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Tumor Blood Flow: I. Blood Flow in Transplantable Tumors During Growth
Tumor Blood Flow: I. Blood Flow in Transplantable Tumors During Growth WAID ROGERS, M.D., Ph.D. * RICHARD F. EDLICH, M.D. t DARREL V. LEWIS, B.A.t J. BRADLEY AUST, M.D., Ph.D., F.A.C.S.§
Over 100 years ago, Virchow35 and Thiersch32 demonstrated by injection techniques a unique capillary network supplying the stroma of tumor tissue. Subsequent studies of the anatomy of blood vessels in spontaneous and transplantable malignant tumors revealed an inadequate blood supply with a defective and dilated vasculature. 3 , 8, 28 Abnormalities in tumor vasculature were further clarified by Goldman 17 in 1907 using intra-arterial injections of bismuth in oil in human tumors and' India ink injections in tumor-bearing small animals. Abundant proliferation of irregular branching small vessels was noted throughout small tumors, while large tumors showed central necrosis with a rich peripheral vasculature. U sing more refined techniques, subsequent investigators have corroborated these vascular changes in transplantable tumors during growth. 9 , 16, 30, 37 The first attempt to quantitate tumor blood flow was reported by From the Department of Surgery, University of Minnesota Medical School, Minneapolis, Minnesota This work was supported in part by a research grant (T-156) from the American Cancer Society. ·Instructor in Surgery, University of Minnesota Medical Center tResident in Surgery, University of Minnesota Medical Center
! University of Minnesota Medical School §Professor of Surgery and Chairman of Department, South Texas Medical School, San Antonio, Texas
Surgical Clinics of North America- Vol. 47, No.6, December, 1967
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WAID ROGERS, ET AL.
Gullino and Grantham. 19. 20 Blood flow was measured by venous outflow of organs totally replaced by tumor and rubidium-86 or potassium-42 clearance techniques in subcutaneous implants. Blood flow per gram of tissue in tumors of all sizes was 15- to 20-fold lower than in normal tissues. Cataland et al,u also confirmed the low perfusion rate of large tumors, but demonstrated that small tumors had a significantly higher blood flow. Similarly, we have reported different levels of tumor flow in transplantable tumor systems. 12• 29 These conflicting results may be explained by changes in flow during tumor growth. For this reason, the purpose of this study was to quantitate the blood flow changes in several transplantable hamster tumors during growth. The first part of the study was devoted to the correlation of tissue blood flow measurements, using antipyrine- l3lI, with microscopic sections of the whole tumor with advancing age. Secondly, the distribution of blood flow within tumor was then exaInined by comparing the uptake of an intravenous vital dye, lissamine green, and antipyrine- l3l 1. Finally, radioactive microspheres were employed as another tissue blood flow reference material to provide an independent estimate of the distribution of flow in tumor.
METHODS
Tissue Blood Flow Techniques ANTIPYRINE- l3l1. * The estimation of tissue blood flow with antipyrine_ 1311 depends on the physical characteristics of the parent compound. Johnson et al. 22 • 23 have shown that antipyrine parallels D 2 0 by its rapid diffusion into tissue. This characteristic permits the application of the Fick principle in calculating tissue blood flow with this substance. Previous work in this laboratory has demonstrated that antipyrine tagged with 1311 is a simple and reliable reference material for the indirect measurement of blood flow at the tissue level. 27 We have modified the flow formula to calculate blood flow in the tissues of small animals: 13 A F=-----
(Caf/2 - A/2)t
where F equals flow in ml./Inin./gm., A equals tissue concentration in cpm/gm. of antipyrine- 131 I, t equals time in minutes, Caf/2 is an approximation of the mean arterial concentration obtained by dividing the final arterial concentration, C af, in cpm/ml. of antipyrine- 13 lJ by 2, and A/2 is an estimate of the mean venous concentration obtained by dividing the tissue concentration, A, in cpm/gm. of antipyrine- 1311 by 2. LISSAMINE GREEN DYE. t This vital stain is a triphenylmethane dye which has an easily detectable green color in blood and tissues, and rapidly distributes in interstitial spaces where blood is circulating. ls 'Antipyrine- I31 I: 4-iodo-antipyrine purchasetl from Abbott Laboratories tLissamine Green Dye: Lissamine green, triphenylmethane dye, V200, purchased from I.e.!. Organics, Inc., Providence, R. I.
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51Cr-Microspheres.'" These tiny ceramic spheres, 12 to 15 JL in diameter, are composed of zirconium phosphate labeled with chromium-51. Quantitation of tissue blood flow using microspheres assumes their even distribution in arterial blood and utilizes their complete extraction in vessels of smaller than 16 JL in all tissues being perfused.
