Zeitschrift fiir Krebsforschung 65, 87--98 (1962)

From the Department of Pathology, The Medical Institute of Sasaki Foundation, Tokyo, Japan (Director: To~Izo YOSmDA)

Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat ascites hepatoma -- Studies on the mechanism of tumor metastasis. II

By

TAKASHI YAMADA

With 14 Figures in the Text

(Received January 27, 1962)

I n the previous paper (YA~ADA 1962) i t was reported t ha t the t r ea tmen t of ra t ascites hepa toma islands in vitro with Tween solutions, resulted in effective dissociation of the bonding of t umor cells. This effect varied not only with the strains of ascites hepatoma, bu t also with the biological condit ion of each tumor

strain. The present paper deals with the physicochemical analysis of: 1. the , ,Tween

80 effect" and 2. the negat ive electrical charge of cell surfaces and the m u t u a l adhesiveness between the tumor cells of hepa toma islands dur ing one transfer

generation.

Material and method

In the present study, 13 different strains of rat ascites hepatoma, AH 62, 601,322, 66, 127, 149, 602, 49, 7974, 13, 414, 66F, 62F, which were being kept by serial animal passage in this laboratory, were used (YOS~IDA 1960) (Fig. 13 and 14). The frequency of free cells and islands of various sizes is indicated in Table 1. This frequency, in general, is constant in every trans- plant generation. For the transplantation of the ascites hepatoma, Japanese common albino rats, weighing 90 to 100 grams, were employed during the experiment.

Table 1. Frequency o/ the occurrence o/single cells and various sized islands o/cells in 13 rat ascites hepatoma

Stra,ins

AH 66F AIt 62 F AH 13 AH 414 AI~ 66 All 62 AH 127 AH 7974 AK 49 AK 601 AK 602 AK 322 AH 149

Freqncncies(%) of islands composed of Single

%

100 98.6 97.4 96.8 39.8 22.9 20.0 17.7 8.8 6.5 6.5 6.1 5.3

2 cells

o2 2.5 2.2

13.6 7.4

11.7 28.6 8.4 8.1 3.1 6.2 7.5

3 cells 4 cells

o 0.1 0.4 0.3 7.9 4.7 3.6 1.3 3.9 2.8

13.0 7.6 3.6 1.9 4.5 3.4 1.2 0.9 3.5 1.7 3.5 3.5

l~1ore th~n 5--10 cells 11 cells

0 0 10.5 0

lF0O o 0.1 17.5 16.7 15.1 51.9 25.7 35.8 16.1 16.9 16.9 60.4 18.8 58.9 12.3 75.9 11.8 70.1 32.1 48.2

In all strains every 500 cells, obtained from 4 rats 4 days after intraperitoneal inoculation, were counted for the analysis of frequency percent among the 4 specimens (2000 cells) were indicated. In this counting every single cells and hepatoma cells, composed of more than 2 cells, were evaluated as one units.

88 TAKASHI YAMADA :

The negative electrical charge of cell surfaces was determined by the colloid titration method introduced by Terayama for both quantitative and qualitative analysis of various kinds of polymer ions (polyelectrolyte). Recently, TEI~AYAMA applied this method for the determination of the negative electrical charge of ascites hepatoma cells. The procedure of the titration is as follows (Fig. 1): 5 ml of a 0.018% protamine-sulfate solution are placed in a small Erlenmeyer flask; 0.1--10 ml of various concentrations of sodium hydroxide or hydro- chloric acid (0.1--0.001 N) are added to adjust the pH. Then, 0.5 ml of hepatoma cell suspen- sion in 0.25 M sucrose solution are added. After shaking for a few minutes, 0.03 to 0.01 ml of

Pro~am:n- sulphate Pol~uv/n,~l,~u//;h,~te kczliu~ ~Ip~)ill'Y~ large mol@oH/aP /'on)

Q Csll

>

|

(ne~a/iv8 l~ge molecul~: /hm)

$ e@ ~) er

r

~ suspens/on

e e e e ( ] ~ e ee

Blue colouf" of/o/u/'din chamge8 /0 :ed#/sh puz'p/e Fig. 1. Schematic presentation shewing the principle of colloid titration method (TERAYAMA)

a 0.1% toluidine blue is added as an indicator. Following this, 0.0006--7 N PVSK (poly- vinyl sulfate kalium) solution is added titrimetrically until the blue color of the indicator is changed into reddish purble. At the same time, a sudden precipitation of the complex of hepatoma cells-protamine-sulfate-PVSK occurs. The blank titration is done in the same manner without the addition of the hepatoma cell suspension. The titration volumes are estimated on the basis of the nitrogen content of the tumor cell suspension employed. The tumor cell suspension is prepared as in the previous report, except thet a 0.25 M sucrose solution is utilized as a suspended medium instead of phys. saline. The evaluation of the "Tween effect" is similar to that described in detail in the previous report (Table 2 and 3).

