Differential tumor growth of blood-borne B16 ... - Springer Link

CLIN. EXPL. METASTASIS,

1989, VOL. 7,

NO. 1, 1--14

D i f f e r e n t i a l t u m o r g r o w t h o f b l o o d - b o r n e B16 m e l a n o m a v a r i a n t s i n c e r e b r a l d u r a m a t e r is r e l a t e d to tumor-host cell reactions TAKANORI KAWAGUCHIt, MICHIKO KAWAGUCHI$, ' T H O M A S M . L E M B O w a n d G A R T H L. N I C O L S O N w 1 8 2 '~:Second Department of Pathology, and ~ First Department of Anatomy, Fukushima Medical College, Fukushima 960, Japan, and wDepartment of T u m o r Biology, The University of Texas System Cancer Center, M.D. Anderson Hospital and T u m o r Institute, Houston, Texas 77030, U.S.A.

(Received 23 November 1987, accepted 21 January 1988) Intracarotid injection of B16-B14b or B16-B15b melanoma cells, previously established from B16-F1 melanoma by in vivo selection fourteen- or fifteentimes, respectively, for brain surface colonization, preferentially produced tumor nodules in mice at brain surface sites, most frequently in the dura mater, followed by the leptomeninges and cerebral cortex. There was a marked difference, however, in tumor growth at these sites using the two B16 sublines. Intracarotid injection of B16-B14b cells rarely produced visible tumors, whereas B16-B15b cells formed deeply pigmented tumors up to 7ram in diameter in the brain meninges of almost all mice examined. Histologic and electron microscopic investigation revealed that B16-B14b tumors evoked dramatic immunocyte cell infiltration and granulomatous reactions, while B16-B15b tumors were accompanied by much less tumor-host cell reactions. Splenectomy or laparotomy 1-2 weeks before or after intracarotid injection of B16-B14b cells dramatically enhanced tumor growth in the dura mater without extensive tumor host cell reactions. The results suggest that the differential growth of B16-B 14b and B16B15b tumor cells in the cerebral dura mater is based, in part, on the abilities of these melanoma cells to elicit host cell reactions.

Introduction B r a i n m e n i n g e a l m e t a s t a s i s o c c u r s in c a r c i n o m a s , m e l a n o m a s a n d l e u k e m i a s [5, 17, 21]. T h e m e c h a n i s m s i n v o l v e d in m e n i n g e a l m e t a s t a s i s are l a r g e l y u n k n o w n , a n d the t h e r a p e u t i c o u t c o m e is u s u a l l y p o o r [6, 9, 25]. F o r the d e v e l o p m e n t of n e w t h e r a p e u t i c strategies, it m a y be n e c e s s a r y to e l u c i d a t e t h e t u m o r cell a n d h o s t p r o p e r t i e s i m p o r t a n t in m e n i n g e a l m e t a s t a s i s . T h i s will to s o m e d e g r e e d e p e n d on the d e v e l o p m e n t of a d e q u a t e t u m o r m o d e l s for m e n i n g e a l m e t a s t a s i s [1,7, 8, 13, 20, 22, 27]. W e have r e p o r t e d t h a t b r a i n - c o l o n i z i n g B16 m e l a n o m a v a r i a n t s u b l i n e s can be e s t a b l i s h e d f r o m B 1 6 - F 1 cells [3] b y in vivo selection for b r a i n surface t u m o r c o l o n y f o r m a t i o n [1]. A f t e r ' f o u r t e e n ( B 1 6 - B 1 4 b ) o r fifteen ( B 1 6 - B 1 5 b ) s e q u e n t i a l selections for b r a i n t u m o r f o r m a t i o n [13], the B16 v a r i a n t s s h o w e d i n c r e a s e d p r e f e r e n c e for c o l o n i z a t i o n of the d u r a m a t e r , l e p t o m e n i n g e s a n d c e r e b r a l c o r t e x [7]. I n a d d i t i o n , t h e r e was a m a r k e d difference in t u m o r g r o w t h in t h e b r a i n m e n i n g e s b e t w e e n t h e two v a r i a n t s u b l i n e s w h e n these cells w e r e i n j e c t e d into t h e c a r o t i d a r t e r y , a n d we 82To whom correspondence should be addressed. 0262-0898/89 $3'00 9 1989 Taylor & Francis Ltd.

