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Effect of dietary linoleic acid level on lodgement, proliferation and survival of mammary tumor metastases
44 (1989) 117-125
Cancer Letters,
Elsevier Scientific Publishers
117
Ireland Ltd.
Effect of dietary linoleic acid level on lodgement, proliferation and survival of mammary tumor metastases N.E. Hubbard and K.L. Erickson Department
of Human Anatomy,
University of California, School of Medicine, Davis, CA 95616 (U.S.A.)
(Received 24 September 1988) (Revision received 1 November 1988) (Accepted 4 November 1988)
Summary
Keywords:
High levels of dietary linoleic acid (18: 2) have been shown to increase the spontaneous metastasis of line 4526 mouse mammary tumors. In this report, the influence of 18: 2 on specific events of tumor metastasis,
Introduction
dietary fat; mammary metastasis; Iinoleic acid.
namely, lodgement, proliferation and survival, were studied using spontaneous and experimental metastasis assays with line 4526 cells. A significantly greater number of radiolabeled tumor cells lodged in the lungs of mice fed 4, 8 and 12% 18: 2 when compared with mice fed lower levels of 18 : 2. The effect of dietary 18 : 2 appeared to be on the host tissue (lungs) and not the tumor cells. Lodgement of tumor cells first cultured in serum of mice fed 18 : 2 then injected into mice fed 1% 18: 2 was not affected. There were no significant differences in the percentage of /3-f]thymidine labeled metastatic cells from lungs of mice fed different levels of 18: 2. However, the number of surface lung nodules that appeared in mice 21 days after injection of unlabeled line 4526 cells increased in mice fed 8 and 12% 18 : 2 compared with those fed lower levels of 18: 2. Thus, dietary 18 : 2 may increase metastasis by influencing the lodgement, implantation and survival but not proliferation of line 4526 mouse mammary tumor cells. ‘Correspondence to; K.L. Erickson. 0304-3835/89/$03.50 0 1989 Elsevier Scientific Publishers Published and Printed in Ireland
tumors;
The successful metastasis of malignant tumor cells to sites distant from the primary neoplasm is dependent upon the cell’s ability to complete a complicated series of sequential steps in the metastatic cascade. The events that normally occur for successful metastasis include: (1) detachment of tumor cells from the primary neoplasm, either singly or in clumps; (2) penetration of blood vessels or lymphatics for passive dissemination to distant sites; (3) lodgement and adhesion to blood vessels in host organs; (4) extravasation and subsequent survival and proliferation to form new tumor foci. The ability of tumor cells, then, to successfully metastasize, is dependent upon an interplay between the intrinsic characteristics of the cell and several host factors [4]. Modulation of cell characteristics or host factors could influence the degree to which tumor cells form distant metastases. We have recently reported that spontaneous metastasis from transplantable mammary fat pad tumors grown in mice fed high fat (HF, 20%, w/w) diets that contained high (12%) w/w) levels of linoleic acid (18 : 2) was increased when compared with mice fed
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118
HF diets containing 8% or less 18 : 2 [8]. Since all diets were equal in the total amount of fat, and body weight did not vary among groups, we concluded that those affects on increased metastasis were due to changes in the dietary level of 18 : 2. In addition we showed that oral administration to mice of an eicosanoid synthesis inhibitor, indomethacin, completely inhibited the 18 : 2-mediated increase in metastasis. Results of those studies support the hypothesis that arachidonic acid (20 : 4) metabolites, such as prostaglandins may play a role in 18 : 2 enhancement of mouse mammary tumor metastasis. It is possible that several of the events in the metastatic cascade may be affected by a modulation of eicosanoid synthesis through dietary 18 : 2. For example, the lodgement of tumor cells in the blood vessels of host organs may be dependent upon platelet aggregation and the status of the hemostatic system [12]. Moreover, the survival of lodged tumor cells probably depends on the status of the immune system, which can be modulated by eicosanoids [1,9,13]. Though our results show that eicosanoids may play an important role in the 18 : 2-mediated increase in metastasis, the exact mechanism(s) is not known. In this study, we have investigated ‘the effects of dietary 18 : 2 levels on important steps, lodgement and survival, of the metastasis of line 4526 mammary tumor cells. In particular, we have determined whether dietary 18 : 2 influences the supportive tissue and modulates the implantation of the metastatic cell or whether it influences the metastatic cell itself. To correlate changes of tumor cell implantation and dietary 18 : 2, the fatty acid composition of the primary site of metastasis, the lung, was determined. In addition, we report the effects of various levels of dietary 18 : 2 on the proliferation of tumor cells within metastases. Materials and methods
Animals and diets Female BALB/cAnN mice 5 weeks old and
Table 1.
