Zeitschrift fiir Krebsforschung 65, 75--86 (1962)

From the Department of Pathology, The Medical Institute of Sasaki Foundation, Tokyo, Japan (Director: Prof. Dr. TOMIZO YOSHIDA)

Mutual adhesiveness of tumor cells in ~,hepatoma islands,~ of the rat ascites hepatoma - - Studies on the mechanism of tumor metastasis. I*

By TAKASHI YAMADA

With 12 Figures in the Tex~

(Received January 27, 1962)

I t is not unusual to see a cancer ease, in which the tumor cells spread all over the patient's body, although the primary tumor is too small to be found. In addition, commonly the frequency or the distribution of the metastases vary in cancers of similar histologie appearances. These variations may be caused by several factors in the biology of the tumor cells concerned with their metastatic formations: 1. decreased mutual adhesiveness between tumor cells (CoMA~ 1944, 1954, 1955), 2. active motility (Vlnc~IOW 1863, ElgTERLINE 1950, Hlno~o 1958), 3. high negative electrical charge of cell surface (AN[BI~OSE et al. 1956) and 4. in- vasive character due to three or more of the above properties (Co~AN1954, ZmDMA~ 1957).

I t is possible that the biological properties of tumor cells are significantly different from those of the original cells. These properties cannot necessarily be considered as unique to malignant cells from a cytological point of view, however, because blood cells and inflammatory emigrating cells may have similar properties. Therefore, the question might be asked what role all these properties play in the emigration of malignant cells into vascular canals. Unfortunately up to now no exact information exists to answer this question.

As emphasized in this paper, of special interest is the fact, that the mutual adhesiveness of cancer cells is greater than that of non-malignant cells. The liberation of tumor cells from the primary tumor may be the first step in meta- static processes (CoMAN, Z~IDMa~) and may represent one of the most funda- mental problems of metastatic formation. Therefore, the mutual adhesiveness of rat ascites hepatoma islands (Fig. 7) was analyzed, with special interest paid to their metastasis.

Materials

Nine different strains of the rat aseites hepatoma were used; they were being kept by serial animal passages in this laboratory (u 1960). They were the ascites hepatoma AH 601, 62, 322, 66, 127, 149, 49, 602 and 7974. These tumors, prepared by means of the aseitie conversion of aminoazo-dye induced hepatoma in rats, were composed of conglomerated hepatoma cells and isolated hepatoma cells proliferating in the ascitic fluid. The conglomerated hepatoma cells were called "hepatoma islands". They were clusters or groups of pure

* This investigation was supported in part by a grant from Japan Cancer Society. Z. Krebsforsch. Bd. 65 6

76 TAKAsHIY.xMADA:

h e p a t o - c e l l u l a r c a r c i n o m a cells c o n t a i n i n g n o o t h e r k i n d of cells (Fig. 7). T h e

i s l a n d s v a r i e d in size a n d s h a p e i n e a c h t u m o r s t r a i n . F o r t h e t r a n s p l a n t a t i o n

of t h e a sc i t e s h e p a t o m a , J a p a n e s e c o m m o n a l b i n o r a t s , w e i g h i n g 90 t o 100 g r a m s ,

were e m p l o y e d d u r i n g

Table 1. t h e e x p e r i m e n t s .

Dissociation o/ hepatoma island8 into /see heaptoma cells after E x p e r i m e n t 1. T h e treatment with various agents in vitro; Ascites hepatoma AH 601

A g e n t s

Trypsin . . . . . . Hyaluronidase . . . Hyaluronidase

§ EDTA . . . . EDTA (Disodium

ethylendiamine tetraacetate) . . .

Saline of various osmotic pressure .

Buffer solution chlorate . . . . . acetate . . . . . phosphate . . . .

Nitrogen mustard N-oxide . . . . .

C.M.C. (Carbox- methylcellulose) .

Surface active agents Span 20 . . . . . Span 60 . . . . . Span 85 . . . . . Tween 20 . . . . Tween 40 . . . . Tween 60 . . . . Tween 80 . . . . Tween 85 . . . .

Steroid hormon Progesteron (E-P hormon) . . Esteron (Ova-

hormon) . . . . Androsteron

(Durabolin) . Testosteron

(Enarmon) . . Adrenoeortical

hormon (Inte- renin) . . . . .

