An Experimental Study of Necrosis in Tumors"

WILLIAME. SCHATTEN

(The Ferst Research Laboratories, Piedmont Hospital, Atlanta, Georgia)

SUMMARY Factors that regulate the amount of viable tumor an animal can support have been studied in rats bearing Walker carcinoma 256. There is a maximum amount of viable- tumor an animal can support, but continued growth of tumor resulting in increase in the amount of necrotic tumor occurs until death of a rat bearing Walker carcinoma 256. The number and location of tumors in an animal do not significantly influence the per centage of the total amount of tumor tissue that is necrotic except in instances in which tumors are very small. In rats with Walker carcinoma 256 there is a positive relation ship between the percentage of the total amount of tumor tissue that is necrotic and the percentage of host weight occupied by tumor. Extensive necrosis occurred in Walker 256 tumors in rats placed under a barometric pressure of 8 inches of mercury for 5 hours. There is rapid growth of tumor that remains viable if the tumor-host equilibrium is disturbed by decreasing the amount of viable- tumor by placing a tumor-bearing rat under reduced barometric pressure.

Central necrosis is a common consequence of a rounding tissue, weighed, and the entire tumor neoplasm's growth. In a previous study, the author was sliced to permit separation of necrotic tissue and co-workers (5) demonstrated that availability from viable tissue by sharp dissection. The necrotic of oxygen to tumor cells was important in the and viable portions were weighed separately. production of central necrosis. The influence of Xecrotic and viable portions of Wralker 256 tu systemic factors on the development of necrosis in mors are sharply demarcated and can be identified tumors was implied. The present report is a study easily, since viable tissue is firm and gray and of factors that regulate the amount of viable tu necrotic tissue is soft and reddish-brown (Fig. 1). mor an animal can support. One group of tumor-bearing rats was subjected to a low oxygen environment at normal atmos MATERIALS AND METHODS pheric pressure. These rats were placed in desicca Female, albino Sprague-Dawley rats, weighing tor jars into which a 95:5 helium :oxygen mixture approximately 250 gm., were housed in individual flowed continuously. After 5 hours of exposure to suspended basket-type cages and fed Purina this 5 per cent oxygen mixture, rats were returned Laboratory Chow and water ad libitum. Each rat to their cages. These animals were sacrificed 24 was weighed twice weekly. Only those animals hours after treatment. which had gained weight at comparable rates for Another group of tumor-bearing rats was sub 2 weeks were used for the experiments. Trans jected to a low oxygen environment by being plants of pieces of Walker carcinoma 256 were placed under a reduced barometric pressure of 8 introduced into the flexor muscles of the thighs or inches of mercury. At this negative pressure, into the subcutaneous tissues of the flank by tro oxygen tension in the inspired air is approximately car technic. equal to that in a 95:5 helium :oxygen mixture. Tumor-bearing animals were sacrificed at dif Approximately 20 minutes are necessary to reduce ferent intervals of time following transplantation. the barometric pressure slowly to 8 inches of Each tumor to be studied was dissected from sur- mercury; if this is done, rats tolerate this reduced * This work was supported by Grant No. C-4746 from the pressure without difficulty. After 5 hours in a Division of Research Grants, National Cancer Institute, U.S. vacuum jar, rats were returned to their cages. Public Health Service. These animals were sacrificed 24 hours after treat Received for publication August 15, 1961. ment. Another group of rats were treated in a 286

vacuum jar in this manner but were not sacrificed mors than in those bearing five tumors (P < 0.001). until 96 hours after treatment. In rats with larger tumors there was no significant The data on proportion of necrotic tumor and differences between the percentages of total tumor per cent of host weight occupied by tumor were tissue that was necrotic in animals bearing two analyzed according to formal analysis of variance tumors and those bearing five tumors (P < 0.1). and covariance procedures. Chart 3 shows the relationship between the per centage necrosis in individual tumors in rats bear RESULTS ing five tumors and the per cent of host weight An animal can support only a certain amount of occupied by the total amount of tumor. The wide viable tumor, even though the total amount of range in the percentage of necrosis in each of five tumor continues to increase until death of the animal (Chart 1). After a tumor comprised ap proximately 12 per cent of the weight of its host, ÃŽJ2 Tumors further growth of tumor resulted in increase in the 5 Tumors amount of necrotic tumor. There was no greater O NO. Rats Treated amount of viable tumor in animals with 75 gm. of ÃŽStand Dev. tumor than in animals with 30 gm. of tumor. With the increase in each per cent of host weight occupied by tumor, the mean increase in the amount of necrotic tumor was 2.26 + 0.22 gm.

