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The microcirculation in two transplantable melanomas of the hamster I. In vivo observations in transparent chambers
Cancer Letters, 4 (1978) 109--116 © Elsevier/North-Holland Scientific Publishers Ltd.
109
THE M I C R O C I R C U L A T I O N IN TWO T R A N S P L A N T A B L E M E L A N O M A S O F THE H A M S T E R I. IN VIVO O B S E R V A T I O N S IN T R A N S P A R E N T CHAMBERS
B.A. WARREN
Department of Pathology, University of Western Ontario, London (Canada)
P. SHUBIK, R. WILSON, H. GARCIA and R. FELDMAN Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68105 (U.S.A.)
(Received 20 August 1977) (Revised version received 14 November 1977) (Accepted 28 November 1977-)
SUMMARY Twenty-eight transparent chambers were inserted into the cheek pouches of hamsters and daily serial observations made of the changing vasculature of transplants of 2 varieties (A-Mel-4B32, ZGYP1) of amelanotic melanomas. The t u m o r A-Mel-4B32 grew at a rate which covered 50% of the viewing area in 3--4 days, while the t u m o r ZGYP1 covered less than 10% of the viewing area at this time and did n o t cover 50% of the viewing area until 9.5 days after transplantation. The difference in growth rate was not reflected in any differences in the m o r p h o l o g y of the vascular n e t w o r k and distribution of the venous arcades.
INTRODUCTION The membrane of the hamster cheek p o u c h has been used extensively to s t u d y transplantation and microcirculatory p h e n o m e n a [4]. A number of different tumors have been observed growing on the cheek pouch membrane including attempts at growing grafts o f human cancer [1]. The purpose of the present investigation was to correlate the stage of growth of the melanoma transplant with the structure and distribution o f the blood vessels within various parts of the t u m o r and underlying pouch membrane using the-technique of implantation of a transparent chamber [5] with serial daily in vivo observations both by gross examination and transillumination in vivo microscopy.
110 MATERIALS AND METHODS
Animals Cheek pouch chambers were inserted into the left pouch of:re,hale Syrian golden hamsters (Mesocricetus auratus). They were kept singly in plastic cages and given a diet of Wayne Lab-blox (Allied Mills, Inc.) and water containing terramycin (oxytetracycline HCI) Pfizer Agricultural Division, Chas. Pfizer and Co., Inc., New York, New York 10017, U.S.A.) in the proportion 7 g/1 distilled water ad libitum.
Tumor transplants Two amelantoic melanomas of the hamster were studied. A-Mel-4B32 was obtained from the Sloan Kettering Institute in 1969 and maintained since then in our laboratories. ZGPY1 was a spontaneous tumor that arose in our colony in 1970. These tumors were carried serially in the laboratory by transplantation into the dorsal subcutaneous tissue of the animals. In an attempt to transplant consistent volumes of tissue a 1 mm 3 space was machined out of the jaws of a needle holder. A 1 mm 3 of tissue was removed from the tumor mass on closing the jaws.
Experimental procedure The methods involved in the transparent chamber technique for the hamster cheek pouch have been detailed previously [3]. The chamber consists of upper and lower plates. The lower plate fits into the cavity of the animal's pouch. It holds the membrane in place by means of pegs which pass through the pouch membrane into the upper plate. The upper plate is a flanged ring and a coverslip. There is thus a well in the chamber through which the pouch can be observed. The skin of the animal over the pouch fits into the flange. The lower plate is introduced into the cleaned pouch of the anesthetized hamster and the shaved skin overlying the plate incised. Some of the areola tissue is removed from the surface of the pouch .presented in the wound. Incisions over the pegs of the base plate allow these to penetrate the membrane. The tumor transplant is then placed centrally on the pouch membrane. A few drops of normal saline are added in an attempt to prevent air bubbles. The top plate is lowered onto the pegs of the base plate and the pegs fixed in place with a glue (Eastman 910 Adhesive, Eastman Chemical Products, Inc., Kingsport, Tennessee, U.S.A.).