Composition of experimental diets. g/ 100 g in following concentrations of 18 : 2 in diet
Ingredient
1%
2%
4%
8%
12%
Casein”
24.8
24.8
24.8
24.8
24.8
Salt mixb*c
6.4
6.4
6.4
6.4
6.4
Vitamin mixCsd
1.7
1.7
1.7
1.7
1.7
Cellulosee
5.5
5.5
5.5
5.5
5.5
Cerelose (Dextrose)
41.6
41.6
41.6
41.6
41.6
Coconut oil’
19.2
17.9
15.2
9.9
4.5
0.8
2.1
4.8
10.1
15.5
Safflower oils
aAlldiets provided 28% of energy from protein. bProvided at 1.4 g/kcal of gross energy. See [2] for composition. dProvided at 0.3 g/kcal of gross energy. ‘Provided at 1.1 g/kcal of gross energy. Provided by Capital City Products (Columbus, OH). gProvided by California Fats and Oils (Richmond, CA).
previously fed a stock diet (Purina Mouse Chow, Ralston Purina, St. Louis, MO), were obtained from Charles River Laboratories (Kingston, NY). Mice were immediately segregated by weight then randomly placed into separate groups in order that the average weight per group was approximately the same. After receiving the stock diets for 48 h, mice were switched to the experimental diets (Table 1). All diets were isocaloric and contained at least the minimum level of recommended nutrients with a constant amount per kilocalorie of casein, salts, vitamins and fiber [ 151. Differing mixtures of coconut and safflower oil were added to make 20% fat (w/w) diets that contained either 1, 2, 4, 8 or 12% (w/w) 18 : 2. The fatty acid composition of the oil mixtures has been previously reported [8]. These two oils were chosen because they have comparable levels of oleic acid. Also, the final oil blends added to each of the experimental diets
119
contained approximately the same levels of oleic acid [8]. Animals received fresh food every other day and were fed the diets for the duration of the experiment. Experiments were not begun until the mice were fed the diets for at least 4 weeks. Body weights of mice were monitored weekly, and based on these measurements, no significant (P > 0.05) differences were observed in the mean body weight increases of mice from the different experimental diet groups (data not shown).
Mouse mammary tumor line 4526 is a cloned subpopulation from a metastatic nodule that appeared in a mouse growing the 10th in vivo passage of a spontaneous mammary adenocarcinoma in a BALB/cfC3H mouse [ 141. The cell line was obtained from Drs. G. Heppner and A. Fulton. All media and antibiotics were obtained from Whittaker MA Bioproducts, Inc. (Walkersville, MD), with the exception of the calf serum, which was obtained from HyClone (Logan, UT). Tumor cells from frozen stock were thawed and maintained in culture (5% CO,, 37OC) with Eagle’s minimum essential medium with Earle’s salts supplemented with 5% calf serum, 2% essential vitamin mixture, I % L-glutamine, I % nonessential amino acids, 1% sodium pyruvate and 50 pg/ml gentamicin.
13H]thymidine (spec. act., 6.7 Ci/mmol). Mice were injected 3 times at 8 h intervals, and received a total of 45 FCi each. Mice were killed 8 h after the last injection and lungs with metastases prepared for autoradiography. Five mice per dietary group were used for this experiment. Briefly, lungs with metastases were insufflated, using a 21 gauge needle, with 6 ml of 0.2 M glutaraldehyde fixative in phosphate buffer, removed, and placed in fresh fixative for 5 min. Lungs were then diced and placed in fixative overnight at 4OC. After rinsing twice in phosphate buffer for 30 min, the tissues were dehydrated in a graded series of ethanols, infiltrated and embedded in JB-4 embedding medium. Sections were cut at 1 pm, fixed onto glass slides which were then coated with Kodak NTB-3 emulsion (Eastman Kodak Co., Rochester, NY). Slides were placed in light-tight boxes at 4OC for 4 weeks. Slides were then developed, fixed, stained lightly with toluidine blue, and observed using a light microscope equipped with an oilimmersion objective and an intraocular grid. Fields containing metastases were randomly selected from each slide and the number of labeled (4 or more grains above or near the nucleus) and unlabeled cells per field were counted. Values were expressed as percent labeled cells of total cells counted and based on about 100 cells per field.
