Effect of oxygen and carbon dioxide concentration upon the development of hematogenous metastases and transplants

Effect of Oxygen and Carbon Dioxide Concentration upon the Development of Hematogenous Metastases and Transplants J. W E S L E Y A L E X A N D E R , M.D., Sc.D., and W. A. A L T E M E I E R , M.D.,

University oJ"Cincimmti College of Medicine

The factors responsible for the successful development of hematogenous metastases of malignant tumors have not yet been precisely established. Many authorities believe that mechanical factors are of primary importance in the success or failure of malignant cellular metastasis whereas others feel that the innate susceptibility of a given tissue is of greater consequence. This latter belief is based in part upon the demonstrated fact that certain tissues and organs are more fi'equently the site of metastatic involvement than are others, DifFerences in metabolic requirements within the varied tissue environments might be at least partially responsible. When it was lbund that injury of a tissue would selectively enhance its susceptibility to the development of experimental hematogenous metastases(-" 4 the question arose as to whether either a relative insumciency of oxygen in healing tissues or a relative increase in the local carbon dioxide concentration might be of significance. In one experiment, :~ligation of the gastrosplenic vessels in I7 animals two days before an intra-aortic tumor inoculation resulted in gastric metastases in 47 per cent: whereas only 5.2 per cent of 58 animals undergoing similar operations when these vessels

From the Department of Surgery, University of Cincimmti College of Medicine, and the Cincinnati General Hospital, Cincinnati, Ohio, Dr. Alexander is an Adwmced Clinical Fellow, American C:~ncer Society, This work was supported by American Cancer Society Grant No. IN79. Submitted for publication September 23, 1964,

90

were not ligated developed gastric metastases. This finding emphasized t;m'ther the possible role of relative tissue oxygen and carbon dioxide concentrations in the development of hematogenous metastases. The following experiments were designed in an attempt to study this problem further. M E T I t O D S AND MATERIALS IDomestic rabbits of a mixed breed, weighing 2,5 to 3.5 kg., were used for all studies. Tumor inoculations were made using a freshly prepared suspension of VX-2 carcinoma cells prepared as described p r e v i o u s l y : T h e intraaortic inoculations were given by rapid injection of 1.0 rot. of a tumor suspension into the thoracic aorta through a small polyethylene catheter passed retrograde through the femoral artery. A total of four experiments using 110 animals were carried out. E x p e r i m e n t 1. Five groups of animals were used to study the effect of hyperoxia on the development of hematogenous metastases, Standard surgical incisions were made aseptically in the abdominal wall of each animal within these groups. Each test animal and a paired control were simultaneously inoculated by the intra-aortic route 48 hours after the surgical incisions were. made. All surviving animals were sacrificed after three weeks, and the number and location of metastases were carefully determined and recorded. Each test animal was given 100% oxygen by mask during the inoculation and until it could be transported to an oxygen chamber designed from an iron lung, where each was caged

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.ISR -- Vol. V. N o . 2 -- F e b r u a r y 1965

