616-006.42 Rattus

IMMUNITY TO JENSEN’S RAT SARCOMA PRODUCED BY TUMOUR EXTRACTS.

HELEN CHAMBERS* and GLADWYS M. SCOTT. From the Barnato Joel Laboratories, Middlesex Hospital, London.

IT has been known for some years that two apparently opposite biological effects can be induced in animals susceptible t o tumour growth by the inoculation of tumour material. An effective resistance can be acquired, or the animals may become more sensitive so that tumours grow in them a t a faster rate than the normal. It is common knowledge that acquired immunity to transplanted tumours is in some way associated with the absorption of tumour tissue and can be produced in a variety of ways which injure tumour cells without causing necrosis, such as exposure to X-rays or radium. Immunity thus caused is the only form of protection known t o be effective against an established tumour and which persists through an animal’s life; for many years it has been attributed without explanation to the absorption of living tumour cells. The opposite phenomenon, increased sensitiveness, was first described by Haaland (1910) after treating animals with mechanically disintegrated tissue ; it occurs after the absorption of the products of tumour autolysis, but there are other agents which are said to cause it as well.

In previous papers (1924, 1926) we have put forward the hypothesis that the production of both tuiriour immunity and increased sensitive- ness could be explained by supposing that two different substances were formed from tumour cells by the action of intra-cellular enzymes during the processes of disintegration. It was suggested that the substance causing immunity was an early cleavage product, an unstable body soon destroyed by further enzyme action and therefore difficult to detect : that the substance causing increased sensitiveness was an end cleavage product, relatively stable and therefore easily detected in autolysed tissue. It was also suggested that neither of these bodies existed in the living tumour cell, and whsther either of them were absorbed when tumour tissue was inoculated would depend upon the condition of the material inserted and the conditions a t the site of inoculation. There was no evidence that actively growing lumour cells could produce immunity.

* Full-time investigator for the Medical Research Council. 288

804 H. CHAMBERS AND G. M. SCOTT

Our paper published in 1926 dealt with the subject of a growth- promoting factor in autolysed tumour tissue. W e can now report experiments which show tha t tuniour tissue deprived of its blood supply and kept at blood heat undergoes a change of relatively ahort duration. Turnour extracts prepared within this period have immunising properties. The change is detectable soon after excision of the tumour ; under anaerobic conditions at 3’7°C. t he immunising property has practically disappeared at the end of two hours. The change in the tumour is probably due to defective oxygenation.

General technique. Strain of turnour and breed of rat. The strain of the Jensen rat sarcoma

used for these experiments has been in continuous propagation i n these laboratories since 1912 and its characters are well known. The technique of inoculations has been the same as that which has been in use since 1920 and has been carried out entirely by G. M. S. The experiments have all been done with T stock, a breed of rat which we have used since 1927 which forms about 80 per ccnt. growing tumours at the end of three weeks. The variations in the growth of the tumour and the influence of technique have been discussed in a previous paper (1930). All the rats used for experimental and control series have been grouped so that each series was as nearly identical as possible. Usually young rats have been used. A tumour extract for the primary inoculation was prepared and 1 C.C. inoculated through a narrow bore needle into the axilla and 1 C.C. into the groin on the left side of each rat of an experimental series (about 20 rats). Two to eleven days later all the experimental animals and the controls have received a test inoculation into the opposite flank. The tissue used for the test inoculations has been chosen with great care and only healthy tissue from actively growing tumours used. Every small piece of tissue used for inoculation has been equally divided between the controls and experimental animals and the tissue has been kept as short a time as possible outside the body. A record has been kept of every animal and the tumours measured twice a week with dividers and a millimetre scale.

Prepamtion of the extracts. Tumours were excised and the healthy-looking tissue used, necrotic or obviously degenerate tissue being discarded. The tissue was minced with a Haaland mincer and mixed with four times its volume of normal saline and incubated a t 37°C. for various times without shaking. The flask containing the mixture was then shaken, and the mixture at once filtered through glass wool and the filtrate used as the extract. The glass wool was made dense enough to prevent visible tumour particles from passing. The extracts obtained could be readily inoculated through a narrow bore ncedle without blocking.

