is their shape. Cells in suspension are rounded or spheroidal; cells attached to plastic are fiat and spread out. Inhibition of growth in the spheroidal state is characteristic of the majority of normal (untransformed) fibroblasts and epithelial cells. Cells of hematopoietic origin are exceptions and can proliferate while spheroidal and in suspension culture.
213 a
I00
LIJ
J J
t !
90~ 80,
Surface
-J
70 ~
z
5O
~ 3o ~- 20 13-
•
7.
Suspension XL
--X-
- -X
O ', ' ' ' ' ' ' X ~ X ,
O
5
IO
20
q
30 50
SERUM CONCENTRATION (per cent)
Fig. 1. Response of BHK21 fibroblasts in layer culture and in Methoce! suspension culture, after exposure to increasing concentrations of serum. Cells were labelled with [aH]thymidine. From Clark et al. [4].
Diploid cells whose growth is inhibited in suspension culture, can escape this inhibition if they are permitted to stretch out on glass fibrils or flatten out on glass beads suspended in soft agar or methyl cellulose. Glass beads 75 ffm in diameter allowed ample room for cell attachment and proliferation into colonies. Silica particles averaging 5 ffm in diameter seemed to provide insufficient surface area for cell spreading and proliferation [2]. It is of interest that the mean area utilized by a normal mouse fibroblast in the flat state is approximately 2 ' 103 ffm 2 [5]. A bead of 75 # m diameter has a surface area of approximately 1.7" l04 ffm 2, enough to accommodate up to 8 cells, but a bead of 5 # m has a surface area of only 7.8 • 10 u m 2, not enough for the spreading of even one cell. Glass fibrils up to 500 # m in length also acted as rafts to which normal cells attached and proliferated while the fibrils themselves were floating in methyl cellulose. In similar studies [6], collagen fibrils have been used to support fibroblast growth in suspension culture, and Horng and McLimans [7] used sephadex beads in suspension, to support growth of pituitary cells. Stoker has applied the term "anchorage dependence" to this phenomenon, implying that an untransformed cell must anchor itself to a rigid surface before it can divide. This concept appears to be supported by Boone's recent demonstration [8], that 3T3 cells which grow well on plastic, will not grow on teflon, presumably because they cannot stick. However, Stoker's B H K cells, freely suspended, were able to go through one division without anchorage, yet could not proliferate further. This suggests a block in the phase during preparation for cell division. Thus, anchorage may be only one component of the flat state which is prerequisite for cell proliferation. Other components must be considered. Plasma membrane surface area is one possible component. When a normal cell changes from spheroidal to flat shape, its total surface area may increase approximately ten-fold. [Assume a cell of 13 ffm diameter
214 in suspension. Its surface area (as a sphere) is approximately 527 #m 2. If a mouse fibroblast when plated [5] can occupy 2600 # m 2, then its total plasma membrane surface area would be approximately 5200 #m2.] Another feature of the fiat state may be an increase in permeability of the cell membrane in comparison to the spheroidal state. Furthermore, when a cell changes from the spheroidal to the flat configuration, a variety of receptors may change position [9]. Finally, the net surface tension distributed over the plasma membrane of a spheroid cell is much greater than in a flat cell. All five phenomena may work in some cooperative fashion, or one among them may be the critical variable, or some other as yet unrecognized feature of the flat state may be important (See Theoretical Considerations).