â€¢VIABLE TUMOR Â£80 A NECROTIC TUMOR 2 Ã• 70 ca * 60 ce o Ã„& â€že. i 50 Ã›A â€ž o H 40 er o ^ 30 0 5 10 15 20 20 PERCENT HOST WEIGHT U OCCUPIED BY TUMOR CD ÃŽ10 CHART2.â€”Comparison between animals bearing two tu mors (bilateral intramuscular) and those bearing five tumors (two bilateral intramuscular and three subcutaneous) ; relation 5 10 15 20 25 30 35 ship between the proportion of necrotic tumor tissue and per cent of host weight occupied by tumor. PERCENT HOST WEIGHT OCCUPIED BY TUMOR CHART 1.â€”Intramuscular tumors; relationship between tumors in the same rat is seen in this scatter graph. amounts of necrotic and viable tumor and per cent of host However, in these rats with five tumors there is a weight occupied by tumorâ€”tumor weight/total weight positive relationship between the percentage of the (carcass plus tumor). Each point represents the total amount of necrotic or viable tumor in an animal. total amount of tumor tissue that is necrotic and per cent of host weight occupied by tumor (Chart Chart 2 shows that the number and location of 4). With the increase in each per cent of host tumors in an animal do not significantly influence weight occupied by tumor, the mean increase in the percentage of total tumor tissue that is necrotic the percentage of necrotic tumor was 3.60 Â±0.57. except in instances in which tumors are very Chart 5 shows the results of exposing rats with small. In rats with tumors occupying 23-7^ per cent bilateral intramuscular tumors to a 5 per cent of host weight the percentage of necrotic tumor oxygen mixture for 5 hours. This treatment did was significantly greater in those bearing two tu not effect a significant increase in the percentage

of necrotic tumor except in the group of rats with tumors occupying 7|-12Â§per cent of host weight 100- â€¢INTRAMUSCULAR TUMORS (P < 0.001 for this group). A SUBCUTANEOUS TUMORS Treatment of rats bearing bilateral intramuscu 90- lar tumors by placing them in a vacuum jar with a cn negative pressure of 22 inches of mercury for 5 Â§ 80 hours significantly increased the percentage of necrotic tumor in all groups (P < 0.001) for all Â¿ 'a* Ã• 70 groups (Chart 6). Treatment of rats bearing five Ã› A Â¿ small tumors in a vacuum jar also significantly Ã•â€¢' .A ' iÃ¬ ' ' increased the percentage of necrotic tumor (P < f 6O 0.001, Chart 7), but it can be seen by comparison 5 ;â€¢:':?â€¢;â€¢:Â«1-â€¢ Z - 50

D 95/5 He/02 for 5 Mrs. g 40 0 No Rx Ã¼ â€¢!i'"'-**' O NO. Rats Treated â€¢f-Ã» â€¢ K 30 Stand. Dev. Z ÃœJ 8 â€¢Â«.** oc 20 :t if.

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5 10 15 20 25 30 35 PERCENT HOST WEIGHT OCCUPIED BY TUMOR CHART3.â€”Rats bearing five tumors (two intramuscular and three subcutaneous); relationship between percentage necrosis in individual tumors and the per cent of host weight occupied by the total amount of tumor.