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T. Kawaguchi et al.

suggested that this difference in tumor growth might be related to their differing abilities to elicit host response mechanisms [7, 8]. We show here that differential growth of B16-B14b and B16-B15b tumors in the cerebral dura mater was modulated by procedures that impaired host-response mechanisms.

Materials and methods

Animals Barrier-raised 4-6-week-old female C57BL/6 mice were certified Sendai virusfree and obtained from Charles River Co. (Wilmington, MA). The mice were received in filtrated cages and were quarantined for two weeks before use. All animals were maintained under guidelines established by the National Institutes of Health and the University of Texas System Cancer Center. For each experiment, the animals were age (6-8 weeks old) and weight (25-30 g) matched.

Cells The murine B16 melanoma sublines B16-B14b and B16-B15b were used [13]. T u m o r cells were maintained in vitro in Falcon tissue culture plates in Dulbeccomodified Eagle's medium ( D M E ) without antibiotics and containing 5 per cent fetal bovine serum and 1 per cent non-essential amino acids. Cells were grown in a humidified gas mixture of 5 per cent CO 2 and 95 per cent air at 37~ and were harvested by overlaying the cells with a calcium- and magnesium-free phosphate buffer solution containing 2 m M E D T A . Suspended, single cells were washed twice by centrifugation, resuspended in cold serum-free D M E at a concentration of 105 cells/ml, and stored at 0~ until use. T u m o r cells were used within ten passages from frozen stocks to eliminate phenotypic drift [13]. Animals were plaeed under mild anesthesia by inhalation of Metofane, and they were injected with the single cell suspension of tumor cells in serum-free D M E . For the intracarotid, injection was performed as follows: the left carotid artery was carefully separated from the N. vagus and other tissues and ligated with thread. Within 30 seconds after ligation 0"2 ml of tumor cell suspension was injected into the carotid artery using a 27-gauge needle.

Histological examination Animals were killed at intervals of 3, 7, or 14 days after the intracarotid injection of B16-B14b or B16-B15b cells, or they were autopsied at the time of death (16 28 days post-injection). T h e brain and other major organs of each animal were fixed in Bouin's solution (brain) or buffered formalin solution for the other organs, respectively, and prepared for histological examination by standard methods.

Electron microscopy B16-B14b or B16-B15b cells (1 • 105 cells per 0"2ml inoculum) were injected into the carotid artery of sex, age, or size matched groups of mice. At 3, 7, or 14 days after injection, the animals were placed under slight Metofane anesthesia, whereupon they received a tail vein injection of heparin solution (0"1 ml, 100 units). T w o to three minutes later, the animals were perfused with Ringer's solution via left ventricle to inferior cava vein for 2rain and then perfused with 1 per cent glutaraldehyde in cacodylate buffer, p H 7"34, using the same route for an additional 2 min. After perfusion, the brains were removed as soon as possible, and they were

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p l a c e d in c o l d (4~ m o d i f i e d K a r n o v s k y ' s fixative. A f t e r fixation for 1 h, t h e b r a i n s w e r e c a r e f u l l y w a s h e d in c o l d 0"1 M c a c o d y l a t e buffer, a n d the d u r a m a t e r was carefully removed. T h e d u r a m a t e r was cut into 2 m m o r s m a l l e r m e m b r a n e s t r i p s a n d fixed again for a n o t h e r 1"2-1"5 h at 4~ A f t e r r i n s i n g in c o l d c a c o d y l a t e buffer t h r e e t i m e s for 10 m i n each, t h e tissues were p o s t - f i x e d in 2 p e r cent o s m i u m t e t r o x i d e in t h e s a m e buffer for 1'2 h at 4~ A f t e r r i n s i n g again in c a c o d y l a t e buffer, t h e tissue f r a g m e n t s w e r e s t a i n e d w i t h 4 p e r cent u r a n y l acetate in 50 p e r c e n t e t h a n o l for 5 m i n at r o o m t e m p e r a t u r e , d e h y d r a t e d in a g r a d e d series o f e t h a n o l , a n d e m b e d d e d in S p u r resin. U l t r a t h i n sections (600 800/.tm thick) w e r e s t a i n e d w i t h 2 p e r c e n t u r a n y l acetate a n d lead citrate a n d were o b s e r v e d in a H i t a c h i M o d e l H U - 1 2 t r a n s m i s s i o n e l e c t r o n microscope.

