J N e u r o s u r g 7 2 : 1 0 2 - 1 0 9 , 1990
Adoptive immunotherapy of intracerebral metastases in mice IAN E.
McCuTCHEON, M . D . , RACHEL A. BARANCO,B.S., DAVID A. KATZ, M . D . , AND
STEPHEN C. SARIS, M . D . Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland us Lymphokine-activated killer (LAK) cells are a heterogeneous population of immune effector ceils that nonspecifically destroy neoplastic cells but not normal ceils. Although parenteral treatment with interleukin-2 (IL-2) alone or a combination of IL-2 and LAK ceils reduces tumor load and prolongs survival in mice with pulmonary, peritoneal, or hepatic metastases, the effect of these treatments on brain metastases has not been studied. To determine in an animal model if intracerebral metastases would be protected by the immunologically privileged status of the brain, intracardiac and intravenous injections of 105 KHT sarcoma cells were performed in C3H mice to create brain and lung metastases, respectively. The mice were treated with adoptive immunotherapy to determine if efficacy seen in an extracerebral site could be reproduced in the brain, and if histological examination of these brains would reveal a significant degree of lymphocyte infiltration and cytolytic activity. Animals were treated with either parenteral IL-2 (7500 U three times daily on Days 3 to 7 after tumor injection), or IL-2 plus LAK cells (7500 U IL-2 three times daily on Days 3 to 7, and 108 LAK cells intravenously on Days 3 and 6 after tumor injection), or IL-2 excipient (three times daily on Days 3 to 7 after tumor injection). As compared to control animals, pulmonary metastases on Day 14 after tumor injection were reduced or eliminated in animals treated with either IL-2 or IL-2 plus L A K cells (p < 0.01). In these same animals, there was no reduction in the number of intracerebral metastases and no evidence of lymphocytic infiltration or cytolytic activity in the brain. This is the first study that reveals an organ-specific resistance to the treatment of metastases with adoptive immunotherapy, and affirms the concern that due to inadequate trafficking of endogenous or exogenous-activated lymphocytes or due to inadequate activation of in situ brain lymphoid precursors, there is no rejection of tumors in the brain. This information suggests that brain metastases in patients with systemic malignancies will not respond to intravenous treatment with LAK cells and IL-2, and that alternative forms of treatment are needed. Furthermore, this modification of a previously existing model of murine brain metastasis provides a method for concurrently evaluating the effectiveness of treatments for intra- and extracranial cancers. KEY W O R D S
~
brain neoplasm
lymphokine-activated
B
killer cell
9 metastasis
RAIN m e t a s t a s e s o c c u r in a p p r o x i m a t e l y 15% o f p a t i e n t s with e x t r a c r a n i a l cancer, 35 a n d are t r e a t e d b y r a d i o t h e r a p y a l o n e o r in c o n j u n c t i o n with surgical excision. Even w i t h t r e a t m e n t , however, the m e d i a n survival t i m e is o n l y 6 m o n t h s , a n d signifi c a n t m o r b i d i t y a c c o m p a n i e s b o t h a p p r o a c h e s . Furt h e r r n o r e , c h e m o t h e r a p y is generally ineffective. L y m p h o c y t e s t h a t d e s t r o y fresh a u t o l o g o u s t u m o r cells b u t n o t n o r m a l cells c a n be g e n e r a t e d b y incubating h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s w i t h the lymp h o k i n e i n t e r l e u k i n - 2 (IL-2). T h e s e c y t o t o x i c effector 102
9 immunotherapy
9 interleukin-2
9
9 mouse
( l y m p h o k i n e - a c t i v a t e d killer, or L A K ) cells express some o f the surface antigens o f natural killer ( N K ) cells a n d classic T l y m p h o c y t e s , 1,9,~~ b u t a t t a c k N K cellresistant targets n o t restricted b y a m a j o r h i s t o c o m p a t ibility c o m p l e x . ~7 W h e n a d o p t i v e l y transferred to tum o r - b e a t i n g m i c e , L A K cells given with IL-2 r e d u c e o r e l i m i n a t e m e t a s t a s e s in several sites, i n c l u d i n g t h e liver, lung, a n d p e r i t o n e a l c a v i t y ) 3.~8,2~ This t h e r a p y is effective against a n u m b e r o f different t u m o r s i n c l u d i n g adenoearcinoma, melanoma, and sarcoma, and works equally well in different strains o f mice.~ 1 I n t e f l e u k i n - 2
J. Neurosurg. / Volume 72/January, 1990
Immunotherapy of murine brain metastasis alone can decrease t u m o r load in those sites, 13'17'18'2z'26 whereas L A K cells alone have relatively little effect.13,18.21 Despite extensive studies of the effectiveness of immunotherapy against tumors in extracranial sites, the effect of L A K cells and IL-2 on metastatic tumors in the brain has not been evaluated. It is known that the administration of IL-2 to mice results in the proliferation and activation of host lymphoid elements and donor L A K cells in the brain, 6,7 and that IL-2 crosses the blood-cerebrospinal fluid (CSF) barrier and perhaps the blood-brain barrier. 