Transplantation, Infusion, and Sampling Techniques Six to eight week old male and female Syrian (golden) hamsters were used in all experiments. The hamster amelanotic melanoma, renal adenocarcinoma, and fibrosarcoma 14 t were prepared by the mince suspension technique and injected into the thigh of separate groups of animals. The first experimental group comprised 61 hamsters with the amelanotic melanoma implanted 10 to 50 days prior to tissue blood flow determination. Under pentobarbital anesthesia, a polyethylene catheter was inserted into the jugular vein of each hamster. For precisely 2 minutes, approximately 3 microcuries of antipyrine j311 diluted in normal saline was continuously infused at 0.68 ml./min. using a Harvard microinfusion pump. At the end of this period, the pump was stopped, the hind limb containing the tumor was immediately amputated, and a final arterial blood sample obtained from the severed femoral artery. The entire tumor, sample of arterial blood, adjacent skin, and gluteus muscle were weighed and the cpm/gm. measured in a well-type counter.! Tissue blood flows of normal and tumor tissues were then calculated using the flow formula. The second group consisted of animals with the amelanotic melanoma, fibrosarcoma, or renal adenocarcinoma of varying sizes. Blood flow determinations were performed 2 minutes after the administration of 1 ml. of a 2 per cent solution of lissamine green dye. After completion of the previously described flow experiment, the tumor was removed and separated into dark green and light green portions and tissue blood flow determined in each sample. The final group contained hamsters with the amelanotic melanoma, renal adenocarcinoma, or fibrosarcoma in different phases of growth. A No. 10 polyethylene catheter was inserted retrograde into the left carotid artery. Approximately one microcurie of 51Cr-microspheres suspended in a I-m!. solution containing 6 per cent dextran in saline (Abbott) and 2 per cent lissamine green was injected over 15 seconds. Two minutes later, the limb was amputated and the uptake of 51Cr in skin, muscle, and tumor determined. Uptake ratios were calculated for each animal as tumor: skin, tumor: muscle, skin: muscle, and dark-stained or lightstained tumors. Following blood flow determinations, routine histological sections were stained with hematoxylin and eosin. ·S1Cr-Microspheres: Purchased from the Minnesota Mining and Manufacturing Co., St. Paul, Minnesota tHamster tumors were obtained from Dr. Joseph Fortner, Sloan-Kettering Institute, New York, N.Y. !Decade Scaler: Curtis Nuclear Corporation,
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Table 1. Total Tumor Blood Flow in Hamster Amelanotic Melanoma During Growth (Antipyrine- 131J Technique) MEAN SKIN FLOW
NO.
10
10 12 14 15
MEAN TUMOR WEIGHT
0.4 1.0 3.5 7.7 20.4
MEAN TUMOR FLOW
0.95 1.02 0.52 0.24 0.23
(±.42)* (±.56) (±'26) (±'10) (±'14)
Leg with tumor 0.15 0.24 0.18 0.23 0.50
(±.06)* (±.19) (±.09) (±.07) (±.25)
Opposite leg
0.21 (±.14)* 0.18 (±'05) 0.20 (±'09)
MEAN MUSCLE FLOW
Leg with tumor 0.07 0.08 0.11 0.17 0.26
(±.04)* (±.04) (±.04) (±.04) (±.14)
Opposite leg
0.12 (±.05)* 0.06 (±'02) 0.09 (±'04)
*Standard Deviation.
RESULTS Group I. Total Tumor Blood Flow During Growth (Antipyrine_ l3l I Technique) To evaluate the changes in mean tissue blood flow with advancing tumor size, the 61 hamsters were divided into five groups according to tumor weight (Table 1). The mean tumor flows of 0.4 and 1.0 gm. tumors were approximately five and ten times higher than skin and muscle blood flow respectively. When the mean tumor weight reached 3.5 gm., the mean tumor blood flow decreased to approximately one-half that of the smaller tumors. As the weight of the tumor increased to a mean of 7.7 to 20.4 gm., a further decrease in mean perfusion to approximately one-fourth of that measured in small tumors was noted. This inverse relationship between tumor blood flow and tumor size is similar to that
Figure 1. Photomicrograph of a 0.44-gm. hamster amelanotic melanoma showing a uniform sheet of tumor tissue. Total tumor blood flow: 0.83 ml./min./gm. (H. & E., x40.)
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Figure 2. Photomicrograph of a 21-gm. hamster amelanotic melanoma showing central necrosis and a peripheral rim of intact tumor tissue. Total tumor flow: 0.19 ml./min./gm. (H. & E., x40.)
Figure 3. Photomicrograph of hamster gluteus muscle adjacent to the 21-gm. tumor in Figure 2. Muscle blood flow: 0.33 ml./min./gm. Note the darkly stained melanoma and inflammatory cells invading the muscle. (H. & E., x 100.)
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WAID ROGERS, ET AL.
reported by Cataland et al,u in mouse tumors. A review of the microscopic sections suggested an explanation for the progressive reduction of tumor blood flow with advancing age. Small tumors were comprised of uniform sheets of tumor cells (Fig. 1), while the .larger tumors contained central necrotic areas with a peripheral rim of intact tumor (Fig. 2). It was interesting to note that the tissue blood flow of muscle and skin adjacent to the large tumors was elevated. The cause of this elevated flow has not been established, but it may be a reflection of the inflammatory and tumor cell invasion readily observed in microscopic sections (Fig. 3).
Group II. Distribution of Blood Flow Within Tumors During Growth (Antipyrine- 131 I - Lissamine Green Technique) Tissue blood flow was estimated separately in the light and dark dye-stained regions of these tumors. Small tumors were more uniformly stained with lissamine green. With an increase in tumor size, light- and dark-stained areas were easily identified within the tumor. As described by Goldacre and Sylven 15 and Owen,25 the tumor periphery was intensely stained and the central region of the tumor remained pale green or white. The uptake of lissamine green was correlated with blood flow within the tumor (Table 2). Mean tissue blood flow of the dark-stained
Table 2. Distribution of Blood Flow Within Hamster Tumors During Growth (Antipyrine j31 I-Lissamine Green Technique) MEAN TISSUE BLOOD FLOW
NO.
MEAN TUMOR WT. (gm.)
(ml./min./gm.)
Stain Intensity Dark
Light
Total Tumor
Amelanotic Melanoma 10
1.6
8
4.3
8
11.1
14
3.3
15
11.0
13
30.8
5
0.5
6
2.9
6
6.2
0.86 (±A5)* Q37 (±.14) 0.39 (±.26)
0.48 (±'21) Q14 (±.09) 0.12 (±.07)
0.71 (±.34) 0.29 (±.14) 0.20 (±.18)
Fibrosarcoma Q38 (±.17)* 0.29 (±.14) 0.25 (±.13)
QI0 (±.02) 0.07 (±.02) 0.02 (±.01)
0.22 (±.11) 0.15 (±.07) 0.05 (±'Ol)
Renal Adenocarcinoma
'Standard Deviation.