Method of observation The transplanted tumor fragment lying on the pouch membrane was observed on a daily basis by light microscopy. This was achieved by illuminating the pouch membrane from below via a light conducting rod introduced into the lumen of the pouch with the animal anesthetized. The whole apparatus was mounted next to a specially adapted microscope which permitted in vivo observation and photography. Macroscopic photographs of the pouch mem-
111 brane in the viewing area of the window were made on a daily basis, and for this the pouch was filled with c o t t o n wool to give a white background to the vacular pattern. The growth of transplants of the 2 tumors were followed both by the macroscopic technique of photography of the whole chamber and by microscopic examination of the transplant. Data sheets were drawn up for each chamber. These recorded the date of the photographs and magnification, the width of the internal well o f the upper plate at the magnification of the photograph, the weight of 100 mm 2 o f p h o t o c o p y paper, t h e weight o f the viewing area cut from this paper, and the weight of the t u m o r outlined on the paper on a daffy basis. Calculation o f the percentage o f viewing area occupied by the tumor The space between the upper plate and the pouch membrane remains constant and permits the calculation of the increase in the size of the t u m o r by overlaying successive dally photographs of the same chamber. All chambers were of the same t y p e and the width of the internal diameter of the upper plate measured 18 mm. The actual calculations of the percentage area were based on the following formula:
Let: x = weight of the photographic paper containing the exact area of t u m o r on the specific day in question, e.g., day 3; y = weight of the photographic paper containing the exact area of the t u m o r on the day following that of x, e.g., day 4; z = weight of the photographic paper comprising the viewing area of the chamber; percentage o f viewing area of chamber occupied by t u m o r on specific day = (xJz) X 100; percentage of viewing area of chamber occupied by t u m o r on specific day + 1 = (y/z) X 100. Since the t u m o r transplant progressively grows, sometimes rapidly, on a day to day basis y is greater t h a n x. The percentage increase is: (y - x / z ) X 100. Thus the percentage o f the viewing area occupied by the growing t u m o r transplants in the individual chambers was recorded and graphed. The results are shown in Figs. 1 and 2. In vivo observations on the microcirculatory bed and its associated vessels were made of the whole hamster cheek pouch membrane and o f the associated t u m o r transplant. A n e s t h e t i c agent The animals were anesthetized by means of an intraperitoneal injection of barbital sodium. The dose was calculated on a body weight basis of 3 mg/40 g b o d y wt.
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Fig. 1 and Fig. 2. Graphs to s h o w the growth o f two amelantoic melanoma tumors on the hamster cheek pouch. Fig. 1. A-Mel-4B32 tumor; Fig. 2. ZGPY1 tumor. The points on the graphs express the progressive growth o f each tumor as an increasing percentage o f the viewing area o f the chambers occupied by the tumors. Each different graphic notation represents an individual chamber. Seven chambers which had received A-Mel-4B32 tumor were suitable for study and this information is incorporated in Fig. 1. Fig. 2 represents the information derived from the study of 10 chambers implanted with ZGPY1 tumor. The much slower growth of the ZGPY1 compared with that o f the A-Mel-4B32 tumor is apparent from these graphs.