Tumor transplantation
Tumor cell survival
Tumor cells
and autoradiography
Monolayers of line 4526 in their exponential growth phase and from the same passage were rinsed with calcium- and magnesium-free Hank’s balanced salt solution (HBSS) then treated with a solution of 0.05% trypsin and 0.025% EDTA in the HBSS. The resultant cell suspension was passed through a 21 gauge needle to break up large clumps, washed, counted, and resuspended in HBSS. Mice were injected in the inguinal mammary fat pad with 1 x 104 tumor cells 4 weeks after being fed the experimental diets. Primary tumors were grown in mice until they reached a size of 12 mm x 12 mm (approx. 45 days), at which time mice were injected i.p. with 15 FCi of
Tumor cells were maintained as above, harvested, and passed through a 21 gauge needle to break up large clumps. An aliquot containing 1 x lo5 cells was injected i.v. into the laterai tail vein of mice fed HF diets containing either 1, 2, 4, 8 or 12% 18: 2. Five mice per dietary group were used. After 3 more weeks, mice were killed, their lungs removed, and total metastases quantitated using a stereoscopic microscope. Tumor cell lodgement
To determine whether dietary fat affects the supportive tissue or the tumor cell itself in the process of lodgement, two assays modified
120
from a technique previously described were used [3]. First, to assay for effects on supportive tissue, tumor cells were maintained in culture with complete medium as described above, then grown in medium for 48 h containing serum from mice fed 1% 18 : 2 before intravenous injection into mice fed the different experimental diets. Second, to assess whether 18 : 2 influences the metastatic cell itself, tumor cells were first maintained in culture as described above under “Tumor Cells”, then switched to media containing sera from mice fed each of the experimental diets before intravenous injection into mice fed the 1% 18 : 2 diet. Hence cells, cultured separately, were maintained in medium containing 5% serum from mice fed HF diets that contained either 1, 2, 4, 8, or 12% 18: 2. Although an alternative procedure of growing primary tumors in dietary-fat manipulated mice and preparing single cell suspensions for injection was tried, it proved to be less satisfactory than the procedure described above. The reasons for that are: first, contamination of the primary tumor digests with other cell types (the primary consisted of 70% tumor cells, 15% inflammatory cells, and 15% other types including fibroblasts; unpublished observations) ; and second, lipids from cells of dietaryfat manipulated mice can flux during the 24 h labeling period and cultures need to be supplemented with autologous mouse serum. When monolayers were 50% confluent, they were rinsed and then incubated in fresh medium that contained 0.5 pCi/ml, 5[lz51]iodo-2’-deoxyuridine (spec. act. 5 pCi/ mg) . In pilot studies, the optimal conditions for labeling without reduction in proliferation were determined. After 24 h, cells were rinsed 3 times using HBSS, harvested, passed through a 21 gauge needle to break up large clumps and aliquots containing 1 x lo5 cells were injected i.v. into the lateral tail vein of mice. Aliquots identical to injected tumor cell suspensions were collected at the same time. After 12 and 24 h post-injection mice were killed, their kidneys, lungs, spleen, liver and heart excised, rinsed in 3 changes of 70% ethanol
for 48 h to dissociate membrane bound label [3], and individually counted in a gamma radiation counter. Radioactivity was calculated on a per cell basis, correcting for counter and isotope background [2]. Five mice per group were utilized for this experiment which was repeated twice. Fatty acid analysis To assay fatty acid composition of tumor cells, line 4526 was grown in medium supplemented with 5 % serum from mice fed either 1, 2,4, 8, or 12% 18 : 2 diets. To assay fatty acid composition of the lungs and in order not to include any micrometastasis, a parallel group of mice fed the experimental diets but not injected with tumor cells were used. Fatty acid composition of cells and tissues was analysed as previously described [8]. Briefly, cell monolayers or lung tissues were homogenized in 1.2 ml of 0.1 M KC1 to which was added 6 ml of chloroform/methanol (2 : 1, v/v). The homogenate was transferred to a glass centrifuge tube, vortexed for 60 s, then centrifuged. The chloroform phase with extracted lipids was run through Na,SO, into a glass tube and the solvent evaporated under a continuous stream of nitrogen. The resultant lipid residues were dissolved in chloroform and stored under nitrogen at - 70°C until further use. Fatty acid methyl esters of samples were dissolved in methylene chloride and analyzed using a Hewlett-Packard 5880A gas-liquid chromatograph. The retention times were compared with those of known standards, and the relative levels of fatty acid in the lipid calculated by dividing the integrated areas of each peak by total area occupied by all peaks on the chromatogram. Statistical analysis Differences in body weight and fatty acid composition among the different groups were tested for statistical significance using one-way analysis of variance. Differences in metastasis were tested for statistical significance using the Wilcoxon rank-sum test for two groups or the Kruskal-Wallis one-way analysis of variance
121 Table 2.
Effect of dietary 18 : 2 level on lodgement
of line 4526 tumor cells=.
