An experimental study of metastasis

AN EXPERIMENTAL STUDY OF METASTASIS.*

By MAKOTO TAKAHASHI, M.D., Tokyo, Voluntary Assistant, ImpeI-ial Cuncer Reseu~ch Fund, London.

(PLATES I.-III.)

IN malignant disease in man, dissemination to distant parts of the body does not generally take place in the early stages of the disease. The same is true of the spontaneous humours of mice and rats, and also of those produced experimentally by subcutaneous implantation in normal animals. Metastasis forniation is a late phenomenon in practically all the yropagable tumours of mice and rats which show it. There is no essential difference between metastasis formation in man and small laboratory animals. I n both dissemination may occur by either the lymphatic or blood vascular system. I t therefore seems justifiable to anticipate that the factors which influence the transportation and growth of tumour emboli in man and animals are similar in nature, and study of one may help to elucidate the other.

In metastasis formation, whether in spontaneous or transplanted tumours, there are two phases. The initial phase is that by which the tumour cells in the primary growth penetrate the endothelial layer from the outside. The transportation of emboli and their arrest are purely mechanical. The second phase, and it is critical for the formation of secondary growths, is that in which the tuniour cells establish organic union with the vessel wall after their arrest, penetrating the endothelial layer from the inside. Before the second phase is achieved the tumour emboli are bathed in blood plasma or serum, if clotting occurs, and in this medium they are exposed to the action of the leucocytes. Endothelial surfaces present great resistance to the spread of growing tumours, as is clearly seen in the frequency of large intravascular growths in the lungs of mice with transplanted tumours of sdme strains, notably the Jensen mouse carcinoma (Haaland, 1 9 0 s 6).

The same condition is also met with when a subcutaneous tumour approaches the peritoneal cavity, and when intraperitoneal inoculation leads to growth. The endothelial layer is stretched over the growing tumour, and only gives way after great distension bas occurred (cf. also Jones and Rous, 1 9 14 7.

The frequency of metastasis formation varies enormously in the Received May 13, 1915.

1--JL. OF PAlII.-VOC. XX.

2 MAKOTO TAKAHASHI

different malignant new growths of nian and animals. The reasons for these differences and their relation to the biological and histological characters are scarcely known. The material of the Imperial Cancer Research Fund comprises transplantable strains which differ widely in this respect, and it i s thus possible to subject these differences to experimental analysis. For this purpose it was necessary to simplify the conditions by eliuimatiug some of the hetors responsible for spontaneous clissemin, J. t ' ion.

As a matter of technical convenieuce the blood vascular system in mice is incom parahly more suitable for experimental investigations than the lymphatic system, and the studies with which this paper deals were therefore carried out by intravenous injection of turnour eiiiulsions into the tail vein of mice. By this means we eliminate a t once one of the factors responsible for na tu~al ly occurring metastasis, namely, the penetration of the tuniour cells through the endothelial wall of the vessels into the blood streaiii in the primary tuinour. The success or failure O F intra,vaecular implantation depends, in these conditions, on the ability of the tumour cells to survive after their arrest in the lung capillaries, and to establish organic uuion with the vessel wall.

Several authors (Levin and Sittenfeld, 1 9 1 0 11, Gussio, 1 9 1 2 5 ,

Werl, 191 3 17) have carried out siinilar investigations on r&s. They iu jected tumour suspensions into the jugular vein and recorded the frequency of macroscopic growths in the lungs. Their results, therefore, are not strictly comparable with those of the present investigation in which the fate of the injected tnmonr cells has been definitely established by microscopical examination of the lungs in serial sections. Heie, again, mice are iuuch more suitable, as the lungs are smaller. I n addition, the greater variety of tumours available in mice enhances the value of the results by permitting a greater variety of experiments.

For inoculation, about 0 2 grin. of healthy tissue was taken and cut up with sharp scissors till a uniforni fine emulsion was olrrtaiued. This was then suspended in about 7 C.C. of norinal saline. This fine hiispension was drawn up into a 1 C.C. all-glass syringe, armed milt11 a tine short glaucoma, needle. The mouse, which must wither t e too young nor too old, is held in a cylindrical wire cage, closed a t the distal end with a plug of cotton-wool, and :tt the proxitnal end with a cork incised at one side for the tail (Fig. 1) The epidermis over the

- - ..