OXYGEN

individually. O x y g e n t e n s i o n was regulated by varying the rate of o x y g e n inflow from p r e s s u r i z e d slorage t a n k s a n d was m o n i t o r e d with an oximeier. A n i m a l care was a c c o m plished t h r o u g h side p o r t s w h i c h c o u l d be o p e n e d and closed. In c o n t r a s t , all o f the control a n i m a l s w e r e given t u m o r i n o c u l a t i o n s while b r e a t h i n g r o o m air, a n d they were kept in an a t m o s p h e r e o f r o o m air t h r o u g h o u t the period o f o b s e r v a t i o n . T h e 33 test animals a n d 33 c o n t r o l s w e r e d i v i d e d into the following subgroups: G r o u p A ( F o u r test animals): F o l l o w i n g the [ntra-aortic i n o c u l a t i o n , the test a n i m a l s w e r e k e p t in an a t m o s p h e r e of 8 0 % o x y g e n for six days. G r o u p B ( F o u r test animals): T h e s e animals w e r e h o u s e d in an a t m o s p h e r e o f 80% o x y g e n for 48 hours, after which lime the o x y g e n c o n t e n t was kept b e l w e e n 50 a n d 6 0 % for the r e m a i n d e r o f ihe three w e e k period. G r o u p C (Six test animals): Eighty p e r c e n t o x y g e n was given for 24 h o u r s , and then d e c r e a s e d to 6 0 % for six days. G r o u p D ( S e v e n test animals): In this s u b g r o u p arl a t m o s p h e r e o f 8 0 % o x y g e n for 24 h o u r s , then 50 lo 6 0 % for the r e m a i n d e r o f the three w e e k s , was used. G r o u p E ( T w e l v e test animals): T h e s e w e r e m a i n t a i n e d in an o x y g e n a t m o s p h e r e o f 8 0 % for 24 hours after i n o c u l a t i o n , t h e n 6 0 % for 24 hours, 4 0 % for 24 hours, and finally w e r e r e m o v e d to r o o m air 72 h o u r s after inoculation. E x p e r i m e n t II. A transient h y p e r c a r b i a was p r o d u c e d to see if a simple i n c r e a s e in blood c a r b o n d i o x i d e c o n t e n t w o u l d result in an increase o f cellular a d h e s i v e n e s s and, therefore, in an i n c r e a s e in m e t a s t a s e s . Standard a b d o m i n a l incisions w e r e m a d e aseptically in 20 rabbits. F o r t y - e i g h t h o u r s later the a n i m a l s w e r e paired into test and control g r o u p s . A s i m u l t a n e o u s intra-aortic i n o c u l a t i o n was m a d e into e a c h test animal and its paired c o n t r o l using 1.0 cc. o f a freshly p r e p a r e d s u s p e n s i o n o f V X - 2 t u m o r cells. S e v e n test a n i m a l s w e r e given a gas mixture, c o n t i n u o u s l y by m a s k , at the rate o f 3 liters p e r m i n u t e , which c o n t a i n e d 5 % c a r b o n dioxide, 2 0 % o x y g e n and 7 5 % nitrogen from 30 m i n u t e s prior to the i n o c u l a t i o n until 45 m i n u t e s following t h e inoculation. T h r e e test a n i m a l s w e r e given a m i x t u r e o f 10% c a r b o n dioxide, 2 0 % o x y g e n a n d 7 0 % nitrogen for a ~ o d f r o m 90 m i n u t e s b e f o r e to 20 m i n u t e s after inoculation. T h e a n i m a l s w e r e t h e n c a g e d individually, a n d a u t o p s i e s w e r e p e r f o r m e d at the time o f sacrifice 21 d a y s later.

AND

HEMATOGENOUS

METASTASES

9!