% 18:2in diet
12h
1 2 4 8 12
2840 3860 4140 3930 4310
Totalb
Spleen
Lung
24h
f f f f f
195’ 275 385 370 430
1000 1040 1520 1340 1450
24h
12 h
f llOd f 9Od + 85 + 100 f 105
400 350 290 300 450
* f + f +
30 25 20 50 80
100 120 140 250 220
24 h
12h
-c + f + +
15d lo’ 5’ 25 20
3900 4800 5130 4930 5290
+ + + + +
300” 400 450 425 415
1190 1280 1800 1690 1820
f: f f f +
150d 125d 195 165 155
‘Labeled tumor cells were grown in medium supplemented with serum from mice fed the 1% 18 : 2 diet and injected into mice fed HF diets that contained either 1, 2, 4, 8, or 12% 18 : 2. Values represent the mean number of cells lodged f S.E. for n = 75. bSum of the tumor cells distributed to 5 major sites: the heart, liver, kidneys, spleen and lungs, in the experimental metastasis assays. Significantly (P< 0.05) less than other dietary groups for this time point. dSignificantly (P< 0.05) less than 4.8 and 12% 18 : 2 dietary groups for this time point. “Significantly (P< 0.05) less than 8 and 12% 18 : 2 dietary groups for this time point.
by ranks. Differences were expressed when the level of significance was 0.05 or less. Statistical analyses were carried out using the Microsoft computer program (Ecosoft, Indianapolis, IN). Results Tumor cell lodgement
To test the influence of 18 : 2 on the tumor cell itself, cells were cultured in media supplemented with serum from mice that were fed various levels of 18 : 2 and injected into host mice fed 1% 18 : 2. No significant (P > 0.05) differences among the groups were observed in the lodgement of tumor cells in the body organs after 12 or 24 h (data not shown). However, in the reverse experiment to determine the influence of 18 : 2 on the supportive tissue, where tumor cells cultured in medium supplemented with serum from mice fed 1% 18 : 2 were injected into host mice that were fed diets contaiing various levels of 18 : 2, significant (P < 0.05) differences were observed (Table 2). To determine the nonorgan-specific effects of 18 : 2 on metastasis, the total number of tumor cells contained in
$
500
z z” P 3
450
2
400
r cz 6
350
z -!? z’
300
c r”
250 I 0
I
I
I
I
I
I
2
4
6
8
10
12
%18:2 Fig. 1.
14
in Diet
Effect of dietary 18 : 2 level on the survival of line 4526 mammary tumor cells. Mice were fed the experimental diets for 4 weeks then injected i.v. with 1 x lo5 unlabeled cells. After 21 days, mice were killed and metastases quantitated. The number of surface lung nodules of mice fed 8 and 12% 18 : 2 diets was significantly (P < 0.05) increased when compared to the number found in the lung of mice fed 1, 2, or 4% 18 : 2. Brackets are S.E. for n = 75.
122
the major sites, heart, liver, kidneys, spleen and lungs, was examined. After 12 h, the total number of cells lodged was significantly less (P < 0.05) in mice fed 1% 18 : 2 compared to mice fed the other diets. As expected, fewer cells were lodged in the organs after 24 h when compared to 12 h. After 24 h, the total number of cells lodged in organs of mice fed 1 or 2% 18 : 2 was significantly (P < 0.05) less than the number of cells lodged in mice fed 4, 8, or 12% 18: 2. At the 12 and 24 h time points, significantly (P < 0.05) fewer cells were lodged in the lungs of mice fed 1% or 1% and 2% 18 : 2, respectively, when compared to mice fed 4, 8 and 12% 18 : 2 diets. With respect to the spleen, no differences were observed in tumor cell lodgment after 12 h, but after 24 h, significantly (P < 0.05) fewer cells were lodged in the spleens of mice fed HF diets that contained 1, 2, or 4% 18 : 2 when compared to groups fed 8 or 12% 18 : 2. No significant (P > 0.05) differences in the number of tumor cells lodged in the heart, liver, or kidney, were observed among the dietary groups (data not shown).
cells within metastases of lungs of mice fed HF diets containing 1, 2, 4, 8, or 12% 18 : 2 was 18.8 f 2,4, 19.3 & 1.9, 22.3 f 3.4, 21.3 f 1.5 and 19.0 f 2.7, respectively. No significant (P > 0.05) differences were observed in the percentage of labeled cells when metastases of lungs from groups of mice fed various levels of 18 : 2 were compared. Tumor cell survival The effect of dietary 18 : 2 level on tumor cell survival in lungs of mice fed the various diets is shown in Fig. 1. There was a linear increase in the number of surface lung nodules with increasing dietary 18 : 2 level. However, the only statistically significant differences (P < 0.05) were the increased number of nodules in the 8 and 12% dietary groups compared to micefed1,2,or4% 18:2. Fatty acid composition
The total fatty acid composition of cells cultured in media supplemented with serum from mice fed various levels of dietary 18 : 2 was significantly (P < 0.05) altered (Table 3). Thus, cells injected i.v. for the lodgement studies differed in their fatty acid composition, depending upon the serum. The relative levels of oleic acid (18 : 1) were significantly (P < 0.05) decreased and the relative levels of
Proliferation
Autoradiography of the lung was performed to study proliferation of tumor cells within the metastatic nodule. The percentage of labeled
Table3. Fatty acid composition Fatty acid
% of total fatty acids with varying dietary 18 : 2 levels 1%
SFAb 16 : 1 18: 1 18 : 2 20:4
of line 4526 cells grown in vitro”.