, I

, . / ..I

FIG. l.--ferforated ziiic cage foi i i i t i s v e r i t , ~ ~ i i i o d a t l o n . (Half.size.)

A N EXPERZMXNTAL STUDY OF METASTASIS. 3

prominent lateral tail vein is scraped away with a sharp scalpel, the surface wiped with a swab moistened with ether, and the needle introduced into the vein. Practice is necessary to hit off every time the exact depth a t which the needle enters the vein, but success is a t once recognised by the displacement of blood by the colourless suspension in the vessel between the needle and the root of the tail. The injection must be made slowly, and it was found that 0.15 C.C. was tolerated without any disturbance. I n a very few cases death from embolism occurred. Three weeks after the injection the mice were killed, the lungs removed and preserved. They were then cut in serial sections and examined, so that even microscopic growths could not be missed.

I n all, fourteen different tuniour strains were used,: three sarcomata and eleven carcinomata. In the different strains success varied between 0 per cent. and 90 per cent. (Table I.).

The table shows that the success of intravascular implantation in the different tumour strains varies within wide limits both in sarcomata and carcinomata. Of the three sarcoma strains studied, one (219) failed to grow in the single short series inoculated, and the other two (37p and 37s) gave 3 3 per cent. and 88 per cent. of success respectively. The eleven carcinoma strains varied between 0 per cent. and 78 per cent. success. The inore differentiated carcinoniata (27, 72, 155, and 630), however, all failed to grow in the lungs. Strains 27, 72, and 155, are papilliferous cystic adeno-carcinomata, and strain 630 is a sqnamous- cell carcinoma. The seven alveolar carcinomata were not uniformly successful, five strains giving from 1 6 to 78 per cent., two strains (113 and 230) 5 per cent. and 0 per cent. respectively. The solid alveolar turnours, therefore, appear to be more capable of intravascular growth than the more diftfkrentiatecl adeno-carcinomata and the caucroid.

The comparison of the biological characters, percentage, and rate of growth on subcutaneous inoculation, with the success of intravascular implantation, can be seen in Table 11. The percentage of success of subcutaneous inoculation is a t once seen to be no guide to the success of intravascular implantation. Tumour strain 27 which never gave growths in the lungs grows in 100 per cent. in nearly every subcutaneous series. For the purpose of comparing the rate of growth the strains have been divided into three groups, a group which glows rapidly, one of medium rate, aucl a group which grows slowly. The rate of growth was arrived a t by taking a representative series, consist- ing of ten or twelve mice and measuring the diameters of the growths obtained ten and seventeen days after inoculation with a uniform dose of 0,015 grni. of tumour. The averages obtained were taken as a mensure of the speed of growth. The Table shows that 37% 63, 37p, 199, and 91 belong to the rapidly growing group, and in them the percentage of intravascular I‘ takes” is high (16 to 88 per cent.). On

4

Number of Series

kt:: Strain. Histology.

MAKOTO TAKAHASHI.

TABLE I.

Number of Percentage “ Takes ” of Lung Im- Lung Im-

~noculated. plantations. plantations. ~~~~~~

of “Takes ” of

1 ~~

SARCOMA.

. _ _ _ _ . _ _ _ _ ~

Polymorph - c e 11- sarcoma . .

R o u n d - c e l l sarcoma.

R

12 I ”) 3

Per cent. 88

33

0

199 . Twort .

63 .

129 . 91 .

113 .

230 .

a .- (630

Ld u

Alveolar . . . .

. . Alveolar, old tum-

ours ncinous in parts . .

CARCINOMA.

Alveolar contain- 1s ing glycogen

Alveolar . .

I’apilliferous cystic adeiio-carcinoma , .

Very papilliferous cystic a d e n o - carcinoma.

Acleno-carcinoma .

Cancroid

9 10

3 3 8 5

8

3 8 9

4 5 6 6

10

3 6 5

11 8

11

7

12 1‘2

78 60

37

24

16

5

0

0

0

0

0

A N EXPERIMZiYTAL STUDY OF METASTASIS. 5

Percentage of Positive Lung

630 . Twort .

TABLE 11.

88 37 33 78 16

24 0

. ; 5

. ! 0 0 0 0 0

60

*~

* I

Subcutaneous Inoculation.

Average Diameter in Centimetres,

ren Days after Inoculation.