E x i ~ e r i m e n t 111. C h r o n i c v e n o u s congestion was p r o d u c e d by ligation of the c o m m o n femoral vein in 12 rabbits. F o r t y - e i g h t hours later, 0.t co. o f a freshly p r e p a r e d t u m o r s u s p e n s i o n was injected into identical sites in both g a s t r o c n e m i u s m u s c l e s o f each animal. All w e r e sacrificed after 15 d a y s and the sizes o f the i n t r a m u s c u l a r g r o w t h s w e r e d e t e r m i n e d and c o m p a r e d by weight. E x p e r i m e n t IV. C h r o n i c arterial i s c h e m i a was p r o d u c e d in 12 animals by a s e p t i c ligation o f the c o m m o n femoral artery. F o r t y - e i g h t hours later, equal a m o u n t s o f a t u m o r susp e n s i o n w e r e injected into similar locations in b o t h g a s t r o c n e m i u s m u s c l e s o f each animal. A c o m p a r i s o n w a s m a d e b e t w e e n lhe size o f the t u m o r t r a n s p l a n t s in t h e ischernic leg a n d the n o r m a l leg w h e n the a n i m a l s w e r e sacrificed t5 d a y s after inoculation. RESULTS E x p e r i m e r l t I. It was quickly learned that rabbits s u b j e c t e d to c o n c e n t r a t i o n s o f g r e a t e r than 6 0 % o x y g e n al a t m o s p h e r i c p r e s s u r e for p r o l o n g e d p e r i o d s would die as a result o f o x y g e n p o i s o n i n g , as has b e e n p r e v i o u s l y o b s e r v e d . " A c c o r d i n g l y , all eight animals in G r o u p s A a n d B died on the fifth or sixth d a y after being placed in the c h a m b e r , b e f o r e any e v i d e n c e o f m e t a s t a s i s could be d e t e r m i n e d . G r o u p C: T h r e e test animals s u r v i v e d the three w e e k period of o b s e r v a t i o n , but w i d e s p r e a d m e t a s t a s e s were found in only o n e pair o f animals. T h e results o b t a i n e d in this g r o u p w e r e thus insufficient for evaluation. G r o u p D: Five o f the s e v e n test a n i m a l s in this s u b g r o u p s u r v i v e d t h e p e r i o d o f o b s e r v a tion. A n insignificant difference in the i n c i d e n c e of m e t a s t a s e s was o b s e r v e d w h e n c o m p a r e d with the control animals (Table 1), but it was noted that the test animals b e c a m e chronically ill and lost weight while in the o x y g e n c h a m b e r , and this m a y possibly h a v e resulted in the r e d u c t i o n o f m e t a s t a t i c g r o w t h s . In o t h e r e x p e r i m e n t s , ~'a ill health o f a n i m a l s had b e e n n o t e d to result in a d e c r e a s e in t u m o r g r o w t h and m e t a s t a s e s . F u r t h e r m o r e , F i s h e r a n d Fishe~ have demonstrated a marked reduction in the size a n d n u m b e r of e x p e r i m e n t a l hepatic m e t a s t a s e s in a n i m a l s fed a diet insufficient in protein. G r o u p E: S e v e n o f the t w e l v e lest animals s u r v i v e d the p e r i o d o f o b s e r v a t i o n , four dying o f o x y g e n p o i s o n i n g a n d o n e s u c c u m b i n g at the time o f inoculation. O n e o f the control a n i m a l s for a s u r v i v i n g test animal died from

92

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ALTEMEIER

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Vol, V, No. 2 -- February 1965

Table 1. Effect of Ilyperoxia ttpo~ ti~.matogenot~,s :~Ietastases

l~oand

'I"

c

Croup I) (5 Animals) 4,0 4,2

Abdomen T C

Spleen and l:'erisple n i,:: 'l'{ss ules T C

10,6

0.2

16.6

0,,t

t,{vcr "t" C

Each Adrenal T C

Intestines T (Z

0,4

1.6

8,8

1,2

2.4

6.0

Muscle (per cubic in¢*h) T C 1.3

2,0

l(ac,h K idnt"y

T

C

13.4 27.8

Group E (7 Animals) 2,4 1,0 12 6.2 0.6 0.0 1,4 0,3 0,6 0.5 I0 ].3 1,3 0,8 10,5 1.7 P<.05 Figure.~ represent average number of metastases per animal. Croup I) received 80% oxygen {'or 24 hours, then 50 to 60% for 20 days. The animals in (;roup l': received 80% oxygen for 24 hours, 60% for 2.1-hours, 40% {'or 24 hours, and therea{'ter w e r e k e p t itt room ;tir, "['h~ c o n t r o l grottps at('. n o t compurable.(T - - T e s | Animal. C -- Control Animal,)