0.78 (±.41)* 0.82 (±.60) 0.42 (±.26)
0.48 (±.29) 0.16 (±.04)
0.78 (±.41) 0.55 (±.30) 0.20 (±.13)
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tumor was 2 to 12 times greater than that of the lighter-stained tumor in the three tumors studied. Group III. Distribution of Blood Flow Within Tumor During Growth (Antipyrine- l3l1- Lissamine Green and 5lCr-MicrospheresLissamine Green Techniques) The distribution of 51Cr-microspheres was compared to the uptake of antipyrine- 131J in skin, muscle, and tumor in separate groups of animals containing the same type of tumor of comparable weight. The uptake of these agents in each individual animal was expressed as a ratio of skin: muscle, tumor: skin, tumor:muscle, and dark-stained: light-stained tumor (Table 3). The uptake of micro spheres and antipyrine in small tumors was uniformly higher than the uptake of these agents in skin and muscle. This is strong evidence, but not unequivocal proof that the blood flow in these small tumors was higher than the flow to skin or muscle. In larger tumors, the distribution of antipyrine and microspheres corresponded to the intensity of the dye. Distribution of these two independent indicators of flow in the peripheral darkstained tumor was considerably higher than the uptakes of these agents in the central light-stained or unstained regions of the neoplasm. Similarly, Shibata and MacLean31 have demonstrated by tissue block analysis that the uptake of micro spheres in the center of large human and rat tumors is lower than in the periphery. Table 3.
Distribution of Blood Flow Within Hamster Tumors During Growth (Antipyrine- 131J-Lissamine Green Technique) MEAN UPTAKE RATIOS
NO.
TAG
MEAN TUMOR WT. (gm.)
Skin Muscle
Tumor Skin
Tumor Muscle
Dark Tumor Light Tumor
Melanoma 17 10
5lCr 131
1
1.3 1.6
2.0 (± 1.2)· 2.2 (± 1.3)
7.8 (± 4.7) 5.0 (± 2.6)
1.5 (± 1.0)· 1.8 (± 1.0) 2.5 (± 1.0) 1.8 (± 1.0)
4.2 (± 2.4) 2.9 (± 1.8) 0.3 (± .01) 0.2 (± .01)
1.4 (± 0.7)· 1.8 (± 1.0)
3.4 (± 2.1) 2.8 (± 1.7)
19.6 (± 12.3)
4.0 (± 3.3)
11.3 (± 6.0)
2.3 (± 1.6)
Fibrosarcoma 4 4 6 10
5lCr 131
1
5ICr 131
1
0.9 1.0 37.4 35.2
7.2 (± 5.7)
8.8 (± 6.0)
4.7 (± 2.9)
0.7 (± 0.6)
5.6 (± 3.0)
133.2 (± 94)
0.3 (± .01)
18.1 (± 10.8)
Renal Adenocarcinoma 6 6
5lCr 131
1
·Standard Deviation.
2.1 2.9
6.6 (± 4.2)
4.9 (± 3.1)
3.5 (± 2.0)
1.7 (± 1.0)
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WAID ROGERS, ET AL.
It is interesting that the mean tumor:normal tissue uptake ratios and dark-stained:light-stained tumor uptake ratios for micro spheres were consistently higher than comparable uptake ratios for antipyrine, while the skin:muscle ratios remained the same for both indicators. This proportionally higher uptake of micro spheres in tumor may be a manifestation of a unique tumor vasculature.
DISCUSSION The vigorous capillary bleeding observed when tumor is incised during operation has led to the popular concept that there is high blood flow in tumors. Evidence in favor of this elevated flow is implied by the more rapid appearance of fluorescein and radiopaque dyes in tumors than adjacent tissue, and increased skin temperature overlying superficial tumors, and a high oxygen tension in venous blood draining neoplasms. 4 , 5, 6, 7 Urbach33 explains these observations on the basis of significant arteriovenous shunting in the periphery of the tumor. Morphological studies by Algire and Chalkleyl, 2 have shown a vascularity in early developing tumors which was double that of surrounding normal tissues. Similarly, our quantitative estimates of tissue blood flow in small hamster tumors have been substantially higher than the flow in adjacent normal tissues. Even during the initial stages of growth, tumor vessels have been noted to be dilated and lacking in normal epithelial layers. 1, 2 But, with subsequent growth, large sinusoids and blood-filled diverticuli develop which end blindly toward tumor centers suggesting obliteration. 21 These morphological observations can be correlated with the progressive decrease in blood flow with increase in tumor size noted in this study. Reduction in flow of the whole tumor is a function of a reduced perfusion in its center, as illustrated by a parallel distribution of dye, microspheres, and antipyrine. The central regions of hamster tumors receiving low blood flow develop progressive necrosis during growth. Goldacre and Sylven16 have demonstrated that these ischemic areas in tumor still contain viable cells. The classical work of Warburg36 demonstrated that tumor cells can survive in the absence of oxygen by converting to anaerobic glycolysis. Furthermore, low oxygen tensions have been noted in a variety of primary and metastatic human and animal tumors. 10, 24, 34 The ability of tumor cells to survive in an anaerobic environment provides an explanation for their resistance to anticancer therapy. Cellular damage produced by ionizing radiation depends upon the concentration of oxygen in the tissues at the time of exposure. 18 The few remaining viable cells in the center of the tumor may account for failure of radiotherapy to produce a cure. 26 Furthermore, the access of blood-borne chemicals and antitumor sera to all cells of a solid tumor would be affected by the progressive reduction of tumor flow during growth. Until a method of enhancement of central perfusion can be developed, the cure of large malignant tumors, short of surgical excision, is unlikely.
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SUMMARY 1. Total tissue blood flow in small hamster tumors estimated by means of the antipyrine- 131 1 technique was five- and ten-fold higher than adjacent skin and muscle. Blood flow of the whole tumor markedly decreased during growth. This decrease in perfusion in tumor was associated with the progressive development of central necrosis. 2. The distribution of lissamine green dye in three types of hamster tumors correlated with the uptake of antipyrine- l31 I. Dark-stained tissue blood flow, while the light-stained central necrotic areas showed peripheral regions of histologically intact tumor, demonstrated a high perfusion. 3. The distribution of 5lCr-microspheres and antipyrine- 1311 in tumor was found to be similar. This suggests that our quantitative estimates of tumor flow are reasonable. ACKNOWLEDGMENT
The authors wish to express their sincere gratitude to Mr. Bruce Benson and Mr. Wilfred Carlson for their valuable technical assistance.