Concentration

1--5 mg/cm 3 5000/~/em 3

5000/~/em 3 § 0.2 %

0.04---O.5 %

Aq. dis -- 10 %

1/10 ~f

0.001--1 rag/era a

0 .1--2%

1%

10 %

0.2 rag/era a

10 rag/era a

0.5 rag/era s

0.25 mg/cm '~

pH

7.1 5--6

5--6

5--6

5--6

1--3 5--6 7--9

2--5

5--6

5- -6

D i s - s o c i a t i o n

effect

+ + +

+ §

§ + +

§ + + §

•

d i s s o c i a t i o n of h e p a t o m a

i s l a n d s i n t o f ree h e p a -

t o m s cells p r o d u c e d b y

v a r i o u s r e a g e n t s .

Procedures. The ascites hepatoma AH 601, which was characterized by relati- vely large islands as well as a few free tumor cells as indicated in Fig. 7, was used in this experiment. The reagents studied for their dissociating capacity were 21 substances includ- ing 8 different surface active agents. They and their concentrat ion are shown in Table 1. The tumor ascites was obtained by a glass capillary pipette from the 5 to 7-day-old tumor-bearing animal and then heparinized. The blood and other normal nat ive cells in the ascites were separated by slow eentri- fugation (500--1,000 rpm., 3 or 4 times) and removed. The tumor material then was suspended in physio- logic saline. The ratio of the tumor material to the saline in the suspension was 1 : 4 per volume (20 % coneent, suspension).

To 0.5 cm s of the tumor suspension in a tes t tube, 2.0 cm s of each reagent was C for 30 minutes. Then i t added and mixed. The test tube was kept s ta t ionary at 370

was shaken for 30 minutes at 370 C by an automatic shaking" device. This precaution in- sured t h a t the tube in each instance was being handled in the same wsy. Immediately after the shaking, the tumor material was examined under the microscope to see whether the hepatoma islands were suceesfully disintegrated into free hepatoma cells.

Results. A m o n g t h e 10 k i n d s of r e a g e n t s so f a r t e s t e d , t h e w a t e r so lub le n o n -

p o l a r su r f ace a c t i v e a g e n t s , T w e e n 20, 40, 60 a n d 80, d i s s o c i a t e d t h e h e p a t o m a

i s l a n d succe s s fu l l y i n t o f ree cells (see T a b l e 1). S i m i l a r r e s u l t s were o b t a i n e d w i t h

t r y p s i n a n d a n a l k a l i n e s o l u t i o n , as r e p o r t e d a l r e a d y b y E s s ~ o t a l . , b u t wore

Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat aseites hepatoma. I 77

no t ob ta ined wi th e i ther e thy lend iamine t e t r a - a c e t a t e (EDTA), a chela t ing agent , nor wi th a mix tu re of E D T A and hya luronidase .

The appea rance of d issoc ia ted h e p a t o m a cells p roduced f rom h e p a t o m a is lands wi th Tween 80 is shown in Fig. 8 and 9. The surface of the freed t u m o r cells is no t so th ickened and presents a r a the r fine i r regular line, a p ic ture different f rom t h a t of the cells f reed wi th t ryps in (Fig. 10). The Tween t r e a t e d cells were s ta inable wi th nigrosin, suggest ing an increased pe rmeab i l i t y of the i r surface (Fig. 11 and 12). W h e n the h e p a t o m a is land was t r e a t e d wi th con- cen t r a t ed Tween solut ion (5 - -10% solution), cyto- lysis of the freed h e p a t o m a cells was noted.

The ey to ly t i c effect m a y no t be due to the osmotic pressure of Tween solutions, because the freezing depress ion po in t of each Tween solut ion examined (5% in dis t i l led water) was exceedingly low. Namely , - -0 .050 C for Tween 80, - - 0 . 0 7 o C for Tween 20, - - 0 . 0 4 o C for Tween 40, - - 0 . 0 1 ~ C for Tween 60 and - - 0 . 5 6 5 o C in physiologic saline.