(2) In Viro Many phenomena in tissue culture have no counterpart in vivo. However, the general rule that flatness permits proliferation in a normal cell while roundness inhibits it, does have at least two analogues in experimental animals. One of these is tumor production by implantation of cells coated on glass beads, and the other is foreign body carcinogenesis. (a) Implantation of glass beads. Balb/3T3 cells proliferate as a monolayer on plastic or glass surfaces in vitro but will not grow in suspension [2]. Neither will they form tumors when injected subcutaneously into histocompatible mice. However, Boone [10] showed that tumors appeared regularly if 3T3 cells were allowed to attach to glass beads (3 m m diameter) before implantation. The inoculation of cells alone or beads alone produced no tumors. All mice receiving beads coated with cells developed tumors within 8 weeks. Cells from these tumors were capable of producing lethal tumors on subsequent transplantation in the absence of glass beads. This implies that the original 3T3 cells were not completely transformed and not tumorigenic by themselves prior to the first implantation on glass beads. However, attachment to glass beads permitted them to take the final step necessary to be a successful tumor. Again, we are unable to escape the conclusion that the flat state was an essential feature contributing to the appearance of a clone of neoplastic cells. (b) Foreign body carcinogenesis (smooth surface carcinogenesis). It has been known since 1941 [11] that implantation of a variety of smooth surfaces into mice arid rats will induce sarcomas. Glass, plastic and steel all have a similar effect. This subject has been well reviewed [12,13]. In the usual experiment, a plastic or glass coverslip is implanted subcutaneously and a sarcoma appears at the site after a long latent period, i.e., approximately 9 months. Several conclusions are convincingly established. Physical form is important; chemical composition is not. A variety of polymer sheets and films will produce tumors, but powders from these materials will not. Surface area is important. Statistical evaluation indicates a direct relationship between tumor incidence and surface area. Discs of less than 1 c m 2 rarely induce a tumor while discs of 2 cm 2 or greater, are highly carcinogenic [14]. Furthermore, tumor incidence decreases and eventually reaches zero when implants are perforated with holes in increasing number or with increasing diameter. Also,
215
SMOOTH SURFACE CARCINOGENESIS
T6
~Jr:,,
Fig. 2. Fibroblast-like cells spread out and proliferate on plastic film implanted into CBA/H-T6 mouse. When film is later transferred to a CBA/H mouse, the tumor which develops is from the T6 donor. This is our diagrammatic summary of Brand's data [16].
the fibrous capsule which forms around the implant, i.e., the foreign body reaction, is important, possibly as a source of the cells which will eventually be involved in carcinogenesis. If the fibrous capsule is removed early, along with the implant, tumors will not develop. On the other hand, tumors may still develop if only the implant is removed late in the course. Perhaps the most elegant studies in regard to etiology have been done by Brand and his co-workers [15,16]. They used two CBA mouse strains differing only by the marker gene T 6. After implantation of a plastic surface, clones of cells which later became attached to the film, began to proliferate. When the film was transplanted to the mouse of different genetic make-up, the tumor which arose was always of the donor type. (Fig. 2). Here again is a strong analogy to fiat tissue culture. Fibloblastlike cells move from the fibrous capsule and spread out on the plastic and continue to proliferate. Brand suggests that these cells are not true fibroblasts, but may derive from pericytes or endothelial cells of the microcirculation. At this writing their origin remains unknown. The pre-neoplastic cells stick to the plastic film and are carried over to the recipient animal. Burnet [17] has proposed that the continuous proliferation which occurs on the plastic promotes the selection of an intrinsic mutant which then accumulates as a clone of neoplastic cells. Recently, Buoen et al. [18] have shown that pre-neoplastic cells can be recovered from the plastic film at an early stage after implantation, and grown in tissue culture. After such a clone was expanded in vitro, cells were reimplanted into recipient animals
216 of the T6 different partner. Again, the resultant tumor had the gene marker of the donor animal. The latent period before the appearance of tumors in recipients, varied in relation to the number of passages in vitro, and also with the method of implantation. Most intriguing was the observation that cells from early culture passages, which were attached to plastic films at the time of implantation, reached neoplastic autonomy earlier than did the same cells inoculated into the animals as a suspension or pellet. This is reminiscent of the story of 3T3 cells on glass beads. So-called "pre-neoplastic" cells which have not yet acquired all the characteristics necessary to produce a tumor when inoculated as a pellet of free cells, can grow into a tumor when implanted while attached to a smooth surface. Another example of foreign body carcinogenesis is observed after exposure to asbestos. A large smooth surface is not necessary; fibers of appropriate length are sufficient. Asbestos fibers produce malignant mesotheliomas in man and in experimental animals. Maroudas et al. [19] showed that asbestos fibers from 20-320/~m in length produced mesotheliomas after injection into the pleural cavities of rats. Also BHK21 and 3T3 fibroblasts in suspension culture, attached themselves to glass fibers of this size and proliferated. Maximum growth was obtained on lengths of 200 # m or greater; no growth occurred below 20 #m. Also, fibers less than 20 #m did not cause mesotheliomas. Glass and aluminum oxide fibers similar to the long asbestos fibers, also produced mesotheliomas and supported fibroblast growth in suspension culture. An inconsistency appears to exist when plastic (or glass) film carcinogenesis is compared to carcinogenesis by asbestos or glass fibers. In the former, a large smooth area is necessary, while in the latter, only fiber length is important. This problem can be resolved by considering the large area of film as necessary for the generation of enough clones from proliferating cells, to permit one or more to reach the "preneoplastic" state during the lifetime of the animal. On this statistical basis, the number of fibers is as important as the area of film. A very low number of glass or asbestos fibers would probably not cause a tumor for the same reason that a small area of film would not be adequate. However, from the perspective of the individual cell, it appears not to matter exactly how a "flat" configuration is achieved. Whether the fibroblast is spread out in two dimensions, or stretched out in one dimension, (i.e. extended), the signal is the same: permission to proliferate. In fact, isolated fibroblasts in agar, if they are allowed to stretch in one dimension, will grow on glass fibers as thin as 0.05 # m [20]. This approaches the dimensions of collagen fibrils.
