[CANCER RESEARCH 26, 797-802,May 1966]

Growth-promoting Effect of Acid Mucopolysaccharides

on Ehrlich Ascites Tumor

JUN TAKEUCHIDepartment of Pathology, A'agoya University School of Medicine, Nagoya, Japan

Summary

The effect of acid mucopolysaccharides on the growth of hypo-tetraploid Ehrlich ascites tumor was investigated using chon-

droitin sulfate and crude hyaluronic acid.Chondroitin sulfate solution was given s.c. into the right flank

of SM mice, followed immediately by Ehrlich tumor. Averagetumor weights in chondroitin sulfate-treated groups on Days 3and 8 after tumor inoculation were 2-3 times as great as incontrols. The survival time of chondroitin sulfate-treated micewas shorter than nontreated ones. By means of the same procedure it was also demonstrated that crude hyaluronic acid solution promoted significant growth of the tumor.

The tumor growth-promoting effect was also studied quantitatively with various amounts and concentrations of chondroitinsulfate and was compared with that of other substances withsimilar physicoehemical properties. The same effect was alsoobserved with the chorioallantoic membrane of embryonatedchicken eggs and with ascitic tumor in the abdominal cavity ofthe mice. Chondroitin sulfate and crude hyaluronic acid enhanced tumor growth; the significance of acid mucopolysaccha-ride activity is discussed. The results appear to indicate that theproliferation of young connective tissue which produces acidmucopolysaccharides is favorable to the growth of cancer cells.

Introduction

Almost all epithelial tumor cells grow more or less interlacedwith connective tissues, in which acid mucopolysaccharides,collagen, and others can be observed. Many workers have reported (2, 11, 12, 15) that the hyaluronidase-like spreading factorcontained in malignant tumor tissues might break down the connective tissue, thus helping the invasion and implantation of thetumor cells. It was recently reported (10) that cancer of the skinproduces not only enzymes capable of depolymerizing acid mucopolysaccharides, but also substances with a cytostatic effect onthe proliferation of cellular elements of connective tissue. Thegrowth-inhibitory action of heparin (one of the acid mucopolysaccharides) on Ehrlich ascites tumor in vivo was described (9).

On the other hand, Vasiliev (21) reported that the formation ofyoung connective tissue around a neoplasm may be important inmany cases for the invasive growth of malignant cells, and recently Black (1) concluded that there is a definite tendency fortumor métastasesto form in active granulation tissue. Growth-promoting activity of acid mucopolysaccharides was demon-

Keceived for publication August 9, 1965.

strated (8,13) in vitro with the embryonic fibroblast and humancarcinoma.

In a previous paper, this author demonstrated (16, 19) thegrowth-promoting effect of chondroitin sulfate on Brown-Pearcerabbit carcinoma and solid Ehrlich ascites tumor.

This paper reports further observations on the growth-promoting effect of chondroitin sulfate and also of crude hyaluronicacid on Ehrlich ascites tumor, as measured in vivo in terms oftumor weight, survival time, cell counts, and histologie observa-tioas of tumor tissue.

Materials and Methods

Tumor cells used in this study were Ehrlich hypotetraploidstock (Kaziwara 4N) (6) maintained in adult male SM micethrough serial i.p. transplantation at 7- or 8-day intervals in thislaboratory.

Animals used throughout this experiment were male SM1 miceabout 100 days old, obtained from the center supplying laboratory animals in this medical school. They were fed with a standard pellet diet (CA-1, Nihon Clea Co., Ltd., Tokyo) and givendrinking water ad libitum.

In the 1st series of experiments, chondroitin sulfate or crudehyaluronic acid solution was injected s.c. into the right flank ofmice, immediately followed by 0.1 ml of the tumor ascites fluidcontaining 5 X IO6cells. In the control, isotonic saline was in

jected before the tumor inoculation. Some of the animals weresacrificed on the 3rd day and the rest on the 8th day after tumorinoculation, and any solid tumor which developed subcutane-ously was excised and weighed. The results of the experimentswere evaluated on the basis of the average weight of tumor tissuein the experimental as compared with the control group. Inanother part of the experiment, survival time was comparedwith that of mice given the same treatment.

The 2nd series involved the i.p. injection of ascites tumor. Theascites (5 X IO6cells) fluid was inoculated i.p.; on days 1, 3, 5

and 7 after tumor inoculation, 1 ml of 2% chondroitin sulfatesolution was given i.p. On the 8th day, the mice were sacrificedand the total tumor cell number was calculated for each mousefrom 5 cell countings by the rinse technic (5).