Af te r b reak ing up the h e p a t o m a is lands in to free t u m o r cells wi th Tween 80 (1%), free cells were p icked up wi th the a id of the micromani - pu l a to r and t r a n s p l a n t e d in to no rma l rats . The n u m b e r of t u m o r cells in each inoculum was 32 to

Table 2. Results of transplan- tation of free tumor cells isolated

from hepatoma islands alter Tween 80 treatment

N o of cells " T a k e " ( § ) o r t r a n s p l a n t e d "Non-take" (--)

32 90

126 197 198 200 234

§ § §

§

Ascites hepatoma AH 601 was used (1% Tween 80 solu- tion, 30 min. incubation, 10 rain. agitation).

234 as i nd i ca t ed in Table 2. Pos i t ive " t a k e s " were ob ta ined in 4 out of the 7 rec ip ient animals . This means t h a t the Tween solut ion can b reak the m u t u a l bond be tween t u m o r cells of the h e p a t o m a is land wi thou t affect ing the i r v iabi l i ty . L i t t l e is know abou t the mecha- nism of ac t ion of Tween excep t of i t s ab i l i t y to increase the a f f in i ty of wa te r to oil, and to decrease the surface tens ion of wa te r (Atlas Comp. 1950, YAM),MOTO 1954). Therefore, factors were s tud ied which perhaps are concerned in this " T w e e n e f fec t " , i .e . the causes of the dissocia t ion of the m u t u a l adhesiveness of h e p a t o m a is land cells. Deta i l s are descr ibed in the following pa rag raph .

Exper iment 2. Ana lys i s of the " T w e e n effect" .

Table 3. Standard error in counting the ]requency (%) o/ single and aggregated tumor cells (island)

o/ the ascites hepatoma

Spe- Single cimen tumor No. cells

16

10 5 16

Islands composed of

2 cells

15 13 10 15 10

3 cells

8 6 7

10 3

more than 3 cells

62 65 65 65 71

66 ~: 1.46

(Each specimen was prepared simultaneously from one and the same tumor ascites of 5-day-old AH 601 animal.)

Procedures. 2.0 cm a of various concentrations of Tween 80 in phys. saline were mixed with 0.5 cm 3 of 20% AH 601 phys. saline suspension in test tube. The latter suspension was prepared as described above. The concentration of the Tween 80 used, the incubation temperature, and the duration of shaking differed in each test. These comparative studies were designed to clarify the role played by the variables in the Tween effect.

6*

78 TAKASHI YAMADA :

Immediately after the agitation, the tumor material was examined under the micro- scope. The frequency of individually isolated tumor cells (as well as of hepatoma islands) was ascertained and compared with that of the control samples, i.e. the material before the agitation. Thus, the Tween effect was estimated by the grade of increase in the frequency of free tumor cells. The error in this counting was minimal; i.e., the standard error in 5 eountings of one specimen is indicated in Table 3.

:,7%

7ool- I- / - -

"t / / 5 %

[ r I

8O

~0

~0 7o %

O /530 80 780 nz/n J00r 75 30 80 180 nT/n 300 Zncobotlon t/iHe /ncobat/~n t/~e

Fig. 1. Variability in "Tween 80 effect" by various factors. (1)Concentration of iween 80: AH 601. Agitation time : 10 rain

1. Regarding the concentration o~ Tween 80. The concentration of Tween 80 tested ranged from 0. l% to 10.0%. The time of agitation was 10 minutes. The incubated time of the

O 1 I I I I I I I I I 35 37 38 q7 t'C

fncubal/on lernperalur~

Fig. 2. Variability in "Tween effect" by various factors. (2) Temperature of incubation: AH 601. Incubation time: 30 rain. Agitation time: 30 min

material before the agitation varied from 15 minutes to 5 hours in each test case. The incubation and the agitation were done at 37 o C. The frequency of free hepatoma cells counted is graphically shown in Fig. 1. The higher the concen- tration of Tween, the more rapidly the dissociation of hepatoma islands occurred. When the material was treated with 5--10% Tween 80, eytolytic change of the freed hepatoma cells was marked. The changes were especially severe in the 10 % concentration, where amorphous cellular substances increased and the number of the tumor cells decreased, as indicated in Fig. 1.