Table 2. The "Tween 80 effect" on the isolation el single cells/rein islands o] hepatoma cells o] 9 different strains (Tween 80 1%, Incubation time: 60 min)

5-day-old tumor cells

Increase percent of single cells Increase percent of single cells (Grade of Tween effect) (Grade of Tween effect)

Strains Agitation time (min) Strains Agitation time (rain)

30 60 90 30 50 90

AH 62 86, 65, 68 76, 53, 55 76, 34, 62 I AH 149 23, 47, 46 38, 31, 39 33, 44, /

I

AH 601 71, 45, 86 66, 58, 71 57, 66, 57 ] AH 49 28, 16, 50 18, 35, 55 28, 30, 38 AH 322 51, 58, 44 53, 42, 42 34, 53, 46 ] AH 602 19, 15, 20 21, 35, 19 24, 41, 25 AH66 37,49,37 37,53,44 47,50,41 ]AI-I7974 10, 2, 7 12,11,12 14, 9, / AI~ 127 31, 45, 45 32, 51, 36 27, 38, 37 I

All tumor cells used are 5-da T-old. Increase percent of single cells was estimated by these procedures: The frequency of sin le cells of every specimen was counted before (A) and after (B) treating with Tween 80 in same conditions. And the differences (B--A) between both frequencies were indicated as Grade of Tween effect. This is the results obtained from every different 3 specimens. Details were described in the previous report (YA~ADA 1962).

Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat ascites hepatoma. I I 89

Table 3. The ':Tween 80 effect" on the isolation ol single cells lrom islands o] hepatoma cells o/ 9 di]ierent strains (Tween 80 1%, Incubation: 60 rain)

10-day-old cells

Increase percent of single cells (Grade of Tween effect)

Strains Agitation time (min)

30 60 90

AI162 AH601 AI-I322 AIt66 AIt127

57,65 14,17 10,47 14, 4 57,12

56,60 12,24 59,53 18, 0 49,14

Strains

Increase percent of single oens (Grade of Tween effect)

Agit~Mon time (min)

30 60 90

I 35,59 AI-I149 I 11,10 27,16 AH49 I 51,29 63,58 AI-I602 14,17 46,22'-1/ AR 7974 8, 9

8, 4 53, 28 12, 24 10, - - 1

All tumor cells used are of 1O-day-old. The procedure is same as the Table 2.

7, 8 59, / 27, 16 11, 1

Exper iment 1. The mechanism of ac t ion of Tween 80 in the dissociat ion of m u t u a l bonds of cells in ascites hepa toma .

1. "Tween 80 e]/ect" and the negative electrical charge o/cell sur/aees. Five -day - old t u m o r cells were examined b y compara t i ve tests , as shown in Table 2. The "Tween effect" was de t e rmined in nine di f ferent s t ra ins and the nega t ive electr ical charge of cells f rom corresponding t u m o r s t ra ins were also de te rmined . B y "Tween effect" , we mean the degree of mu tuM bond ing be tween cells which can be d i s rup t ed b y means of the Tween agent . It is felt t h a t ~his is a ref lect ion of the l ip id subs tances which are responsible for the m u t u a l bonding. The resul ts are shown in Fig . 2 and Table 2.

I t can be seen t h a t the nega t ive electr ical charge of the surface membranes was highest in those strMns which were s epa ra t ed mos t r ead i ly b y Tween 80, whereas i t was t he smMlest in A H 7974. Of all the t u m o r s t ra ins examined the l a t t e r s t rMn was sepa ra t ed wi~h the mos t d i f f icu l ty b y Tween 80. The remain ing tumors l ay inbetween. However , AI-I 602 had a r e l a t ive ly h igher nega t ive charge, a l though i t was sepa ra t ed b y Tween 80 wi th diff iculty.