Splenectomy and laparotomy S p l e n e c t o m y a n d l a p a r o t o m y were p e r f o r m e d u n d e r light M e t o f a n e a n e s t h e s i a . T h e a n i m a l s r e c e i v e d an i n t r a c a r o t i d i n j e c t i o n o f B 1 6 - B 1 4 b or B 1 6 - B 1 5 b cells (5 x 104 cells) one to two weeks before, one or t w o weeks after s p l e n e c t o m y , a n d one w e e k after l a p a r o t o m y .

Results

Incidence and gross appearance of brain meninges tumors W h e n the a n i m a l s were k i l l e d 3 or 7 d a y s after i n t r a c a r o t i d i n j e c t i o n o f B 16-B 14b or B 1 6 - B 1 5 b cells (5 • 104cells p e r 0"2ml i n o c u l u m ) , d i s t i n c t t u m o r s w e r e n o t o b s e r v a b l e in any organs, i n c l u d i n g the b r a i n a n d b r a i n m e n i n g e s . H o w e v e r , g r o s s t u m o r s were f o u n d in the a n i m a l s k i l l e d 14 d a y s after i n j e c t i o n a n d in t h o s e t h a t d i e d b e c a u s e of gross t u m o r s 16-18 d a y s p o s t - i n j e c t i o n . I n t h e s e a n i m a l s , t u m o r s w e r e a p p a r e n t in the brain, b r a i n m e n i n g e s a n d lungs. T h e b r a i n m e n i n g e a l t u m o r s caused b y B 16-B 14b cells were quite small (less t h a n 1 m m d i a m e t e r ) , u s u a l l y n o n - p i g m e n t e d , a n d difficult to i d e n t i f y w i t h the u n a i d e d eye. H i s t o l o g i c e x a m i n a t i o n of o v e r one h u n d r e d t h i n sections p e r b r a i n r e v e a l e d t i n y

Figure 1. A grey-white subdural tumor is visible (arrow) 14 days after intracarotid injection of B16-B14b melanoma cells. This was the only visible meningeal tumor in the B 16-B14b group. The animals were killed 14 days after intracarotid injection of B16B14b melanoma cells. B a r = 0"2 cm.

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T. Kawaguchi et al.

Figure 2. Two large, deep-black, pigmented tumors are visible in this brain of an animal that died 16 days after intracarotid injection of B16-B15b melanoma cells. Bar=0"2 cm.

tumors in eight of fourteen animals examined (figure 1). T h e intracarotid injection of B16-B15b cells produced large (up to 7 - 1 0 m m diameter), well-defined, deeply pigmented tumors in the dura mater in all animals examined (figure 2). T h e average n u m b e r of B16-B14b and B16-B15b, gross brain meningeal tumors was 0 and 1"8 (range 1-4), respectively. T h e y were found mainly in the dura mater or subdural space covering the bulbus olfactorius, left cerebral hemisphere, fissura transverse cerebri, cerebellum and cisterna interpeduncularis. T h e tumors formed by B16B15b cells caused depression of the brain; alternatively, some of these tumors appeared to grow laterally on the brain surface and invaded into the brain parenchyma. B16-B15b tumor in the cisterna interpeduncularis depressed the cranial nerves and sometimes involved the pituitary gland.

