effect of portal branch ligation on experimental tumor of the liver—immunological aspect

7
Effect of Portal Branch Ligation on Experimental Tumor of the Liver Immunological Aspect Yoshiro MATSUMOTOand Rokuro ASAKUMA ABSTRACT: Walker carcinosarcoma 256 was implanted in the middle or the left lobe of the liver of inbred Wister rats. Ligation of the portal vein branching to the lobe housing the tumor resulted in degeneration and atrophy of the implanted tumor. The challenge inoculation after tumor degeneration was rejected more than two times as rapidly as the allograft. The enhanced host resistance through activation of anti-tumor resistance by ligating the portal branch may be responsible for the suppression of the metastasis. The effect of portal branch ligation is mainly due to the inter- ruption of the portal blood supply to the corresponding lobe, but also may be attributed to the activation of the anti-tumor resistance of the host. KEY WORDS: portal branch ligation, Walker carcinosarcoma 256, anti- tumor resistance, challenge inoculation, "take", viable cell, allograft reac- tion, survival time, autochthonous host, ligation and release method. ~NTRODUCTION A radical resection of cancer of the liver is extremely limited because of concomitant cirrhosis or intrahepatic multiple metastasis. Experimentally, ligation of a portal branch in rabbit results in atrophy of the corresponding lobe 6. Honjo applied this observation to the treatment ofunresectable hepatic cancer, and also reported a series of studies on the effect of portal branch ligation. 3 By using transplantable tumors in the rat liver, Hirono reported 2 that ligation of portal branch resulted in prolongation of survival time and in regression of the main tumor as well as suppression of metastasis. Our clinical experience indicates that suppression of metastasis and angiographically confirmed disappearance of the tumors in unligated lobes occasionally take place after interruption of portal blood to the lobe housing the main tumor. Humoral and/or immunological factors may possibly be responsible for the suppression of metastasis and the disappearance of the remaining tumors. This report is to present immunological observation following portal branch ligation in rats with transplantable liver tumor. MATERIALS AND METHODS Male Wister rats weighing 200 to 250 grams supplied by the inbred Animal Center of Kyoto University were used throughout the experiment. Isogenic homogenicity of the strain of the rats was tested by reciprocal skin grafting. Rats were fed synthetic diet and drinking water ad libitum. Walker carcinosarcoma 256 supplied by the Research Center of Takeda First Department of Surgery , Kyoto University Medical School, Shogoin-Kawaramachi, Sakyo-ku, Kyoto, Japan. JAPANESE JOURNAL OF SURGERY, VOL. 3, No. 2, pp 106-112, 1973

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Page 1: Effect of portal branch ligation on experimental tumor of the liver—Immunological aspect

Effect of Portal Branch Ligation on Experimental Tumor of the Liver Immunological Aspect

Yoshiro MATSUMOTO and Rokuro ASAKUMA

ABSTRACT: Walker carcinosarcoma 256 was implanted in the middle or the left lobe of the liver of inbred Wister rats. Ligation of the portal vein branching to the lobe housing the tumor resulted in degeneration and atrophy of the implanted tumor. The challenge inoculation after tumor degeneration was rejected more than two times as rapidly as the allograft. The enhanced host resistance through activation of anti-tumor resistance by ligating the portal branch may be responsible for the suppression of the metastasis. The effect of portal branch ligation is mainly due to the inter- ruption of the portal blood supply to the corresponding lobe, but also may be attributed to the activation of the anti-tumor resistance of the host.

KEY WORDS: portal branch ligation, Walker carcinosarcoma 256, anti- tumor resistance, challenge inoculation, "take", viable cell, allograft reac- tion, survival time, autochthonous host, ligation and release method.

~NTRODUCTION

A radical resection of cancer of the liver is extremely limited because of concomitant cirrhosis or intrahepatic multiple metastasis. Experimentally, ligation of a portal branch in rabbit results in atrophy of the corresponding lobe 6. Honjo applied this observation to the treatment ofunresectable hepatic cancer, and also reported a series of studies on the effect of portal branch ligation. 3 By using transplantable tumors in the rat liver, Hirono reported 2 that ligation of portal branch resulted in prolongation of survival time and in regression of the main tumor as well as suppression of metastasis. Our clinical experience indicates that suppression of metastasis and angiographically confirmed disappearance of the tumors in unligated lobes occasionally take place after interruption of portal blood to the lobe housing the main tumor. Humoral and/or immunological factors may possibly be responsible for the suppression of metastasis and the disappearance of the remaining tumors. This report is to present immunological observation following portal branch ligation in rats with transplantable liver tumor.