30However, it is unclear whether exogenously administered L A K cells travel to targets in the brain, and whether tumors in the brain respond to i m m u n o t h e r a p y with L A K cells and/or IL-2 differently from tumors elsewhere in the body. The effectiveness of L A K cells and IL-2 against intracerebral metastases has relevance to understanding i m m u n e responses within the central nervous system and to improving the current treatment of metastatic brain tumors. Patients with cerebral metastases are currently excluded from therapy with IL-2 because treatment-induced increases in tumoral and peritumoral vessel permeability m a y cause neurological deterioration due to increased cerebral e d e m a ? '2v If successful treatment were accomplished in an animal model, the criteria for admission to clinical protocols that use L A K cells and IL-2 to treat patients with disseminated cancer might be broadened. For these reasons, an animal model of metastatic brain cancer was used to evaluate concurrently the effectiveness of administering IL-2 and L A K plus IL-2 against tumors in the brains and lungs of C 3 H mice. In this way, the effect of therapy was studied in two sites within a single animal, of which one was immunologically privileged and one was not. Materials and Methods
Animals and Tumor Cells Female C 3 H / H e N mice, aged 10 to 12 weeks, were used in all experiments.* A K H T sarcoma,t a weakly immunogenic cell line that arose spontaneously in a C3H mouse, was used in these experiments. All tumors were cultured in complete m e d i u m comprising RPMI1640 with 10% fetal calf serum, 0.05 m M 2-mercaptoethanol, 0.l m M nonessential a m i n o acids, 0.1 lzM sodium pyruvate, L-glutamine (300 ug/ml), penicillin (100 U/ml), streptomycin (100 ug/ml), gentamicin sulfate (50 ug/ml), and amphotericin (0.25 ug/ml). Cells were incubated in flasks at 37~ in an atmosphere of 5% CO2, grown to confluence, and harvested using standard trypsinization techniques.
* Animal breeding colonies provided by National Institutes of Health, Hazelton, Maryland. t KHT sacroma generously provided by Dr. Frances Conley, Stanford, California.
J. Neurosurg. / Volume 72/January, 1990
Animal Preparation and Tumor Model Cerebral metastases were created by intracardiac injection of a t u m o r cell suspension. 2 After induction of anesthesia with methoxyflurane, the anterior thoracic wall was exposed and a No. 27 needle was introduced through the second intercostal space into the left ventricle. K H T cells (105 cells in 0.2 ml H a n k s ' balanced salt solution (HBSS)) were injected as a bolus. Pulmonary metastases were created by tail-vein injections of 105 K H T cells in 0.5 ml HBSS. To determine the reproducibility o f the brain-tumor model and the time at which animals should be sacrificed in later experiments, two groups of animals (14 in the first group and 13 in the second group) were given intracardiac injections of 105 K H T cells and followed until death. Two additional groups of animals (25 in the first group and 17 in the second group) were given intracardiac injections of 103 K H T cells and sacrificed after 14 days to determine the percentage o f animals that developed intracerebral metastases. A final group of 20 animals was given an intravenous injection of l05 K H T cells to confirm growth in the lung. Preparation of L A K cells The spleens were removed aseptically f r o m retired C 3 H breeder mice and crushed in HBSS. The resulting cell suspension was filtered through a Nitex mesh.~ After osmotic lysis o f the erythrocytes by A C K lysing bufferw and further washing with HBSS, the splenocytes were placed in flasks containing complete m e d i u m (5 x l08 cells/175 ml) and IL-2 (10 3 U/ml). The highly purified recombinant IL-2II used in these trials was produced in Escherichia coli transfected with a gene for IL-2 isolated from the Jurkat cell line. 24 After incubation in an atmosphere of 5% CO2 at 37"C for 72 hours, the cells were passed through a gradient of Lympholyte-M,* washed, and resuspended in HBSS at l08 cells/ml for intravenous injection. Effect of L A K Cells Against K H T In Vitro In short-term chromium-release assays, L A K cells were tested against fresh and cultured K H T targets. Fresh t u m o r targets were grown in the subcutaneous space of C 3 H mice. Two weeks after subcutaneous inoculation o f l05 K H T cells, palpable t u m o r s were excised, minced, and digested in a solution o f collagenase, hyaluronidase, and deoxyribonuclease. The cell suspension was filtered through Nitex mesh, washed in HBSS, and resuspended in complete m e d i u m . These fresh cells were used as targets in chromium-release Nitex mesh manufactured by Lawshe Industrial Co., Bethesda, Maryland. wMedia kit manufactured by National Institutes of Health, Bethesda, Maryland. UIL-2 kindly supplied by the Cetus Corporation, Emeryville, California. * Lympholyte-M supplied by Cedarlane Laboratories, Hornby, Ontario, Canada. 103
I. E. McCutcheon, et al. assays performed in triplicate. Targets (5 • 10 6 cells) were labeled by incubation with 400 lzCi of Na[5'Cr]O4 for 2 hours. Effector cells (either L A K cells or splenocytes) were mixed with washed labeled target cells in effector:target cell ratios of 100:1, 25:1, and 6.25:1, and incubated for 4 hours. To test m a x i m u m lysis, 0.1 N HC1 was added to labeled target cells; spontaneous lysis was tested by adding cell-free complete medium. The plates were then spun at 175 G and the supematants collected with a Skatron collection system.~ Filters containing the supernatants were counted in a g a m m a counter. The percentage of specific lysis was calculated by the formula: 100(experimental c p m - spontaneous cpm) m a x i m u m cpm - spontaneous c p m Chromium-release assays were performed in the same manner, with K H T harvested from culture flasks by trypsinization.