2. Regarding the temperature o] in- cubation. The temperature of incubation

tested varied from 35 o C to 440 C. The concentration of Tween 80 was 1%, and 30 min. incubation and 30 min. agitation were adopted in this test. The results are shown in Fig. 2. The Tween effect, as estimated by the increase of free hepatoma cells, was almost directly proportional to the higher temperature. The higher the temperature of incubation, the greater was the Tween effect.

3. Regarding the time o/agitation. The time interval for agitation of the material varied from 15 minutes to 2 hours. The Tween used was a 1% concentration. A 60 minutes in- cubation was used at 37 o C. The grade of dissolution of hepatoma islands reached the maximum in the sample agitated for 60 minutes as indicated in Fig. 3.

Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat ascites hepatoma. I 79

Experiment 3. Different "Tween effect" with different T w e e n s - 20, 40, 60,

and 80.

Procedures. About 107 tmnor cells of the aseites hepatoma AH 601 were transplanted intraperitoneMly into 16 rats. After the transplantation, one or two rats were sacrificed every day. The ascitie fluid was collected from the animal and a 20% AH 601 suspension was prepared in phys. saline. The effects of 4 different Tweens, i.e. Tween 20, 40, 60 and 80, were examined on this tumor suspension. The concentration of each Tween used was 1%. Sixty minutes incubation and 30 minutes agitation of the suspension were selected at 370 C.

Results. The results are summarized in Fig. 4. The effects of the 4 Tweens differed as indicated by the grade of increase of freed cells. Tween 60 showed

the least effect, whereas Tween 80 gave 7o~ the most marked effect on every day

' % ~ of examinat ion. The effects of the

20 ~ 20

] I I I I I I l I I O ~ 0 30 60 rain 720 0 2 q 6 8 70 7B

Ajit~tion l/me 00~ts after/'noculalion

Fig. 3 Fig. 4

Fig, 3, Variabi l i ty in " T w e e n 80 e f f ec t " by var ious factors. (3) Time of agi ta t ion: AI t 60i. E v e r y line indicates repee t ive ly each resul t in the different exper iments util ized with different old t u m o r cells.

Incnba t ion t ime: 1 hour

Fig. 4. Increase percent of single cells isolated f rom islands af ter t r ea t ing wi th surface act ive agents in vitro: At t 601. Incuba t ion : 60 rain. Agi ta t ion: 30 min

remain ing 2 were between those of the former. The rise and fall of the effects in each curve demons t ra ted a similarity. Thus, for example, the effect increased day by day in the mater ia l obta ined from an early stage of tumor growth, and reached the m a x i m u m at the 4 or 5-day-old tumor and decreased thereafter, except in the case of Tween 60.

Experiment 4. "Tween effect" in various growth stages of the tumor.

I t was an unexpected bu t significant fact t ha t the "Tween effect" varied dur ing the tumor growth as described as above. Therefore, experiments were carried out to re-examine the correlation between the age of the tumor and the "Tween effect"

Procedures. 16 rats received intraperitoneal transplantants of 107 tumor cells of Att 601, and thereafter one or two animals were saerified daily. The ascitic fluid taken from the sacrificed rats was used for the examination of the "Tween" effect, for the counting of the totM number of cells, as well as for the measuring of the size of the hepatoma islands.

The "Tween effect" was examined by the same procedure as described in experiment 3. However, only Tween 80 was used. The count of the term tumor cells in the ascitic fluid was obtained by the following way: The ascites hepatoma rat was killed and its abdominal cavity

Z. Krebsforseh. Bd. 65 6a

80 TAKASm YA3~ADA:

was irrigated with physiologic saline. The irrigation was repeated and all fluid (highly diluted tumor ascites)was collected. Then a saturated aqueous solution of oxalic acid (6.63 g/dl) was added to an aliquot of the tumor fluid. The ratio of the oxalic acid to the tumor fluid was 20:1 per volume. The mixed fluid was kept at 370 C for 30 minutes and then agitated severely for 30 minutes. After this t rea tment , all of the nucleated cells in the mixture had lost their cytoplasm and only naked nuclei remained. The naked of ~umor cells per mm 3 were counted under the microscope after a differential analysis of nuclei of non-tumorous origin. From this the total number of tumor cells in the whole fluid was estimated.

7oop % !