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113 RESULTS
N u m b e r o f cheek pouch membranes (enclosed in chambers) examined Thirty-four transparent chambers were inserted into the cheek pouches of hamsters. In twenty-eight, serial observations were made on the growth of the t u m o r transplants b y in vivo light microscopy. Pieces of t u m o r tissue from A-Mel-4B32 were implanted into hamster cheek pouch chambers and the pouch membrane with the t u m o r removed at 3 days (2 chambers), 4 days (2 chambers), 6 days (3 chambers), 7 days (4 chambers) and 8 days (2 chambers). In a similar fashion pieces of ZGPY1 t u m o r tissue were implanted into hamster cheek p o u c h chambers and the membranes with the t u m o r removed at 3 days (2 chambers), 7 days (6 chambers), 8 days (4 chambers), 10 days (2 chambers) and 13 days (1 chamber). Transparent chambers were inserted into 2 animals and no t u m o r transplanted. These were kept for 4 days and 12 days. There was almost no change in the pattern of the venous and arterial arcades o f the membrane. After the animals were killed, the pouch membranes were processed and examined by light microscopy to provide normal control specimens. During the experimenta! period 2 chambers became partially eroded out of the p o u c h and these animals were p r o m p t l y killed. Two further chambers became severely infected and the preparations were unsuitable for examination. The rate o f growth o f the tumor transplants (Figs. 1 and 2) Every a t t e m p t was made to ensure that only 1 mm 3 of t u m o r tissue was transplanted onto each chamber. There is general uniformity of the growth rate o f each t u m o r transplant within its own group. As can be seen b y comparing Figs. 1 and 2, the A-Mel-4B32 t u m o r grew much faster than the ZGPY1 tumor. The former grew rapidly after a lag period of only 2--3 days and usually covered 50% of the viewing area in 3--4 days. The ZGPY1 t u m o r had a longer lag period which usually was 3--4 days and even after that did not cover more than 30% of the viewing area 8 days after transplantation. The graphs are based on the fact that the tumors grow as even plates extending out from the transplantation site, so that an increment in the mass of the t u m o r is represented by the increase in the area occupied b y the t u m o r within the viewing area multiplied b y the thickness of the t u m o r 'plate'. This is the distance between the coverslip o f the t o p ring of the chamber and the dorsal surface of the pouch membrane. The 'viewing area' is the well of the chamber and is a circle of. 18 mm diameter. The tumors did not c o m m e n c e growing until blood was observed flowing through them. Static blood in capillary sprouts in the t u m o r transplants was observed a day before the sprouts contained flowing blood. The extent of the t u m o r cell plate became obvious following vascularization because of the red cell content of the capillaries. The microvascular pattern o f the cheek pouch o f the host and its variation in the presence o f a growing tumor (Fig 3) The pattern of the arteriolar loops remained constant. The walls o f the arte-
114
Fig. 3. The i n c o r p o r a t i o n o f a major vein of the viewing area in the advancing edge o f the t u m o r (ZGPY1). A m a j o r venous arcade on the p o u c h m e m b r a n e ( V A ) is shown in Fig. 3a close to the finely vascular edge (E) o f the t u m o r transplant (T). This is 7 days after transp l a n t a t i o n o f 1 m m s of t u m o r substance o n t o the central area o f the p o u c h . Fig. 3b is 8 days after transplantation and shows the i n c o r p o r a t i o n of the venous arcade (VA in Fig. 3a) i n t o the edge (E) o f the t u m o r and t h e great d i m i n u t i o n of direct through flow in this a r c a d e Alternative p a t h w a y s have o p e n e d up in area C. The venous arcade labelled B remains constant. T w o arterioles A 1 and A 2 are constant in position. Fig. 3a, X 6.75; Fig. 3b, X 6.
115
rioles remained parallel and the bifurcation pattern remained the same as it was on the day of transplantation. Some increase in width was noted in some arterioles which appeared to supply the capillary bed of the tumors. The arteriolar pattern of the chamber actually became clearer in some instances after the t u m o r had almost completely covered the viewing area (in A-Mel-4B32 tumors). Thin capillaries containing rapidly flowing blood were present in the t u m o r tissue overlying the arterioles. This had the effect of making the t u m o r tissue less opaque than when dilated venous sinusoidal vessels were present over the arterioles. These thin, rapidly flowing capillaries after a short length fed into the slowly flowing sinusoidal vessels. In contrast to the constancy of the arteriolar pattern, the pattern of the venous arcades and the flow of blood through the capillary modules of the p o u c h