Similar studies were conducted using the chorioallantoic membrane (CAM) of the embryonated eggs of the White Leghorn

1 In all mice of this strain, solid Ehrlich ascites tumors devel

oped at the site of the inoculations; the growth of the tumor wasgreater in males than in females of this age, as described in aprevious paper (20).

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chickens, Hi-line obtained from Hattori Chicken Yard, Nagoya,Japan. A small disk, 8 mm in diameter and 3 mg in weight, madeof Spongel (Yamanouchi Pharm. Co., Ltd., Tokyo) was sterilizedin an autoclave and then was soaked with solutions of variousconcentrations of acid mucopolysaccharides. For the control,isotonic saline was used. After the disk containing the solutionwas placed on the Petri dish, 0.01 ml of tumor ascitic fluid containing 5 X IO6cells was dropped on it. Then the disk, which

contained both tumor cells and the solution, was implanted onthe CAM of the embryonated egg on the 6-7th day of its incuba

tion. The embryonated eggs were sacrificed on the 8th day aftertumor implantation, and the solid tumor that had developed inand around the Spongel disk on the CAM was excised andweighed.

Heparin, collagen, polyvinylpyrrolidones, methyl cellulose,fibrinogen, and dextran sulfate were similarly checked for theeffect on Ehrlich tumor growth by s.c. injection or by the diskmethod.

Tumor tissues were fixed in formol and embedded in paraffin,and were stained with hematoxylin-eosin.

TABLE 1EFFECTOF2% CHONDROITINSULFATEONTHEGROWTHOFSOLID

EHRLICHASCITESTUMORON THE SRDDAY AFTERs.c. INOCULATIONIN MICE

One ml of 2% chondroitin sulfate solution was injected s.c.into mice, immediately followed by tumor inoculation into thesame site. After 3 days, the mice were sacrificed and solid tumorwas excised and weighed.

Control2%chondroitinsulfateTumor

sizeincontrols= 100No.

OFMICEExpe

rimen t166322Experiment21010266Totals1616295'TUMOR

WT.[mean±S.E.(mg)]128.7

±9378.1±32P"<0.001

0 Statistical significance of difference in tumor weight betweenexperimental and control. The evaluation of it was based on theStudent's t-test.

bAverage.

The following materials were used: Chondroitin sulfate C:average molecular weight about 50,000; Kaken Yakukako Co.,Ltd., Tokyo, Japan. Crude hyaluronic acid: extracted fromhuman umbilical cord by Egami's technic (4) (essentially an

extract of dry acetone powder of human umbilical cord with 2%phenol solution precipitated with ethyl alcohol saturated withpotassium acetate). The fibrous substance thus produced wasdried with acetone, ethanol, and ethyl ether. The crude hyaluronic acid was dissolved in isotonic saline. Collagen: obtainedfrom tendon (Sigma Chem. Co., Ltd.); suspended in isotonicsaline. Heparin sodium (100 units/mg), methyl cellulose, polyvinylpyrrolidones (PVP K30), and fibrinogen (bovine) (Kata-yama Chem. Co., Ltd., Osaka, Japan) were dissolved in isotonicsaline. Dextran sulfate (average M.W. 20,000) (Kowa Co., Ltd.,Nagoya, Japan).

Results

Effect of Chondroitin Sulfate on the Subcutaneous Growth of SolidEhrlich Ascites Tumor in Mice

Chondroitin sulfate solution was injected s.c. into the rightflank of mice, immediately followed by tumor inoculation intothe same site. The solid tumor that developed was excised, andthe tumor weights were compared between experimental andcontrol mice. Tables 1 and 2 show that the injection of 1 ml of 2%chondroitin sulfate solution prior to tumor inoculation promotesthe growth of solid tumors. The average tumor weight in experimental groups was about 3 times as great as that in controls onthe 3rd day after tumor inoculation, and about 2 times as greaton the 8th day; the difference of average tumor weight betweenexperimental and control was statistically significant (P <0.001). Microscopic examination revealed that the increase intumor weight is due to the increase in number of the tumor cells.On the 3rd day after inoculation, the tumor growth was moreinfiltrative with less necrosis in experimental groups than that incontrol groups.