Effect of L A K Cells and~or IL-2 Against K H T
In Vivo In all experiments, animals were prepared with tumor as described below, and then r a n d o m l y assigned to one of three groups (Fig. 1). G r o u p 1 (IL-2 alone) received an intraperitoneal injection of 7500 U IL-2 three times daily from Days 3 to 7 inclusive after t u m o r injection. G r o u p 2 was injected intraperitoneally with 7500 U IL2 three times daily on Days 3 to 7 after t u m o r injection, and also received an intravenous injection of 108 LAK cells on Days 3 and 6 after t u m o r injection. Group 3 (control) was treated with an intraperitoneal injection of 0.5 ml IL-2 excipient three times daily on Days 3 to 7 after t u m o r injection. This treatment schedule was chosen to allow comparison with experiments performed with extracranial metastases. 13'17'18,23 Furthermore, this schedule yields IL-2 serum levels and biological effects that are comparable to or greater than those seen with intravenous therapy. 8 Three treatment experiments were performed, and each was repeated once (Fig. 1). Experiment 1 assessed treatment efficacy in animals with p u l m o n a r y tumors. Twenty-four animals received a tail-vein injection of 105 K H T cells; this experiment was repeated with 21 animals. Experiment 2 assessed treatment efficacy in animals with brain tumors. Eighteen animals received an intracardiac injection o f 105 K H T cells; this experim e n t was repeated with 28 animals. Experiment 3 assessed treatment efficacy in animals with brain and lung tumors. On the same day, 22 animals received intracardiac and intravenous injections of 105 K H T cells; this experiment was repeated with 24 animals. In each experiment, after preparation with tumor, the animals were randomly assigned to a treatment group, treated, and sacrificed after 14 days for quantitation of brain and/or lung metastases. t Skatron collection system manufactured by Skatron, Inc., Sterling, Virginia. 104
Quantitation of Brain Metastases Immediately after sacrifice, the brains were removed and frozen in isopentane (-70~ Coronal sections (20 um thick) were cut on a cryomicrotome at -16"C. Starting in the anterior frontal cortex and ending in the posterior temporal cortex, six sequential sections were taken from each brain at 700-urn intervals. The sections were m o u n t e d on glass slides, air-dried, fixed in a solution o f a m m o n i u m bromide (2%) in 6% formalin, and stained with hematoxylin and eosin. The sections were photographed through a green filter (for high contrast) and enlarged (• 10). Metastases were counted from the photographs in blinded fashion with the assistance of a magnifying glass. If more than 250 metastases were present in the six sections, the number of brain metastases in that animal was assigned a value of 250.
Quantitation of Lung Metastases Immediately after sacrifice, the animals' lungs were insuffiated by intratracheal injection of 15 % India ink and placed in Fekete's solution. 34 Metastases (visible as distinct white nodules on the black background of normal lung parenchyma) were counted on all external lobar surfaces with the assistance of a magnifying glass. If more than 250 metastases were present, the number of lung metastases in that animal was assigned a value of 250.