30 ~1 , I - W , I , , [ I r ~ ' ~

0 ~ t~ 8 8 10 12 74 t

0 8 z/ 6 8 10 1Z 7z/

760 r-

"~ 700

V , 2 4 z 8 8 70 7~ 7g

Dags af'/er /nooulation Fig. 5a--e. "Tween 80 effect" and growth of ascites hepatoma AH 601. a Increase of single cells isolated from "islands" of various growth stages of tumor after treating w%h Tween 80 in vitro. Incubation: 60 rain, Agitation: 30 rain. Each line indicates respectively each result in the different experiment with same condition, b Total tumor cell nnmber in tumor ascites of various stages, c Size of

islands of various growth stages

T h e t u m o r cell of a se i t e s

h e p a t o m a A H 601 p r o l i f e r a t e s

i n t h e a c c u m u l a t i n g ase i t i e

f l u id a f t e r t h e i n t r a p e r i t o n e a l

t r a n s p l a n t a t i o n . I n f i l t r a t i v e

g r o w t h s in t h e p e r i t o n e a l

t i s sue s a re r a r e l y f o u n d 10

d a y s a f t e r t h e t r a n s p l a n -

t a t i o n .

After the count of to ta l tumor cells, the "growth r a t e " (R) of tumor cells was calculated by the formula: R = A / B ; A was the average number of total tumor cells in the ascitie fluid obtained at the t ime the 1 or 2 tumor- bearing animals were sacrificed; B was the average number of tumor cells (obtained in the ani- mals) on the preceeding day.

The "average d iameter" (d) of hepatoma islands served as an indicator of the size of the islands. I t was calculated as follows. Hepatoma islands in tumor ascites were examined under the phase- contrast microscope. By the for-

d a + b (a and b being the mula, =

measured max. and min. dia- meters) a mean value was cal-

culated. The "average d iameter" is the simple ari thmetical mean of the values calculated on 100 different islands.

R e s u l t s . T h e r e s u l t s o b t a i n e d a re s h o w n i n Fig . 5 a n d 6. T h e a se i t e s h e p a t o m a

cells p r o l i f e r a t e d v e r y a c t i v e l y i n t h e a sc i t i e f lu id d u r i n g t h e e a r l y s t a g e s of t h e

t u m o r g r o w t h a n d t h e i r n u m b e r r e a c h e d t h e m a x i m u m 5 d a y s a f t e r t r a n s p l a n -

t a t i o n (Fig. 5). T h e c u r v e of t h e i r n u m b e r , h o w e v e r , d id n o t r i se t h e r e a f t e r ,

p r e s e n t i n g a n a l m o s t h o r i z o n t a l l ine . T h i s m a y m e a n t h a t t h e asc i t i c f l u i d or t h e

h o s t c o n d i t i o n b e c o m e s u n f a v o r a b l e for t h e m u l t i p l i c a t i o n of t h e cells d u r i n g t h e

l a s t s t age . T h e c u r v e of t h e " T w e e n e f f e c t " s h o w e d a r ise d u r i n g t h e a c t i v e p e r i o d

of p r o l i f e r a t i o n of t h e t u m o r cells. I t r e a c h e d a p e a k in t h e m a t e r i a l o b t a i n e d

4 or 5 days after transplantation, and showed a fall thereafter. The rise and fall

in the curves of "Tween effect", as well as the growth rate of tumor cells, as

d e m o n s t r a t e d i n Fig . 6, s h o w c o n s i d e r a b l e pa r a l l e l i sm . T h i s sugges t s t h a t d u r i n g

Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat ascites hepatoma. I. 81

the actively growing period of the aseites hepatoma, the tumor ceils are success- fully freed from their clusters (hepatoma islands), by means of the Tween treat- ment. This may also imply that the mutual adhesiveness of ascites hepatoma cells in hepatoma islands varies during their growth and tha t this adhesiveness decreases to a minimum at the most actively grow- ing stage of the tumor. As shown in Fig. 5, however, no significant relation between "Tween effect" and the size of the hepatoma islands could be revealed.

Experiment 5. " T w e e n effect" in different aseites

0 0

I I I I I I I I ] I I I I 2 q 6 3 10 72 74

Days after inoculat/on

8

-I

Table 4. "Tween 80 e//ect" on the isolation o/ single cells /rein islands o/hepatoma cells el 9 di/]erent strains (Tween 80, 1%, Incubation time: 60 min. Agitation time: 30 min.)