45.0 3.6 27.2 12.3 6.4
2% f -t f -c f
1.2 0.5 1.2 0.6 0.9
47.7 2.7 24.0 12.9 4.7
4% * k f f f
1.1 0.5 0.9 0.5 0.7
48.4 2.5 25.8 15.8 4.5
12%
8% + f f f +
2.1 0.3 1.1 0.7d 0.6
45.9 3.0 21.8 20.2 5.1
f f + -c f
1.5 0.6 0.5 0.9 0.5
47.5 3.2 20.9 21.3 4.8
* f f + *
2.1 0.4 0.9 0.9 0.4
Walues represent the mean relative percent f S.E. for n = 15. Cells were grown in medium supplemented with 5% serum from mice fed either 1, 2, 4, 8, or 12% 18 : 2 diets, then extracted, derivatized, and analyzed by gas-liquid chromatography. bSaturated fatty acids (SFA) include 12 : 0, 14 : 0, 16 : 0 and 18 : 0. cSignificantly (P < 0.05) decreased when compared with 1, 2 and 4% 18 : 2 dietary groups for this fatty acid. dSignificantly (P< 0.05) greater than 1 and 2% 18 : 2 dietary groups for this fatty acid. -Significantly (P < 0.05) greater than 1, 2 and 4% dietary 18 : 2 groups for this fatty acid.
123
Table 4.
Fatty acid composition
Fatty acid
46 of total fatty acids with varying dietary 18 : 2 levels 1%
SFAb 18 : 1 18 : 2 20 : 4 Others
of lung tissue”.
51.4 22.6 3.5 10.3 4.7
4%
2% f 0.1 f 1.1 f 0.2 f 0.5 f 1.1
52.3 20.7 6.1 10.0 6.6
f f f f +
0.6 0.7 0.2 0.1 1.2
8%
50.4 17.4 10.4 10.8 6.8
-c f * f f
0.7 1.2d 0.4’ 0.4 1.2
45.8 17.2 18.7 9.2 5.1
12% f f f -c k
0.7= l.ld 1.4’ 1.1 1.6
44.1 14.1 24.3 9.3 4.3
+ f k -c f
0.4 0.3 0.5’ 0.7 0.9
*Values represent the mean relative percent of total f SE. for n = 30. Lungs were obtained from mice fed the experimental diets but not injected with tumor cells. bSaturatedfatty acids (SFA) include 12 : 0,14 : 0, 16 : 0 and 18 : 0. cSignificantly (P < 0.05) decreased when compared with 1, 2 and 4% dietary groups for this fatty acid. dSignificantly (P < 0.05) decreased when compared with 1 and 2% dietary groups and significantly (P < 0.05) increased when compared with 12% dietary group for this fatty acid. Significantly (P < 0.05) decreased when compared with other dietary groups for this fatty acid. Significantly (P < 0.05) increased when compared with group(s) containing less dietary 18 : 2.