Â¡80 â€¢S70 0 5 10 15 20 25 30 35 40 PERCENT HOST WEIGHT OCCUPIED BY TUMOR o 60 CHART5.â€”Bilateralintramusclar tumorsâ€”comparison be ~o tween animals exposed to a 5 per cent oxygen mixture for 5 o 50 hours and controls; relationship between the proportion of necrotic tumor tissue and per cent of host weight occupied by Â¡40 tumor.

o 30 of Charts 6 and 7 that small tumors were not af fected by the reduced barometric pressure nearly as much as were larger tumors. <Â¿IO There was rapid growth of tumor that remained viable following treatment of rats with bilateral 5 IO 15 20 25 30 35 intramuscular tumors by placing them under re PERCENT HOST WT. OCCUPIED BY TUMOR duced barometric pressure (Chart 6). The per centage of necrotic tumor decreased significantly CHABT4.â€”Rata bearing five tumors (two intramuscular between 24 and 96 hours after treatment of tu and three subcutaneous); relationship between percentage mors comprising 2Jâ€”7|per cent of the host weight necrosis of total amount of tumor in each rat and per cent of host weight occupied by tumor. (P < 0.01) and tumors comprising 7j-12f per cent

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1962 American Association for Cancer Research. SCHATTENâ€”NecrosisinTumors 289 of the host weight (P < 0.001). In the former group tumor that was necrotic only when there were very the percentage of necrotic tumor 96 hours after small tumors. Studies of the blood supply of ex treatment was still significantly greater than in perimental neoplasms by Algire and Legallais (1) control rats (P < 0.005). However, in the latter and of human neoplasms by Bierman and co- group the percentage of necrotic tumor 96 hours workers (2) show solid malignant tumors to have after treatment was no greater than that in un an abnormal vascular system. The major arterial treated rats. supply to transplanted tumors has been shown to be peripheral and to be distributed circumfer- DISCUSSION entially to capillaries that penetrate the globular These data demonstrate that there is a maxi mum amount of viable tumor an animal can sup 50 r~] 22" Hg. VACUUM port; but continued growth of tumor resulting in K O *â€”'FOR 5 HOURS increase in the amount of necrotic tumor occurs 40- until death of a rat bearing Walker carcinoma 256. Â£3 NO Rx There are two determinants of the amount of O MO RATS TREATED O 30 et T STANO DEV. 22" Hg. Vacuum FJSacrificed 24 Hrs. o VacuumiNoRx.u for 5 Hours Cafter Rx. Â¡n 20- Hrs.ONO. ^Sacrificed96 10090ftO70-60SO403020IOr-]Vacuum1Rats Treated Cafter Rx in LÃœ O e Stand.Dev.iÂ®^(2 10-

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0 5 IO 15 20 25 PERCENT HOST WEIGHT OCCUPIED o BY TUMOR ce o ::-:+:S CHART7.â€”Rats bearing five tumors (two intramuscular LU 0e<&.Â®/IB):â€¢:â€¢ Ã•9:i(23)li:|lH-, and three subcutaneous)â€”comparison between animals placed in a vacuum jar with a negative pressure of 22 inches of mer cury for 5 hours and controls; relationship between the pro O portion of necrotic tumor tissue and percentage of host weight ce iÃÃteS^1I Lu occupied by tumor. O. mass. Nutrients are therefore available to central ly located cells in very small tumors but not in larger tumors. This explains the findings in this 0 5 10 15 20 0 5 10 15 study that small tumors in rats bearing five tu PERCENT HOST WEIGHT OCCUPIED BY TUMOR mors had less necrosis than larger tumors in rats CHART 6.â€”Bilateral intramuscular tumorsâ€”comparison bearing two tumors when the total amount of between animals placed in a vacuum jar with a negative pres tumor in each group was approximately equal. sure of 22 inches of mercury for 5 hours and controls; relation Also, these small tumors were not affected as ship between the proportion of necrotic tumor tissue and per much by treatment in a vacuum jar as were larger cent of host weight occupied by tumor. The last two bars show tumors. The vascular pattern developed by an a comparison between sacrificing animals 24 hours and 96 hours after treatment. individual tumor plays a role in the determination of the amount of necrosis in that particular tu necrotic tumor in an animal: (a) systemic factors mor, as evidenced by the wide range in the per that govern the amount of viable tumor that can centage of necrosis in individual tumors in rats be supported and (6) the growth potential of the bearing five tumors. However, in these rats with tumor. Walker carcinoma 256 has a dominant multiple tumors the total amount of necrotic tu growth potential. Regressions are extremely rare, mor is determined by systemic factors regulating and the growth rate is reliable, reproducible, and the amount of viable tumor the rat can support rapid (7). and by rate of growth of the tumor. Local factors played a significant role in deter Goodman (3) has shown that mice with multiple mining the percentage of the total amount of tumors live the same length of time as mice with