Composition of the extracts. When living tumour tissue is minced and mixed with saline and the mixture shaken and filtered at once through glass wool, the filtrate consists of saline, tumour juices, a certain amount of blood and tumour cells. Such an extract when inoculated forms tumours in a large per- centage of rats. If the extract is made with saline at blood heat and used a t once for inoculation, growing tumours will form, but if there is any delay the percentage number of successful inoculations will gradually fail and at the end of half an hour there is an obvious drop. When the mixture of tumour tissue and saline is incubated before filtration, the extracts contain in addition to the above contents more turnour juices, and numbers of isolated and apparently intact tumour cells which have wandered out from the substance of the tumour particles. The number of these cellsvaries with the fineness of the mincing,

RAT SARCOMA 285

thc length of time of incubation, the density of the filter and other factors. The cells, however, can be readily counted with a Thoma-Zeiss counting slide. They settle out fairly rapidly from the extract and if the inoculum is to be uniform it must be mixed for every rat inoculated.

The colour of a tumour mixture incubated in air remains almost unaltered for the first hour or two but rapidly changes as autolysis advances. The rate of change depends upon a number of factors such as the condition of the tumour, the ratio of tuniour to saline, the extent of aeration, the amount of blood and serum present and whether the mixture is shaken or not. Advanced autolysis is indicated by the tumour tissue becoming opaque and white instead of semi- transparent, and the fluid becoming greenish yellow and fluorescent ; this stage corrrsponds to the formation of the substance causing increased sensitiveness but it is not reached in the experiments described in this paper. The immunity phase takes place and is passed before there is any very distinct cliango in colour, but on shaking in air the fluid will become greenish and slightly fluorescent indicating that changes of autolysis have begun. There appears to be no test for the presence of the irnmunising body other than the biological one.

l'emperature. The two chief factors which control the disintegration products found in a mixture undergoing autolysis are the teniperature and time. In our first experiments (2651, 2663) the flask containing the tuinour mixture was started at room temperature and slowly warmed to blood he& Most of tho latcr expeririients were done by adding h i d a t 37'C. to utensils which were already warmed to that temperature, and the flasks placed in a water bath. The time of incubation starts from tlie addition of the warm saline.

Aemtion. I n a mixture consisting of minced tumour tissue and saline at 37" C. the oxygen supply for the living cells is very soon exhaubted. The amount of oxygen present a t first must be uncertain and dcpends upon the amount in the tumour itself and the blood i t contains ; there are also trxes of sugar from which tho cells may obtain oxygen for a very limited time. Even in an open flask with a large surface freely exposed to air, and only a very sh,illow depth of fluid, diffusion of oxygen is unable to meet the needs of tlie cells and most of them rapidly die. Efforts to create more coniplete anaerobic conditions have only slightly affected the results. When the mixture has had hydrogen bubbled through and then been incubated with the flask sealed, very little diifercnce has been noted in the results. Some experiments were madc in closed Hanscn's flasks filled as completely as possible with the mixture, and in these anaerobic conditions were more rapidly obtained. The rate of change was hastened but not much. The fluid consists only of saline and tissue juices, and tumour cells will remain alive in it for several days at 37' C. provided the physical conditions for tissue cultures are established, and the cells are allowed to form a thin film spread out on a surface in contact with air. In all these tests the chief factor creating the anaerobic state is that there is not enough oxygen present to supply the needs of such a large number of cells. During the short time of incubation with which we arc concerned there is no material change in the pH of the fluid.

The extract has formed a tumour in 34 cases and all but two of these have grown turnours from the test inoculation. In the rest of the series there has been no evidence at all of any growing tumour from the primary inoculation ; there has generally been a teniporary swelling due to intlammatory conditions.

Table I gives the protocols of nine experiments showing tli0 difference in the number of tumours in the experirrieiital rats compared with the controls a t the end of three weeks. The technique in all

286 H. CHdMBERS AND G. M. SCOTT

Number with

tumours 1st inoc.

11 3

0 2

0

10

0

1

0 0

...