p n e u m o n i a during t h e p e r i o d o f o b s e r v a t i o n . T h e r e m a i n i n g test a n i m a l s ail r e m a i n e d h e a l t h y a n d did not lose weight, in c o n t r a s t to the a n i m a l s in G r o u p 13, m e t a s t a s e s w e r e i n c r e a s e d in t h o s e a n i m a l s that had r e c e i v e d o x y g e n t h e r a p y , g e n e r a l l y b e i n g a l m o s t d o u b l e that s e e n in the c o n t r o l a n i m a l s (D<0.05) ( T a b l e 1). T h i s i n c r e a s e in all l i k e l i h o o d reflects a n e e d f o r a d e q u a t e o x y g e n a t i o n during the early development of metastases. E x p e r i m e n t I L T h e effect o f h y p e r c a r b i a u p o n the d e v e l o p m e n t o f h e m a t o g e n o u s met a s t a s e s w a s s t u d i e d in 10 pairs o f animals. T w o o f the t e s t a n i m a l s died f o l l o w i n g the i n s u l a t i o n , and their c o n t r o | s w e r e d i s c a r d e d , F i v e o f t h e s e v e n test a n i m a l s that w e r e g i v e n 5 % c a r b o n d i o x i d e a n d t h e t h r e e test a n i m a l s that w e r e given 10% c a r b o n d i o x i d e s u r v i v e d t h e p e r i o d o f o b s e r w i t i o n , T a b l e 2 s h o w s the a v e r a g e n u m b e r o f m e t a s t a s e s f o u n d in the m a j o r o r g a n s and t i s s u e s . N o significant d i f f e r e n c e in m e t a s t a s e s w a s f o u n d b e t w e e n a n y o f the t e s t a n i m a l s a n d their c o n t r o l s , Experiment III (Ligation of Femoral V e i n ) . T h e c o m b i n e d w e i g h t s o f the t u m o r g r o w t h s r e m o v e d f r o m the legs in w h i c h v e n o u s

ligation h a d b e e n d o n e w a s 11,55 gin, c o m p a r e d to a c o m b i n e d weight o f 9,85 gin. o f the t u m o r s r e m o v e d f r o m the n o r m a l legs, a n d the a v e r a g e w e i g h t o f t u m o r s f r o m t h e legs w i t h v e n o u s ligation w a s 0 , 9 6 gin. c o m p a r e d to 0 , 8 2 gm. in t h e n o r m a l legs, T h e g r o w t h f r o m t h e leg with v e n o u s ligation w a s larger in f o u r animals, while it w a s larger in the n o r m a l Ieg in t h r e e a n i m a l s . In five o f the a n i m a l s the n e o p l a s m s w e r e the s a m e size in b o t h legs. O n l y o n e o f the a n i m a l s had a c o n s i d e r a b l e d i f f e r e n c e in size b e t w e e n the t w o sides, the t u m o r b e i n g larger in t h e leg with v e n o u s ligation, T h e r e s u l t s s u ~ e s t e d that t h e o v e r all d i f f e r e n c e in g r o w t h rate w a s insignificant ( P - 0.3), Experiment IV ( L i g a t i o n of Femoral Artery,). T h e t u m o r s w e r e larger in t h e n o r m a l leg in 10 a n i m a l s , o f c o m p a r a b l e size in o n e animal, a n d slightly larger in the ische~mic leg o f o n e animal. T h e c o m b i n e d w e i g h t o f the t u m o r s f r o m t h e i s c h e m i c legs w a s 8,43 gin, an'd it w a s I 9 , 8 3 gin. f r o m the n o r m a l legs. T h e a v e r a g e w e i g h t o f t u m o r s f r o m t h e legs with arterial i s c h e m i a w a s 0 . 7 0 gin, c o m p a r e d to 1.65 gm, f r o m the n o r m a l legs, o r 4 2 p e r

Table 2. Effect o / I t y p e r c a r b i a upo?* ltenmtoge~ous M e t a s t a s e s

Wound "r c: Average Me.t,,stases 7.,1 7.0

Abdomen T C

Spleen and Perisplenic Tissues T C

|Aver T C

22

0.1

0.9

25

0.4

0,4

Each Adrenal T C 3.3

3.8

Figures represent average number of metastases per animal. T - T e s t

Intestines T C

Muscle (per cubic inch) T C

Each Kidney T C

4.3

1.3

34

2,4

2,1

Animal, C - C o n t r o l Animal.