1.8 1.5 1.3 1'3 1 '4

0 '5 0 '3

0.3 0.2 0 '2 0 '1 0.3 0.5 0.3

Average Diameter in Centimetres, Seventeen Days after

Inoculation.

2 '1 2'1 1.7 3 '0 1 '6

1 '2 1 '2

0.6 0'5 0.6 0 '3 0'6 0.6 0'6

Average per Cent.

__-

100 95

100 100

90

a5 75

61 95 40 35 95 90 95

the contrary, the tuinours of slow growth (113,72,27,155,630, Twort, and 230) have low percentages (5 t o 0 per cent.), with the exception of Twort (60 per cent.). Strains 129 and 219 are intermediate in rate of growth, but while 129 gave 24 per cent. of lung takes, 219 gave 0 per cent. As a general result it may be concluded that the tuniours of rapid growth grow better and in a higher percentage in the lung capillaries than the strains which grow slowly. The results of the microscopic examination of emboli in the first days following injection will be seen to indicate the reasons for this difference.

Among the tumour strains used for these experiments, some had produced metastasis after subcutaneous inoculation, while others had produced none through many (fifty to sixty) generations. It is ini- portant to correlate the success of intravenous implantation with the frequency of metastasis occurring naturally, since a general correspond- ence would indicate the importance of the factors operative a t the site of arrest, while a discrepancy would signify a predominant iniportartce of those responsible for the entry of tumour cells into the vessels in the primary tumour. It will be seen that strains 37s, 37p, 63, 199, 91, Twort, and 113 produced metastases of themselves, and in all of them lung implantation was successful. On the other hand, the strains which did not metastasise to

This information is set out in Table 111.

the lungs naturally, failed to grow on intravenous inociilation, with the single exception of strain 129. The percentage of successful intravenous inoculations is, however, not very high in this strain. The conclusion seeins justified that the absence of metastases after subcutaneous inoculation of many mouse turnours, is due to the inability of the turnour cells to survive after arrest in the lung capillaries, and establish organic union with the vessel wall. Whether these tuinours are also incapable of penetrating into the blood vessels cannot be de- termined, but unless this depends on the same properties of the cells as subserve their growth in the vessels (which is not, improbable), it must be of less importance.

TABLE In.

Natural Metastasis. Strain,

37s . .

199 Twort 91 . . 113 . . 129 . . 230 . . 72 . . 195 . . 27 . . 219 . . 630 . .

;;P . ’ . .

~ . . , + . . + . . + . . + . . I + . . + . . + ’ . I

- - . . - . . . - . . - . . - . . ~ . .

Dnring ten genciations 111 e x l i tumoor strain-- 90 to 120 caaes in each.

~ -- .- - - -~~ -

M. B. Schmidt ( 1 9 0 3 15) showed by study of the lungs in cases of generalised cancer in man that iiiariy emboli die in the vessels, and, therefore, do not give rise t o secoudary growths. He pointed out the iiuportniice of the organisation of thrombus in providing il new vascular coIinectiori between the tunionr cells and the vessel wall, iixid showed that failure or delay of this process sufficed to ensure the death of transported tuniour cells. Levin and Sittenfeld (1 9 1 0 11) iiijected saline suspensious of two rat sarcomata aiid a mouse tuniour into the jugular vein. A l l these tunionrs grew well on subcutaneous inoculation, but macroscopic lung noduIes were \ ery rarely seen after intravenous injection. They concluded froiu their findings that Schmidt was right iri believing that the tuniour cells were destroyed in tJhe blood vessels, and in the present investigation, 011 esaniining the lungs of inice inoculated with strains which failed to grow, iiisny hyaline emholi

A N EXPERIMENTAL STUDY OF METL4STASIS. 7

were found. Others were already fibrous, with occasionally a few included turnour cells much altered, but still recognisable. Such an embolus in a late stage of absorption is shown in Plate I. Fig. 1, in a branch of the pulmonary artery filled with new connective tissue. I n contact with the large clear tumour cells is a group of macrophages with spongy protoplasm. The internal elastic lamina is not quite complete, but is still recognisable.