2. The changes o/ electrical charge o/ hepa- toma islands ( A H 601) under the e/leer ol

7q ~ 70-q

ff/~g//

Y l l / ,r ..,4 ;'

# I I i

, / ~<tr +

i J l ._L__

6 7 6 3 pH

Fig. 2. Colloid titration curves of 7 rat ascites hepatomas of different island celI types in 5-day-old tumor

~ 8

Tween 80. I n this s tudy , 5-day-o ld t u m o r t r ansp lan t s were used. Two ml of a 1% Tween 80 were mixed wi th 0,5 cm a of t u m o r cell suspension respec t ive ly in 20 tubes , hav ing a n i t rogen conten t of 1 - - 2 mg/cmK The tubes were ag i t a t ed under the same condi t ions for per iods of 10, 30, 60, 90 and 120 minutes . F o r each per iod of t ime, one half of these were cent r i fuged and the s u p e r n a t a n t f luid examined t i t r imet r i ea l ly . The remain ing tubes were also cent r i fuged b u t the s u p e r n a t a n t f luid was d iscarded, and af te r washing the t u m o r cells adheren t to the glass wall wi th 0.25 M sucrose solution, the t u m o r cell sed iment was examined b y the t i t r a t i on method . I n this pa r t i cu l a r exper iment , the resul ts for nega t ive

90 TAKASttI YA~A1)A :

electrical charge were determined on the basis of nitrogen content (mg) of tumor cells for both supernatant fluid and tumor sediment. In addition, a similar experiment was carried out using trypsin (0.5 mg/cm 3 in pH 5.6 acetate buffer solution) and 0.25 M sucrose (alkali solution).

As can be seen in Fig. 3, the negative electrical charge of the cell surface increased temporarily during the dissociation of ascites hepatoma cells by

• 10 -~ - - - - - - supernalant /I

27 ,, tumor sediment i / dr I .~ ~s 4 / " - ' - . . -

.~ . i . i i sz- : z . i /

70 80 60 80 mill 7ZO Agital:bn T/me

Fig. 3. "Tween 80 effect" and change of volume of colloid titration: AH 601 (5-day-old tumor)

xTo-,l e ~ T:ypsin(O,5:::g/co.,pll ~8)

Zr

~ M/~ Sucrose

e ol , "7 , , I 70 30 #0 SO rn /n 7Z O

A@itotion ~me

Fig. 4. T r y p s i n effect a n d changes of vo lume of colloid t i t r a t i o n : A H 601 (5 -day-o ld t u m o r )

Tween 80, while the negative charge in the supernatant fluid increased lineally. Parallel morpho- logic investigation revealed that the intercellular dissociation was slight after ten minutes of agita- tion, moderate after 30 minutes, and severe after 60 minutes of agitation. However, the temporal increase of negative electrical charge of the cell surface was not seen in trypsin or alkali solutions although these also dissociated the cellular bonds (Fig. 4). Similar results of a temporal increase in the negative electrical charge by Tween 80 were seen in another strain of ascites hepatoma (AH 62).

3. Chemical analysis o/ super- natant /luid o/ tumor cell suspen- sion a/ter treating with Tween 80 (AH 601). In order to examine the phenomenon in the above experiments, the substances de- pleted by Tween 80 from the sur- faces of the hepatoma cells (AH 601, 5-day-old tumor cells) were analy- sed. From reports of DANIELLI a n d E A S T : f , it was anticipated that Tween 80 removed ]ipoprotein from

the cell surface. As an indicator of lipid removal, we measured cholesterol electro- photometrically (BLooR). The determination of cholesterol was chosen, because Tween 80 is itself a fa t ty ester (polyoxethylene sorbitan mono-oleate) and the quanti tat ive differentiation among neutral fat, fa t ty acid, phospholipid and Tween 80 was technically difficult. As an indicator of protein breakdown, nitrogen was measured by the micro-Kjeldahl method.

As shown in Table 4, the depletion rate of cholesterol into the supernatant fluid was much greater than that of nitrogen. This indicates that the depletion of fat by Tween 80 was greater than tha t of protein.

Experiment 2. The change of negative electrical charges of cell surfaces in islands of ascites hepatoma during one transfer generation.