38

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OXYGEN AND HEMATOGENOUS

cent as large. It is interesting to note that the animals used their legs normally during the period of observation, and there was no e v i d e n c e of gangrenous change within any of the involved legs. T h e results indicated that ischemia induced by arterial ligation of the main arterial supply of a recipient extremity d e c r e a s e d the rate of growth of the transplanted t u m o r (0+01 < P<0,02),

DISCUSSION T h e rote of tissue oxygen and carbon dioxide concentration in growth and development of malignant neoplasms in vlvo has been a subject of considerable discussion. Warburg TM expressed the opinion that malignant neoplasms may actually arise from d a m a g e d tissues as an adaptation to relatively anaerobic conditions, and showed that neoplasms have an increased glycolysis under anaerobic conditions w h e n c o m p a r e d to normal tissues, suggesting that cancers might grow more readily u n d e r relatively hypoxic conditions. Kieter and Biczralso found an increase in the rate of anaerobic glycolysis in solid tumors with an increase in carbon dioxide c o n c e n t r a t i o n in vitro, but this effect w a s abolished by the addition of fibroblasts or adenosine triphosphate to the medium. In other experiments an increase in carbon dioxide concentration inhibited both cellular respiration and maximal energy production. Barach and g i c k e r m a n ~found that transplanted sarcoma I80 had a decreased rate of growth when implanted into hypoxic C+57 mice. O u r studies indicate that there was a decreased rate of growth of tumors trm~splanted by injection into the limbs of animats having a relative ischemia produced by ligation of the major arterial supply of the extremity. An attempt was made to determine the actual oxygen concentration in the tissues of the ischernic and normal limbs in vivo with a needle o x i m e t e r (Clarke electrode) and Beckman gas analyzer, but the values were so erratic that interpretation of the results was impossible. One can assume, h o w e v e r , that a relative ischemia was produced in the areas into which the tumors were inoculated after the femoral arterial ligation. T h e decreased rate of growth of the tumors in the limbs following arterial ligation was quite significant, while no significant difference was d e m o n s t r a t e d in the rate of growth of tumor inoculated into limbs

METASTASES

93

after ligation of the femoral vein. T h e s e studies suggest that + hypoxia will decrease the rate of growth of transplanted malignant lU +

tours.

T h e effect of increased arterial blood flow upon the d e v e l o p m e n t of hepatic metastases was studied by Lee, Fishes" and F i s h e r y T h e y produced an aortic-vena caval fistula with a reversed Eck fistula in t~tS tO increase the arterial flow through the liver. A significant increase in hepatic metastases was produced by the intraportal injection of Walker 256 t u m o r cells in these animals c o m p a r e d to controls. An increase in both the serum glutamic oxaIoacetic transaminase and serum glutamic pyruvic transaminase levels, h o w e v e r , indicated that hepatic damage had occurred+ Since tissue damage in itself will result in an increase in the number of h e m a t o g e n o u s metastases,~'+the effect of any increase in oxygen tension cannot be evaluated separately in their preparation+ In another study'~they produced hepatic congestion by vena caval constriction above the liver and found an increased susceptibility to hepatic metastases. Again, there was associated liver damage which they concluded a c c o u n t e d for the increase in metastases. Willis ~ has suggested that lhe live+" might be more susceptible to lhe development of metastases because of a relatively high rate of glycolysis and a relativety p o o r oxygen supply. In Experiments 1 and I I we had+ hoped to explore w h e t h e r or not a relative increase in total oxygen or carbon dioxide tension would produce a change in the incidence of metastases, if hypoxia in itself could increase the numbbr or rate o f growth of metastases+ then hyperoxia might be e x p e c t e d to d e c r e a s e it. This was not found to be true. At concentrations which did not produce oxygen poisoning, an increase in oxygen tension in the respired air resulted in an increase in hematogenous metastases. Prolonged periods of exposure to greater than 0.6 a t m o s p h e r e of oxygen has resulted in the death of most experimental animMs,:+a fact which has caused considerable limitation on the degree of hyperoxia that could be maintained in our studies. It is pertinenl that a relative anoxia p r o d u c e d a decrease in the rate of tumor growlh in the studies o f Barach and Bickerman. a T h e role of hyperbaric oxygenation in the development of hematogenous metastases would be of considerable interest but remains to be determined. W h e n increased a m o u n t s of carbon dioxide were administered to the animals during the