Pathology All brains and lungs in Experiment 3 were evaluated in blinded fashion. All sections were reviewed by the same pathologist (D.A.K.) with attention given to the metastatic lesions, as well as to the noninvolved parenchyma. A semiquantitative estimate was m a d e of the number o f lesions present in a given brain section (few: _< 25 lesions; moderate: 25 to 50 lesions; many: > 50 lesions). The lungs were evaluated in a similar fashion. Group |
I
IL2
Group 2
I
IL2 § LAK
I
Group 3
I
excipient
I
5
4
DAYS
I
T
2
TUMOR
T
S
T
]
6
TREATHENT
7
T
8
9
I0
II
12
13
14
T
SACRIFICE
experiment 1 : | ( ~ c e l ] s iv experiment 2 : 11~cel|s ic experiment 3 : 105cells ic and iY
FIG. 1. Experimental protocol. On Day 1, animals were prepared with tumor and then randomly assigned to one of three treatment groups: interleukin-2 (IL-2) alone, IL-2 plus lymphokine-activated killer cells (IL2 + LAK), and control (IL-2 excipient). On Day 14, the animals were sacrificed for quantitation of tumors. In Experiment 1, intravenous (iv) tumor injections were performed to create pulmonary metastases; in Experiment 2, intracardiac (ic) tumor injections were performed to create brain metastases; and in Experiment 3, both intravenous and intracardiac injections were performed in each animal.
J. Neurosurg. / Volume 72/January, 1990
Immunotherapy of murine brain metastasis Statistical Analysis
Immunotherapy of Brain Metastases
Data from the chromium-release assays were compared to Student's two-tailed t-test. Counts of metastases from the treatment experiments were compared using the nonparametric Kruskal-Wallis test statistic.
In Experiment 2, animals given intracardiac injections of t u m o r showed no response to treatment with either IL-2 alone or L A K cells plus IL-2. There was no difference between the number of brain metastases in animals treated with IL-2 and in control animals (p = 0.44), or between animals treated with L A K cells plus IL-2 and control animals (p -- 0.19). The mean n u m b e r of brain metastases in the IL-2-treated group, the IL-2 plus L A K cell-treated group, and the control group was 203, 237, and 193, respectively. This experiment was repeated once with similar results 9The combined data from the two experiments are shown in Fig. 4.
Results
Tumor Model Mice given 105 cells by intracardiac injection died on Days 13 to 20; median survival was 17 days (Fig. 2). Greater than 200 intracerebral metastases were present in 19 (76%) of the first group of 25 and in 15 (88%) of the second group of 17 animals sacrificed on Day 14 after intracardiac t u m o r injection. The remaining animals had no intracerebral tumors. Pulmonary metastases were successfully induced in 19 (95%) of 20 animals killed on Day 14 after tail-vein injection.
Lysis of K H T by LAK Cells In Vitro In 4-hour chromium-release assays at effector:target ratios of 100:1, 25:1, and 6.25:1, the percent-specific lysis for flesh t u m o r targets was 45.8%, 23.8%, and 11.2%, respectively (Fig. 3). Lysis induced by L A K cells was significantly greater than that caused by splenocytes at all ratios (p < 0.05). There was no difference between lysis of cultured and fresh t u m o r targets (48.0 versus 45.8 at 100:1, 19.7 versus 23.8 at 25:1, and 7.0 versus 11.2 at 6.25:1).
~
5O "~ 40' 3020' 10. 0'
LAK (fresh tumor) LAK (cultured tumor) Splenocytes (fresh tumor) Splenocytes (cultured tumor)
-10 EFFECTOR/TARGET RATIO
Immunotherapy of Lung Metastases In Experiment 1, animals with pulmonary metastases responded to treatment both with IL-2 alone and with IL-2 plus L A K cells. As compared to control animals, the number of pulmonary metastases was markedly reduced in both treatment groups (p < 0.01), and tumor-free lungs were present in 54% of treated animals. A similar result was obtained when the experiment was repeated.
FIG. 3. Chromium-release assay for lymphokine-activated killer (LAK) effector ceils against fresh and cultured KHT targets. The ordinate expresses the release of 5]Cr as a percent of the maximum release seen. The two LAK curves are statistically indistinguishable from each other, as are the two splenocyte curves. In six assays LAK cells produced greater lysis than splenocytes (three assays) at all ratios (p < 0.0 I).
T R E A T M E N T OF K H T B R A I N M E T A S T A S E S >250
loo
.~
o 9149 9 ooo nov 9
200
80'
9149 9149149
Z
9
9 go 9
|
150 e3 N=13 40 ' a.
9 N=14
Z I00 ~o
20"
0
0 10 20 TIME (DAYS)
30
FIG. 2. Survival of mice after receiving intracardiac injections of KHT sarcoma cells. In two separate experiments, animals were given intracardiac injections of 105 cells in 0.2 cc of Hanks' balanced salt solution, and were followed until death. Histological examination of the brains of two subsequent, similarly treated groups of mice revealed tumors in 76% and 88% of animals.