18 : 2 were significantly (P< 0.05) increased in the 8 and 12% dietary groups when compared with cells grown in medium supplemented with serum from mice fed HF diets containing 1, 2, or 4% 18 : 2. Cells cultured in medium containing serum from mice fed 4% 18 : 2 had significantly (P < 0.05) greater levels of 18 : 2 than the 1 and 2% groups and significantly (P < 0.05) lower levels when compared to the 8 and 12% groups. To assess whether the fatty acids of the lungs, the major site of metastasis, changed as the level of dietary 18 : 2 varied, composition was assessed. Lung tissues of mice fed various levels of dietary 18 : 2 were also significantly altered (Table 4). The levels of saturated fatty acids (12: 0, 14: 0, 16: 0 and 18 :0)were decreased (P < 0.05) in animals fed 8 and 12% 18 : 2 when compared with the 1, 2 and 4% 18 : 2 dietary groups. Oleic acid was significantly (P < 0.05) decreased in mice fed 4, 8 and 12% 18 : 2 when compared with the 1 and 2% 18 : 2 groups and the relative levels of 18 : 2 significantly (P< 0.05) increased with increases in dietary availability of 18 : 2. Discussion
Metastasis is a complex multistep event in
the progression of malignant tumors that may be influenced by many systemic and local host factors as well as the properties of the tumor cell. We have previously shown that dietary 18 : 2 influences spontaneous metastasis in general. We now wish to determine whether 18 : 2 influences some of these specific events such as implantation, proliferation and survival. We have observed a modulation in the lodgement and survival of line 4526 mammary tumor cells by changing dietary 18 : 2 concentrations. When dietary 18 : 2 has an effect on the lodgement and implantation of the tumor cell it was on the host tissues rather than on the tumor cells themselves, because we observed significant increases in the lodgement of similar cells all cultured in 1% mouse sera from dietmanipulated mice, and no differences in lodgement of lipid-manipulated cells injected into similar mice all fed 1% 18 : 2 diets. Lack of an effect when the fatty acid composition of the sera changed was not due to lack of incorporation because our analysis indicated an increase in 18 : 2 levels and a decrease in oleic acid levels in the tumor cells as dietary 18 : 2 levels increased. The level of initial tumor cell lodgment in the lung appears to be directly related to the level of 18 : 2 found in that tissue. In our longer term assay using
124
autoradiograpy, we observed no difference in the rate of proliferation with the established metastatic nodule. Although we would also like to determine whether dietary fat has a direct influence on only the tumor cell, the microenvironment, or both, in the process of cell proliferation at a metastatic site, that could not be assessed because the tumor cell would change its fatty acid composition once placed in a different lipid environment, e.g. in dietaryfat manipulated mice. In a short period of time, tumor cell fatty acid composition would be similar to the fatty acid levels in the diet and the serum. For that reason, only short term experimental metastasis assays were used and only effects observed with a short term assay may be associated with changes in the tumor cell or the microenvironment. Fatty acid analysis of tumor cells injected into mice showed that changes in the serum were associated with changes in the lipid composition of the cells. Results of the tumor cell survival study indicate that 18 : 2 increased survival of tumor cells that lodged, since there were more surface lung metastases 21 days postinjection in mice that were fed diets containing higher levels of 18 : 2 (8 and 12%). Moreover, the increase in the number of surface lung nodules observed here and in other studies, was probably not due to an increase in the proliferation of tumor cells within the nodule, since we observed no differences in the percentage of radiolabeled cells among the dietary groups. The mechanism(s) involved in the 18 : 2mediated increases in lodgement and survival of line 4526 mammary tumor cells is not known. However, there are a few possible explanations. Increasing the level of 18 : 2 in the diet may serve to alter the synthesis of eicosanoids [5,11]. Eicosanoids can effectively modulate mechanisms of the immune system [9,10,13] and alter events in hemostasis [6,7,12]. Lymphocytes and macrophages could be suppressed by dietary fat and prostaglandins [ 1,9,13] resulting in an increase in the number of tumor cells that survive to form secondary foci.
In this study, we observed decreased tumor cell lodgement cells after 12 h of mice fed a low level of 18 : 2 (1%). After 24 h, we observed decreased lodgement in mice fed 1% and 2% 18 : 2. After 21 days, we observed a decrease in the number of experimental lung nodules in mice fed 1, 2 and 4%. In other studies [8] we observed increased spontaneous metastasis of primary line 4526 tumors from the mammary fat pad of mice fed 12% 18 : 2. These results suggest that increasing the level of 18 : 2 in the diet, first affects the number of cells that lodge. This may occur either by suppressing the immune system such that fewer cells are removed from the circulation, or by increasing the probability that cells will lodge by increasing platelet aggregation. Second, the level of dietary 18 : 2 can affect the number of cells or clumps that ultimately become secondary tumor foci. This may occur by modulating mechanisms of immune response. Acknowledgements
The authors wish to thank Dr. Steve Lanning of Capitol City Products for providing the coconut oil. This work was supported in part by grants from the Gustavus and Louise Pfeiffer Research Foundation and the National Dairy Board. Administered by the National Dairy Council. References Erickson, K.L. (1986) Mechanisms of dietary fat modulation of tumorigenesis: changes in immune response. In: Progress in Clinical and Biological Research, pp. 555586, Editors: C. Ip, D.F. Bitt, A.E. Rogers and C. Mettlin. Alan R. Liss, New York. Erickson, K.L., Schlanger, D.S., Adams, D.A., Fregeau, D.R. and Stern, J.S. (1984) Influence of dietary fatty acid concentration and geometric configuration on murine mammary tumorigenesis and experimental metastasis. J. Nutr., 114,18341842. Fidler, I.J. (1970) Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with i251-5iodo-2’-deoxyuridine. J. Natl. Cancer Inst., 45, 778782. Fidler, I.J. (1978) Tumor heterogeneity and the bilogy of cancer invasion and metastasis. Cancer Res., 38, 26512660.