Histologic and electron microscopic observations Most of the brain meningeal tumors formed by B16-B14b cells contained relatively few melanoma cells and extensive infiltrations of lymphoid cells and fibrous elements (figure 3). These tumors infrequently invaded the brain parenchyma or the skull. In contrast, the tumors formed by B16-B15b cells were constituted predominantly of B16 melanoma cells with slight infiltrations of lymphoid cells and abundant vascularization (figure 4). T h e B16-B15b tumors in the dura mater usually extended into the subdural space and depressed the brain. These subdural tumors were covered, in large part by a meningothelial cell lining (figure 4), and this same covering was obscure and difficult to recognize in the B16-B14b tumors. Electron microscopic observations on the implantation and invasion of B16B14b and B16-B15b cells in the dura mater appeared to be similar to the histologic findings. T u m o r cells proliferated expansively or migrated into the dural tissue by infiltrating through collagen bundles and cellular components of the dura mater until reaching the meningothelial cell linings (figures 5 and 6). T h e y breached this structure and caused cellular retraction, probably by extension of t u m o r cell

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Figure 3. Histology of subdural B16-B14b t u m o r shown in figure 1. T h e t u m o r contained extensive granulomatous tissue; a few m e l a n o m a cells can be seen in the periphery of the tumor (arrow, inset), x 100 (inset, x 250).

Figure 4. Histology of subdural B16-B15b t u m o r shown in figure 2. T h e t u m o r was welldefined by the meningothelial cell lining (arrows, inset), x 100 (inset, x 225).

Figure 5. A B16-B14b cell appears to be migrating in the dura mater 14days after intracarotid injection. SDS, subdural space. • 2800.

Figure 6. Growing foci of B16-B15b cells in the dura mater 14 days after intracarotid injection of tumor cells. Some melanoma cells have migrated through adjacent collagen bundles (arrows). x 2800.

~q

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cytoplasmic processes or protrusions, and eventually, some of the t u m o r cells migrated to subdural positions (figures 7 and 8). Defects in the meningothelial cell linings caused by invading tumor cells appeared to be repaired by neighboring meningothelial cells. During the process of intradural or subdural t u m o r growth, host cellular reactions were observed. I m m u n o c y t i c and granulomatous reactions were usually distinct in or around the B16-B14b t u m o r nodules. Except in rare cases, the B16B14b tumors were accompanied by extensive immunocyte cell infiltrations, including lymphocytes, plasma cells and monocytes (figure 9), and some of the tumors were completely surrounded by granulomatous tissue. L y m p h o c y t i c responses against the B16-B14b cells appeared to cause some degeneration and necrosis of tumor cells. In contrast, the B16-B15b tumors were accompanied by much less infiltration of host cells, and granulomatous reactions were rarely found in these tumors (figure 10). The covering of subdural tumor nodules by meningothelial cells was easily observed in the B16-B15b tumors, but this was obscure in the B16B14b tumors, though partial extension of meningothelial cells into the tumors was occasionally observed.

Figure 7. Two B16-B15b melanoma cells (T) are seen between the meningothelial cell lining (M) and underlying connective tissue (C). The meningothelial cell lining is retracted at the arrow, x 2100.

Figure 8. Apparent migration of a melanoma cell (T) into a subdural position (S) 14 days after intracarotid injection of B 16-B 15b cells. A defect in the meningothelial cell lining caused by an invading tumor cell appears to be covered by a cytoplasmic process from a neighboring meningothelial cell (arrow). x 3000.

Figure 9. A subdural t u m o r seen 14days after intracarotid injection of B16-B14b cells. T w o t u m o r cells (T) surrounded by several l y m p h o i d cells. T h e m e l a n o m a cells contain a few melanosomes. M C , meningothelial cell. x 3800.

Figure 10. A subdural t u m o r seen 14days after intracarotid injection of B16-B15b cells. T h e m e l a n o m a cells contain melanosomes, especially at their cell peripheries. S o m e of the t u m o r cells at the front of the dural tissue are in close contact with a meningothelial cell (M) (single arrow) or submeningothelial connective tissue (C) (two arrows). A few l y m p h o i d cells are visible around but not in the growing t u m o r foci. • 3100.

