Zeitschrift fiir Krebsforschung 66, 109--114 (1964)

From the Department of Pathology and Surgery, The Medical Lastitute of Sasaki Foundation, Tokyo, Japan (Director: Prof. Tomzo YosmDA)

Mutual adhesiveness of tumor cells in "hepatoma island" of the rat ascites hepatoma. III. The influence of nitrogen mustard N-oxide.

By

TAKASH1 YAMADA and H m o s m SA'roo

With 3 Figures in the Text

(Received August 23, 1963)

The an t icancer drugs have been repor ted to exer t an inh ib i to ry effect on fo rma t ion of t u m o r metas tases under bo th expe r imen ta l and clinical condi t ions (SATO [1, 2]). I n our exper imen ta l resul ts , t he f requency of the appea rance of t he r a t asei tes h e p a t o m a cells in t he local venous b lood was s ignif icant ly decreased under t he influence of n i t rogen m u s t a r d I~-oxide (HN2-O), if i t was given in vivo a t a dosis over the min ima l effective dosis to each h e p a t o m a s t ra in (Table 1).

Table 1. Influence o/HN~-O on the/requeney o/the appearance O/the rat ascites hepatoma cells in the local vein after inoculation into the right thigh muscle o/rats

Control r a t s Ra t s t r ea t ed wi th HN~-O

Strains

Att 13 A]t 601 AH7974

Tota l No. of T u m o r cell T u m o r cell r a t s tes ted posi t ive smears posit ive ra t s

10 I 36/50 ] 9/10 20 27/91 11/20 18 9/89 9/18

Tota l No. of ra ts tes ted

10 10 10

T u m o r cell T u m o r cell )ositive smeaxs posit ive ra t s

3/50 3/10 0/50 0/10 3/47 3/lO

Each ~nimM received ~n i.p. iajection of 107 of tumor cells, suspended in abou~ 0.3--0.4 em a physiologicM saline, into the right thigh muscle. In the case of AI:[ 13, HN2-O was given i.p. on the 3rd and 4th day and aspiration of blood from the locM vein was carried out on the fifth day of experiment. In the cases of AH 601 and AH 7974, HN~-O injection was given i.p. on the 8th and 9th day and blood was taken on the 10th day. Because the recipient rats receiving AH 13 could not survive over 7 days. The dosis of HN2-O was I rag• for AH 13 and AH 601, 3 mg • 2 for A l l 7974 (minimal effective dosis of ItN2-O for AH 7974 is 5 rag/100 g B.W.). The venous blood was aspirated directly from the vena cava inferior on laparotomy. The venous bloods were put into several hematoerit tubes, and centrifuged at 3,000 r.p.m. for 10 minutes. The superficial white layer of the sediment in the tube, usually containing most of the nucleate cells, was smeared on 5 glass slides.

Frequency of the appearance of tumor cells was expressed by ratio of positive slides to the all slides examined.

The effects have been genera l ly a t t r i b u t e d to the d i rec t i nh ib i to ry ac t ion of the drugs upon the t u m o r cells in circulat ion, and, in consequency, should be para l le l wi th t he chance of ind iv idua l t u m o r cells coming in con tac t wi th the drugs. However , mul t ip le factors are invo lved in the fo rma t ion of metas tas i s . A n y effects on one of these factors such as l ibera t ion vascular , pe rmea t ion , lodge- m e n t a n d pro l i fe ra t ion in t h a t sessile stage, could resul t in a l t e ra t ion of m e t a s t a t i c r a t e of a s t ra in of ma l ignan t t u m o r cells in a s t ra in of animals .

Z. Krebsforseh., Bd. 66 8

110 T ~ s m YASVIADA and H~,OSHI S~To~:

I n order to ana lyse t he i nh ib i t o ry effect of NH2-O on the emigra t ion of the r a t asci tes h e p a t o m a cells in to t he local vein, an a t t e m p t was made to tes t t he influence of NId~-O on the m u t u a l adhesiveness in the h e p a t o m a is lands.

T h e resul ts are suggest ive of the poss ib i l i ty t h a t the m u t u a l adhesiveness of t u m o r cells becomes s t ronger when pro l i fe ra t ion of the t u m o r cells is suppressed b y an t i cancer drugs, a n d t h a t in consequence, t he an t i cancer drugs m a y inh ib i t the emigra t ion of t u m o r cells.