MATERIALS AND METHODS

Male Wister rats weighing 200 to 250 grams supplied by the inbred Animal Center of Kyoto University were used throughout the experiment. Isogenic homogenicity of the strain of the rats was tested by reciprocal skin grafting. Rats were fed synthetic diet and drinking water ad libitum. Walker carcinosarcoma 256 supplied by the Research Center of Takeda

First Department of Surgery , Kyoto University Medical School, Shogoin-Kawaramachi, Sakyo-ku, Kyoto, Japan.

JAPANESE JOURNAL OF SURGERY, VOL. 3, No. 2, pp 106-112, 1973

Page 2: Effect of portal branch ligation on experimental tumor of the liver—Immunological aspect

Volume 3 Number 2 Immunological Effect of Portal Branch Ligation 107

Chemical Industory Co. Ltd. (Osaka), was used. Anesthesia was induced with ether. Through a midline laparotomy a 1.5 mm 3 of the tumor was implanted in the middle or the left lobe of the rat liver by means of the trocar technique. The abdomen was opened again one week after implantation. The growth of the tumor was confirmed and the portal vein to the tumor housing lobe was ligated and severed. Two or three weeks after the operation the rats received subcutaneously challenge inoculation on their backs (group A). The material for the inoculation was prepared by mincing the tumor and homogenizing them with Potter's homogenizer. The viable cells were counted by trypanblue staining method. Cell suspensions containing from 10 s to 10 s of the tumor cells were made up for the chal- lenge inoculation. This inoculation was judged to be "taken" when the nodule attained 5 mm or more in its diameter. In order to exclude the influence of allograft reaction or of the surgical procedures, control experiments were done in the following manner. The tumor was implanted subcutaneously in the axillary fossas of rats and 2 or 3 weeks after partial excision of the tumor, the rats received the challenge inoculation (group B). The portal vein branching to the middle and left lobes was ligated and the rats received the

�9 challenge inoculation 2 or 3 weeks after the surgical procedure (group C). A simple lapa- rotomy was done and the rats received the challenge inoculation in 2 or 3 weeks later (group D).

RESULTS

In rats weighing 200 to 250 grams, the liver occupies 6.5 per cent of the body weight and the middle and left lobes 65 per cent of the whole liver. Five weeks after ligation of the portal branch, the middle and left lobes showed remarkable atrophy and the unligated lobes did conspicuous regenerative hypertrophy (Table 1). Ligation of the portal branch re- duced the total liver weight, but increased the weight of the unligated lobes prominently to about 80 per cent of the whole liver. The complication due to portal branch ligation was as rare as simple laparotomy. As was shown by Hirono 2, significant prolongation of the survival time was observed in group A. Seventy per cent of rats in group A survived more than six weeks after implantation of the tumor in the liver. In the other three groups, the tumor killed almost all of the hosts within four weeks (Table 2). Group B should be com- posed of those rats which received partial excision of the tumor implanted in the liver. But

Table 1.

Control (Non-ligated) Portal branch

ligation

Number of Total liver weight rats used Body weight

Change of weight of liver and spleen after portal branch ligation in rats*

Weight of non-ligated lobes Spleen weight Body weight Body weight

20 6.51 • 3.0-4-0.68 0.40•

18 5.40• 4.1 • 0.58i0.06

*Rats were killed 36 days after portal branch ligation. **Mean ztz Standard Deviation

The weight of the unligated lobes increased prominantly to about 80 per cent of the total liver weight in normal rats. Increase of the spleen weight in portal branch ligated rats may be caused by congestion of portal blood.

Page 3: Effect of portal branch ligation on experimental tumor of the liver—Immunological aspect

Jap. J. Surg. 108 Matsumoto and Asakuma June 1973

Table 2. Survival days of portal branch ligated group and other three control groups in rats

01 I I I: ; T u m o r Death

12 S a c r i f i e d

i

5{

40

m 30

20

I0

Groups A

Liver tumor & Portal Branch

Ligation

B Su~utaneOus

tn~r Implantation

C : Portal

Branch Ligation

Alone

�9 A O

e k A

D No

Pretrontment

Seventy per cent of rats in group A survived m o r e than 6 weeks after imp lan ta t ion of the

t u m o r in the liver. In o ther three groups , the

imp lan t ed tumors killed a lmost all of the hosts

wi th in 4 weeks.

tumors imp lan t ed in the l iver kil led the host wi th in two weeks, and , therefore, subcutaneous

imp lan t a t i on was given subst i tu t ional ly to observe the influence of the al lograft react ion.

The resistance of the hosts is shown in T a b l e 3. In g roup C and D, less than 106 of the t u m o r

cells were enough to be taken by 100 per cent. T h e cr i t ical numbe r s of the cells were 3 to

5 X 10 7 in g roup B. M o r e than 7.5 • 10 7 were d e m a n d e d to be taken successfully in g roup A,

and some rats showed resistance to even this n u m b e r o f the t u m o r cells.