REFERENCES 1. Algire, G. H., and Chalkley, H. W.: Vascular reactions of normal and malignant tissue in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic transplants. J. Nat. Cancer Inst., 6:73-85, 1945. 2. Algire, G. H., and Chalkley, H. W.: The vascular supply of mammary gland carcinomas. In Symposium on Mammary Tumors in Mice. Spec. Publ., Am. Assoc. Adv. Sc., 22:47, 1945. 3. Apolant, H.: Arb. Inst. Exp. Ther. (Frankfurt), 1:7,1906. 4. Bierman, H. R., Kelly, K. H., Dod, K. S., and Byron, R. L., Jr.: Studies on the blood supply of tumors in man. I. Fluorescence of cutaneous lesions. J. Nat. Cancer Inst. 11 :877890,1951. 5. Bierman, H. R., Byron, R. L., Jr., Kelly, K. H., and Grady, A.: Studies on the blood supply of tumors in man. III. Vascular patterns of the liver by hepatic arteriography in vivo. J. Nat. Cancer Inst., 12:107-131, 1951. 6. Bierman, H. R., Kelly. K. H., and Singer, G.: Studies on the blood supply of tumors in man. IV. The increased oxygen content of venous blood draining neoplasms. J. Nat. Cancer Inst., 12:701-707, 1952. 7. Bierman, H. R., Gilfillan, R. S., Kelly, K. H., Kuzma, O. T., and Noble, M.: Studies on the blood supply of tumors in man. V. Skin temperature of superficial neoplastic lesions. J. Nat. Cancer Inst., 13: 1-16,1952. 8. Borst, M.: Die Lehre von den Geschwulsten mit einem mikroskopischen Atlas. Wiesbaden, J. F. Bergman, 1902. 9. Braitwaite, J. L.: Arterial supply of benzypyrine-induced tumors in the rat. Brit. J. Cancer, 12:75, 1958. 10. Carter, D. B., Grigson, C. M. B., and Watkinson, D. A.: Changes of oxygen tension in tumors induced by vasoconstrictor and vasodilator drugs. Acta Radiol. 58:4Cl-434, 1962. 11. Cataland, S., Cohen, C., and Sapirstein, L. A.: Relationship between size and perfusion rate of transplanted tumors. J. Nat. Cancer Inst., 29:389-394,1962. 12. Edlich, R. F., DeShazo, C. V., Jr., and Rogers, W.: Drug-induced selective blood flow in the hamster melanoma (Abstract). Fed. Proc., 24:494, 1965. 13. Edlich, R. F., Rogers, W., DeShazo, C. V., Jr., and Aust, J. B.: Effect of vasoactive drugs on tissue blood flow in the hamster melanoma. Cancer Res., 26: 1420-1424, 1966. 14. Fortner, J. R., Maby, A. G., and Schrodt, G. R.: Transplantable tumors ofthe Syrian golden hamster. Cancer Res. 21 :161-234, 1961. 15. Goldacre, R. J., and Sylven, B.: A rapid method of studying tumor blood supply using systemic dyes. Nature (London) 184:63-64, 1959.
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16. Goldacre, R J., and Sylven, B.: On the access of blood-borne dyes to various tumor regions. Brit. J. Cancer 16:306-322, 1962. 17. Goldman, E.: Growth of malignant disease in man and the lower animals with special reference to vascular system. Proc. Roy. Soc. Med., 1: 1, 1907. 18. Gray, L. H., Conger, A. D., Ebert, M., Homsey, S., and Scott, O. C. A.: Concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Brit. J. Radiol., 26:638-648, 1953. 19. Gullino, P. M., and Grantham, F. H.: Studies on the exchange of fluids between host and tumor. I. A method for growing "tissue-isolated" tumors in laboratory animals. J. Nat. Cancer Inst., 27:679-709,1961. 20. Gullino, P. M., and Grantham, F. H.: Studies on the exchange of fluids between host and tumor. II. The blood flow of hepatomas and other tumors in rats and mice. J. Nat. Cancer Inst. 27:1465-1491, 1961. 21. Ide, A. G., Baker, N. H., and Warren, S. L.: Vascularization of the Brown-Pierce rabbit epithelioma transplant as seen in the transparent ear chambers. Am. J. Roentgen. 42:891, 1939. 22. Johnson, J. A., Cavert, H. M., and Lifson, N.: Kinetics concerned with distribution of isotopic water in in'solated perfused dog heart and skeletal muscle. Am. J. Physiol. 171 :687-693. 23. Johnson, J. A., Gott, V., and Weiland, F.: Perfusion rates of brain, intestine, and heart under conditions of total body perfusion. Am. J. Physiol. 200:551-556, 1961. 24. Kruuv, J. A., Inch, W. R, and McCredie, J. A.: Blood flow and oxygenation of tumors in mice. I. Effects of breathing gases containing carbon dioxide at atmospheric pressure. Cancer 20:51-59, 1967. 25. Owen, L. N.: A rapid method of studying tumor blood supply using lyssamine green. Nature (London) 187:795-796, 1960. 26. Powers, W. F., and Tolmach, L. J.: A multicomponent x-ray survival cure for mouse lymphosarcoma cells irradiated in vivo. Nature (London) 197:710-711,1963. 27. Reller, C. R, Jr., Sheridan, J. D., and Aust, J. B.: Tissue blood flow during varied perfusion conditions. Am. J. Physiol., 207:1354-1360,1964. 28. Ribbert, H.: Das Karzinom des Menschen sein Bau, sein Wachstum, sein Enstehung. Deutsch. Med. Wschr. 30:801,1911. 29. Rogers, W., Reagon, T. R, and Aust, J. B.: Tissue blood flow in the mammary carcinoma of the mouse estimated with antipyrine- 131 1 (Abstract). Proc. Am. Assoc. Cancer Res. 4:58, 1963. 30. Sampson, J. A.: Blood supply of uterine myomata: Based on the study of one hundred injected uteri containing these tumors. Surg. Gynec. Obst. 14:215, 1912. 31. Shibata, H. R, and MacLean, L. D.: Blood flow to tumors. Progress in Clinical Cancer, Vol. II. I. M. Ariel, Ed. New York, Grune & Stratton, 1966,33-47. 32. Thiersch,C.: Der Epithelialkrebs namentlich der Haut mit Atlas. Leipzig, 1865. 33. Urbach, F.: Anatomy and pathophysiology of skin tumor capillaries. Conf. on Biology of Cutaneous Cancer, Nat. Cancer Inst. Monograph No. 10, pp. 539-559. 34. Urbach, F., and Noell, W. K.: Effect of oxygen breathing on tumor oxygen measured polarographically. J. Applied Physiol., 13:61-65, 1958. 35. Virchow, R: Die Krankhaften Geschwulste. Betlin, August Hirschwald, 1863. 36. Warburg, 0.: The Metabolism of Tumors (English translation). London, Arnold Constable, 1930. 37. Waters, H. G., and Green, J. A.: Vascular system of two transplantable mouse granulosacell tumors. Cancer Res., 19:326, 1959.