membrane, within the viewing area of the chamber, changed daffy with the growth o f the t u m o r transplants. We have reported this in an earlier paper [6]. An example of the incorporation o f parts of a major venous arcade of the chamber into the advancing edge of the t u m o r transplant is shown in Fig. 3. A segment of the venous arcade is incorporated into the tumor-edge venous vessels. Diminution of flow has occurred in this particular instance. Reversal o f flow in such vessels was c o m m o n as was an increase in flow once channels from the t u m o r itself flowed freely into the incorporated vein. Daily change in the venous pattern of the pouch membrane occurred and there were parallel changes in the extent of flow through various capillary networks. In Fig. 3 a dilatation of the neighboring capillary bed is evident following the diminution of flow through the incorporated vein. DISCUSSION
This paper presents in vivo observations on 2 transplantable tumors detailing the time from transplantation and the growth curve of the whole t u m o r following transplantation. The m e t h o d utilizing the hamster cheek pouch possesses a number of advantages for the close study o f the growth of the t u m o r transplant and its m e t h o d o f vascularization. The t u m o r grows as a thin,plate between the coverslips, and its vascularization is obvious because o f the red cell content of the blood vessels. In this technique the exact 'age' of the transplant is known, as well as the in vivo alterations of the vascular pattern surrounding the transplant f r o m the time of its introduction, and these are combined in a preparation that permits easy access for the preparation o f tissues for electron microscopy. The changes in the vascular architecture that ensue, following transplantation o f melanoma onto the cheek pouch, have been reported using a hand lens [ 2 ] . A state o f congestion o f the vascular pattern around the t u m o r graft was noted from the second d a y after transplantation. There was a peritumoral capillary engorgem e n t with occasionally small peripheral hemorrhages. From the 4th to the 6th d a y (but sometimes as late as the 8th day) the venous and capillary pattern was seen to enlarge and vascularization of the t u m o r became complete. From this time the t u m o r was n o t e d to grow regularly in the form of a spherical mass.
116
The morphology of the melanoma vessels in thin, cleared sections of the tumors after opacification of the vascular system of the animal with carbon particles (India ink) was studied. Three zones were found: (1) a central zone of avascular necrosis; (2) a midzone which consisted of t u m o r cells interlaced b y vascular channels o f capillary t y p e which were thin at times and extensively anastomosed with each other. These were irregular and showed local dilations and tortuosities and they isolated cords of t u m o r cells. (3) A peripheral zone where vessels of large caliber surrounded the tumor. Our results described above are in agreement with these findings. In addition, it was possible to observe great alteration in the venous system of the pouch membrane and the t u m o r transplant after vascularization with growth of the t u m o r transplant. The arterioles of the pouch membrane remained in their original pattern and form. The vascular netw o r k o f t u m o r tissue implanted onto host tissue depends on (a) time from transplantation, (b) the nature of the response of the host tissue. However, the rate of growth does n o t effect the pattern of the vessels. ACKNOWLEDGEMENTS
The w o r k reported here was supported b y a grant from The National Cancer Institute of Canada and U.S. Public Health Service grant Nos. 5R31 CA-1476602. Our thanks are due to Dr. L. Malik, Miss L. Love and to Mr. W. Chauvin for assistance with the preparations and photography. REFERENCES 1 Chute, R.N., Sommers, S.C. and Warren, S. (1952) Heterotransplantation of human cancer. II. Hamster cheek pouch. Cancer Res., 12,912--914. 2 Delarue, J., Mignot, J. and Caulet, T. (1963) Modifications vasculaires de la poche jugale du hamster dor~ au cours du d~veloppement de greffes d'une tumeur m~lanique. C.R. Soc. Biol., 157, 69--71. 3 Greenblatt, M., Choudari, K.V.R., Sanders, A.G. and Shubik, P. (1969) Mammalian microcirculation in the living animal: Methodologic considerations. Microvasc. Res., 1, 420--432. 4 Handler, A.H. and Shepro, D. (1968) Cheek pouch technology: uses and application. In: The Golden Hamster, Its Biology and Use in Medical Research, p. 195. Editors: R.A. Hoffman, P.F. Robinson and H. Magalhaes. The Iowa State University Press, Iowa. 5 Sanders, A.G. and Shubik, P. (1964) A transparent window for use in the Syrian hamster. Isr. J. Exp. Med., 11,118 (abstract). 6 Warren, B.A. and Shubik, P. (1966) The growth of the blood supply to melanoma transplants in the hamster cheek pouch. Lab. Invest., 15,464--478.