Five mill ions of the v iable cells were i m p l a n t e d in the rats of g roup A and B as the

inoculat ion. T a b l e 4 shows tha t only one ra t among 10 " t o o k " the inocula t ion in g roup A,

whereas, in g roup B, 11 a m o n g the 13 " t o o k " the inocula t ion wi th in 5 days, and in 6 rats of

which the inocula ted tumors con t inued increas ing thei r size and the rest showed regression.

His tological examina t ion on the 7th day revealed fibrosis and r o u n d cell inf i l t ra t ion in

the pe r iphe ra l a rea o f the t u m o r in the l iga ted lobe, and with the mix tu re of dead and v iab le

tumorceUs in the cent ra l a r ea of the tumor . In five weeks, no v iab le cells were detec ted , and

necrot ic mass was su r rounded by fibrous m e m b r a n e wi th r e m a r k a b l e inf i l t rat ion of r o u n d

cells a c c o m p a n i e d by p la sma cells (Fig. 1, 2 and 3).

Page 4: Effect of portal branch ligation on experimental tumor of the liver—Immunological aspect

Volume 3 Number 2 Immunological Effect of Portal Branch Ligation

T a b l e 3. Comparison of host resistance in rats against tumor challenge in portal branch ligated group and other three control groups

109

80

70 o x

6o

.~ 5O

4O

3O

.o 20

10

Groups

0 0

O0

0

~ o o ~

A Liver Tumor & Portal Branch

Ligation

I I O O O

o o

B i �9 �9 a o a

B Subcutaneous

Tumor Implantation

C Portal Branch

Ligation Alone

�9 Take

0 Not Take

dl I I �9 I I

D

No Pretreatment

Critical number of the cells on the allograft reac- tion is 3 to 5X107. More than 7.5• are demanded to be taken successfully in group A. In group C and D, less than 106 of the tumor cells are well enough to be taken by 100 per cent.

T a b l e 4. Host resistance in rats against tumor challenge in portal branch ligated group and non-ligated group

Groups Days after tumor challenge 5 7 8 21

Liver tumor (A) &

Portal branch ligation

(B) Subcutaneous tumor implantation

1/10* 1/10

11/13 6/13 6/13

* No, of " take"

Total No. of rats

Five millions of viable cells of Walker carcinosarcoma 256 were challenged 3 weeks after portal branch ligation. In group A, one rat among 10 " took" the inoculation within 5 days, and in 6 rats of which the inoculated tumors continued increasing their size and the rest showed regression.

Page 5: Effect of portal branch ligation on experimental tumor of the liver—Immunological aspect

Ja#. J. Surg. 110 Matsumoto and Asakuma June 1973

Fig. 1. The ligated lobe one week after ligation of left portal vein, showing peripheral fibrosis and mixture of dead and still viable tumor cells in the central area.

H-E stain • 100

Fig. 2. The ligation lobe 45 days after ligation of left portal branch, showing necrosis surrounded by fibrous tissue.

H-E stain X 100

Page 6: Effect of portal branch ligation on experimental tumor of the liver—Immunological aspect

Volume 3 Number 2 Immunological Effect of Portal Branch Ligation 111

Fig. 3. The fibrous tissue in Fig. 2, showing remarkable in- filtration of round cells.

H-E stain x 200

DISCUSSION

The hepatic artery has been taken for the primary nutrient vessel for tumor of the liver. Matsumura s and Suzuki 7 reported that the hepatic artery mainly covered the central area of the tumor, whereas the portal vein has a great influence at the peripheral area of a rapidly growing hepatic tumor. Therefore, it is reasonable that interruption of the portal blood supply to the tumor will reduce the tumor size and prolong the survival time, but it does not explain sufficiently the suppression of metastasis and the regression of the tumors in the unligated lobes. Apart from metastatic tumors, intrahepatic metastasis is not in- frequent in cases with primary hepatic cancer. Fear was expressed that portal branch ligation would bring rapid growth of the tumors in the unligated lobes as a result of the regeneration and hypertrophy of the lobes. However, no such clinical evidence has been observed. Goto 1 also denies this discouraging apprehension with his experimental studies.