Department of Surgery University of Minnesota Medical School Minneapolis, Minnesota 55455
Over 100 years ago, Virchow35 and Thiersch32 demonstrated by injection techniques a unique capillary network supplying the stroma of tumor tissue. Subsequent studies of the anatomy of blood vessels in spontaneous and transplantable malignant tumors revealed an inadequate blood supply with a defective and dilated vasculature. 3 , 8, 28 Abnormalities in tumor vasculature were further clarified by Goldman 17 in 1907 using intra-arterial injections of bismuth in oil in human tumors and' India ink injections in tumor-bearing small animals. Abundant proliferation of irregular branching small vessels was noted throughout small tumors, while large tumors showed central necrosis with a rich peripheral vasculature. U sing more refined techniques, subsequent investigators have corroborated these vascular changes in transplantable tumors during growth. 9 , 16, 30, 37 The first attempt to quantitate tumor blood flow was reported by From the Department of Surgery, University of Minnesota Medical School, Minneapolis, Minnesota This work was supported in part by a research grant (T-156) from the American Cancer Society. ·Instructor in Surgery, University of Minnesota Medical Center tResident in Surgery, University of Minnesota Medical Center
! University of Minnesota Medical School §Professor of Surgery and Chairman of Department, South Texas Medical School, San Antonio, Texas
Surgical Clinics of North America- Vol. 47, No.6, December, 1967
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Gullino and Grantham. 19. 20 Blood flow was measured by venous outflow of organs totally replaced by tumor and rubidium-86 or potassium-42 clearance techniques in subcutaneous implants. Blood flow per gram of tissue in tumors of all sizes was 15- to 20-fold lower than in normal tissues. Cataland et al,u also confirmed the low perfusion rate of large tumors, but demonstrated that small tumors had a significantly higher blood flow. Similarly, we have reported different levels of tumor flow in transplantable tumor systems. 12• 29 These conflicting results may be explained by changes in flow during tumor growth. For this reason, the purpose of this study was to quantitate the blood flow changes in several transplantable hamster tumors during growth. The first part of the study was devoted to the correlation of tissue blood flow measurements, using antipyrine- l3lI, with microscopic sections of the whole tumor with advancing age. Secondly, the distribution of blood flow within tumor was then exaInined by comparing the uptake of an intravenous vital dye, lissamine green, and antipyrine- l3l 1. Finally, radioactive microspheres were employed as another tissue blood flow reference material to provide an independent estimate of the distribution of flow in tumor.
METHODS
Tissue Blood Flow Techniques ANTIPYRINE- l3l1. * The estimation of tissue blood flow with antipyrine_ 1311 depends on the physical characteristics of the parent compound. Johnson et al. 22 • 23 have shown that antipyrine parallels D 2 0 by its rapid diffusion into tissue. This characteristic permits the application of the Fick principle in calculating tissue blood flow with this substance. Previous work in this laboratory has demonstrated that antipyrine tagged with 1311 is a simple and reliable reference material for the indirect measurement of blood flow at the tissue level. 27 We have modified the flow formula to calculate blood flow in the tissues of small animals: 13 A F=-----
(Caf/2 - A/2)t
where F equals flow in ml./Inin./gm., A equals tissue concentration in cpm/gm. of antipyrine- 131 I, t equals time in minutes, Caf/2 is an approximation of the mean arterial concentration obtained by dividing the final arterial concentration, C af, in cpm/ml. of antipyrine- 13 lJ by 2, and A/2 is an estimate of the mean venous concentration obtained by dividing the tissue concentration, A, in cpm/gm. of antipyrine- 1311 by 2. LISSAMINE GREEN DYE. t This vital stain is a triphenylmethane dye which has an easily detectable green color in blood and tissues, and rapidly distributes in interstitial spaces where blood is circulating. ls 'Antipyrine- I31 I: 4-iodo-antipyrine purchasetl from Abbott Laboratories tLissamine Green Dye: Lissamine green, triphenylmethane dye, V200, purchased from I.e.!. Organics, Inc., Providence, R. I.
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51Cr-Microspheres.'" These tiny ceramic spheres, 12 to 15 JL in diameter, are composed of zirconium phosphate labeled with chromium-51. Quantitation of tissue blood flow using microspheres assumes their even distribution in arterial blood and utilizes their complete extraction in vessels of smaller than 16 JL in all tissues being perfused.