(B) Transformed cells (1) In vitro Transformed cells have in a sense escaped the restriction that geometry imposes on the growth ofuntransformed cells in suspension. Transformed cells can proliferate equally well in the flat state or the spheroidal state; i.e., while plated on a culture dish
217 or suspended in spinner culture or soft agar. Of all the characteristics acquired during malignant transformation, this may be of the most fundamental importance to understanding neoplasia. At the moment we have only a dim perception of how this change might come about, and no explanation at all at the molecular level. For the purpose of this paper, transformation is important from another perspective. Transformed cells, having escaped one level of geometrical growth control, form colonies in suspension culture. However, these colonies do not grow indefinitely; they are subject to another form of growth control by geometry. This is the control of cell populations. Cell populations may be considered as either two-dimensional or three-dimensional, depending upon the configuration of their growth. When transformed cells are grown in flat tissue culture, growth continues only in two dimensions [21,22]. The third dimension, or thickness of the cell population, becomes constant. It is a thin layer, rarely more than 6-8 cells thick [5]. Transformed cells in this fiat configuration will increase their number indefinitely, provided two requirements are met: (1) there must always be sufficient open space, i.e., the dish must be continually enlarged or the population periodically subdivided and transferred to a new dish; (2) there must be sufficient fresh nutrient, i.e., a continuous or frequent change of media. Under these conditions, two-dimensional growth is not self-limited. Cultures have been passaged for many years with continuous increase in cell number. Depending on the space available, such a cell culture may exceed 108 cells in a few weeks. When the same cells are grown in suspension culture (soft agar or methocel), the population expands in all three dimensions. However, expansion does not continue indefinitely despite the provision of sufficient open space and frequent exchange of nutrient. The total population does not increase beyond l 0 6 cells even after months of continuous incubation, although the cells remain viable [21]. Therefore, growth in three dimensions is self-limited, in contrast to two-dimensional growth which is not! The study of three-dimensional growth was initiated by suspending transformed cells in soft agar [21]. As soon as a colony became visible it was removed to a separate flask. The diameter of each spheroid was measured every other day by projection of its image. Each spheroid was transferred every 2-3 days with a wide-bore pipette to a new flask containing 10 ml fresh agar. This procedure prevented any significant changes in pH. Approximately 10000 measurements were made over a period of 1 year. All spheroids enlarged along an approximately linear growth curve for 5-23 weeks before reaching a critical diameter beyond which there was no further expansion. This was termed the stage of"population dormancy". For L-5178Y (mouse leukemia) cells the diameter of the dormant phase was 3.8 mm ± 0.5 mm at approximately 25 days (Fig. 3). For V-79 (hamster lung) cells, the dormant diameter was 4.0 mm -k 0.8 mm at 175 days (Fig. 4). For B-16 (mouse melanoma) cells it was 2.4 mm :k 0.4 mm reached at approximately 100 days. These are the maximum diameters which can be reached by any single spheroid of a given cell line. When multiple spheroids
218 SPHEROIDS OF LSI78y CELLS
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DAYS
F i g . 3. D i a m e t e r a n d s t a n d a r d d e v i a t i o n o f 50 i s o l a t e d s p h e r o i d s o f L - 5 1 7 8 Y cells. E a c h s p h e r o i d w a s m a i n t a i n e d in 10 ml. soft a g a r a n d t r a n s f e r r e d e v e r y 2 - 3 d a y s to a n e w flask. F r o m F o l k m a n a n d H o c h b e r g [21].
V-79
SPHEROIDS
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