109
THE M I C R O C I R C U L A T I O N IN TWO T R A N S P L A N T A B L E M E L A N O M A S O F THE H A M S T E R I. IN VIVO O B S E R V A T I O N S IN T R A N S P A R E N T CHAMBERS
B.A. WARREN
Department of Pathology, University of Western Ontario, London (Canada)
P. SHUBIK, R. WILSON, H. GARCIA and R. FELDMAN Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68105 (U.S.A.)
(Received 20 August 1977) (Revised version received 14 November 1977) (Accepted 28 November 1977-)
SUMMARY Twenty-eight transparent chambers were inserted into the cheek pouches of hamsters and daily serial observations made of the changing vasculature of transplants of 2 varieties (A-Mel-4B32, ZGYP1) of amelanotic melanomas. The t u m o r A-Mel-4B32 grew at a rate which covered 50% of the viewing area in 3--4 days, while the t u m o r ZGYP1 covered less than 10% of the viewing area at this time and did n o t cover 50% of the viewing area until 9.5 days after transplantation. The difference in growth rate was not reflected in any differences in the m o r p h o l o g y of the vascular n e t w o r k and distribution of the venous arcades.
INTRODUCTION The membrane of the hamster cheek p o u c h has been used extensively to s t u d y transplantation and microcirculatory p h e n o m e n a [4]. A number of different tumors have been observed growing on the cheek pouch membrane including attempts at growing grafts o f human cancer [1]. The purpose of the present investigation was to correlate the stage of growth of the melanoma transplant with the structure and distribution o f the blood vessels within various parts of the t u m o r and underlying pouch membrane using the-technique of implantation of a transparent chamber [5] with serial daily in vivo observations both by gross examination and transillumination in vivo microscopy.
110 MATERIALS AND METHODS
Animals Cheek pouch chambers were inserted into the left pouch of:re,hale Syrian golden hamsters (Mesocricetus auratus). They were kept singly in plastic cages and given a diet of Wayne Lab-blox (Allied Mills, Inc.) and water containing terramycin (oxytetracycline HCI) Pfizer Agricultural Division, Chas. Pfizer and Co., Inc., New York, New York 10017, U.S.A.) in the proportion 7 g/1 distilled water ad libitum.
Tumor transplants Two amelantoic melanomas of the hamster were studied. A-Mel-4B32 was obtained from the Sloan Kettering Institute in 1969 and maintained since then in our laboratories. ZGPY1 was a spontaneous tumor that arose in our colony in 1970. These tumors were carried serially in the laboratory by transplantation into the dorsal subcutaneous tissue of the animals. In an attempt to transplant consistent volumes of tissue a 1 mm 3 space was machined out of the jaws of a needle holder. A 1 mm 3 of tissue was removed from the tumor mass on closing the jaws.
Experimental procedure The methods involved in the transparent chamber technique for the hamster cheek pouch have been detailed previously [3]. The chamber consists of upper and lower plates. The lower plate fits into the cavity of the animal's pouch. It holds the membrane in place by means of pegs which pass through the pouch membrane into the upper plate. The upper plate is a flanged ring and a coverslip. There is thus a well in the chamber through which the pouch can be observed. The skin of the animal over the pouch fits into the flange. The lower plate is introduced into the cleaned pouch of the anesthetized hamster and the shaved skin overlying the plate incised. Some of the areola tissue is removed from the surface of the pouch .presented in the wound. Incisions over the pegs of the base plate allow these to penetrate the membrane. The tumor transplant is then placed centrally on the pouch membrane. A few drops of normal saline are added in an attempt to prevent air bubbles. The top plate is lowered onto the pegs of the base plate and the pegs fixed in place with a glue (Eastman 910 Adhesive, Eastman Chemical Products, Inc., Kingsport, Tennessee, U.S.A.).