Transplantation, Infusion, and Sampling Techniques Six to eight week old male and female Syrian (golden) hamsters were used in all experiments. The hamster amelanotic melanoma, renal adenocarcinoma, and fibrosarcoma 14 t were prepared by the mince suspension technique and injected into the thigh of separate groups of animals. The first experimental group comprised 61 hamsters with the amelanotic melanoma implanted 10 to 50 days prior to tissue blood flow determination. Under pentobarbital anesthesia, a polyethylene catheter was inserted into the jugular vein of each hamster. For precisely 2 minutes, approximately 3 microcuries of antipyrine j311 diluted in normal saline was continuously infused at 0.68 ml./min. using a Harvard microinfusion pump. At the end of this period, the pump was stopped, the hind limb containing the tumor was immediately amputated, and a final arterial blood sample obtained from the severed femoral artery. The entire tumor, sample of arterial blood, adjacent skin, and gluteus muscle were weighed and the cpm/gm. measured in a well-type counter.! Tissue blood flows of normal and tumor tissues were then calculated using the flow formula. The second group consisted of animals with the amelanotic melanoma, fibrosarcoma, or renal adenocarcinoma of varying sizes. Blood flow determinations were performed 2 minutes after the administration of 1 ml. of a 2 per cent solution of lissamine green dye. After completion of the previously described flow experiment, the tumor was removed and separated into dark green and light green portions and tissue blood flow determined in each sample. The final group contained hamsters with the amelanotic melanoma, renal adenocarcinoma, or fibrosarcoma in different phases of growth. A No. 10 polyethylene catheter was inserted retrograde into the left carotid artery. Approximately one microcurie of 51Cr-microspheres suspended in a I-m!. solution containing 6 per cent dextran in saline (Abbott) and 2 per cent lissamine green was injected over 15 seconds. Two minutes later, the limb was amputated and the uptake of 51Cr in skin, muscle, and tumor determined. Uptake ratios were calculated for each animal as tumor: skin, tumor: muscle, skin: muscle, and dark-stained or lightstained tumors. Following blood flow determinations, routine histological sections were stained with hematoxylin and eosin. ·S1Cr-Microspheres: Purchased from the Minnesota Mining and Manufacturing Co., St. Paul, Minnesota tHamster tumors were obtained from Dr. Joseph Fortner, Sloan-Kettering Institute, New York, N.Y. !Decade Scaler: Curtis Nuclear Corporation,
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Table 1. Total Tumor Blood Flow in Hamster Amelanotic Melanoma During Growth (Antipyrine- 131J Technique) MEAN SKIN FLOW
NO.
10
10 12 14 15
MEAN TUMOR WEIGHT
0.4 1.0 3.5 7.7 20.4
MEAN TUMOR FLOW
0.95 1.02 0.52 0.24 0.23
(±.42)* (±.56) (±'26) (±'10) (±'14)
Leg with tumor 0.15 0.24 0.18 0.23 0.50
(±.06)* (±.19) (±.09) (±.07) (±.25)
Opposite leg
0.21 (±.14)* 0.18 (±'05) 0.20 (±'09)
MEAN MUSCLE FLOW
Leg with tumor 0.07 0.08 0.11 0.17 0.26
(±.04)* (±.04) (±.04) (±.04) (±.14)
Opposite leg
0.12 (±.05)* 0.06 (±'02) 0.09 (±'04)
*Standard Deviation.
RESULTS Group I. Total Tumor Blood Flow During Growth (Antipyrine_ l3l I Technique) To evaluate the changes in mean tissue blood flow with advancing tumor size, the 61 hamsters were divided into five groups according to tumor weight (Table 1). The mean tumor flows of 0.4 and 1.0 gm. tumors were approximately five and ten times higher than skin and muscle blood flow respectively. When the mean tumor weight reached 3.5 gm., the mean tumor blood flow decreased to approximately one-half that of the smaller tumors. As the weight of the tumor increased to a mean of 7.7 to 20.4 gm., a further decrease in mean perfusion to approximately one-fourth of that measured in small tumors was noted. This inverse relationship between tumor blood flow and tumor size is similar to that
Figure 1. Photomicrograph of a 0.44-gm. hamster amelanotic melanoma showing a uniform sheet of tumor tissue. Total tumor blood flow: 0.83 ml./min./gm. (H. & E., x40.)
BLOOD FLOW IN TRANSPLANTABLE TUMORS DURING GROWTH
1477
Figure 2. Photomicrograph of a 21-gm. hamster amelanotic melanoma showing central necrosis and a peripheral rim of intact tumor tissue. Total tumor flow: 0.19 ml./min./gm. (H. & E., x40.)
Figure 3. Photomicrograph of hamster gluteus muscle adjacent to the 21-gm. tumor in Figure 2. Muscle blood flow: 0.33 ml./min./gm. Note the darkly stained melanoma and inflammatory cells invading the muscle. (H. & E., x 100.)
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WAID ROGERS, ET AL.
reported by Cataland et al,u in mouse tumors. A review of the microscopic sections suggested an explanation for the progressive reduction of tumor blood flow with advancing age. Small tumors were comprised of uniform sheets of tumor cells (Fig. 1), while the .larger tumors contained central necrotic areas with a peripheral rim of intact tumor (Fig. 2). It was interesting to note that the tissue blood flow of muscle and skin adjacent to the large tumors was elevated. The cause of this elevated flow has not been established, but it may be a reflection of the inflammatory and tumor cell invasion readily observed in microscopic sections (Fig. 3).
Group II. Distribution of Blood Flow Within Tumors During Growth (Antipyrine- 131 I - Lissamine Green Technique) Tissue blood flow was estimated separately in the light and dark dye-stained regions of these tumors. Small tumors were more uniformly stained with lissamine green. With an increase in tumor size, light- and dark-stained areas were easily identified within the tumor. As described by Goldacre and Sylven 15 and Owen,25 the tumor periphery was intensely stained and the central region of the tumor remained pale green or white. The uptake of lissamine green was correlated with blood flow within the tumor (Table 2). Mean tissue blood flow of the dark-stained
Table 2. Distribution of Blood Flow Within Hamster Tumors During Growth (Antipyrine j31 I-Lissamine Green Technique) MEAN TISSUE BLOOD FLOW
NO.