125 5
6
7
8
9
10
Galli, C. (1980) Dietary influences on prostaglandin synthesis. In: Advances in Nutritional Research, pp. 95126. Editors: H.H. Draper. Plenum Press, New York. Gorelik, E., Wiltrout, R.H., Okumura, K., Habu, S. and Herberman, R.B. (1982) Role of NK cells in the control of metastatic spread and growth of tumor cells in mice. Int. J. Cancer, 30, 107-112. Honn, K.V., Bockman, R.S. and Marnett, L.J. (1981) Prostaglandins and cancer: a review of tumor initiation through tumor metastasis. Prostaglandins, 21.833-864. Hubbard, N.E. and Erickson, K.L. (1987) Enhancement of metastasis from a transplantable mouse mammary tumor by dietary linoleic acid. Cancer Res., 47, 6171-6175. Johnston, P.V. and Marshall, L.A. (1985) Dietary fat, prostaglandins and the immune response. Prog. Food Nutr. Sci., 8.3-25. Lala, P.L., Parhar, R.S. and Singh, P. (1986) Indomethatin therapy abrogates the prostaglandin-mediated supression of natural killer activity in tumor-bearing mice and prevents tumor metastasis. Cellular Immunol., 99, 108118.
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12
13
14
15
Mathias, M.M. and DuPont, J. (1985) Quantitative relationships between dietary linoleate and prostaglandin (eicosanoid) biosynthesis. Lipids, 20, 791-801. Menter, D.G., Hatfield, J.S., Harkins, C., Sloane, B.F., Taylor, J.D., Crtssman, J.D. and Honn, K.V. (1987) Tumor cell-platelet interactions in vitro and their relationship to in uiuo arrest of hematogenously circulating tumor cells. Clin. Exp. Metastasis, 5, 65-78. Merttn, J., Stackpoole, A. and Shumway, S. (1985) Nutrition and immunity: the immunoregulatory effect of n-6 essential fatty acids is mediated through prostaglandin E. Int. Archs. Allergy Appl. Immunol., 77,390-395. Miller, F.R., Miller, D.E. and Heppner, G.H. (1983) Characterization of metastatic heterogeneity among subpopulations of a single mouse mammary tumor: heterogeneity in phenotypic stability. Invasion Metastasis, 3, 2231. National Research Council (1978) Nutrient Requirements of Laboratory Animals, 3rd edn. National Academy of Sciences, Washington, DC.
Cancer Letters,
Elsevier Scientific Publishers
117
Ireland Ltd.
Effect of dietary linoleic acid level on lodgement, proliferation and survival of mammary tumor metastases N.E. Hubbard and K.L. Erickson Department
of Human Anatomy,
University of California, School of Medicine, Davis, CA 95616 (U.S.A.)
(Received 24 September 1988) (Revision received 1 November 1988) (Accepted 4 November 1988)
Summary
Keywords:
High levels of dietary linoleic acid (18: 2) have been shown to increase the spontaneous metastasis of line 4526 mouse mammary tumors. In this report, the influence of 18: 2 on specific events of tumor metastasis,
Introduction
dietary fat; mammary metastasis; Iinoleic acid.