~[utual adhesiveness of tumor cells in "hepatoma islands" of the rat ascites hepatoma. I I 91

I n a previous r epor t i t has been no ted t h a t the "Tween effect" var ies in pro- por t ion to the growth ra te of h e p a t o m a cells dur ing one t ransfer genera t ion (Fig. 5). This phenomenon was exami- ned by an eva lua t ion of the nega t ive electr ical charge of the corresponding t u m o r cell. The asei tes t u m o r cells, in general , prol i fera te most m a r k e d l y dur ing the 4th to 7th day , whereas the i r proli- fera t ion ra te decreases dur ing the 8th to 10th d a y and decreases even fur ther in sub-

Table 4. Chemical analysis o/supernatant fluid o i tumor cell suspension after treating with Tween 80: AH 601,

5-day-old tumor

Nitrogen Cholesterol :-kgitation

time

rain

0 10 30 60 90

Increase Increase quantity rate rng/em a

(o.o6) 0.44 0.65 1.48 1.00 1.54 1.84 1.84

Increase quantity

y/era ~

.G0 410 9.0

69.1

Increase rate

1.43 2.25 7.67

Cell suspension of 15 cm 3 having the nitrogen con- tent of 5.07 mg in every specimen, and 1% Tween 80 in 0.25 M sucrose solution of 100 em a were mixed and agitated under the same condition for 10--90 minutes. After that chemical analysis of the every supernatant fluid was carried out.

sequent days. Similar f indings were observed b y KLEIN. Because of this, we chose to inves t iga te the five- and t en -day-o ld t u m o r cells in order to es t imate the re la t ionship of the "Tween 80 effect" and the ra te of cellular prol i fera t ion.

1. "Tween 80 effect" on the 5- eo and lO-day-old tumor cells ( A H 601), and the correlation with negative electrical charges o i the cell sur/aces. The increase of electr ical charge of 5- and 10-day-old t u m o r cells under the "Twcen 80 effect" was es t ima ted , as out l ined in the experi- men t above.

As shown in Fig. 6 and 7, the negat ive electr ical charge of bo th cell sed iment and s u p e r n a t a n t f luid of 5-day-o ld t u m o r cells Was g rea te r t han t h a t of lO-day-old t u m o r cells.

Furthermore, the electrical charge

of tumor cells of different ages

(AH 601) was measured without

the "Tween 80 effect" and is illu-

strated in Fig . 8. As can be seen, the nega t ive electr ical charge of 5-day-old , 1 - a n d 2-day-old , and 10-dayold t u m o r cells decreased in t h a t order. Such decrease paral le ls t h a t seen wi th the "Tween 80 effect" in Fig. 5.

z. Krebsforsch. ]~d. 65

%

gg

gO

0 2 q 8 0 70 Oa#8 utter inoculation

Fig. 5. Increase percent of single cells isolated from islands of ascites hepatoma AH 601 treated with Tween 80 ("Twcen effect"). ~ treated with Tween-80. ------ treated with phys. saline (control).

(Refer to Fig. 5 and 6 in the previous paper)

JeT0 -s~

78

N

&dw-o ld turnur usll~

I I I I I gO 30 80 ,gO lnin ]gO

A#i&lion 77rne Fig. 6. "Twoen 80 effect" and change of volume of colloid titration on different ages tumor ceils

(AH 601) (supernatant)

92 TAKASItI YAMADA :

2 ? - x]O -#

78 [r

N s

5- dau-old /urnor cells

I q I I J 70 30 60 9g rn/n IZ0

,4#l/all'on 5"me

" T w e e n 80 e f fec t " and change of vo lume of t i t r a t ion on d i f ferent ages t u m o r cells (AH 601)

( tumor sed iment )

x 70 -q /

5 6 7 8 0

2// Fig. 8. Colloid t i t r a t i on curve of ra tase i t es h e p a t o a m (AH 601). x x 5-day-old t u m o r cells; o o

1 § 2-day-old t u m o r cells; �9 �9 10-day-old t u m o r cells

Fig. 7. colloid

2. The "Tween 80 e//ect" on the various kinds o/ tumor cells and the electrical charge o/ cell sur/aces during cellular proli/eration. The "Tween 80 effect" on 5-day- and ]0-day-old tumor cells was measured with various strains of ascites hepatoma (island type) in the same manner as given in the previous report (Table2 and 3, Fig. 9 and 10). The "Tween 80 effect" varied not only with the age of the tumor cell but also with each strain. Hepatoma strains may be classified into three types, as distingiushed by the "Tween 80 effect": ]. the cell disso- ciation produced is marked in tumor islands of either age (AH 62 and 322); 2. cell dissociation is marked only in the 5-day-old tumor islands (AH 601, 66 and 149); 3. cell dissociation occurs only to a minor degree in both age groups (AH 602 and 7974).