MEAN TUMOR WT. (gm.)
(ml./min./gm.)
Stain Intensity Dark
Light
Total Tumor
Amelanotic Melanoma 10
1.6
8
4.3
8
11.1
14
3.3
15
11.0
13
30.8
5
0.5
6
2.9
6
6.2
0.86 (±A5)* Q37 (±.14) 0.39 (±.26)
0.48 (±'21) Q14 (±.09) 0.12 (±.07)
0.71 (±.34) 0.29 (±.14) 0.20 (±.18)
Fibrosarcoma Q38 (±.17)* 0.29 (±.14) 0.25 (±.13)
QI0 (±.02) 0.07 (±.02) 0.02 (±.01)
0.22 (±.11) 0.15 (±.07) 0.05 (±'Ol)
Renal Adenocarcinoma
'Standard Deviation.
0.78 (±.41)* 0.82 (±.60) 0.42 (±.26)
0.48 (±.29) 0.16 (±.04)
0.78 (±.41) 0.55 (±.30) 0.20 (±.13)
1479
BLOOD FLOW IN TRANSPLANTABLE TUMORS DURING GROWTH
tumor was 2 to 12 times greater than that of the lighter-stained tumor in the three tumors studied. Group III. Distribution of Blood Flow Within Tumor During Growth (Antipyrine- l3l1- Lissamine Green and 5lCr-MicrospheresLissamine Green Techniques) The distribution of 51Cr-microspheres was compared to the uptake of antipyrine- 131J in skin, muscle, and tumor in separate groups of animals containing the same type of tumor of comparable weight. The uptake of these agents in each individual animal was expressed as a ratio of skin: muscle, tumor: skin, tumor:muscle, and dark-stained: light-stained tumor (Table 3). The uptake of micro spheres and antipyrine in small tumors was uniformly higher than the uptake of these agents in skin and muscle. This is strong evidence, but not unequivocal proof that the blood flow in these small tumors was higher than the flow to skin or muscle. In larger tumors, the distribution of antipyrine and microspheres corresponded to the intensity of the dye. Distribution of these two independent indicators of flow in the peripheral darkstained tumor was considerably higher than the uptakes of these agents in the central light-stained or unstained regions of the neoplasm. Similarly, Shibata and MacLean31 have demonstrated by tissue block analysis that the uptake of micro spheres in the center of large human and rat tumors is lower than in the periphery. Table 3.
Distribution of Blood Flow Within Hamster Tumors During Growth (Antipyrine- 131J-Lissamine Green Technique) MEAN UPTAKE RATIOS
NO.
TAG
MEAN TUMOR WT. (gm.)
Skin Muscle
Tumor Skin
Tumor Muscle
Dark Tumor Light Tumor
Melanoma 17 10
5lCr 131
1
1.3 1.6
2.0 (± 1.2)· 2.2 (± 1.3)
7.8 (± 4.7) 5.0 (± 2.6)
1.5 (± 1.0)· 1.8 (± 1.0) 2.5 (± 1.0) 1.8 (± 1.0)
4.2 (± 2.4) 2.9 (± 1.8) 0.3 (± .01) 0.2 (± .01)
1.4 (± 0.7)· 1.8 (± 1.0)
3.4 (± 2.1) 2.8 (± 1.7)
19.6 (± 12.3)
4.0 (± 3.3)
11.3 (± 6.0)
2.3 (± 1.6)
Fibrosarcoma 4 4 6 10
5lCr 131
1
5ICr 131
1
0.9 1.0 37.4 35.2
7.2 (± 5.7)
8.8 (± 6.0)
4.7 (± 2.9)
0.7 (± 0.6)
5.6 (± 3.0)
133.2 (± 94)
0.3 (± .01)
18.1 (± 10.8)
Renal Adenocarcinoma 6 6
5lCr 131
1
·Standard Deviation.
2.1 2.9
6.6 (± 4.2)
4.9 (± 3.1)
3.5 (± 2.0)
1.7 (± 1.0)
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WAID ROGERS, ET AL.
It is interesting that the mean tumor:normal tissue uptake ratios and dark-stained:light-stained tumor uptake ratios for micro spheres were consistently higher than comparable uptake ratios for antipyrine, while the skin:muscle ratios remained the same for both indicators. This proportionally higher uptake of micro spheres in tumor may be a manifestation of a unique tumor vasculature.
DISCUSSION The vigorous capillary bleeding observed when tumor is incised during operation has led to the popular concept that there is high blood flow in tumors. Evidence in favor of this elevated flow is implied by the more rapid appearance of fluorescein and radiopaque dyes in tumors than adjacent tissue, and increased skin temperature overlying superficial tumors, and a high oxygen tension in venous blood draining neoplasms. 4 , 5, 6, 7 Urbach33 explains these observations on the basis of significant arteriovenous shunting in the periphery of the tumor. Morphological studies by Algire and Chalkleyl, 2 have shown a vascularity in early developing tumors which was double that of surrounding normal tissues. Similarly, our quantitative estimates of tissue blood flow in small hamster tumors have been substantially higher than the flow in adjacent normal tissues. Even during the initial stages of growth, tumor vessels have been noted to be dilated and lacking in normal epithelial layers. 1, 2 But, with subsequent growth, large sinusoids and blood-filled diverticuli develop which end blindly toward tumor centers suggesting obliteration. 21 These morphological observations can be correlated with the progressive decrease in blood flow with increase in tumor size noted in this study. Reduction in flow of the whole tumor is a function of a reduced perfusion in its center, as illustrated by a parallel distribution of dye, microspheres, and antipyrine. The central regions of hamster tumors receiving low blood flow develop progressive necrosis during growth. Goldacre and Sylven16 have demonstrated that these ischemic areas in tumor still contain viable cells. The classical work of Warburg36 demonstrated that tumor cells can survive in the absence of oxygen by converting to anaerobic glycolysis. Furthermore, low oxygen tensions have been noted in a variety of primary and metastatic human and animal tumors. 10, 24, 34 The ability of tumor cells to survive in an anaerobic environment provides an explanation for their resistance to anticancer therapy. Cellular damage produced by ionizing radiation depends upon the concentration of oxygen in the tissues at the time of exposure. 18 The few remaining viable cells in the center of the tumor may account for failure of radiotherapy to produce a cure. 26 Furthermore, the access of blood-borne chemicals and antitumor sera to all cells of a solid tumor would be affected by the progressive reduction of tumor flow during growth. Until a method of enhancement of central perfusion can be developed, the cure of large malignant tumors, short of surgical excision, is unlikely.