Klein 4 studied induced immunity against tumors in the primary autochthonous host by using 20-methylcholanthrene-induced sarcoma in mice. Takeda and his coworkers reported 8 that they had demonstrated production of transplantation-immunity in the autoehthonous hosts against 20-methylcholanthrene-induced subcutaneous sarcomas in the rat in an extreme rate and at a high intensity by the "ligation and release method". The induced tumor of a suitable size was tightly ligated with a circular rubber band to form a pedunculated nodule for interception of blood circulation and the strangulation was released in approximately 24 hours later. Owing to the temporal interception of the blood supply, the ligated tumor fell into necrosis, most part of which regressed and was absorbed gradually in two or three weeks to become a native antigen and the rest covered with crusted skin sloughed off in a few weeks. Walker carcinosarcoma 256 successively transplanted to inbred rats was taken by 100 per cent in that liver, but the tumors submitted degeneration and were surrounded by fibrous tissue one week after ligating the portal branch. At this

Page 7: Effect of portal branch ligation on experimental tumor of the liver—Immunological aspect

Jap. J. Surg. 112 Matsumoto and Asakuma June 1973

t ime, however , one ha l f of the t umor cells still had the i r v iabi l i ty . In five weeks, the per i -

phera l a rea of the t u m o r and the sur rounding l iver cells submi t t ed fibrosis comple te ly and

the cent ra l a r ea fel l into necrosis. I n t e r rup t i on of 70 per cent of the por ta l b lood supply

to the lobe housing a t u m o r degenera tes the pe r iphe ra l a rea of the r a p id ly growing t u m o r

and the degenera ted t u m o r cells are absorbed to become the ant igen . This is suspected to

result in suppression o f the g rowth of the r ema in ing t u m o r and the metastasis. In our exper iment , in g roup A, the chal lenge inocula t ion , 7.5 X 107 of the t umor cells

given subcutaneous ly a t 2 to 3 weeks after l iga t ing the por ta l b r anch , was taken bu t even

this n u m b e r of the cells were not enough to be taken in all ra t s in this group. In g roup

C and D, the cells of 106 were enough to be taken by 100 per cent suggesting the procedures

in these groups have no th ing to do with t r ansp lan ta t ion or pro l i fe ra t ion of the t umor cells.

In g roup B, chal lenge inocula t ion of 5 X 107 cells was taken wi th in 5 days, bu t one ha l f of

the tumors degene ra t ed a n d d i sappea red in 10 days. A n e n h a n c e d host resistance m a y be

responsible for this observat ion. In g roup A, 5 X 107 of t u m o r cells were not taken a t all.

T h e difference of the cri t ical numbers , 7.5 X 107 in g roup A and 3 to 5 X 107 in group B,

suggests definite difference be tween the enhanced host resistance in rats wi th the por ta l

b ranch l igat ion and the al lograf t react ion, which is l ikely to be due to an t i - t umor resistance

induced by the l igat ion of the por ta l vein b r a n c h i n g to the t u m o r housing lobe. P rob lem concerning the genet ic difference be tween cancer a n d host should be ul t i -

ma te ly solved by an invest igat ion on whe ther or not a u t o i m m u n i t y can be induced agains t

na t ive tumors in the au toch thonous host. Fu r the r , T a k e d a s r e p o r t e d tha t the an t ibody

involved in the t u m o r a u t o i m m u n i t y was de tec ted in l y m p h o i d cells bu t no t in sera of

a u t o i m m u n e rats. Therefore , in our next exper iment , the a n t i b o d y to the nat ive t u m o r

must be detec ted .

ACKNOWLEDGEMENT

T h e authors a re grea t ly indeb ted to Prof. Ich io Honjo , d i rec tor of this depa r tmen t ,

for his va luab le suggestion t h roughou t the exper iments and for rev iewing the manuscr ip t .

(Received for publication on July I7, 1973)

References

1. Goto, A.: The 2nd Annual Meeting of Japan Hepatic Association.July, 1966, Tokyo.

2. Hirono, T. : Effect of segmental interruption of portal venous blood supply in implanted tumor in the liver of rats. Arch. Jap. Chir., 33: 526-560, 1964.

3. Honjo, I. & Suzuki, T.: Portal branch ligation for hepatoma. J. Jap. Can. Clin., 16: 567-573, 1970. (in Japanese)

4. Klein, G., Sj6gren, H. O., Klein, E., Hellstrom, K. E. : Demonstration of resistance against methylcholanthrene induced sarcomas in the primary autochthonous host. Cancer Res., 20: 1561-1572, 1960.

5. Matsumura, H. : Vascular distribution of ex- perimental hepatomas. Arch. Jap. Chir., 34: 1~3, 1965.

6. Rous, P. & Larimore, L. D. : Relation of the portal blood to liver maintenance. J. Exp. Med., 31 : 609-632, 1920.

7. Suzuki, T. & Honjo, I.: Selective angio- graphy of the liver and pancreas. J. Clin. Surg. 24: 317-326, 1969. (in Japanese).

8. Takeda, K., Aizawa, M., Kikuchi, Y., Yama- waki, S. & Nakamura, K.: Tumor auto- imunity against methylcholanthrene induced sarcomas of the rat. Gann, 57 : 221-240, 1966.