J. Neurosurg. / Volume 72/January, 1990
9 EXCIPIENT
9 IL-2
9149149 11--2 4" L A K
FIG. 4. Antitumor efficacy of interleukin-2 (IL-2) or IL-2 plus lymphokine-activated killer (LAK) cells on KHT brain metastases. Experimentally induced KHT brain metastases were treated with LAK cells (108 cells intravenously on Days 3 and 6 after tumor injection) plus IL-2 (7500 U intraperitoneally, three times daily), IL-2 alone, or IL-2 excipient. Each point representing the number of brain metastases refers to an individual mouse. 105
I. E. McCutcheon, et al. TREATHENT )25O
OF K H T L U N G H E T A S T A S E S
o 9 g= 99
T R E A T H E N T OF K H T B R A I N i'IETASTASES 9 9 9 9149 oee 99149149149149 1 4 9 1 4 9 1 4 9 99149149 9149 99 e,~ 9
9
200
>25(
Adoptive immunotherapy of intracerebral metastases in mice IAN E.
McCuTCHEON, M . D . , RACHEL A. BARANCO,B.S., DAVID A. KATZ, M . D . , AND
STEPHEN C. SARIS, M . D . Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland us Lymphokine-activated killer (LAK) cells are a heterogeneous population of immune effector ceils that nonspecifically destroy neoplastic cells but not normal ceils. Although parenteral treatment with interleukin-2 (IL-2) alone or a combination of IL-2 and LAK ceils reduces tumor load and prolongs survival in mice with pulmonary, peritoneal, or hepatic metastases, the effect of these treatments on brain metastases has not been studied. To determine in an animal model if intracerebral metastases would be protected by the immunologically privileged status of the brain, intracardiac and intravenous injections of 105 KHT sarcoma cells were performed in C3H mice to create brain and lung metastases, respectively. The mice were treated with adoptive immunotherapy to determine if efficacy seen in an extracerebral site could be reproduced in the brain, and if histological examination of these brains would reveal a significant degree of lymphocyte infiltration and cytolytic activity. Animals were treated with either parenteral IL-2 (7500 U three times daily on Days 3 to 7 after tumor injection), or IL-2 plus LAK cells (7500 U IL-2 three times daily on Days 3 to 7, and 108 LAK cells intravenously on Days 3 and 6 after tumor injection), or IL-2 excipient (three times daily on Days 3 to 7 after tumor injection). As compared to control animals, pulmonary metastases on Day 14 after tumor injection were reduced or eliminated in animals treated with either IL-2 or IL-2 plus L A K cells (p < 0.01). In these same animals, there was no reduction in the number of intracerebral metastases and no evidence of lymphocytic infiltration or cytolytic activity in the brain. This is the first study that reveals an organ-specific resistance to the treatment of metastases with adoptive immunotherapy, and affirms the concern that due to inadequate trafficking of endogenous or exogenous-activated lymphocytes or due to inadequate activation of in situ brain lymphoid precursors, there is no rejection of tumors in the brain. This information suggests that brain metastases in patients with systemic malignancies will not respond to intravenous treatment with LAK cells and IL-2, and that alternative forms of treatment are needed. Furthermore, this modification of a previously existing model of murine brain metastasis provides a method for concurrently evaluating the effectiveness of treatments for intra- and extracranial cancers. KEY W O R D S
~
brain neoplasm
lymphokine-activated
B
killer cell
9 metastasis
RAIN m e t a s t a s e s o c c u r in a p p r o x i m a t e l y 15% o f p a t i e n t s with e x t r a c r a n i a l cancer, 35 a n d are t r e a t e d b y r a d i o t h e r a p y a l o n e o r in c o n j u n c t i o n with surgical excision. Even w i t h t r e a t m e n t , however, the m e d i a n survival t i m e is o n l y 6 m o n t h s , a n d signifi c a n t m o r b i d i t y a c c o m p a n i e s b o t h a p p r o a c h e s . Furt h e r r n o r e , c h e m o t h e r a p y is generally ineffective. L y m p h o c y t e s t h a t d e s t r o y fresh a u t o l o g o u s t u m o r cells b u t n o t n o r m a l cells c a n be g e n e r a t e d b y incubating h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s w i t h the lymp h o k i n e i n t e r l e u k i n - 2 (IL-2). T h e s e c y t o t o x i c effector 102
9 immunotherapy
9 interleukin-2
9
9 mouse
( l y m p h o k i n e - a c t i v a t e d killer, or L A K ) cells express some o f the surface antigens o f natural killer ( N K ) cells a n d classic T l y m p h o c y t e s , 1,9,~~ b u t a t t a c k N K cellresistant targets n o t restricted b y a m a j o r h i s t o c o m p a t ibility c o m p l e x . ~7 W h e n a d o p t i v e l y transferred to tum o r - b e a t i n g m i c e , L A K cells given with IL-2 r e d u c e o r e l i m i n a t e m e t a s t a s e s in several sites, i n c l u d i n g t h e liver, lung, a n d p e r i t o n e a l c a v i t y ) 3.~8,2~ This t h e r a p y is effective against a n u m b e r o f different t u m o r s i n c l u d i n g adenoearcinoma, melanoma, and sarcoma, and works equally well in different strains o f mice.~ 1 I n t e f l e u k i n - 2