Turnour strains having been obtained, in which intravascular implantation was very successful, while in others i t was uniformly negative, the changes immediately following implantation mere studied by inoculating il number of animals with each strain, and killing two or more each day for a week. The strains chosen were 37s (spindle-cell sarcoina), giving 8 0 t o 9 0 per cent. of success, and 27 (adeno-carcinoma papilliferum), which had always been negative. One lung from each mouse was preserved in Zenker’s fixative, and the other in platino- chromosmic acetic acid mixture (Borrel’s formula). The lungs were cut in serial sections and examined microscopically.

In the lungs of mice inoculated with the tunzour which never grew (27) fresh thrombi were easily found three to five days after inoculation. Tumour cells could seldom be recognised except as hyaline structureless masses. When the lungs were preserved twenty-four hours after inoculation, however, tumour cells could be found with ease. While many already showed profound degenerative changes, others were more healthy. The nucleus (Plate I. Fig. 2) still stains well, but the protoplasm is vacuolated, especially a t the periphery. Here the vacuoles appear stalked, and forni a complete fringe all round the cell. It is easy to see that as soon as these vacuoles burst, the complete disintegration of the cell iiiast take place very rapidly. This, in all probability, accounts for the almost complete absence of recognisable tumour cells after three to five days. Numbers of leucocytes gather around the arrested cells, but the endothelium of the vessel shows no reactive changes.

The appearances seen when tumour 37s is inoculated differ in important details. This strain produces metastases after subcutaneous inoculation, and grows well on intravenous implantation. Twenty-four hours after inoculation the cells of the tumour emboli are seen lying in the artery surrounded and separated by polymorpho-nuclear leucocytes (Plate 11. Fig. 3). The surface protoplasm is seen to be vacuolated, but t o a much less extent than is the case in tumour 27. The staining reactions are unaltered. After three days, if the embolus is going to survive, a considerable amount of growth has already taken place, and the vessel lumen is almost completely filled (Plate TI. Fig. 4). The tumour cells have resumed their spindle form, and very few leucocytes are visible. Where the tumour cells are not in contact with the vessel wall, the endothelium presents a curious vacuolation. The protoplasm of the endothelial cells is transforiiled into a foamy

8 MAKOTO TAKAHASHL

Right side ofneck tumour, size of a cherry.

structure in which the nuclei are embedded, distorted, and indented by the snrroundiug vacuoles. I n Plate 111. Fig. 5 the condition after five days is represented. It differs froni the three-day stage in the increasing distension of the vessel, diminution in the vacuolation of the endothelium, and entrance of capillaries froin the intima into the young tuinour. When the vascular imlilants of this tumour fail to establish themselves, an intense vacuo- latioii of the timiour cells and of the intima takes place (Plate 111. Fig. 6). In this figure, showing a degenerating embolus three to four days after inocnlation, the tuinonr cells are faintly stained, and vacuolated all over the surface. The vacuolation of the endotheliuni is extiwnely intense, the endothelinl nuclei being distorted by the pressure of the snrronnding vacuoles. The cause of this endotlielial change is not quite clear. It has never been seen in the preparations froni inice injected with turnour 27, and it may be a special peculiarity of the cells of tnmonr 37s setting up a specific irritation, which is favourable to the settling down of the tunour cells.

The later stages present nothing of note.

-

Experiments 37s/179 B (October 22, 1914), 37 s/182 A (November 21, 1914), and 37 s/l'i9 E (October 28, 1914).-~ntr,zvenous inoculation of 0.15 C.C. saline suspension of tumour emulsion. All mice killed twenty-one days after inoculation.

Lung.

Several pinhead nodules (Text Fig. 2 ) .

Two p i n h e a d nodules (Text Fig. 3).

Xmiy pinhead nodules.

Many pinhead nodules.

T n o sniall nodules.

hfany small nodules

3nbciitaneous andl in Muscles.

r/i 1 -- .__

One tumoui less than 1 em. in diameter.

Kidney.

[ n t r a v a s c u l a r ni i c P o s en p i e nodules.

-

-

-

-

-

Other Sites.

Iesenteric tumoiir, size of cherry.

rumour on deep surface of a te r - num, size of a cherry.

...

...

...

Iesenteric tuiiiouu. size of a cherry.

While the ma<jority of the growths following intravenous implantation ha,ve been situated i n the lungs, in some instances tumours have developed in other sites involving transport by the greater circnl a t ' ion.