Strains

A H 62 A H 601

A H 322 A H 66 A H 127 A H 149 A H 49

A H 602 A H 7974

Percent of single cells before and

after t reated with Tween 80 in vitro

before (control)

4 4

6 25

6 2 5

14 4

after

77 71

57 66 46 37 36

32 9

§

All t u m o r cells used are 5-day-old. I n eve ry s t ra in , t he ave rage va lue among 6 cases was ind ica ted .

Percent of single cells Grade of

after shaking Tween in phys. saline effect

in vitro

14 § 2 4 7

+ +

strain to strain. For this reason the "Tween effect" in each tumor strain was examined.

Procedures. The tumor material of each strain was suspended in phys. saline (20% content.) and was treated with the 1.0% Tween 80 solution. The material was shaken very slowly for 60 minutes at 370 C and then severely agitated for 30 minutes. Each procedure was performed under the same conditions.

oft hese ascites hepatomas originated in separate animals. They were, naturally, common in their ancestry, being deri- ved from the liver cell. Ho- wever, comparative studies of their general characteris- tics, such as growth tempo, metastasizability, aseitie pic- ture, chromosomal constitu- tion, and response to the same chemotherapeutic treat- ment, demonstrated tha t they never were exactly alike.

Different ascitie pictures, represented mainly by diffe- rent frequencies of hepatoma islands and free hepatoma, appeared to suggest tha t the mutual adhesiveness of aseites hepatoma cells differed from

Fig. 6. "Growth r a t e " of t~lmor cells in ascitic fluid and "Tween 80 effect" in vitro: AH 601. "Tween effect"

o ~ r v e , - - - - ~ ~' ~ - r o w t h r a t e " c ~ 2 r v e hepatoma of the rat.

Nine different strains of the ascites hepatoma in rats, i.e. AH 601, 62, 322, 66, 127, 149, 49, 602 and 7974, were employed for the comparative studies. Five-day- old tumor aseites hepatomas were utilized exclusively. The pr imary tumors

82 TAKASHI YAMADA :

Results. The results obtained are shown in Table 4. The Tween effect, re- presented by the increase of free hepatoma cells after treating the tumor material with Tween 80, was greatest with AH 62 and 601, whereas with AH 7974 it was the least of all the tumor strains examined. The remaining tumor strains were between these extremes and showed various grades of Tween effect. This implies that the different strains of ascites hepatoma also gave different Tween effects. Nevertheless, the differences could not always be related to the size of tumor islands.

Discussion 1. The union between epithelial cells, both normal and malignant

Numerous reports have presented interesting data on the mechanisms or structures which are responsible for the bonding of mammalian epithelial cells. These reports were primarily concerned with the protoplasmic products of epithelial cells, with special morphologie characteristics, with the intercellular cementing substances, and with physicoehemical properties of the surface of epithelial cells.

Recent electron microscopic studies (CoMA~ 1954, F~WCETT 1955, BE~WICK 1959,UsuI and KAZlW~A 1960, VOGEL 1958) disclosed that neighboring epithelial cells were bonded to each other by their variously shaped processes, especially by an "interlocking formation". This type of interdigitation was demonstrated in various epithelial cells, including human carcinoma cells and hepatic cells, and the rat ascites hepatoma cells. Special attention was paid to the "terminal bar desmosome" which was recognized as a region of increased electron density (osmophilic plaque) at the surface of the epithelial cell. KAZlW~A and UscI reported that the terminal bar was situated at the sticky, adhering sites of hepat- oma island cells observed during the trypsinizations of them.

Among the various chemical substances responsible for the bonding of epi- theliM cells, calcium received the most attention. A decreased adhesiveness of carcinoma cells with a reduction in the content of calcium was demonstrated by many workers (SgEAI~ 1933, BaV~SgV~G and D~Z~HAM 1946, CA~a~ZTgEI~ 1946, DUNgA~ et al. 1946, ZEID~AS 1947, DE LO~C~ ctal . 1950). According to STEI~- BERG and others, calcium or magnesium salts, located between the surface membranes of adjacent cells, were the most important substances for maintaining mechanical cross linkage between the cells, tI6BER emphasized the role of a calcium ion as a stabilizer for the protein of the cell membrane, or for the inter- cellular cements, in which it influenced a sol-gel transition. Other workers con- sidered that the calcium ion reduced the repulsion of the negative electrical charges of cellular surfaces (B~:~cGE~B~aO D~ J o s e 19r CurTIS 1960).