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Effect of splenectomy and laparotomy on meningeal tumor growth S p l e n e c t o m y p e r f o r m e d o n the a n i m a l s b e f o r e or after i n t r a c a r o t i d i n j e c t i o n of t u m o r cells d r a m a t i c a l l y e n h a n c e d b r a i n m e n i n g e a l t u m o r g r o w t h of B 16-B 14b cells (table 1 a n d figures 11 a n d 12). O f t h e s p l e n e c t o m y e x p e r i m e n t s , one w e e k after t u m o r cell i n j e c t i o n was m o r e effective; six o f n i n e a n i m a l s e x a m i n e d h a d large, p i g m e n t e d t u m o r s in t h e b r a i n m e n i n g e s (figure 11). I n b o t h s p l e n e c t o m y g r o u p s (1-2 weeks b e f o r e a n d 2 weeks after t u m o r cell i n j e c t i o n ) sizeable m e n i n g e a l t u m o r s w e r e f o u n d in t h r e e of eight a n d f o u r of e l e v e n a n i m a l s , r e s p e c t i v e l y . O b s e r v a b l e d u r a l t u m o r n o d u l e s were also f o u n d in the l a p a r o t o m y g r o u p (three o f seven a n i m a l s ) (table 1).

Figure 11. A large, deeply pigmented subdural tumor and two small, black tumors are seen in the brain of an animal (splenectomized one week before tumor cell injection) that died 19 days after intracarotid injection of B16-B14b cells. Bar=0'25 cm.

Figure 12. A large, deeply pigmented brain surface tumor in an animal (splenectomized one week after tumor cell injection) that died 21 days after intracarotid injection of B16B14b cells. Bar = 0-2 cm.

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T a b l e 1.

T. Kawaguchi et al.

Effect of splenectomy

Splenectomy or laparotomy before/after i.c. injection of tumor cells Untreated animals Splenectomized animals 1-2 weeks before 1 week after 2 weeks after Laparotomized animals 1 week after

or l a p a r o t o m y o n t u m o r g r o w t h o f B16-B14b c e l l s i n t h e d u r a m a t e r a.

No. of brain surface tumors per mouse b

Median (range)

No. of lung surface tumors per mouse b

Median (range)

0,0,0,0,0,0,0,0 0,0,0,0,0,0,0,0 (0/14)

0 (0-0)

0,0,0,0,0,0,0 0,0,0,0,0,0,0 (0/14)

0 (0-0)

0,0,0,1,2,6,0,0 0,0 (3/8) 0,0,0,1,1,1,1 2,2 (6/9) 0,0,0,0,0,0,0 1,1,3,6(4/11)

1"12 (0-6) 0'9 (0-2)

0,0,0,0,0,1,3 10 (3/8) 0,0,0,0,0,1,2 3,4 (4/9) 0,0,0,0,0,0 3,3,58 (4/11)

1'75 (~10) 1-1 (0-4) 5"9 (0-58)

0,0,0,0,1,1,1 (3/7)

0.4 (0-1)

0,0,0,0,0,0,3 (1/7)

0-4 (0-3)

1"0

(0-6)

a A 0"5 ml of tumor suspension containing 5 x 104 B 16-B 14b cells was injected into the left carotid artery. bThe counting of tumor nodules by unaided eye was performed 16-28 days after intracarotid injection.

Figure 13. Histology of the tumor seen in figure 12. A well-defined subdural tumor has compressed the brain. About half of the tumor is necrotic. D, dura mater; L, leptomeninges. • 20.

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Figure 14.

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Perivascular tumor cell infiltration is seen in a splenectomized animal that died 18 days after intracarotid injection of BI6-B14b cells, x 75.

T h e location and gross appearance of the B16-B14b meningeal tumors after splenectomy or laparotomy resembled those of B16-B15b tumors. T h e y were usually well defined from the surrounding tissues of the brain and skull (figure 13), but marked perivascular tumor infiltration was sometimes found in the brain (figure 14). I m m u n o c y t i c cell infiltrations and granulomatous reactions were dramatically reduced in these tumors (figures 13 and 14). Observable t u m o r nodules in the lung also increased in the splenectomized or laparotomized animals, but tumors were not found in other organs (table 1).