Method of observation The transplanted tumor fragment lying on the pouch membrane was observed on a daily basis by light microscopy. This was achieved by illuminating the pouch membrane from below via a light conducting rod introduced into the lumen of the pouch with the animal anesthetized. The whole apparatus was mounted next to a specially adapted microscope which permitted in vivo observation and photography. Macroscopic photographs of the pouch mem-
111 brane in the viewing area of the window were made on a daily basis, and for this the pouch was filled with c o t t o n wool to give a white background to the vacular pattern. The growth of transplants of the 2 tumors were followed both by the macroscopic technique of photography of the whole chamber and by microscopic examination of the transplant. Data sheets were drawn up for each chamber. These recorded the date of the photographs and magnification, the width of the internal well o f the upper plate at the magnification of the photograph, the weight of 100 mm 2 o f p h o t o c o p y paper, t h e weight o f the viewing area cut from this paper, and the weight of the t u m o r outlined on the paper on a daffy basis. Calculation o f the percentage o f viewing area occupied by the tumor The space between the upper plate and the pouch membrane remains constant and permits the calculation of the increase in the size of the t u m o r by overlaying successive dally photographs of the same chamber. All chambers were of the same t y p e and the width of the internal diameter of the upper plate measured 18 mm. The actual calculations of the percentage area were based on the following formula:
Let: x = weight of the photographic paper containing the exact area of t u m o r on the specific day in question, e.g., day 3; y = weight of the photographic paper containing the exact area of the t u m o r on the day following that of x, e.g., day 4; z = weight of the photographic paper comprising the viewing area of the chamber; percentage o f viewing area of chamber occupied by t u m o r on specific day = (xJz) X 100; percentage of viewing area of chamber occupied by t u m o r on specific day + 1 = (y/z) X 100. Since the t u m o r transplant progressively grows, sometimes rapidly, on a day to day basis y is greater t h a n x. The percentage increase is: (y - x / z ) X 100. Thus the percentage o f the viewing area occupied by the growing t u m o r transplants in the individual chambers was recorded and graphed. The results are shown in Figs. 1 and 2. In vivo observations on the microcirculatory bed and its associated vessels were made of the whole hamster cheek pouch membrane and o f the associated t u m o r transplant. A n e s t h e t i c agent The animals were anesthetized by means of an intraperitoneal injection of barbital sodium. The dose was calculated on a body weight basis of 3 mg/40 g b o d y wt.
¸
I
2 3 4 DAYS FOLLOWING
/
/
/
/
5 6 IMPLANT OF
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/
/
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A-MEL-4B32
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Fig. 1 and Fig. 2. Graphs to s h o w the growth o f two amelantoic melanoma tumors on the hamster cheek pouch. Fig. 1. A-Mel-4B32 tumor; Fig. 2. ZGPY1 tumor. The points on the graphs express the progressive growth o f each tumor as an increasing percentage o f the viewing area o f the chambers occupied by the tumors. Each different graphic notation represents an individual chamber. Seven chambers which had received A-Mel-4B32 tumor were suitable for study and this information is incorporated in Fig. 1. Fig. 2 represents the information derived from the study of 10 chambers implanted with ZGPY1 tumor. The much slower growth of the ZGPY1 compared with that o f the A-Mel-4B32 tumor is apparent from these graphs.