The electrical charge of corres- ponding tumor cells was determined in p i t 5--9 solutions in three kinds of the above strains, and the results are indicated in Fig. 9 and 10. In AH 149, the electrical charge of

5-day-old hepatoma islands increased more than in the 10-day-old hepatoma islands. Similar results were found with At t 601 as shown in Fig. 8. In con-

!

,: 70-~'1 gH zOZV '? zel-

~ 0 P ) I I 5 6 7 8 3 5

pH

trast, in AH 7974, no diffe- A//1r rence was noted, whereas

in AH 62 and AH 602 the difference was also slight in p i t 5--7 solutions. These results indicate a o

parallel relationship bet- �9 �9 ween electrical charge and

the "Tween 80 effect". / - , i o I t m u s t b e pointed o u t

J �9 ~ I / / o however, that the tumor ~176 p .... I islands grow in size after

7 s s inoculation and the size of P// 5-day-old hepatoma islands

is smaller than that of Fig. 9. Colloid t i t r a t i on cu rve of r a t asci tes h e p a t o m a (island

type) . - - 5-day-old cell; - - - - - - 10-dayold cell

Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat ascites hepatoma. II 93

10-day-old islands. The in- =io_~ fluenee of size of the hepa- 7q toma islands must be not [elm## neglected in the deter- ~ 72 mination of the electrical ~ 70 charge. Furthermore, it .~ 8 was noted in the previous t report that any signi- ~ s ficant relation between ~ the "Tween effect" and '~

~5 2 the size of hepatoma is- lands could not be revealed in AH 601. However, a exact comparative analysis of the size of islands between various strains ~/o-~/ is difficult because the r./~7~I frequency and the size of ~'/"'~"|.% 7z[

islands, vary in every .~ 7ol strain (Table 1).

Consequently, the vari- .~ e ation of the electrical ~ charge was analyzed in ~ q ascites hepatoma of the N free-cell type (AH 13,414, ~ 66F and 62F) avoiding ~" the complication of diffe- 5 rent island sizes. The result of such a study, as shown

- AN 62 /

/

O O

5 # 7 8 3 p//

Fig . 10. Col lo id t i t r a t i o n c u r v e of type) . - - 5 - d a y - o l d cel l ;

A// 8S F /

�9 / 0 o /

_ / i I I y �9 ~ /

/ " ." U�9 / � 9 o / /'go

/ /

e / / ."o

/ o I I I I 8 7 8 3 E

p//

,4//8Y2

o

p - d # 7 B 8

p// r a t asc i tes h e p a t o m a ( i s l and - - - - - - 10 -day-o ld cell

- A//#2 f

../Q, ./o o

/ /

//o o -/

l l ~ l 8 7 8 ,Y

p// Fig . I1 . Co l lo id t i t r a t i o n c u r v e of r a t a sc i t e s h e p a t o m a (free celt

type) . 5 - d a y - o l d cel l ; - - - - - - 10 -day -o ld cell

in Fig. 11 and 12, indicates =10.~/ that the difference of the ,4//13 negative electrical charge [r between the different age ~ lz~ . groups also was found an N 7oh �9 y �9 the free-cell type, where ~ ~ ,~ the size of the cell was the .~ 8- % / � 9 t "~ same throughout one trans- ~ # - / ~ " _

t~ i o o fer generation. This indi- ( ~- / ~ % o o cares that the negative N /// electrical charge of the '~ % z z / cell surface of tumors ~- ~/ F , , ]

_ ~ o

�9 o/f

- / o

W e - /~

I I I ]

varies in proportion to the ~ 8 7 8 8 s 6 7 8 8 growth of the tumor strain P// P//