Discussion T h e present experiments demonstrate that the murine melanoma variant sublines B16-B14b and B16-B15b, which were established by sequential in vivoselection for brain surface t u m o r colony formation from B16-F1, had differing potentials to form brain meninges tumors. Intracarotid injection of B16-B14b cells rarely formed visible tumors, while injection of B16-B15b cells produced deeply pigmented, meningeal tumors up to 7 m m in diameter. A m o n g the possible explanations for the increased ability of selected B16 cell lines to form metastatic nodules in certain organs [16,24,28] three have been proposed as extremely important: (1) an increased rate of t u m o r cell implantation in the target organ [16, 18]; (2) a decreased death rate of the tumor cells arrested in the target organ [18, 24, 28]; and (3) an increased growth response of t u m o r cells in the target organ [17]. In the case of B16-B14b and B16-B15b, both sublines possessed the ability to colonize brain meninges because several tiny B16-B14b dural tumors were detected histologically. This suggests that the difference between these sublines is not at an early stage of implantation, such as the lodgement and extravasation of tumor cells,

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but is related to the growth of extravasated tumor cells in the dura mater. Distinct differences in implantation and invasion between these sublines were not found, but they did differ in their growth properties in vivo. One of the possible causes of differential tumor growth in the dura mater between the two B16 sublines that we observed was different host cell reactions towards these tumors. In B16-B14b tumors, immunocyte cell infiltrations with slight granulomatous reactions usually occurred, whereas such host reactions were rare in B16-B15b tumors. This difference suggests that the growth properties of the brain meningeal tumors were related to elicitation of host-response mechanisms. In support of this possibility, Miner et al. [12, 14] recently showed that the brainselected B16 melanoma sublines were more resistant to cytotoxic and cytostatic effects of activated syngeneic macrophages. Our splenectomy and laparotomy experiments also suggest that host defense mechanisms might be involved in restricting tumor growth in the dura mater. Fisher and Fisher [4] found that surgical trauma resulting in liver injury had a profound effect on the growth of hepatic metastases. Since operative trauma is known to suppress host defense mechanisms, especially immune responses, laparotomy could have resulted in an enhancing effect on B16-B14b brain meningeal tumor growth. T h e effect of splenectomy on tumor growth has been controversial; some investigators have proposed an enhancing effect of splenectomy on tumor growth, while others have demonstrated an inhibitory effect of splenectomy [2, 11]. Recent studies indicate that effect of splenectomy on tumor growth is dependent on the time of splenectomy (prior to or after tumor cell inoculation), the number of tumor cells inoculated, and the immunogenicity of the tumor cells [22, 23]. T h e condition of the spleen cells as effector and suppressor cells and the time of splenectomy are also considered important in determining the effect [23]. Therefore, it is likely that the enhancing effect of splenectomy on brain meningeal tumor growth of B16-B14b tumors may be due to the removal of effector cells or appearance of circulating suppressor cells. We also noticed the covering of subdural tumor nodules by meningothelial cells. This was found usually in B16-B15b tumors and rarely in B16-B14b tumors. T h e covering of tumors by meningothelial cells resembles endothelialization of tumor cells arrested in blood vessels [10], enclosure of tumor cells by endothelial cells [26], and their covering by mesothelial cells [15]. T h e enclosure of tumors by mesenchymal cells and its possible effect on tumor growth is unclear, although it has been suggested that this process might protect the tumors against host defense mechanisms [10, 15]. Finally, the brain meningeal cells may release specific growth factors upon injury that could be important in stimulating the growth of B16 cells [19].

Acknowledgments We thank K. M. Dulski for technical assistance. These studies were supported by U.S. National Cancer Institute grant R35-CA44352(OIG) and a grant from the Meadows Foundation to G. L. Nicolson.

References [1] BRUNSON,K. W., BEATTIE,G., and NICOLSON,G. L., 1978, Selection and altered tumor cell properties of brain-colonizing metastatic melanoma. Nature (London), 272, 543-545.

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