Table 3 shows the dose-response relation between chondroitinsulfate and tumor growth. Three ml of 2% chondroitin sulfateaccelerated more markedly the growth of tumor than did 1 ml.The correlation between chondroitin sulfate dose and the increase in tumor growth was statistically significant.

Table 4 shows the tumor growth-promoting effects of chon-

TABLE 2EFFECTOF 2% CHONDROITINSULFATEONTHE GROWTHOF SOLIDEHRLICHASCITES

TUMORONTHESTH DAY AFTERs.c. INOCULATIONIN MICE

The experimental procedure was the same as that given in Table 1. The mice were sacrificed on the8th day after tumor inoculation.

Control2%chondroitinsulfateTumor

size in controls = 100NO.

OFMICE1«8817321515184312122054101022551010218Totals5555191"TUMORWT.[mean±

S.E.(mg)]1005.1

±601922.0±104p<0.001

" Experiment number.6Average

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Mucopolysaccharides and Ehrlich Ascites Tumor

TABLE 3EFFECT OF DIFFERENT AMOUNTSOF CHONDROITINSULFATE ON

SOLID EHRLICH ASCITES TUMOR ON THE STH DAYAFTER INOCULATION

Two % chondroitiu sulfate solutions were injected s.o. followedby tumor inoculation. The mice were sacrificed on the 8th dayand the tumors excised and weighed.

Control (1 ml ofsaline)1ml of 2% chondroitinsulfate3ml of 2% chondroitin sulfateNo.

ofmice6C6Tumor

wt.[mean ±S.E.

(mg)]998

±941900±963030±200p<0.001°<0.001<>

0 Statistical significance of difference in tumor weight between

experimental (1 ml of 2% chondroitin sulfate) and control.6 Statistical significance of difference in tumor weight between

the 2 groups of experimental animals.

TABLE 4

EFFECT OF DIFFERENT CONCENTRATIONSOF CHONDROITINSULFATE ON SOLID EHRLICH ASCITES TUMOR ON THE

STH DAY AFTER INOCULATION

One ml of chondroitin sulfate solution was injected s.c. intothe right flank of mice followed by tumor inoculation in the samesite. After 8 days, the mice were sacrificed and the tumors excisedand weighed.

Control2%chondroitin

sulfate0.3%chondroitin

sulfate0.03%chondroitin

sulfateNo.

ofmice10101010Tumor

wt.[mean ±S.E.(mg)]932

±692104±1871776

±1531539

± 191Tumor

size incontrols

=100225190165P"<0.001<0.0010.01

< P < 0.02

" In each case the significance given is that of the difference intumor weight between the experimental animals and the controls.

droitin sulfate solutions injected in different concentrations priorto the tumor inoculation. The tumor growth tends to be accelerated as the concentration of chondroitin sulfate is increased.

Effect of Crude Hyaluronic Acid cm the Subcutaneous Growth ofSolid Ehrlich Ascites Tumor in Mice

By means of the same experimental procedure as that usedwith the chondroitin sulfate solution, the effect of crude hyalu-ronic acid on tumor growth was investigated. Table 5 shows thatthe injection of 1 ml of 1% crude hyaluronic acid solution prior totumor inoculation accelerated the growth of solid tumor. Theaverage tumor weight in the experimental group was about 3times as great as that of the control on the 3rd day and about 1.6times as great on the 8th day, and the difference in tumor weightbetween experimental and control was statistically significant(P < 0.001).

Effect of Injection of Chondroitin Sulfate and Crude HyaluronicAcid on Survival Time of Mice

Table 6 shows the mean survival time of mice which were given1 ml of either 290 chondroitin sulfate or 1% crude hyaluronic acidsolution s.c. prior to the tumor inoculation. The survival time ofmice was shortened significantly by treatment with chondroitinsulfat«and crude hyaluronic acid.

Effect of Chondroitin Sulfate on the Growth of Ehrlich AscitesTumor in the Abdominal Cavity of Mice

The effect of chondroitin sulfate on tumor growth is reported interms of the total number of free ascites tumor cells per mouse.No effect was detected on tumor growth with 1 ml of 2% chondroitin sulfate i.p. when given prior to i.p. tumor inoculation,but as shown in Table 7, when 1 ml of 2% chondroitin sulfatewas injected i.p. on the 1st, 3rd, 5th, and 7th days after tumorinoculation, an increase in the number of tumor cells in chondroitin sulfate-treated mice was observed to some extent on the

8th day, compared with controls given injections of saline.Ascitic fluid in mice which were given chondroitin sulfate wasmore viscous, and in 12 of 18 chondroitin sulfate-treated mice,jelly-like tumor masses were observed. The real cell number in

TABLE 5

EFFECT OF CRUDE HYALURONICACID ON THE GROWTH OF SOLID EHRLICH ASCITESTUMOR ON THE 3RD AND 8TH DAYS AFTER INOCULATION

One ml of 1% crude hyaluronic acid solution was injected s.c., followed by tumor inoculation into thesame site. On the 3rd and 8th days after tumor inoculation, the mice were sacrificed and the tumorsexcised and weighed.