BLOOD FLOW IN TRANSPLANTABLE TUMORS DURING GROWTH
1481
SUMMARY 1. Total tissue blood flow in small hamster tumors estimated by means of the antipyrine- 131 1 technique was five- and ten-fold higher than adjacent skin and muscle. Blood flow of the whole tumor markedly decreased during growth. This decrease in perfusion in tumor was associated with the progressive development of central necrosis. 2. The distribution of lissamine green dye in three types of hamster tumors correlated with the uptake of antipyrine- l31 I. Dark-stained tissue blood flow, while the light-stained central necrotic areas showed peripheral regions of histologically intact tumor, demonstrated a high perfusion. 3. The distribution of 5lCr-microspheres and antipyrine- 1311 in tumor was found to be similar. This suggests that our quantitative estimates of tumor flow are reasonable. ACKNOWLEDGMENT
The authors wish to express their sincere gratitude to Mr. Bruce Benson and Mr. Wilfred Carlson for their valuable technical assistance.
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16. Goldacre, R J., and Sylven, B.: On the access of blood-borne dyes to various tumor regions. Brit. J. Cancer 16:306-322, 1962. 17. Goldman, E.: Growth of malignant disease in man and the lower animals with special reference to vascular system. Proc. Roy. Soc. Med., 1: 1, 1907. 18. Gray, L. H., Conger, A. D., Ebert, M., Homsey, S., and Scott, O. C. A.: Concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Brit. J. Radiol., 26:638-648, 1953. 19. Gullino, P. M., and Grantham, F. H.: Studies on the exchange of fluids between host and tumor. I. A method for growing "tissue-isolated" tumors in laboratory animals. J. Nat. Cancer Inst., 27:679-709,1961. 20. Gullino, P. M., and Grantham, F. H.: Studies on the exchange of fluids between host and tumor. II. The blood flow of hepatomas and other tumors in rats and mice. J. Nat. Cancer Inst. 27:1465-1491, 1961. 21. Ide, A. G., Baker, N. H., and Warren, S. L.: Vascularization of the Brown-Pierce rabbit epithelioma transplant as seen in the transparent ear chambers. Am. J. Roentgen. 42:891, 1939. 22. Johnson, J. A., Cavert, H. M., and Lifson, N.: Kinetics concerned with distribution of isotopic water in in'solated perfused dog heart and skeletal muscle. Am. J. Physiol. 171 :687-693. 23. Johnson, J. A., Gott, V., and Weiland, F.: Perfusion rates of brain, intestine, and heart under conditions of total body perfusion. Am. J. Physiol. 200:551-556, 1961. 24. Kruuv, J. A., Inch, W. R, and McCredie, J. A.: Blood flow and oxygenation of tumors in mice. I. Effects of breathing gases containing carbon dioxide at atmospheric pressure. Cancer 20:51-59, 1967. 25. Owen, L. N.: A rapid method of studying tumor blood supply using lyssamine green. Nature (London) 187:795-796, 1960. 26. Powers, W. F., and Tolmach, L. J.: A multicomponent x-ray survival cure for mouse lymphosarcoma cells irradiated in vivo. Nature (London) 197:710-711,1963. 27. Reller, C. R, Jr., Sheridan, J. D., and Aust, J. B.: Tissue blood flow during varied perfusion conditions. Am. J. Physiol., 207:1354-1360,1964. 28. Ribbert, H.: Das Karzinom des Menschen sein Bau, sein Wachstum, sein Enstehung. Deutsch. Med. Wschr. 30:801,1911. 29. Rogers, W., Reagon, T. R, and Aust, J. B.: Tissue blood flow in the mammary carcinoma of the mouse estimated with antipyrine- 131 1 (Abstract). Proc. Am. Assoc. Cancer Res. 4:58, 1963. 30. Sampson, J. A.: Blood supply of uterine myomata: Based on the study of one hundred injected uteri containing these tumors. Surg. Gynec. Obst. 14:215, 1912. 31. Shibata, H. R, and MacLean, L. D.: Blood flow to tumors. Progress in Clinical Cancer, Vol. II. I. M. Ariel, Ed. New York, Grune & Stratton, 1966,33-47. 32. Thiersch,C.: Der Epithelialkrebs namentlich der Haut mit Atlas. Leipzig, 1865. 33. Urbach, F.: Anatomy and pathophysiology of skin tumor capillaries. Conf. on Biology of Cutaneous Cancer, Nat. Cancer Inst. Monograph No. 10, pp. 539-559. 34. Urbach, F., and Noell, W. K.: Effect of oxygen breathing on tumor oxygen measured polarographically. J. Applied Physiol., 13:61-65, 1958. 35. Virchow, R: Die Krankhaften Geschwulste. Betlin, August Hirschwald, 1863. 36. Warburg, 0.: The Metabolism of Tumors (English translation). London, Arnold Constable, 1930. 37. Waters, H. G., and Green, J. A.: Vascular system of two transplantable mouse granulosacell tumors. Cancer Res., 19:326, 1959.
Department of Surgery University of Minnesota Medical School Minneapolis, Minnesota 55455