4o-
50-
90
~" t~
i--L
113 RESULTS
N u m b e r o f cheek pouch membranes (enclosed in chambers) examined Thirty-four transparent chambers were inserted into the cheek pouches of hamsters. In twenty-eight, serial observations were made on the growth of the t u m o r transplants b y in vivo light microscopy. Pieces of t u m o r tissue from A-Mel-4B32 were implanted into hamster cheek pouch chambers and the pouch membrane with the t u m o r removed at 3 days (2 chambers), 4 days (2 chambers), 6 days (3 chambers), 7 days (4 chambers) and 8 days (2 chambers). In a similar fashion pieces of ZGPY1 t u m o r tissue were implanted into hamster cheek p o u c h chambers and the membranes with the t u m o r removed at 3 days (2 chambers), 7 days (6 chambers), 8 days (4 chambers), 10 days (2 chambers) and 13 days (1 chamber). Transparent chambers were inserted into 2 animals and no t u m o r transplanted. These were kept for 4 days and 12 days. There was almost no change in the pattern of the venous and arterial arcades o f the membrane. After the animals were killed, the pouch membranes were processed and examined by light microscopy to provide normal control specimens. During the experimenta! period 2 chambers became partially eroded out of the p o u c h and these animals were p r o m p t l y killed. Two further chambers became severely infected and the preparations were unsuitable for examination. The rate o f growth o f the tumor transplants (Figs. 1 and 2) Every a t t e m p t was made to ensure that only 1 mm 3 of t u m o r tissue was transplanted onto each chamber. There is general uniformity of the growth rate o f each t u m o r transplant within its own group. As can be seen b y comparing Figs. 1 and 2, the A-Mel-4B32 t u m o r grew much faster than the ZGPY1 tumor. The former grew rapidly after a lag period of only 2--3 days and usually covered 50% of the viewing area in 3--4 days. The ZGPY1 t u m o r had a longer lag period which usually was 3--4 days and even after that did not cover more than 30% of the viewing area 8 days after transplantation. The graphs are based on the fact that the tumors grow as even plates extending out from the transplantation site, so that an increment in the mass of the t u m o r is represented by the increase in the area occupied b y the t u m o r within the viewing area multiplied b y the thickness of the t u m o r 'plate'. This is the distance between the coverslip o f the t o p ring of the chamber and the dorsal surface of the pouch membrane. The 'viewing area' is the well of the chamber and is a circle of. 18 mm diameter. The tumors did not c o m m e n c e growing until blood was observed flowing through them. Static blood in capillary sprouts in the t u m o r transplants was observed a day before the sprouts contained flowing blood. The extent of the t u m o r cell plate became obvious following vascularization because of the red cell content of the capillaries. The microvascular pattern o f the cheek pouch o f the host and its variation in the presence o f a growing tumor (Fig 3) The pattern of the arteriolar loops remained constant. The walls o f the arte-
114
Fig. 3. The i n c o r p o r a t i o n o f a major vein of the viewing area in the advancing edge o f the t u m o r (ZGPY1). A m a j o r venous arcade on the p o u c h m e m b r a n e ( V A ) is shown in Fig. 3a close to the finely vascular edge (E) o f the t u m o r transplant (T). This is 7 days after transp l a n t a t i o n o f 1 m m s of t u m o r substance o n t o the central area o f the p o u c h . Fig. 3b is 8 days after transplantation and shows the i n c o r p o r a t i o n of the venous arcade (VA in Fig. 3a) i n t o the edge (E) o f the t u m o r and t h e great d i m i n u t i o n of direct through flow in this a r c a d e Alternative p a t h w a y s have o p e n e d up in area C. The venous arcade labelled B remains constant. T w o arterioles A 1 and A 2 are constant in position. Fig. 3a, X 6.75; Fig. 3b, X 6.
115
rioles remained parallel and the bifurcation pattern remained the same as it was on the day of transplantation. Some increase in width was noted in some arterioles which appeared to supply the capillary bed of the tumors. The arteriolar pattern of the chamber actually became clearer in some instances after the t u m o r had almost completely covered the viewing area (in A-Mel-4B32 tumors). Thin capillaries containing rapidly flowing blood were present in the t u m o r tissue overlying the arterioles. This had the effect of making the t u m o r tissue less opaque than when dilated venous sinusoidal vessels were present over the arterioles. These thin, rapidly flowing capillaries after a short length fed into the slowly flowing sinusoidal vessels. In contrast to the constancy of the arteriolar pattern, the pattern of the venous arcades and the flow of blood through the capillary modules of the p o u c h membrane, within the viewing area of the