J. Neurosurg. / Volume 72/January, 1990
Immunotherapy of murine brain metastasis alone can decrease t u m o r load in those sites, 13'17'18'2z'26 whereas L A K cells alone have relatively little effect.13,18.21 Despite extensive studies of the effectiveness of immunotherapy against tumors in extracranial sites, the effect of L A K cells and IL-2 on metastatic tumors in the brain has not been evaluated. It is known that the administration of IL-2 to mice results in the proliferation and activation of host lymphoid elements and donor L A K cells in the brain, 6,7 and that IL-2 crosses the blood-cerebrospinal fluid (CSF) barrier and perhaps the blood-brain barrier. 30However, it is unclear whether exogenously administered L A K cells travel to targets in the brain, and whether tumors in the brain respond to i m m u n o t h e r a p y with L A K cells and/or IL-2 differently from tumors elsewhere in the body. The effectiveness of L A K cells and IL-2 against intracerebral metastases has relevance to understanding i m m u n e responses within the central nervous system and to improving the current treatment of metastatic brain tumors. Patients with cerebral metastases are currently excluded from therapy with IL-2 because treatment-induced increases in tumoral and peritumoral vessel permeability m a y cause neurological deterioration due to increased cerebral e d e m a ? '2v If successful treatment were accomplished in an animal model, the criteria for admission to clinical protocols that use L A K cells and IL-2 to treat patients with disseminated cancer might be broadened. For these reasons, an animal model of metastatic brain cancer was used to evaluate concurrently the effectiveness of administering IL-2 and L A K plus IL-2 against tumors in the brains and lungs of C 3 H mice. In this way, the effect of therapy was studied in two sites within a single animal, of which one was immunologically privileged and one was not. Materials and Methods
Animals and Tumor Cells Female C 3 H / H e N mice, aged 10 to 12 weeks, were used in all experiments.* A K H T sarcoma,t a weakly immunogenic cell line that arose spontaneously in a C3H mouse, was used in these experiments. All tumors were cultured in complete m e d i u m comprising RPMI1640 with 10% fetal calf serum, 0.05 m M 2-mercaptoethanol, 0.l m M nonessential a m i n o acids, 0.1 lzM sodium pyruvate, L-glutamine (300 ug/ml), penicillin (100 U/ml), streptomycin (100 ug/ml), gentamicin sulfate (50 ug/ml), and amphotericin (0.25 ug/ml). Cells were incubated in flasks at 37~ in an atmosphere of 5% CO2, grown to confluence, and harvested using standard trypsinization techniques.
* Animal breeding colonies provided by National Institutes of Health, Hazelton, Maryland. t KHT sacroma generously provided by Dr. Frances Conley, Stanford, California.
J. Neurosurg. / Volume 72/January, 1990
Animal Preparation and Tumor Model Cerebral metastases were created by intracardiac injection of a t u m o r cell suspension. 2 After induction of anesthesia with methoxyflurane, the anterior thoracic wall was exposed and a No. 27 needle was introduced through the second intercostal space into the left ventricle. K H T cells (105 cells in 0.2 ml H a n k s ' balanced salt solution (HBSS)) were injected as a bolus. Pulmonary metastases were created by tail-vein injections of 105 K H T cells in 0.5 ml HBSS. To determine the reproducibility o f the brain-tumor model and the time at which animals should be sacrificed in later experiments, two groups of animals (14 in the first group and 13 in the second group) were given intracardiac injections of 105 K H T cells and followed until death. Two additional groups of animals (25 in the first group and 17 in the second group) were given intracardiac injections of 103 K H T cells and sacrificed after 14 days to determine the percentage o f animals that developed intracerebral metastases. A final group of 20 animals was given an intravenous injection of l05 K H T cells to confirm growth in the lung. Preparation of L A K cells The spleens were removed aseptically f r o m retired C 3 H breeder mice and crushed in HBSS. The resulting cell suspension was filtered through a Nitex mesh.