Material and Method Material used in the experiment are rat ascites hepatomas, At t 601, AH 62 and AH 322,

M1 of which are composed mainly of conglomerated hepatoma eel]s, called islands, and a small number of isolated tumor cells proliferating in the aseitie fluid. The hepatoma islands consist purely of hepatocellular carcinoma cells containing no other kind of cells. The islands vary in size and shape in the three tumor strains, and detMled cytological characteristics of each of them have been fully analysed. Difference in biological properties of the tumor strains have Mso been known: for example, they differed in the sensitivity to HN2-O. The minimum effective doses in r ive of HN~-O to AI-I 601, AH 322 and AH 62 are 0.1 rag/100 g (B.W.)% 5 rag/100 g (B.W.) and 2 rag/100 g (B.W.) respectively (YOSnlI)k 1960).

Seventy-eight hybrid rats, weighing 100--120 g, received about 107 tumor cells intra- peritoneally. The animals were divided in four groups, and 0.5 mg of Id!~2-O was injected intraperitoneMly once or twice on the 3rd and 4th day after inoculation. The total HN2-O dosis for each group was 0.5 rag, 1.0 mg and 2.0 rag, respectively.

On the 5th day the asci~es fluid was collected and suspended in physiological saline by slow centrifugation to make a 20 volume % suspension. Two cm 3 of the tumor suspension was put into 8 cm a of 1% Tween 80 in physiological saline, and the mixture was incubated for 30 minutes and agitated for 30 mimltes at 370 C. Before and after treating with Tween 80, the percentage of free cells in the tumor suspension was counted: The degrees of increase in percentage of the free cells in the tumor suspension after above procedure were estimated as the grade in the looseness of mutual adhesiveness (YAMADA 1962). The control experiments were performed under the same conditions without previous administration of HN~-O. The experiments, using A_I-I 601, were repeated four times on different dates.

Result

No signif icant change in the r a t io of t he free cells and is lands in the asci t ic fluids, nor in the genera l appea rance of the is lands has occurred af ter the ad- min i s t r a t i on of HN2-O in a n y expe r imen ta l series, a l though the i r i nd iv idua l componen t s have become swollen. Bu t , when the in r i v e p r e t r e a t e d t u m o r cons t i tuen t s were t e s t e d for Tween 80 effects in v i t ro , r e m a r k a b l e changes have occurred, especia l ly in AI-I 601. As ind i ca t ed in Fig. 1, r epea t ed exper imen t s i n v a r i a b l y r evea led a decrease in d i s in tegra t ion oi t he h e p a t o m a is lands in to free i nd iv idua l cells when p u t in to Twcen 80 a l t e r t tN2-O t r e a t m e n t . The larger the dosis of I tN2-O in vivo, the less conspicuous the freeing of t u m o r cells b y Tween 80 t r e a t m e n t f rom the i s land format ion . The resul ts in A H 601 are suggest ive of t he increased m u t u a l adhesiveness , in para l le l wi th the previous dosis of HN2-O

administration.

W h e n the o the r s t r a in of r a t asci tes h e p a t o m a A H 62 a n d A H 322, s t ra in wi th n a t u r a l res is tance aga ins t t IN2-O, were p r e t r e a t e d wi th the same dosis of HN~O, and t e s t ed for Tween 80 effects, no s ignif icant change occurred in the free celt popu l a t i on ra t io as i nd i ca t ed in Fig . 2. I t is no t i ced t h a t , while for A H 601

i B.W. Body weight of the recipient rat.

~u~bual adhes ivenes s o~ t u m o r cells in " h e r p a t o m a i s l and" . I I l 111

the ItN~-0 doses used are over the minimal effective dosis (1.0 mg/kg), they are far below for AH 62 and AH 322. This result suggest that the small amount of HN~-O less than the minimal effective dosis can not induce significant change in the mutual adhesiveness of ascites hepatoma islands.

gO

qO ~0

3O 3~. I 2~ 20

7~ ~o

o ) o con~, ~S 710 Y~5 "too 2~0 Fig. 1

I J I I I ~'ont. o,~- ,Tg 7,~" mg ;,o

F i g . 2

Fig. 1. Influence of HN~.-O on the mutual adhesiveness in the ra t aseites hepatoma islands (AH 601). Coordinate: Increase percentage of single cells after treating with Tween 80 in vitro. Abscissa: Total

dosis of injected HN2-O

Fig. 2. Influence of HN~-O on the mutua l adhesiveness in the ra t ascites hepatoma islands (AH 62 and AH 322). Coordinate: Increase percenta~ge of single cells after ?~reating with Tween 80 in vitro.