A N EXPERIMENTAL STUDY OF METASTASIS. 9

These observahions, besides being interesting in themselves, deserve detailed consideration because of the analogy they present to some types of dissemination in the human subject. As is well known in some cases of prostate carcinonia in man, multiple metastases are found in the bones without any involvement of the lungs (v. Hecklinghausen, 1 8 9 1 13, Kauffmann, 1 9 0 2 8). The two possibilities which are offered in explanation of this distribution are-First, that tumour emboli pass through the lung capillaries ; and, second, that a retrograde lyniphatic spread has taken place. U p to the present no experimental observations have been made which would help to decide between these alternatives. The particular experiments now under consideration involved six animals belonging to several series, all inoculated in the tail vein with tuniour 37~, a spindle-cell sarcoma. The animals were killed twenty-one days after inoculation, and, in addition to the lungs, the spleen, kidney, and liver were examined microscopically. Condensed protocols of tbe cases will be found on page 8.

From the small size of the lung nodules, as compared with the systemic, in these cases, it is practically certain that the emboli giving

._... v

FIG. 2.-Systemic dissemination of spindle- FIG. 3.--Systemic dissemination in an- cell-sarcoma after intravenous inocula- other series after intravenous inoeala- tion, No. 1 in tahle. Experiment tion with the same tuniour, No. 2 3 7 , 4 7 9 ~ ; inocnlntedOctober 22, 1914 ; in table. Experiment 37/182 A ; in- killed November 12, 1914. oculated November 21, 1914 ; killed

Deceniber 10, 1914.

rise to the latter traversed the lung a t the time of inoculation, and found niore favourable conditions for growth in the domain of the systemic circulation than are presented by the vessels of the lungs. The small number of cases in which dissemination occurred beyond the

10 M A K O T O TAKAHASUI.

lungs (six cases out of 2 2 8 mice injected, of which sixty-three were positive), shows how eecient a filter tlie liing capillaries are.

Two other features are significant: the restriction of transpulmonary disseniination to one strain of sarcoina, and the limited number of sites iu which the growths mere found. Three turnours were situated in the thigh, two in the neck, two in the mesentery. To the sternal tuniour in the sccond inouse of the table inust be added two further cases of disseiiiination of 37s, one in the s a n e situation on tlie deep surface of the steriiuni, and another on the outer surface. The mice of this experinienl are not strictly comparable with the others, as for another purpose they were inoculated subcutaneously with a suspension of lecithin. The limitation in the number of sites selected by the tumour cells in these experiments preseiits an exact parallel to the phenomenon of selective metastasis in the hutnan subject already referred to (Schmidt, 1 9 0 3 Is). I t points to delicate cliEerences in the conditions favouring growth, peculiar to individual turnours, which cannot be more precisely defined a t present.

Incidentally, it may be pointed out that these observations have iiot confirnied the opinioii of Kretz (1912 that emboli which reach the heart by the inferior vena cavil tend to be distributed to the lower pulmonary lobes, those from the superior vena cava to the upper lobes. Walter Georgi (1912 4), also, could not confiriii Kretz's observations from his experimental investigations, and in the present material the distribution of the nodules in the fifty-four cases of positive lung inipl~liitation was qnite irregular.

I n recent years considerable attention has been directed to the r61e of certain lipoids, principally cliolesterin and lecithin, in health and in pathological conditions (Levy, 1 9 0 5 12). A definite favourable action of lecithin on growth has been claimed by Danilewsky (1895 3, and Bain (1 9 1 2 l). Numerous experiments have shown also that prolonged feeding with cliolesterin produces atheroiiiatous changes in the intinia of blood vessels (Leiiioine, 1 9 11 lo), Tliacher and Hueclr, 1 9 13 16), and T. B. Robertson and J. C. Burnett (1 9 13 I*, 1 9 1 4 ">, following Danilewsky's results, have published experiments to prove that lecithin and cholesterin have an important influence on the growth of rat tumouis. The effects observed were, that while subcutaneous iiijections of lecithin eniulsions retarded growth aiid dmiinishecl the freqrreiicy of metastases, botli these processes were augmented by cholesterin.

In the light of the intirnal changes recorded above, in connection with recciitly arrested emboli, experiments were carried out 011 the tumour stiains, whose behavioiir on intravascu1:tr iiiiplantation was known, to see if the oliservations of these authors could he coufirniecl.