CUaTIS'S hypothesis on the cohesion of epithelial cells was very interesting and was based on the Verwey-Oberbeek's colloidM interaction theory. He stated that the cells might be kept in a stable cohesion when their intercellular distance was within 100--200 A, and that this was due to the balanced interaction between the molecular attraction and the repulsive action of the negative charge in the neighboring cellular membrane. The distance between the neighboring hepatic cells, as well as between the cells of the hepatoma island was about 100--200 A (FAWOETT 1955, UsuI and KAJIWX~A 1960). TERAYAMA reported that the negative elelctricM charge of the natural island-forming aseitie hepatoma cells was always higher than of free-cell ascites hepatoma as measured with the colloid titration method.

i~{utuul adhesiveness of tumor cells in "hepatoma islands" of the rat aseites hep~toma. I 83

2. Mutual adhesiveness o~ ascites hepatoma cells in hepatoma islands

Although the terminal bar and the interlocking formation of the island constituents have been demonstrated, as described, no one has been able to confirm those functional characteristics in the living. That these structural features are closely related to the bonding of aseites hepatoma cells has not been established beyond dispute.

C o M ~ stressed the role of the calcium ion for the bonding of the rat liver cells. He observed dilated inter-hepatocellular spaces caused by a loosening of each hepatic cell after perfusing the liver with ethylenediamine tetraacetate (EDTA). However, preparations with this substance or with hyaluronidase mixed with EDTA failed to cause separation of the constituent cells of the hepatoma cells of hepatoma islands.

The fact tha t the islands were successfully dissociated into free hepatoma cells by Tween presented another clue to this problem. I t has been said that Tween increased the affinity of water to lipid and decreased the surface tension of water (Atlas Comp. 1950, YA~rAMOTO 1954). This perhaps means that the substance which is responsible for the bonding of the tumor cells includes lipids. This assumption may be supported by some previous studies. For example, WEISS confirmed the necessity of lipoprotein for the adhesion of cells in tissue culture cells. The Tween treated cells were well stained with nigrosin, suggesting an increased permeabili ty of their surface. I t is possible tha t the Tween takes out the lipid substance from the cells and induces a change in the cellular surface. Thus, the increased permeability of tumor cells and the isolation of free cells from hcpatoma islands may be explained by the same mode of action of the Tween.

E s s s s g et al. suggested after successful dissociation of the islands with trypsin, a proteolytie enzyme, tha t the ability of the cementing substances in maintaining the integrity of the hepatoma islands may depend largely upon the presence of pro- tein. But the effect of Tween is not in such a proteolytic action. The free tumor cells isolated with Tween differ in features from those with trypsin, especially in their surface characteristics (Fig. 8, 9 and 10). The intercellular substance in hepa- toma islands could never be demonstrated by electronmicroscopy (UsuI and KAzI- WAgA 1960).

3. Mutual adhesiveness o/tumor cells and metastasis

In the mechanism of metastasis, the first step is the liberation of tumor cell(s) from the site where a tumor has developed primarily. This has been well analyzed by COMAX, ZmDMA~, Oo~a and S~To. This process, the liberation of tumor cells, is the main concern in this discussion.

That the liberation of tumor cells from their nests seemed to be correlated with the mutual adhesiveness of the cells is agreed. The motility of tumor cells m a y rely on the stickiness of their membranes (AMBROSE 1956, KLEIN 1957). According to HI~ONO, the more free tumor cells an ascites hepatoma has, the more motile are the cells. In this relation it was worthy to note tha t in 9 strains of the ascites hepatoma which are endowed each with different abilities for island formation, metastasizabili ty and invasiveness, different adhesiveness of the tumor cells in the hepatoma island could be shown.