__-

these cases has been much the same except that in the first experiment the niixture was merely shaken for a minute, then filtered and used a t once for inoculation; the only form of incubation which it received was that the saline was a t 3'7°C. when it was added to the tumour tissue. The inoculations were not finished until 30 minutes after the saline had been added; by that time the immunising effect was becoming apparent. In the other experiments the mixture of saline and tumour has been incubated for varying periods, sometimes in air and sometimes in hydrogen.

-__.__

Number with

tumours test iuoc

11 3

11 10

7

10

7 4

1 7

57 - 37%

__-_

TABLE I.

tefernnc number.

~

2713

2742

2690 2745

2791

2822

2876

2651

2663

Total (

Preparation of extract.

Mixture made at 37°C. filtered and used right away (first 2 cages)

%me (last caged)' 50 min. at 37 C. in

hydrogen . . 60 min. at 37" in air . 65 min. at 37°C. in

hydrogen 75 min. at 37" C. in hyl

drogen, 75 min. at room temperature.

85 min. at 37°C. in hydrogen

90 min. at 37°C. in hydrogen . .

Put into incubator at room temperature 120 min. in air .

Do. do.

;eluding first experimen

Days oetween 1st and test in-

xulation

3 3

4 11

4

3

7

5

2 3

I .

Experimental rats.

Num' iiioc late

14 8

20 21

20

21

17

17

18 21

155

__

Controls. __ _ _ _

Rats in- xulated.

___

22 22

21 21

21

21

12

16

21 25

158

- -.

rumour:

__

20 20

21 21

21

16

12

11

20 25

147 - 93%

The figures refer to the results 3 weeks after test inoculation. In every group the tumours growing in the experimental rats were smaller than the control tumours.

Taking the experiments in table I as a whole, excluding the first (where the immunising property has hardly appeared), 14'7 out of 158 control rats (93 per cent.) had tumours a t the end of three weeks, while only 57 out of 155 (37 per cent.) inoculated rats had tumours and the average size in every group was considerably smaller than the controls.

The graph (experiments 2651, 2663, 3135, 2690, 2742, 2745, 2822, 2876) shows the rise in the immunising power of the extract with

RAT SARCOMA 287

incubation. The figures are based on the relative percentage of growing tumours in the experimental rats compared with the controls. Seven rats which grew tumours at the site of the first inoculation are not included. The two points which fall nowhere near the curve represent experiments 2822 and 2663, in both these cases the material used for the immunising dose was the edge of large tumours with necrotic centres, and the results are probably influenced by this; for all the other experiments younger tumours were used.

The strongest immunising effect has been obtained when the mixture has been incubated in air for 120 minutes after the saline

had been added a t room temperature; under these conditions the contents of the flask reached 3'1" C. in about three-quarters of an hour (see experiment 2651) and only one tumour grew out of 18 rats inoculated. When the extract has been centrifuged a t low speed (table 11) the top fluid (cell content 2200 per c.mm.) has shown a slight fall in its immunising power (3132); when the extract has only been allowed to settle there has been only a slight difference in the immunising power of the sediment (32,000 per c.mm.) and top fluid (4500perc.mni.) (3100). Inexperiment 2882 the extract wascentrifuged a t high speed and the deposit, which was washed repeatedly, retained its immunising properties. When either formaldehyde 1 in 5000 or 0.25 per cent. phenol were added to the extract the immunising effect was destroyed, but with 0.1 per cent. phenol it was not entirely lost. The immunising property was also lost when the extract was filtered

288 U. CHdMBERS AND G. M. SCOTT

through closely packed asbestos wool which had been washed repeatedly in boiling water and found to be neutral.

TABLE 11.

leference lumber.

- 2879

2882

3132

3135

3100

3103

3062

Preparation of extract.

95 min. at 37" C. in hy- drogen (5% blood).

Same as 2859; cen- trifuged deposit washed then saline added . . .

95 min. at 37" C. in air, centrifuged at low speed, top fluid used. 2200 cells perc.mm. . .

Sameas 3132; notcen- trifuged,whole fluid used. 14,400 cells perc.mm. . .

105 min. at 37°C. in air,allowed to settle, top fluid used. 4500 cells per c.mm. .

Same as 3100; sedi- ment used. 32,000 cells per c.mm. .