I t is unnecessary to go into details of these experiments, which were vitiied in many ways, since it was found that in no case did the growth of the tumours respond to treatment mith lecithin with retnrdation, nor to cholesterin treatment with acceleration. In an experiment with

A N EXPERIMENTAL STUDY O F METASTASIS. 11

the Flexner-Jobling rat tumour no effect could be discovered either on the growth of the subcutaneous tuniours, or ir, the direction of favouring metastasis in the lungs. As it is well known that the intinis changes following cholesterin injections take some time t o develop, in one series of thirteen mice cholesterin was injected repeatedly (eight times) for a month before intravenous inoculation of a tumour strain 129, which in the earlier experiments gave 24 per cent. of lung-"takes." Twelve normal untreated mice were injected a t the same time its the contyol. The result of the exniniiiation of the hugs after three weeks showed no difference (25 per cent. control, 23 per cent. cholesterin- treated). It is possible that a more prolonged treatment might have shown a different result, as in these animals no changes could be de- tected in the aorta and other vessels on microscopical examination.

SUMMARY.

Carcinonia and sarcoma are equally capable of producing growths in the lungs when injected intravenously. The tumour strains of both kinds examined differ widely, however, in the frequency with which intravenous implantation results in growth. In general it may be said that the more differentiated glandular mammary carcinomata give much lower percentages of success than the solid alveolar carcinomata. A highly differentiated cancroid used in these experiments was always negative. No relation could be established with the percentage of success on subcutaneous implantation; in fact, the majority of the strains used grow in 1 0 0 per cent. under the skin, but as a general rule success was proportional to the rapidity of growth. The cause of the variations in the production of metastases by the several strains is not so much the inability of the cells to make their way into the blood stream, as their inability to survive and gtow after arrest in the vessels of the lungs.

A large proportion of the cells introduced into the blood stream disappear after undergoing vacuolation, surrounded by leucocytes. I t was noticed that the endothelium of the pulmonary vessels showed a peculiar vacuolation also in aninials injected with a sarcoma strain, which grew well in the vessels. This intinial change may be of inzpoi t- ame for the successfill settlenient of the tuniour emboli in the vessels.

Although the lung capillaries form a very efficient filtei-, and arrest the majority of the tumour cells introduced into veins, some cells are able to pass through the lungs, and, being distributed by the systemic circulation, give rise to growths in various purts of the body. Apparently all tumours cannot disseminate in this way, arid the strain which did so exhibited a predilection for a limited number of sites.

I take this opportunity to express my gratitude t o the Executive Committee of the Imperial Cancer Research Fund for permission to work

1 2 MAKOTO TAKANASUI.

in their Laboratory, and to the meillhers of the Scientific Staff for advice and assistance in carrying out this investigation.

R E F E R E K C E R

1. ~YILLIAM BAIN . . . . “ Pharniacology and Therapeutics of Lecithin and Phytin, Lancet, London, 1912, vol. i. p. 918.

2. T. C. BURNETT . . . . ‘’ Noteon the Re-establishment of a Tendency to Mctnstasise in a Flexner-Jobling Car- cinoma,” P~oc. Soc. Exper. Biob. and itfed., New York, 1914, vol. xii. p. 33.

3. M. C. DAKILEWSKY. . [‘lk l’iiiflucnce de la lbcithiiie sur la crois- caiice et la inultiplication des orgaiiismes,” C o q l t . Tend. Acad. d . Sc., Paris, 1895, tome cxxi. 1) 1167.

4. W. GEOIZOI . . . . . Kxperinientelle Untersnchungen zur Ern- holielolralisatioii in der Lunge,” Ueitr. 2.

5 . GUSSIO. . . . . . . 6. &I. HL4ALANn. . . . .

7. 1;. S. JONES AXD I’EYTON

8. E. KAUFMANS . . . . Rous

9. KRETZ . . . . . . .

10. &I. LEMO~SE, . . . . . 11. T. LEVIN ASD 31. J. SIT-

TENFELI)

13. F. LEVY . .

13. v. RECRCISGHAUSEN . .

14. ;1. E. ROBERTSON A m T. C. RLTRNETT

15. 31. C. SCIIXIDT

p t k . Anat. u. Z. allg. Path.; Jena, 1912, Ed. liv. S. 401.

Tumori, Roma, 1912, tome i. (6) p. 701. ‘‘ Les Tnnieurs de la Souris,” Ann. de l’lnst.