Why the mutual adhesiveness of tumor cells varies during their growth remains unexplained. The present study demonstrates tha t the ascites hepatoma ceils may be freed best from hepatoma islands a t ~heir most actively pro/iferating

84 TA~ASm YA~'rADA :

Fig. 7. Phase contrast microscopic view of the ascites hepatoma AH 601. Tumor cells agg'regate and form "hepatoma islands". • few single tumor cells are also found (5-day-old tumor)

Fig. 8. Individually isolated tumor cells from "hepatoma islands" of the ascites hepatoma AI~ 601 after Tween 80 treatment in vitro. 5-day-old tumor. Phasecontrast

Fig. 9. The tumor cells are the same as that in Fig. 8, but fixed with I :I ether-alcohol and stained with hematoxylin -cosin

Fig. I0. Individually isolated tumor cells from "hepatoma islands" of the aseites hepatoma AII 601 after trypsin treatment in vitro. 5-day-old turner. Phasecontrast

Fig. ii and 12. Nigrosin stainability of "hepatoma islands" of AH 601 during Tween 80 treatment in ~;itro. 5-day-old tumor. Fig. 11. Several minutes after the beginning of treatment. Slightly nigrosin-stained islands. Dissociation of the islands is not seen at the time. Fig. 1"2. Sixty minutes after the beginning of treatment. DissoCiation of the isla~ids into single cells is almost eoml~lete.

All the dissociated ceils stained well with nigrosin

Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat aseites hepatoma. I 85

stage. This may imply tha t the liberation of tumor cells from their nests, i.e., the first step of metastat ic spread, can take place most frequently at the stage of the most active growth. However, the liberated cells may differ from each other in their fate, and in their metastat ic processes, by their different behaviors as well as by the various host environment.

Summary The aseites hepatoma cells of rats proliferate in the ascitic fluid by forming

cell aggregates (hepatoma islands). By trying to dissociate the islands into free hepatoma cells with various reagents in vitro, the mutual adhesiveness of the ceils in tile islands was studied. Among 21 reagents tested, Tween 20, 40, 60 and 80, caused successful dissociation of the islands. Trypsin and an alkaline buffer solution showed a similar effect. However, ethylene diamine tetraacetate, a chelating agent, and hyaluronidase failed to disintegrate the islands. The effect of Tween varied with various factors, such as concentration of the reagent, incubation temperature and agitation time. The effect was significantly high in tumors of the most actively growing stage.

Nine different ascites hepatomas, varied in the mutual adhesiveness of their cells. I t was assmned tha t substances which were responsible for maintaining the

integrity of hepatoma islands may contain some lipids besides protein.

Der gegenseitige Zusammenhang der Tumorzellen in ,,Hepatom-Inseln" des Ra~ten-Asci teshepatoms

I, Untersuchungen iiber den Mechanismus der Tumormetastasierung

Zusammenfassung Die Zellen des Aseiteshepatoms der t~atte proliferieren in der Ascitesfl~ssig-

keit unter Bildung yon Zellaggregaten, den sog. Itepatominseln. Der gegenseitige Zusammenhalt der Zellen in diesen Inseln wurde dadurch geprfift, dab man sic mit versehiedenen Reagentien zusammenbrachte und die jeweils beim Schfitteln frei werdenden Zellen auszghlte. Unter den gepriiften 21 Reagentien waren Tween 20, 40, 60 und 80 f~hig, eine Zelldissoziation an den Inseln herbeizufiihren. Trypsin und alkalische Pufferl6sung hatten einen ~hnlichen Effekt. Athylen- diamintetraacetat , ein chelierendes Agens, und ttyaluronidase waren nicht im- stande, die Inseln aufzul6sen. Die St/trke der Wirkung yon Tween h/ingt yon ver- schiedenen Faktoren ab, wie Konzentration, Temperatur der Inkubationsflfissig- keit, geitdauer des Schiittelns ; sic war deutlich hSher bei den in s tarkem Waehs- turn befindlichen Tumoren. Der gegenseitige Zusammenhalt war bei neun auch sonst voneinander verschiedenen Aseiteshepatomen unterschiedlich. Es ist an- zunehmen, dab die Substanzen, die verantwortlich sind ffir die Aufreehterhaltung des Zusammenhaltes im Asciteshepatom auger Proteinen auch Lipoide enthalten.

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Dr. T. YA~IAI)A, The Department of Pathology, The Medical Institute of Sasaki Foundation,

Surugadai 2--2, Chiyoda-ku, Kanda, Tokyo, Japan