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1962 American Association for Cancer Research. 290 Cancer Research Vol. 22, April 1962 single tumors, and at death the total tumor mass is tissue pOj when blood pU2 is decreased. The the same in both groups. Also, excision of one of mechanism of action of negative pressures in en two tumors in a mouse increases the growth rate hancing necrosis in tumors is unexplained. The of the remaining tumor. The present report physiology of flight has been studied extensively, defines this concept further by demonstrating that and work is now being carried out in this labora an animal can support only a certain amount of tory to elucidate the effects of low pressures on viable tumor regardless of the number and location tumors. of implants. Also, there is rapid growth of tumor ACKNOWLEDGMENTS that remains viable if the tumor-host equlibrium The author wishes to express his thanks to Miss Carol is disturbed by decreasing the amount of viable Barnett and Miss Diane Falk for their technical assistance. tumor by placing a tumor-bearing rat under re duced barometric pressure. These data show that, REFERENCES once a host has been altered or conditioned so that 1. ALGIRE,G. H., and LEGALLAIS,F.Y. Vascular Reaction of Normal and Malignant Tissues in Vito. IV. The Effects of it will support a certain amount of viable tumor, Peripheral Hypotension on Transplanted Tumors. J. Nat. removal of a portion of this tumor results in its Cancer Inst., 12:399-421, 1951. rapid growth until it equals the pre-existing viable 2. BIERMAN,H. R.; BYRON,R. L., JR.; KELLY,D. H.; and tumor mass. The author (4) has demonstrated (Ã•RADY,A.Studies on the Blood Supply of Tumors in Man. III. Vascular Patterns of the Liver by Hepatic Arteriogra- previously that there is enhancement of growth of phy in Vim. J. Nat. Cancer Inst., 12:107-32, 1951. mÃ©tastasesfollowing removal of a primary tumor. 3. GOODMAN,G.J. Effects of One Tumor upon the Growth of Sundstroem and Michaels (6) demonstrated Another. Proc. Am. Assoc. Cancer Research, 2:207, 1957. that necrosis of tumors was greater in animals sub 4. SCHATTEN,W.E. An Experimental Study of Postoperative jected to negative pressures than in animals sub Tumor MÃ©tastases.I. Growth of Pulmonary MÃ©tastases Following Total Removal of Primary Leg Tumor. Cancer, jected to low oxygen environments at atmospheric 11:455-59, 1958. pressure. The findings presented in this paper 5. SCHATTEN,W.E.; MANTEL,X.; and MIDER,G. B. A Re demonstrate that exposure to an environment of lationship between Amount of Necrosis in Walker Carci noma 256 and Concentration of Hemoglobin in the Host's 5 per cent oxygen at atmospheric pressure which Blood. Cancer Research, 18:274-278, 1958. results in decreased oxygen tension and alters dif 6. SUNDSTROEM,E.S., and MICHAELS,G.The Adrenal Cortex fusion of oxygen in the blood does not cause nearly in Adaptation to Altitude,. Climate, and Cancer. Mem. as much necrosis in tumors as does exposure to Univ. Calif., 12:201-346, 1942. negative pressures. Urbach and Xoell (8) demon 7. TÃ•LALAY,P.;TARANO,G. M. V.; and HUGGINS,C.Studies on the Walker Tumor. I. Standardization of the Growth of a strated there is a poor response of tumor tissue Transplantable Tumor. Cancer Research, 12:834-37, 1952. pOs to the administration of 100 per cent oxygen, 8. UKBACH,F., and XOELL, W. D. Studies on the Oxygen and results of this study indicate that there is Supply of Tumor Tissue. Proc. Am. Assoc. Cancer Re probably little decrease in the normally low tumor search, 2:257, 1957.

FIG. 1.â€”This photograph demonstrates the demarcation between necrotic and viable portions of Walker 256 tumor. The outer rim of viable tissue is easily distinguished from the necrotic core.

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1962 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1962 American Association for Cancer Research. An Experimental Study of Necrosis in Tumors

William E. Schatten

Cancer Res 1962;22:286-290.

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