Abscissa: Total dosis of injected HN2-O

eq/ g N 78

x !O-~

72

�9 ~ 70

N 8

2

O AH 73

�9 �9 0

I 1 ~ ) ) ] ~ 7 8 7 8 3

?H ~H

76" / , / N/SGF /

" / o / 7'2 / o / / /

I0 / ,i,/o

e -oiled ~ 7 b) ?

Fig. 3. Influence of HNr on the negative electrical charge of hepatoma cell surface (AH 13 and 66 F) (Colloid ti tration method). - - control. - - - - treated with ItN~-O. Coordinate: Capacity of

Protamin binding

R a t s , we igh ing 1 0 0 ~ 1 2 0 g, rece ived asci tes h e p a t o m a s i.p. H N ~ - O of 1 m g was g iven i.p. twice a t t h e 3rd a n d 4 t h d a y af te r inoculat ion. A t 5 t h d a y r a t s were sacrif iced a n d electric nega t i ve charge of t h e t u m o r was e x a m i n e d in v i t ro b y colloid t i t r a t i on m e t h o d , compar ing to t he control (Tv, I~AyA~A 1952)

T h e capac i t y of p ro~amin b i n d i n g indica tes ?~he grade of ~he n e g a t i v e electrical charge of t h e cellular surface. T he sens i t iv i ty of b o t h t u m o r s to H N ~ - O is s a m e [0.1 rag/100 g (B. W.) ] .

8*

112 TAKASm YA~A])A and Hmosm SAvoy:

Discussion

The present experiment indicated an increase of the mutual adhesiveness of the rat ascites hepatom, AH 601, under the influence of HN2-0 over minimal effective dosis in rive. The results are compatible with the data of the previous papers that the mutual adhesiveness and the negative electrical surface charge in both regenerated liver cells and the aseites hepatoma islands of rats varied in proportion to the growth rate (EIsv, N~I~G, B~c-Oa, DOLJA~SX~; YAMAHA).

In the literatures, a close correlation between the mutual adhesiveness of tumor cells and its emigratory property are disclosed (ABEnCHI~OBI~ and AMBrOSe, CV~TIS). For example, it was reported that more invasive malignant cells of mice with higher metastatic property had higher electrical charge in the surface mem- brane (Pv-aDO~r and A~B~os~) and the mutual adhesiveness in the rat ascites hepatoma with higher electrical charge of surface membrane are also looser (~X~A1V~ADA). In addition, our recent study revealed that the frequency of the appear- ance of the rat ascites hepatoma cells in the local vein were found to be well correlated with the mutual adhesiveness in the hepatoma islands except a strain (AH 7914) (Table 2). I t was also found in our data that the surface electrical charge of rat aseites hepatoma of the free-cell-type was reduced markedly byHN~-O(Fig.3). Therefore, the increase in the mutual adhesiveness of the hepatoma island induced by ItN2-O may be one of important factors responsible for the inhibition of emigration of ascites hepatoma cells into the local vein, resulting in diminution of the metastatic formation.

Table 2..Frequenvy o/ the appearance o/ the rat ascltes hepatoma cells in the local vein after inoculation into the right thigh muscle and the mutual adhesiveness o/hepatoma islands

St r a in s

AH 62 AH 601 AH 322 AI-I 66 AH 423 AH 127 AH 149

AH 602 AI-I 7974

~ v e r a ~ e d i a m e t e r of inocuXa%e4

t u m o r

2.8 ]..7 3.0 0.4 2.7 1.0 1.3

0.7 2.4

F r e q u e n c y of a p p e a r a n c e of t u m o r cells i ~ t h e loca l ve in

Trm~or celt pos i t ive p e r

e v e r y 10 r a t s

4/lo 6/lo 4/lo 3/~o 3/10 2/10 2/10 ~/lo 5/lo

T u m o r ce~ pos i t ive s m e a r s

6/45 19/41 5/41 4/44 5/48 3/44 3/47 2/46 6/40

I~crease p e r c e n t of s i n g l e c e l t s b y

T w e e n 80 t e s t (YAMADA 1962)

86, 65, 68 71, 45, 86 51, 49, 44 37, 49, 37 / / /

31, 45, 45 28, 16, 50

19, 15, 20 10,--2, 7

G r a d e of m u t u a l ~dJ:lefflve]2e~s

marked

moderate

shght

The experimental conditions are same as the one of the experiment in Table I. However no I-IN~-O was given. The bloods of all cases were aspirated at the 10th day after inoculation.