Fig . 12. Col lo id t i t r a t i o n c u r v e of r a t asc i tes h e p a t o m a (free cell with and wi thout an epi- type) . - - 5 - d a y - o l d c e l l ; - - - - - - 1 0 - d a y - o l d cell

thelial bonding structure. This fact, that the differences of the negative electrical charge between both 5- and 10-dayold tumors of free-cell type were generally greater than that of the

7*

94 TAKASH~ YAMADA :

F i g . 13 F ig . 1r

F i g . 1 3 a - - c . A s c i t e s p i c t u r e of f ree -ce l l t y p e a s c i t e s h e p a t o m a of r a t s ( W r i g h t - G i e m s a s t a i n ) ( • 900). a A H 6 6 F . b A N 1 3 . c A H 4 1 4

F i g . l ~ a - - o . A s c i t e s picture, of i s l a n d - t y p e a s c i t e s h c ~ a t o m a of r a t s ( A c e t o - g e n t i ~ n v i o l e t s t a i n ) ( • 500). a A t t 6 0 2 . b A H 7 9 7 4 , c A H 6 2

island types of aseites hepatoma, revealed more intimately the correlation between the negative electrical charge and the growth, because the free-cell type generally proliferates after intraperitoneal inoculation more markedly than the island type.

Mutual adhesiveness ot tumor cells in "hepatoma islands" of the rat ascites hepatoma. II 95

Discussion

In the first experiment, it was shown that the dissociating effect of Tween 80 on hepatoma island cells was different from that of trypsin or alkaline solutions. With Tween 80, the distinct phenomenon consisted of a temporal increase of negative electrical charge of the cell surface during the dissociation of cell bonds. This appeared to indicate that the "Tween 80 effect" occurred not only due to the depletion of some substances chemically from the cellular surface, but also due to a physicoehemical action. The greater the negative electrical charges of the cellular surface, the more easily were the hepatoma islands dispersed by the Tween 80 solution (Fig. 2 and Table 2 and 3).

CURTIS has already commented on this inter-relationship between the negative electrical charge of cell surfaces and their mutual adhesiveness. The cell surface structure consists of a bimolecular layer of lipid covered with globular protein on either side (DA~InLLI). Thus, it appears that the relative depletion of lipid from the cell surface by Tween 80 might cause a spatial rearrangement in the negative ions of the remaining proteins of the cell surface. In addition, the mole- cular chains of the remaining proteins may be broken in some parts.

This effect on the protein breaking by Tween 80 may not be profound because the separated tumor cells do not change in form, wehreas the trypsin-separated tumor cells become round (Fig. 8--10 of Report 1). Although some other causes must be considered, it is felt that the ion rearrangement at the cell surface is due chiefly to a temporal increase of negative electricM charges and this is re- sponsible in part for the dissociating effect of Tween 80.

In the studies it was found that in different strains of aseites hepatoma, regardless of the size of the cells, there were differences in negative electrical charge on the surface membrane. The cause of these differences in bhe negative electrical charge is probably not only due to the amount of negative ion on the cell, but also due to variation in the arrangement of the negative ion.

The second set of experiments revealed the significant variability of negative electrical charges on the surface membrane as related to cellular proliferation during one transfer generation: The negative eleetricM charge was significantly higher in tumors of the most actively growing stage. In addition, it was note- worthy that this variability of negative electrical charge showed a specific pattern in each strain and appeared to be related directly to the "Tween 80 effect".

The results indicated that the variability of the "Tween 80 effect" i.e., the mutual adhesiveness of tumor cells, is caused in part by the difference of negative electrical charges in the surface membrane. This may also imply that the tumor cells with high negative electrical charges not only may be more loosely bonded to each other but also may have more motility, resulting in the frequent liberation of tumor cells into vascular canals.

Analogous observation have already been reported by LowlcK and JAMnS employing bacterial systems of aerobacter aerogenes. They pointed out that the inhibitory effect of proflavin and crystal violet on the electrokinetie properties was variable and that this variation was related to the age of the parent inoeulum. They thought that the inhibitory effect was due to differences in the suscepti- bility of the cell membranes to the chemical compounds and that it was also

Z. Krebsforsch. Bd. 65 7a

96 TAKASttI YAI~IADA :

related to differences in age. RAPPAPO~T et al. investigated the role of attachment and out-growth of tissue culture cells to the treated glass surface in a protein-free sythetic medium. They observed a "released tendency" of attached cells from the glass wall during out-growth, and postulated the hypothesis that the protection against this "released tendency" was induced by the secretion of proton which bound with a negative charged substance in the glass surface. However, they did not refer to the variability of electrical negative charge of the cells.