Pasteur, Paris, 1905, tome xix. p. 165. J o w n . Exper. Met?., S e w York, 1914, vol. xx.

p. 404. ‘‘ Pathologische Anatoniie der malignen Neu-

bildungen der Prostata,” Deufsche Chir., S t u t t p t , 1902, Bd. liii. S. 381.

” Denionstration zweier Versuche uber Lolral- iration der Lungenembolien,” Sitzungsb. d. Plt~~sil~al.-iiaedizilz. GesellsclL. Z Z L Wiirzburg, 1912, No. 1, s. 9.

‘. Etude clinique et therapeutiqne,” 1911. “ The Formation of Netsstasis after an Intra-

vascular Injection of Tuniour Emulsions,” Pmc. Soe. Expsi-. Biol. and Med., 1910, vol. viii. p. 114.

“ Ueber den therapeutischen Wer t des Le- cithins uiid der lecithinhaltigen Nahrprae- !mate (Lecitogen),” Berl. ?ilk. Wchzschr., 1905, No. 30, S. 1242.

“Die fibrose oder deforniierende Ostitis, die Osteomalacie und die osteoplastische Car- cinoni in ihren gegenseitigen Eeziehungen,” Festeptschr(ft, Rudolf Virchoio zu seinein 7 1 Geburfstage, 1891, p. 22.

‘( The Influence of Lecithin and Cholesterin upon theGrowth of Tumours,” Journ. Xxper. iMed., New Yorlr, 1913, vol. xvii. p, 344.

(‘ Verbreitungswege der Karzinome und die Reziehung generalisierter Sarkome zii den leulraeinischen Neubildnngen,” Jena, 1903.

hfunclien. med. Wchnschr., 1913, Nr. 38,

17. R. WEIL . . . . . . s. 2097.

“ The Iiitrarascular Iinplantation of Rat Tiimoim,” Journ. Me(?. Besearch, Boston, 1913, vol. xxviii. p. 497.

JOURNAL OF PATHOLOGY.--TOL. XX. PLATE I.

FIG. 1.

FIG. 2 .

JOURNAL OF PATHOLOGY.-VOL. XX. PLATE 11.

F I G . 3.

FIG. 4.

JOURNAL OF PATHOLOGY.-VOL. XX. PLATE 111.

A N EXPERIMENTAL STUDY O F METASTASIS. 13

DESCRIPTION OF PLATES I.-111.

PLATE I.

FIG. 1.-Experiment 113/60 c. Intravenous inoculation, October 6, 1914; killed twenty- one days later. A small branch of the pulmonary artery occluded by a mass of new connective tissue showing a group of degenerating carcinoma cell? partly surrounded by niacrophages. A newly formed capillary is oeen cut twice in the lower part of the vessel. ( x 410.)

FIG. 2. -Experiment 27/88 A. Iiitravenous inoculation, January 4, 1915 ; killed twenty- four hours later. A degeiierating carcinoma cell showing pedunculated vacuoles and nuclear shrinkage is seen surrounded by red and white corpuscles. ( x 1050.)

PLATE 11.

FIG. 3.-Experinient 37/184 A. Intravenous inoculation, December 17, 1914 ; killed twenty-four hours later. Cf. Fig. 2. Mnch less intense superficial vacuolation of sarconia cells, surrounded and separated by polynuclear leucocytep. ( x 1050.)

FIG. &-Experiment 37/189 E. Iiitravenous inoculation, March 3, 1915 ; killed three days later. Commencing growth of a young sarcoma embolus. Vacuolation of endothelial cells is seen a t one point, and early distension of the artery is indicated by flattening of the folds of the elastic lamine. Cells resnming spindle shape. ( x 525.)

PLATE 111.

FIG. 5.--Experimeiit 37/189 E. Intravenous inoculation, March 3, 1915 ; killed five days later. A later stage thaii Fig. 4. Increasing distension of vessel and entrance of capillaries into embolus on left upper surface.

Intravenous inoculation, February 25, 1915 ; killed three to four days later. A mass of degenerating racaolated sarcoma cells separated by blood clot in a pulmonary arteriole. Intense vacuolation of endothelium of vessel. Cf. Figs. 2 and 3. ( x 525.)

( x 525.)

FIG. 6.-Experiment 37/189 B.

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An experimental study of metastasis