115 'min. at 37'C.; in full Hansen's flasks, ends sealed. 16,000 cells per c,mm. .

Days between 1st and test in- culation

___

5

5

9

9

4

4

4 ___

Experimental rats.

0

0

2

4

0

0

1 I

~

Xumber with

umours est irioc

5

4

i

4

8

7

17 -

Controls. ~

%at8 in- culated.

-

20

20

20

20

22

22

16 -

lumoura.

15

15

17

17

15

15

14

The figures refer to results 3 weeks after test inoculation.

General discussion.

I n the experiments detailed, 34 primary inoculations have grown into turnours and in 32 failed to produce immunity; subsequent test inoculations produced tumours in every case but two. Our general experience ie, that the inoculation of tumour cells which actively grow does not have any appreciable immunising effect on subsequent inoculations.

The ability to produce immunity appears concurrently with the inability to produce tumours. The potency of the fluid does not depend on the number of tiimour cells, as in some cases rats have been successfully imrnunised with suspensions containing comparatively few cells while a much higher concentration of cells has failed completely in other cases. The condition of the cells seems to be of much greater importance than the actual number. Filtration through tightly packed asbestos wool removes the power to confer immunity

RAT SARCOMA 289

and this suggests that an active body resulting from the breaking down of the cell may adhere to solid particles or be absorbed by the filter.

The K.C. breed of rats (1930) which gives nearly 100 per cent. progressive tumours is much more difficult to immunise than the less susceptible stock, but they have been made to acquire immunity. T. stock have been used for the experiments recorded in this paper as they are a more delicate indicator of the presence of an immunising factor. The immunity once established is general and apparently permanent ; subsequent inoculations do not grow.

The experiments indicate that conditions for immunity involve enough reduction in the oxygen supply to damage the tuniour cell, but that the immunity can be effectively produced only when the damaged cell or its break down products are a t the right stage of degeneration and in contact with an active circulation. These are artificial conditions which probably do not come into play with an established tumour, as any deficiency in the vascular supply, enough to stop tumour cells growing, is generally lasting and carried to the stage of necrosis but without enough vascular supply to remove inter- mediary bodies. In most of our experiments the immunising potency would have been very soon lost with longer incubation. It may be that absorption of the substance causing immunity only occurs under suitable local conditions and some particular mechanism is necessary for transmission round the body.

The question arises whether it would be possible by artificial means to create these conditions ilz vivo. The attempts which have been made recently to treat malignant growths by reducing the oxygen tension in inspired air scarcely seem to be likely t o succeed in reducing the local oxygen supply enough to effect these changes. Many years ago numerous efforts were made to control tumour growth by obliterating the blood supply; these efforts often resulted in the formation of extensive necrotic areas. I n the light of this work we should expect such treatment to give rise t o increased sensitiveness and not to immunity, and there is some clinical evidence suggesting that this actually did occur.

On the other hand this work points to the possibility that a temporary obliteration, not long enough to damage blood vessels or capillaries so that a return circulation could be established, might provide the conditions for protection. Very little appears to be known as to the duration of life in vivo of tissue cells of different types temporarily cut off from their blood supply. It may be that cells deprived of oxygen live for varying times depending upon their state of activity, rate of division and consequent hunger for oxygen. Tumour cells show signs of damage a t a very early stage after deoxygenation. Only future work and clinical observations can decide questions such as these.

290 H. CHAMBERS AND G. M. SCOTT

Summary.

(1) Experiments are described which show that Jensen’s rat sarcoma, deprived of its blood supply and kept at blood temperature, undergoes a transitory change during which extracts from it have immunising properties.

(2) The immunising property develops for a short time with increasing potency and then disappears; it is apparently due t o changes in the tumour cells set up by defective oxygenation.

REFERENCES. CHAMBERS, If., AND SCOTT, Brit. Journ. Exper. Pathol., v. 1.

CHAMBERS, H., AND SCOTT, Ibbid., vii., 33.

CHAMBERS, H., AND SCOTT, this Journal, xxxiii. 553.

HALLAND, M. (1910) . I . . Proc. Roy. Soc. B., lxxxii. 293.

G. M. (1924)

G. M. (1926)

G. M. (1930)