~ After osmotic lysis o f the erythrocytes by A C K lysing bufferw and further washing with HBSS, the splenocytes were placed in flasks containing complete m e d i u m (5 x l08 cells/175 ml) and IL-2 (10 3 U/ml). The highly purified recombinant IL-2II used in these trials was produced in Escherichia coli transfected with a gene for IL-2 isolated from the Jurkat cell line. 24 After incubation in an atmosphere of 5% CO2 at 37"C for 72 hours, the cells were passed through a gradient of Lympholyte-M,* washed, and resuspended in HBSS at l08 cells/ml for intravenous injection. Effect of L A K Cells Against K H T In Vitro In short-term chromium-release assays, L A K cells were tested against fresh and cultured K H T targets. Fresh t u m o r targets were grown in the subcutaneous space of C 3 H mice. Two weeks after subcutaneous inoculation o f l05 K H T cells, palpable t u m o r s were excised, minced, and digested in a solution o f collagenase, hyaluronidase, and deoxyribonuclease. The cell suspension was filtered through Nitex mesh, washed in HBSS, and resuspended in complete m e d i u m . These fresh cells were used as targets in chromium-release Nitex mesh manufactured by Lawshe Industrial Co., Bethesda, Maryland. wMedia kit manufactured by National Institutes of Health, Bethesda, Maryland. UIL-2 kindly supplied by the Cetus Corporation, Emeryville, California. * Lympholyte-M supplied by Cedarlane Laboratories, Hornby, Ontario, Canada. 103
I. E. McCutcheon, et al. assays performed in triplicate. Targets (5 • 10 6 cells) were labeled by incubation with 400 lzCi of Na[5'Cr]O4 for 2 hours. Effector cells (either L A K cells or splenocytes) were mixed with washed labeled target cells in effector:target cell ratios of 100:1, 25:1, and 6.25:1, and incubated for 4 hours. To test m a x i m u m lysis, 0.1 N HC1 was added to labeled target cells; spontaneous lysis was tested by adding cell-free complete medium. The plates were then spun at 175 G and the supematants collected with a Skatron collection system.~ Filters containing the supernatants were counted in a g a m m a counter. The percentage of specific lysis was calculated by the formula: 100(experimental c p m - spontaneous cpm) m a x i m u m cpm - spontaneous c p m Chromium-release assays were performed in the same manner, with K H T harvested from culture flasks by trypsinization.
Effect of L A K Cells and~or IL-2 Against K H T
In Vivo In all experiments, animals were prepared with tumor as described below, and then r a n d o m l y assigned to one of three groups (Fig. 1). G r o u p 1 (IL-2 alone) received an intraperitoneal injection of 7500 U IL-2 three times daily from Days 3 to 7 inclusive after t u m o r injection. G r o u p 2 was injected intraperitoneally with 7500 U IL2 three times daily on Days 3 to 7 after t u m o r injection, and also received an intravenous injection of 108 LAK cells on Days 3 and 6 after t u m o r injection. Group 3 (control) was treated with an intraperitoneal injection of 0.5 ml IL-2 excipient three times daily on Days 3 to 7 after t u m o r injection. This treatment schedule was chosen to allow comparison with experiments performed with extracranial metastases. 13'17'18,23 Furthermore, this schedule yields IL-2 serum levels and biological effects that are comparable to or greater than those seen with intravenous therapy. 8 Three treatment experiments were performed, and each was repeated once (Fig. 1). Experiment 1 assessed treatment efficacy in animals with p u l m o n a r y tumors. Twenty-four animals received a tail-vein injection of 105 K H T cells; this experiment was repeated with 21 animals. Experiment 2 assessed treatment efficacy in animals with brain tumors. Eighteen animals received an intracardiac injection o f 105 K H T cells; this experim e n t was repeated with 28 animals. Experiment 3 assessed treatment efficacy in animals with brain and lung tumors. On the same day, 22 animals received intracardiac and intravenous injections of 105 K H T cells; this experiment was repeated with 24 animals. In each experiment, after preparation with tumor, the animals were randomly assigned to a treatment group, treated, and sacrificed after 14 days for quantitation of brain and/or lung metastases. t Skatron collection system manufactured by Skatron, Inc., Sterling, Virginia. 104
Quantitation of Brain Metastases Immediately after sacrifice, the brains were removed and frozen in isopentane (-70~ Coronal sections (20 um thick) were cut on a cryomicrotome at -16"C. Starting in the anterior frontal cortex and ending in the posterior temporal cortex, six sequential sections were taken from each brain at 700-urn intervals. The sections were m o u n t e d on glass slides, air-dried, fixed in a solution o f a m m o n i u m bromide (2%) in 6% formalin, and stained with hematoxylin and eosin. The sections were photographed through a green filter (for high contrast) and enlarged (• 10). Metastases were counted from the photographs in blinded fashion with the assistance of a magnifying glass. If more than 250 metastases were present in the six sections, the number of brain metastases in that animal was assigned a value of 250.
Quantitation of Lung Metastases Immediately after sacrifice, the animals' lungs were insuffiated by intratracheal injection of 15 % India ink and placed in Fekete's solution. 34 Metastases (visible as distinct white nodules on the black background of normal lung parenchyma) were counted on all external lobar surfaces with the assistance of a magnifying glass. If more than 250 metastases were present, the number of lung metastases in that animal was assigned a value of 250.