94

ALEXANI)ER AND A L T E M E I E R

period of transplantation, no significant increase or decrease in hematogenous metastases occurred between test animals and control animals. This suggested that attachment of tumor cells to the endothelial lining in areas of injury may be unrelated to any change brought about by an increase in local carbon dioxide concentration. SUMMARY A study of the effect of changes in oxygen and carbon dioxide concenlration in respired air upon the development of hemalogcnous metaslases of the VX-2 carcinoma in rabbits was undertaken after it was found that simple ligation of the gastrosplenic vessels resulted in a striking increase in metastases of this tumor to the area of stomach supplied by these vessels. An increase in respired oxygen resulted in an overall increase in the nunlber of hematogenous metastases in the absence of oxygen poisoning. Transicnt hypercarbia during the period of intra-aortic inoculation failed to cause any significant change in the occurrence of metastases, Arterial ischemia produced by ligation of the femoral artery resulted in a significant decrease in the rate of growth of tumors transplanled to that extremity. In contrast, no significant change in the rate of growth of transplanted tumors was tbund in extremities in which venous congestion was produced by ligation of the femoral vein. No information is available concerning the possible influence of hyperbaric oxygenation upon the development of hemalogenous metastases, but such information would be valuable because of recent enthusiasm for the treatment of malignant tumors using this technique, These findings indicate that a prolonged increase in oxygen concentration promotes both the development of hematogenous metastases and their subsequent growth. Transient hypercarbia during the period of tumor

JSR ~ Vol, V, Na. 2 -

February 1965

inoculation had no appm'ent effect in these experiments, suggesting that the increased susceptibility of injured tissues to the development of hematogenous metastases is I_mrelated to local changes in carbon dioxide concentration, This experimental evidence suggests further that neither carbon dioxide nor oxygen therapy would be of great importance in the development of hematogenous metastases under ordinary clinical circumstances, ltEFERENCES I. Alexander. J. W., Bmunstein, H.. and Altemeier, W. A.: Transplantation s~udies of the venereal sarcoma of dogs. J. Surg, Res., 4:15 l, 1964, 2, Alexander. 3, W., and Altemeier, W. A,: Susceplibilily of h~jured r.issues to hematogenous metas|ases: An experimental study, Ann. Surg, 159:933, i964. 3. t3arach, A, L., ;rod Bickerman, 15, A.: The effe.ct of anoxla on lumor growlh with special reference to sarcoma 181) implanled C-57 mice. Cancer Res, 14:672. 1954. 4 Fisher, B.. and Fisher. E. R,: Experimental studies of Nclors influencing hepatic metastases III. Effect of surgical trauma with special reference to liver injury. Ann. Surge, 150:731, 1959. 5. Fisher. B., and Fisher, E, R,: Experimental studies of faclors influe~cing hepatic metas*,ases VI, Elt'eet of nutrition, Cancer. t4:547. 196 t. 6. Fisher. B., Fisher. F.. R., and Lee, S, H,: The effec~ of akemtion of liver blood flow upon experimental hepalic metastases. StnN.. Gynec. & Obstet., I I2:l I. 196I 7. Kieler, J.. and Bicz, W.: The fimclion of COe in celt metabolism and growlh. In Brennan, M, J,. and Simpson, W. 1.. Eds,: Interactions in Normal and Neoplastic Growth. A Contribution to the t-lostTumor Problem. l,itlle, Brown, and Co,, Boslon, 1962, p. 89. 8. l.ee. S. H.. Fisher. B.. and Fisher. E. R.: Experimental factors influencing hepatic metastases. Xll. Effec~ of increased arterial blood flow. Proc. Soc, Exper. Biol. & Med.. I t2:126, 1963, 9. Ohlsson, W. T. L : A study of oxygen toxicity at atmospheric pressure with special reference '/o the pathogenesis of pulmonary damage and clinical oxygen therapy. Acre reed. scandinav. (Suppl.} |90:1, 1947, 10. Warburg. W.: On the origin of cancer cells, Science, I23:309, ~956. It. Willis, R, A.: Pathology of Tumors. C, V. Mosby Co. St. l.ouis, 1948.