FRIEDMAN and D~VTZ observed that both keratin formation in the epidermal tumor and melanin production in the melanoic tumor cells, which were cultured in vitro, were accelerated by the X-ray irradiation. Increase of serotonin contents in the P-815 ascites mast-cell neoplasm was measured after treatment with cyclo- phosphoramids (M~NG~L). MIDG~s and PIERCE found that both X-ray irradiation and administration of eolehiein induce two or three fold increase in the gonado-

Mutual adhesiveness of tumor cells in "hepatoma island". I I I 113

tropin secretion of human embryonal carcinoma, which was heterotransplanted into the hamster after conditioning with cortison.

If the mutual adhesiveness in the rat ascitcs hepatoma islands may be one of the functional manifestations of the tumor, the present finding is compatible to the above results: HN~-O may provide some alterations in the cellular function of the rat ascites hepatoma.

In the process of tumor emigration into the local vein the influence of HN2-O on the host side should be also considered. However, this concern cannot be discussed in the present experiment.

Summary The disintegration of rat ascites hepatoma island (AH 601) by Tween 80

treatment was markedly enhanced, when nitrogen mustard N-oxide over the mini- mal effective dosis was previously contacted in vivo. The degree of the dis- integration was roughly in inverse relation with the dosis of I-IN 2-O administrated in the present experimental condition.

In consequence, one of the important factors responsible for the inhibitory effect of IIN~-0 on metastasis of the rat ascites hepatoma may be an increase in the mutual adhesiveness. The interrelation between the mutual adhesiveness and the emigratory property of rat hepatoma cells was discussed.

Der gegenseitige Zusammenhang yon Tumorzellen in Hepatominseln des Ascitestumors yon Ratten

III. Der Einflul~ yon Stickstoff-Lost-0xyd

Zusammenfassung Die AuflSsung yon Asciteshepatominseln (AH 601) durch Behandlung mit

Tween 80 wurde wesent]ich gest5rt, wenn Stickstoff-Lost-Oxyd (HN~-O)in einer die minimale wirksame Dosis fiberschreitenden Menge mit ihnen in Berfih- rung kam.Das Ausmal~ dieser AuflSsung stand unter den hier gegebenen experimen- tellen Bedingungen etwa ira umgekehrten Verh/iltnis zu der angewandten Dosis von Stiekstoff-Lost. Einer yon den wichtigen Faktoren, die ffir den Hemmeffekt von IIN2-O auf die Entstehung yon Metastasen des l~atten-Asciteshepatoms ~erantwortlich sind, dfirfte daher die Erh6hung des gegenseitigen Zusammen- hanges der Tumorzellen sein. Auf die Beziehungen zwischen gegenseitigem Zu- sammenhang und tier F/~higkeit zv_r Emigration der 1%atten-Hepatomzellen wird hingewiesen.

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Cancer ires. 22, 525--548 (1962). Cv~s , A. S. G. : Cell contacts. Some physical considerations. Amer. Nat. 94, 37--56 (1960). EisE~Bs,~a, S., S. BE~C-0a and F. DOLJACCSKr: Electro-kinetic properties of cells in growth

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ME~G~r,, C.E., and M. G. K~LzY: Biochemical and morphologic changes in a mast-cell neoplasm during treatment with cyclophosphamide. Cancer Res. 21, 1545--1550 (1961). Z. Krebsforsch., Bd. 66 8a

114 T. u and It . SATOV: Mutual adhesiveness of tumor cells in" hepatoma island". I I I

MIDGLEY, A. 1:~., and G. B. PIERCE : Effect of colchieine and X-radiation on the differentiation of human embryonal carcinoma. Cancer Res. 21, 545--s (1961).

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- - (2) Experimental studies on the role of cancer chemotherapy for prevention of recurrence and metastasis formation in malignant tumor. Acta Un. int. Caner. 16, 764--768 (1960).

TEttAYA~, H. : Method of colloid titration. A new titration between polymerion. J. polymer. Sci. 8, 243--253 (1952).

YAMAD_% T.: Mutual adhesiveness of tumor cells in "hepatoma islands" of the rat ascites hepatoma. - - Studies on the mechanism of tumor metastasis I and II . Z. Krebsforsch. 65, 75--98 (1962).

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Dr. T. YAM~DA, The Department of Pathology, Tokyo Medical and Dental College, School of Medicine

(Yushima 3-1, Bunkyo-kn, Tokyo, Japan)

Dr. H. SArou, The Department of Surgery, The Medical Insti tute of Sasaki Foundation

(Surugadai 2-2, Chiyoda-ku, Tokyo, Japan)