3 days after tumor inoculationControl1%

crude hyaluronic acid8 days after tumor inoculation

Control1%crude hyaluronic acidXo.

ofmice1615

2120Tumor

wt. [means±S.E.(mg)]123.3

±9411.3± 62

1052.3 ±1001697.5± 111Tumor

size incontrols =100313161P"<0.001<0.001

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Jun Takeuchi

the ascitic fluid in experimental mice might have been muchgreater than the one obtained from counting of free cells only.

Effect of Various Substances on the Subcutaneous Growtli of Solid

Tumor in mice

Collagen, dextran sulfate, methyl cellulose, fibrinogen, andpolyvinylpyrrolidones were tested by means of the same experimental procedure as used with chondroitin sulfate. Table 8 shows

TABLE 6EFFECT OF CHONDROITINSULFATEAND CRUDE HYALURONIC

ACIDON THE SURVIVALTIME OF TUMOR-INOCULATEDMICE

One ml of 2% chondroitin sulfate or 1% crude hyaluronic acidsolution was injected s.o., followed by tumor inoculation in thesame site.

Control2%chondroitinsulfateControl1%

crudehyaluronicacidNo.

ofmice10101010Mean

survival±S.E.(days)77.2

±8.444.2±3.872.6

±5.850.1±7.4p0.001

< P <0.0050.02

< P < 0.05

TABLE 7EFFECTOF2% CHONDROITINSULFATEONTHEGROWTHOFEHRLICH

ASCITESTUMORIN THE ABDOMINALCAVITYOF MICETumor cells (5 X IO6)were inoculated i.p. on Day 0, and 1 ml

of 2% chondroitin sulfate solution was injected i.p. on Days 1, 3,5, and 7. On Day 8, the mice were sacrificed and tumor cell numbers/mouse were measured.

Control2% chondroitin sulfateNo.

ofmice12

18Tumor

cell Xo, /mouse(mean ±S.E.)1.73

±0.63 X 10»2.97 ±0.69 X 10»p0.05< P < 0.1

that these substances have no promoting effect on tumor growth,but chondroitin sulfate accelerates the growth; however, some ofthese substances showed an inhibitory effect on tumor growth.

Heparin was tested by the same procedure, but the experiments resulted in lethal hemorrhage at the site of the injection.

Observations on Tumor Transplants on CAM of Embryonated Eggs

Tables 9-11 show the effects of crude hyaluronic acid, heparin,and collagen solutions on the growth of solid tumor on CAM. Itwill be noted that there was a significant increase in tumor sizein 0.1% and 0.01 % crude hyaluronic acid-treated groups, but nosignificant differences were seen in 1% crude hyaluronic acid-,heparin-, and collagen-treated groups. A tendency to inhibit thetumor growth was observed in the 1% collagen-treated group.

A consistent increase in tumor size was observed in the 2, 0.3,and 0.03 % chondroitin sulfate-treated groups and the differencein average tumor weight between experimental and control wasstatistically significant, as shown in a previous paper (19).

Discussion

Present data indicate that chondroitin sulfate and crudehyaluronic acid promote the growth of Ehrlich tumor. Ozzelloet al. reported the growth-promoting activity of acid mucopoly-saccharides, in vitro, on a strain of human mammary carcinomacells (13). They ascribed this action to the negative electriccharge and the viscosity of acid mucopolysaccharides. Rogersreferred to water retention, rate of diffusion, lubrication, macro-ionic function, and other functions, such as the physiologic function of hyaluronate (14). The present study showed that acidmucopolysaccharides have some promoting action on tumorgrowth, and it is quite conceivable that acid mucopolysaccharidesserve not as a nutritional substance but as a supporter which protects the surface of tumor cells and promotes the exchange oftheir metabolites.