Swan stressed in his article that cell contact has an antagonistic relationship to cell proliferation. PURDOM et al. found different electrokinetic properties in four substrains of the MCIM sarcoma of mice. He correlated these with the differences in the ability to metastasize and with the growth rate of each of these strains.

Additional reports (MA~T~EZ 1956, WOOD 1958) have shown that the fre- quency of lung metastases appears to be in proportion to the growth rate of the primary tumor inoculated into the subcutaneous tissues of the tails of mice.

In contrast, AMBROSE has indicated that he was unable to find variation in the e]ectrokinetic properties of Ehrlich ascites carcinoma during one transfer generation. These phenomena in these various reports, may be explained by the findings of the present paper.

Using quantitative electrophoretic analysis, ]~ACHTOLD and GEB~t~RDT deter- mined the pattern of serum utilization during the in vitro growth of monkey kidney cells. They found that the cells utilized mostly the ~2-globulin fraction from the medium for the formation of a cell layer. LIEBER~AN~ and OvE, FIS~E~, Puck and SATO reported similar results (Foetin fraction). Most recent evidences have stressed the necessity of lipoprotein for the adhering ability of cells in tissue culture during proliferation (L. W~Iss 1959 and EASTY and AMBROSE 1960). Thus, the evidences obtained in this reports support the recent opinion.

The lipoprotein of the cell surface membrane of the hepatoma island may change in quality or quanti ty during one transfer generation. This change may be the cause of the alteration of negative electrical charge and of variations in the intercellular adhesiveness.

Summary

The phenomena concerning the mutual adhesiveness of rat ascites hepatoma cells, which were observed in the previous paper, were analysed physicochemically. Under the effect of Tween 80 solution, the negative electrical charge of the hepatoma islands increased temporarily during the dissociation of the cells. When treated with Tween 80, the decrease of cholesterol from the cellular surface of hepatoma islands was more marked than that of nitrogen. The negative electrical charge of the cell surface in several strain of hepatoma islands varied in propor- tion to the proliferation of the tumor cells during one transfer generation. And this relation was also parallel to "Tween 80 effect" in the corresponding hepatoma islands. The variability of the negative electrical charge and mutual adhesiveness of aseites hepatom~ cells during proliferation is discussed with special respect to the growth of tumor cells and their emigration into blood vessels.

Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat ascites hepatoma. I1 97

D e r g e g e n s e i t i g e Z u s a m m e n h a n g de r T u m o r z e l l e n in ,,Hepatom.Inseln" des R a t t e n - A s c i t e s h e p a t o m s

I I . Untersuchungen fiber den Meehanismus der Tumormetas tas i e rung

Zusammenfassung Die in einer vorhergehenden VerSffent l ichung In i tge te i l ten Ph~nolnene hin-

s ichtl ich des gegensei t igen Zusammenha l t e s yon Asc i teshepa tomzel len wurden phys ikochemisch analys ier t . U n t e r der E inwi rkung einer Tween 80-L6sung stieg die nega t ive e lektr isehe L a d u n g der Hepa tominse ln wahrend der Dissozia t ion ihrer Zellen zeitweil ig an. Die A b n a h m e yon Cholesterol an der Zel loberf lache war dabe i mehr ausgesprochen als die yon Stickstoff . Die nega t ive e lektr isehe Ladung der Zelloberfl~che var i i e r te in verschiedenen Linien des Asc i teshepa toms, en t sprechend der Pro l i fe ra t ion der Tumorzel len in einer l~lbertragungsgenerat ion. Diese ]3eziehung liel3 sich auch fiir den Tween 80-Effekt an den t t e p a t o m i n s e l n nachweisen. Die festgestel l te Va r i ab i l i t a t der nega t iven e lekt r i schen Ladung und des gegensei t igen ZusammenhMtes dfirfte Beziehungen haben zum W a c h s t u m und zur E inwande rung der Tumorze l len in Blutgefal~e.