chamber, changed daffy with the growth o f the t u m o r transplants. We have reported this in an earlier paper [6]. An example of the incorporation o f parts of a major venous arcade of the chamber into the advancing edge of the t u m o r transplant is shown in Fig. 3. A segment of the venous arcade is incorporated into the tumor-edge venous vessels. Diminution of flow has occurred in this particular instance. Reversal o f flow in such vessels was c o m m o n as was an increase in flow once channels from the t u m o r itself flowed freely into the incorporated vein. Daily change in the venous pattern of the pouch membrane occurred and there were parallel changes in the extent of flow through various capillary networks. In Fig. 3 a dilatation of the neighboring capillary bed is evident following the diminution of flow through the incorporated vein. DISCUSSION
This paper presents in vivo observations on 2 transplantable tumors detailing the time from transplantation and the growth curve of the whole t u m o r following transplantation. The m e t h o d utilizing the hamster cheek pouch possesses a number of advantages for the close study o f the growth of the t u m o r transplant and its m e t h o d o f vascularization. The t u m o r grows as a thin,plate between the coverslips, and its vascularization is obvious because o f the red cell content of the blood vessels. In this technique the exact 'age' of the transplant is known, as well as the in vivo alterations of the vascular pattern surrounding the transplant f r o m the time of its introduction, and these are combined in a preparation that permits easy access for the preparation o f tissues for electron microscopy. The changes in the vascular architecture that ensue, following transplantation o f melanoma onto the cheek pouch, have been reported using a hand lens [ 2 ] . A state o f congestion o f the vascular pattern around the t u m o r graft was noted from the second d a y after transplantation. There was a peritumoral capillary engorgem e n t with occasionally small peripheral hemorrhages. From the 4th to the 6th d a y (but sometimes as late as the 8th day) the venous and capillary pattern was seen to enlarge and vascularization of the t u m o r became complete. From this time the t u m o r was n o t e d to grow regularly in the form of a spherical mass.
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The morphology of the melanoma vessels in thin, cleared sections of the tumors after opacification of the vascular system of the animal with carbon particles (India ink) was studied. Three zones were found: (1) a central zone of avascular necrosis; (2) a midzone which consisted of t u m o r cells interlaced b y vascular channels o f capillary t y p e which were thin at times and extensively anastomosed with each other. These were irregular and showed local dilations and tortuosities and they isolated cords of t u m o r cells. (3) A peripheral zone where vessels of large caliber surrounded the tumor. Our results described above are in agreement with these findings. In addition, it was possible to observe great alteration in the venous system of the pouch membrane and the t u m o r transplant after vascularization with growth of the t u m o r transplant. The arterioles of the pouch membrane remained in their original pattern and form. The vascular netw o r k o f t u m o r tissue implanted onto host tissue depends on (a) time from transplantation, (b) the nature of the response of the host tissue. However, the rate of growth does n o t effect the pattern of the vessels. ACKNOWLEDGEMENTS
The w o r k reported here was supported b y a grant from The National Cancer Institute of Canada and U.S. Public Health Service grant Nos. 5R31 CA-1476602. Our thanks are due to Dr. L. Malik, Miss L. Love and to Mr. W. Chauvin for assistance with the preparations and photography. REFERENCES 1 Chute, R.N., Sommers, S.C. and Warren, S. (1952) Heterotransplantation of human cancer. II. Hamster cheek pouch. Cancer Res., 12,912--914. 2 Delarue, J., Mignot, J. and Caulet, T. (1963) Modifications vasculaires de la poche jugale du hamster dor~ au cours du d~veloppement de greffes d'une tumeur m~lanique. C.R. Soc. Biol., 157, 69--71. 3 Greenblatt, M., Choudari, K.V.R., Sanders, A.G. and Shubik, P. (1969) Mammalian microcirculation in the living animal: Methodologic considerations. Microvasc. Res., 1, 420--432. 4 Handler, A.H. and Shepro, D. (1968) Cheek pouch technology: uses and application. In: The Golden Hamster, Its Biology and Use in Medical Research, p. 195. Editors: R.A. Hoffman, P.F. Robinson and H. Magalhaes. The Iowa State University Press, Iowa. 5 Sanders, A.G. and Shubik, P. (1964) A transparent window for use in the Syrian hamster. Isr. J. Exp. Med., 11,118 (abstract). 6 Warren, B.A. and Shubik, P. (1966) The growth of the blood supply to melanoma transplants in the hamster cheek pouch. Lab. Invest., 15,464--478.