The growth-inhibitory action of heparin, however, on theEhrlich ascites tumor in mice was reported by Sister MurielLippman (9). The present data also show the nonpromotingeffect of heparin on the growth of tumor with CAM of chicken

TABLE 8EFFECTOF VARIOUSSUBSTANCESON THE GROWTHOF SOLIDEHRLICHASCITESTUMORON

THE STH DAY AFTER INOCULATION

One ml of the substance was injected s.c., immediately followed by tumor inoculation into the samesite. After 8 days, the mice were sacrificed and the tumors excised and weighed.

SubstanceSaline

(control)2%chondroitinsulfate2%dextransulfate1%

collagen1%methylcellulose1%

fibrinogen1%polyvinylpyrrolidonesNo.

ofmice10101010101011Tumorwt. [(mean ±S.E.(mg)]1060

±1221825±1711075±211820±931195±112480±68886±107Tumor

size incontrols

=100172101771124583pao.ooi

< r <0.0050.9<Po.i< r <0.20.4< P <0.5P

<0.0010.3< P < 0.4

" In each case the significance given is that of the difference in tumor weight between the experimentalanimals and the controls.

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Mucopolysaccharides and Ehrlich Ascites Tumor

TABLE 9EFFECTOF CRUDEHYALUBONICACIDON THE GROWTHOF SOLIDEHRLICHASCITES

TUMOHON CAM OF CHICKEMBBYOSpongel disk, which contained both tumor cells (5 X IO5)and crude hyaluronic acid solution, was

implanted on the CAM of the chick embryo. On the 8th day after implantation, the solid tumorthat had developed in and around the disk was excised and weighed.

No. of tumors,controlsNo.of tumors, 1% crude hyaluronicacidTumor

size in controls =100No.of tumors, 0.1% crudehyaluronicacidTumor

size in controls =100No.of tumors, 0.01% crudehyaluronicacidTumor

size in controls = 100Experi

ment1878381306126Experiment26412061333129Totals141196141319129Tumorwt.

[mean ±S.E.(mg)]69.7

±3.167.4±3.991.8

±5.090.5

±4.5pa0.6

< P <0.70.001

< P <0.0050.001

< P < 0.005

" Significance of difference in tumor weight between experimental and control animals.

TABLE 10EFFECTOFHEPARINONTHEGROWTHOFSOLIDEHRLICHASCITES

TUMORON CAM OF CHICKEMBRYOThe experimental procedure was the same as that given in

Table 9.

No. oftumors,controlsNo.

oftumors,0.5%heparinTumor

sizeincontrols= 100Exper

iment18696Experiment210896Totals181494Tumor

wt.[mean ±S.E.

(mg)]71.5

±5.107.7

±5.5p0.6 < P < 0.7

TABLE 11EFFECTOFCOLLAGENONTHEGROWTHOFSOLIDEHRLICHASCITES

TUMORON CAM OF CHICKEMBRYOThe experimental procedure was the same as that given in

Table 9.

Control1%collagen

0.1% collagen0.01% collagenNo.

oftumors1010

1210Tumor

size incontrols

=10078104

106Tumor

wt.[mean ±S.E.

(mg)]68.9

±5.653.9±5.671.9±7.2

73.5 ±7.7p°0.02

< P < 0.050.6 < P < 0.70.6 < P < 0.7

«Significance of difference in tumor weight between experimental and control animals.

embryo. It appears that the action of heparin is somewhat different from that of chondroitin sulfate and crude hyaluronic acid ontumor cells. Costachel el al. noted a cytotoxic action of heparinin vitro on Syrian hamster sarcoma cell cultures (3).

Using the 13ro\vn-Pearce rabbit carcinoma in early studies, this

author demonstrated the growth-promoting activity of chondroitin sulfate (16-18). The author also observed the occurrenceof metastatic tumors induced by femur fracture or implantationof foreign bodies at the site of proliferation of young connectivetissue (7). Recently, Black showed a definite tendency for tumormétastasesto form in active granulation tissue (1). Vasilievthought that proliferating connective tissue might facilitate theestablishment and enhance the growth of tumor cells, and agreedwith the idea of the importance of connective tissue proliferationfor invasive growth of malignant cells (21). The present experiments appear to indicate that the proliferation of young connective tissue which produces acid mucopolysaccharides is favorableto the growth of cancer cells.