References AMRlCOS~, J. E. : Difference between the electrical charge carried by normal and homologous

tumor cell. Nature (Lond.) 177, 567--577 (1956). - - ]). M. EASTY and P. C. JONES: Specific reactions of polyelectrolytes with the surfaces

of normal and tumor cells. Cancer (Philad.) 12, 4 3 9 ~ 4 7 (1958). BACHTOLD, J. G., and L. P. GEBI-IARDT: Utilization of serum proteins by normal and polio-

virus infected monkey kidney cells. Exp. Cell Res. 13, 432~433 (1957). BLOOR, W. R. : The determination of cholesterol in blood. J. biol. Chem. 24, 227--236 (1916.) CVRTIS, A. S. G. : Cell contacts. Some physical considerations. Amer. iNaturalist 94, 37--56

(1960). DA~-IEI~LI, J. F. : The cell surface and cell physiology. In: Cytology and cell physiology (ed.

by G. BOUR~). New York: Oxford Univ. Press 1948. EASTY, G. C., D. M. E~STY and J. E. AMBrose: Studies of cellular adhesiveness. Exp. Cell

Res. 19, 539--548 (1960). FIshER, H. W., T. T. Pvc~ and G. SATo: Molecular growth requirements of single mamma-

lian cells. The action of fetuin on promoting cell a%achment to glass. Proe. nat. Acad. Sci. (Wash.) 44, 4--10 (1958).

J A ~ s , A.M., and P. J. BARRY: The effect of bacteriostatic agents on the electrokinetic properties of aerobacter aerogenes. Biochim. biophys. Acta (Amst.) 1~, 186--193 (1954).

KLnI~, G., and LJ~zL5 RI~vEsz: Quantitative studies on the multiplication of neoplastic cells in vitro. I. Growth curves of the Ehrlich and MCIM ascites tumors. J. nat. Cancer Inst. 14, 229--275 (1953).

LIn~ER~A~, L, and P. OVE: Purification of a serum protein required by a mammalian cell in tissue culture. Biochim. biophys. Acta 25, 449~450 (1957).

LOWICK, J. H. B., and A. M. JAMES: The effect of bacteriostatic agents on the electrokinetic properties of aerobacter aerogenes. II. Crystal violet. Biochim.biophys.Acta 17, 424--433 (1955).

MARTI~Z, C., G. MIROFF and J. J. B1TTna: Effect of size and/or rate of growth of a trans- plantable mouse adenoma on lung metastasis production. Cancer Res. 16, 313--315 (1956).

PU~DOM, L., E. J. AMBROSE and G. KLEIn: A correlation between electrical surface charge and some biological characteristics during the stepwise progression of a mouse sarcoma. Nature (Loud.) 181, 1586--1587 (1958).

98 TAKASttI YAMADA: Mutual adhesiveness of tumor cells in "hepatoma islands". II

I~APPAPORT, C., J. P. POOLE and H. P. Rs Studies on properties on surface required for growth of mammalian cells in synthetic medium. Exp. Cell l~es. 20, 465--510 (1960).

8WA~, M. M.: The control of cell division. A review. Cancer Res. 18, II18--I160 (1960). TERA:fA~A, H. : Method of colloid titration. A new titration between polymer ion. J. Polymer

Sci. 8, 243--253 (1952). -- The substances in surface membrane of cancer cells and metastasis. Jap. J. Cancer Clin.

(Tokyo) 6, 459--460 (1960). WEIss, L. : Studies on cellular adhesion in tissue culture, i. The effect of serum, 2. The

adhesion of cells to gel surfaces. Exp. Cell Res. 17, 499--515 (1959). WooD, J. 8., E. D. HOLYOKE, W. P. C. CLASO~, 8. C. 8OMMEr~S and S. WA~E~: An experi-

mental study of the relationship between tumor size and number of lung metastasis. Cancer (Philad.) 7, 437--443 (1958).

u T.: Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat aseites hepatoma - - Studies on the mechanism of tumor metastasis. I. Z. Krebsforsch. 6fi, 75--86 (1962).

YosttlnA, T.: On the aseites hepatoma. Summary of the results of studies obtained during I0 years from 1951--1960. Tokyo J. reed. Sci. 68, 715--749 (1960).

Dr. T. YAMADA, The Department of Pathology, The Medical Institute of Sasaki Foundation,

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