Pathology All brains and lungs in Experiment 3 were evaluated in blinded fashion. All sections were reviewed by the same pathologist (D.A.K.) with attention given to the metastatic lesions, as well as to the noninvolved parenchyma. A semiquantitative estimate was m a d e of the number o f lesions present in a given brain section (few: _< 25 lesions; moderate: 25 to 50 lesions; many: > 50 lesions). The lungs were evaluated in a similar fashion. Group |
I
IL2
Group 2
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IL2 § LAK
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Group 3
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8
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SACRIFICE
experiment 1 : | ( ~ c e l ] s iv experiment 2 : 11~cel|s ic experiment 3 : 105cells ic and iY
FIG. 1. Experimental protocol. On Day 1, animals were prepared with tumor and then randomly assigned to one of three treatment groups: interleukin-2 (IL-2) alone, IL-2 plus lymphokine-activated killer cells (IL2 + LAK), and control (IL-2 excipient). On Day 14, the animals were sacrificed for quantitation of tumors. In Experiment 1, intravenous (iv) tumor injections were performed to create pulmonary metastases; in Experiment 2, intracardiac (ic) tumor injections were performed to create brain metastases; and in Experiment 3, both intravenous and intracardiac injections were performed in each animal.
J. Neurosurg. / Volume 72/January, 1990
Immunotherapy of murine brain metastasis Statistical Analysis
Immunotherapy of Brain Metastases
Data from the chromium-release assays were compared to Student's two-tailed t-test. Counts of metastases from the treatment experiments were compared using the nonparametric Kruskal-Wallis test statistic.
In Experiment 2, animals given intracardiac injections of t u m o r showed no response to treatment with either IL-2 alone or L A K cells plus IL-2. There was no difference between the number of brain metastases in animals treated with IL-2 and in control animals (p = 0.44), or between animals treated with L A K cells plus IL-2 and control animals (p -- 0.19). The mean n u m b e r of brain metastases in the IL-2-treated group, the IL-2 plus L A K cell-treated group, and the control group was 203, 237, and 193, respectively. This experiment was repeated once with similar results 9The combined data from the two experiments are shown in Fig. 4.
Results
Tumor Model Mice given 105 cells by intracardiac injection died on Days 13 to 20; median survival was 17 days (Fig. 2). Greater than 200 intracerebral metastases were present in 19 (76%) of the first group of 25 and in 15 (88%) of the second group of 17 animals sacrificed on Day 14 after intracardiac t u m o r injection. The remaining animals had no intracerebral tumors. Pulmonary metastases were successfully induced in 19 (95%) of 20 animals killed on Day 14 after tail-vein injection.
Lysis of K H T by LAK Cells In Vitro In 4-hour chromium-release assays at effector:target ratios of 100:1, 25:1, and 6.25:1, the percent-specific lysis for flesh t u m o r targets was 45.8%, 23.8%, and 11.2%, respectively (Fig. 3). Lysis induced by L A K cells was significantly greater than that caused by splenocytes at all ratios (p < 0.05). There was no difference between lysis of cultured and fresh t u m o r targets (48.0 versus 45.8 at 100:1, 19.7 versus 23.8 at 25:1, and 7.0 versus 11.2 at 6.25:1).
~
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LAK (fresh tumor) LAK (cultured tumor) Splenocytes (fresh tumor) Splenocytes (cultured tumor)
-10 EFFECTOR/TARGET RATIO
Immunotherapy of Lung Metastases In Experiment 1, animals with pulmonary metastases responded to treatment both with IL-2 alone and with IL-2 plus L A K cells. As compared to control animals, the number of pulmonary metastases was markedly reduced in both treatment groups (p < 0.01), and tumor-free lungs were present in 54% of treated animals. A similar result was obtained when the experiment was repeated.
FIG. 3. Chromium-release assay for lymphokine-activated killer (LAK) effector ceils against fresh and cultured KHT targets. The ordinate expresses the release of 5]Cr as a percent of the maximum release seen. The two LAK curves are statistically indistinguishable from each other, as are the two splenocyte curves. In six assays LAK cells produced greater lysis than splenocytes (three assays) at all ratios (p < 0.0 I).
T R E A T M E N T OF K H T B R A I N M E T A S T A S E S >250
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FIG. 2. Survival of mice after receiving intracardiac injections of KHT sarcoma cells. In two separate experiments, animals were given intracardiac injections of 105 cells in 0.2 cc of Hanks' balanced salt solution, and were followed until death. Histological examination of the brains of two subsequent, similarly treated groups of mice revealed tumors in 76% and 88% of animals.
J. Neurosurg. / Volume 72/January, 1990
9 EXCIPIENT
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FIG. 4. Antitumor efficacy of interleukin-2 (IL-2) or IL-2 plus lymphokine-activated killer (LAK) cells on KHT brain metastases. Experimentally induced KHT brain metastases were treated with LAK cells (108 cells intravenously on Days 3 and 6 after tumor injection) plus IL-2 (7500 U intraperitoneally, three times daily), IL-2 alone, or IL-2 excipient. Each point representing the number of brain metastases refers to an individual mouse. 105
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