Acknowledgment

The author is greatly indebted to Professor H. Tauchi for hisadvice and encouragement; to Dr. I. Asamoto, Nagoya City University Medical School, for supplying human umbilical cords; andto Dr. M. Kodama, Aichi Cancer Centre Research Institute, forfruitful discussions of the problems.

References

1. Black, J. W. The Localisation of Metastatic Brown-PearceCarcinoma in Granulation Tissue. Brit. J. Cancer, 18: 143-45,1964.

2. Boyland, E., and McClean, D. A Factor in Malignant Tissueswhich Increases the Permeability of the Dermis. J. Pathol.,41: 553-65, 1935.

3. Costachel, ()., Fadei, L., and Nachtigal, M. The Action ofHeparin in Vitro on Syrian-Hamster Sarcoma Cell Cultures.Exptl. Cell Res., 34: 542-47, 1964.

4. Egami, F. Tatorui Kagaku (Chemistry of Polysaccharides),p. 208. (Japanese.) Tokyo: Kyoritsu Shuppan Co., 1955.

5. Hauschka, T. S., Grinnel, S. T., Revesz, L., and Klein, G.Quantitative Studies of the Multiplication of Neoplastic Cellsin Aivo. IV. Influence of Doubled Chromosome Number onGrowth Rate and Final Population Size. J. Nati. Cancer Inst.,19: 13-31, 1957.

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Jun Takeuchi

6. Kaziwara, K. Deviation of Stable Polyploid Sublines from aHyperdiploid Ehrlich Ascites Carcinoma. Cancer Res., ¡4:795-801, 1954.

7. Kojima, K., and Takeuchi, J. Experimental Studies on theBrown-Pearcc Rabbit Carcinoma. (Japanese.) J. Nagoya Mcd.Assoc., 73: 457, 1957.

8. Lasfargeus, K. Y., Dclannay, A., and Daniel, P. Influence ofHuman Cord Extract on Chick Embryonic Fibroblasta Grownin Vitro. Exptl. Cell Res., 10: 188-93, 1956.

9. Lippman, S. M. The Growth-Inhibitory Action of Heparin onthe Ehrlich Ascites Tumor in Mice. Cancer Res., 17: 11-14,

1957.10. Majewska, II., Majewski, C., Radziki, J., and Leja, Z. Acid

Mucopolysaccharides in the Ground Substance of ConnectiveTissue in Cancer of the Skin. Cancer, 18: 388-90, 1965.

11. McCutcheon, M., and Coman, D. R. Spreading Factor inHuman Carcinomas. Cancer Res., 7: 379-82, 1947.

12. Ozaki, M. Malignant Tumours and the Spreading Factor.Kumamoto Med. J., 5: 119-25, 1952.

13. Ozzello, L., Lasfargeus, E. Y., and Murray, M. R. Growth-Promoting Activity of Acid Mucopolysaccharides on a Strainof Human Mammary Carcinoma Cells. Cancer Res., 20: 600-

4, 1960.

14. Rogers, H. J. The Biochemistry of Mucopolysaccharides ofConnective Tissue, Biochemical Society Symposia No. 20,p. 51. London: Cambridge University Press, 1961.

15. Simpson, W. L. In: Asboe-Hansen, G. (ed.) Connective Tissueand Cancer in Connective Tissue in Health and Disease,pp. 225-38. Copenhagen: Munksgaad, 1954.

16. Takeuchi, J. An Effect of Acid Mucopolysaccharide on theTransplantation and Growth of Tumor. Gann, 51 (suppl.):128, 1960.

17. - —. Histochemical and Chemicopathological Investigations on the Interaction of Brown-Pearce Carcinoma Cellswith Intercellular Components. Nagoya J. Med. Sci., £S:415-26, 1961.

18. — —. Influences of the Brown-Pearce Carcinoma Growth onthe Connective Tissue Proliferation. Ibid., 25: 175-79, 1963.

19. — —. Growth-Promoting Effect of Chondroitin Sulphate onSolid Ehrlich Ascites Tumour. Nature, 207: 537-38, 1965.

20. Takeuchi, J., Kano, S., and Tauchi, H. Influences of Age andSex on the Effect of Hydrocortisone upon the Ehrlich Tumourand the Adrenal Gland. Brit. J. Cancer, 19: 353-59, 1965.

21. Vasiliev, J. M. The Role of Connective Tissue Proliferationin Invasive Growth of Normal and Malignant Tissues: A Review. Ibid., 12: 524-36, 1958.

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