namely, lodgement, proliferation and survival, were studied using spontaneous and experimental metastasis assays with line 4526 cells. A significantly greater number of radiolabeled tumor cells lodged in the lungs of mice fed 4, 8 and 12% 18: 2 when compared with mice fed lower levels of 18 : 2. The effect of dietary 18 : 2 appeared to be on the host tissue (lungs) and not the tumor cells. Lodgement of tumor cells first cultured in serum of mice fed 18 : 2 then injected into mice fed 1% 18: 2 was not affected. There were no significant differences in the percentage of /3-f]thymidine labeled metastatic cells from lungs of mice fed different levels of 18: 2. However, the number of surface lung nodules that appeared in mice 21 days after injection of unlabeled line 4526 cells increased in mice fed 8 and 12% 18 : 2 compared with those fed lower levels of 18: 2. Thus, dietary 18 : 2 may increase metastasis by influencing the lodgement, implantation and survival but not proliferation of line 4526 mouse mammary tumor cells. ‘Correspondence to; K.L. Erickson. 0304-3835/89/$03.50 0 1989 Elsevier Scientific Publishers Published and Printed in Ireland
tumors;
The successful metastasis of malignant tumor cells to sites distant from the primary neoplasm is dependent upon the cell’s ability to complete a complicated series of sequential steps in the metastatic cascade. The events that normally occur for successful metastasis include: (1) detachment of tumor cells from the primary neoplasm, either singly or in clumps; (2) penetration of blood vessels or lymphatics for passive dissemination to distant sites; (3) lodgement and adhesion to blood vessels in host organs; (4) extravasation and subsequent survival and proliferation to form new tumor foci. The ability of tumor cells, then, to successfully metastasize, is dependent upon an interplay between the intrinsic characteristics of the cell and several host factors [4]. Modulation of cell characteristics or host factors could influence the degree to which tumor cells form distant metastases. We have recently reported that spontaneous metastasis from transplantable mammary fat pad tumors grown in mice fed high fat (HF, 20%, w/w) diets that contained high (12%) w/w) levels of linoleic acid (18 : 2) was increased when compared with mice fed
Ireland Ltd
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HF diets containing 8% or less 18 : 2 [8]. Since all diets were equal in the total amount of fat, and body weight did not vary among groups, we concluded that those affects on increased metastasis were due to changes in the dietary level of 18 : 2. In addition we showed that oral administration to mice of an eicosanoid synthesis inhibitor, indomethacin, completely inhibited the 18 : 2-mediated increase in metastasis. Results of those studies support the hypothesis that arachidonic acid (20 : 4) metabolites, such as prostaglandins may play a role in 18 : 2 enhancement of mouse mammary tumor metastasis. It is possible that several of the events in the metastatic cascade may be affected by a modulation of eicosanoid synthesis through dietary 18 : 2. For example, the lodgement of tumor cells in the blood vessels of host organs may be dependent upon platelet aggregation and the status of the hemostatic system [12]. Moreover, the survival of lodged tumor cells probably depends on the status of the immune system, which can be modulated by eicosanoids [1,9,13]. Though our results show that eicosanoids may play an important role in the 18 : 2-mediated increase in metastasis, the exact mechanism(s) is not known. In this study, we have investigated ‘the effects of dietary 18 : 2 levels on important steps, lodgement and survival, of the metastasis of line 4526 mammary tumor cells. In particular, we have determined whether dietary 18 : 2 influences the supportive tissue and modulates the implantation of the metastatic cell or whether it influences the metastatic cell itself. To correlate changes of tumor cell implantation and dietary 18 : 2, the fatty acid composition of the primary site of metastasis, the lung, was determined. In addition, we report the effects of various levels of dietary 18 : 2 on the proliferation of tumor cells within metastases. Materials and methods
Animals and diets Female BALB/cAnN mice 5 weeks old and
Table 1.
Composition of experimental diets. g/ 100 g in following concentrations of 18 : 2 in diet
Ingredient
1%
2%
4%
8%
12%
Casein”
24.8
24.8
24.8
24.8
24.8
Salt mixb*c
6.4
6.4
6.4
6.4
6.4
Vitamin mixCsd
1.7
1.7
1.7
1.7
1.7
Cellulosee
5.5
5.5
5.5
5.5
5.5
Cerelose (Dextrose)
41.6
41.6
41.6
41.6
41.6
Coconut oil’
19.2
17.9
15.2
9.9
4.5
0.8
2.1
4.8
10.1
15.5
Safflower oils
aAlldiets provided 28% of energy from protein. bProvided at 1.4 g/kcal of gross energy. See [2] for composition. dProvided at 0.3 g/kcal of gross energy. ‘Provided at 1.1 g/kcal of gross energy. Provided by Capital City Products (Columbus, OH). gProvided by California Fats and Oils (Richmond, CA).
previously fed a stock diet (Purina Mouse Chow, Ralston Purina, St. Louis, MO), were obtained from Charles River Laboratories (Kingston, NY). Mice were immediately segregated by weight then randomly placed into separate groups in order that the average weight per group was approximately the same. After receiving the stock diets for 48 h, mice were switched to the experimental diets (Table 1). All diets were isocaloric and contained at least the minimum level of recommended nutrients with a constant amount per kilocalorie of casein, salts, vitamins and fiber [ 151. Differing mixtures of coconut and safflower oil were added to make 20% fat (w/w) diets that contained either 1, 2, 4, 8 or 12% (w/w) 18 : 2. The fatty acid composition of the oil mixtures has been previously reported [8]. These two oils were chosen because they have comparable levels of oleic acid. Also, the final oil blends added to each of the experimental diets
119
contained approximately the same levels of oleic acid [8]. Animals received fresh food every other day and were fed the diets for the duration of the experiment. Experiments were not begun until the mice were fed the diets for at least 4 weeks. Body weights of mice were monitored weekly, and based on these measurements, no significant (P > 0.05) differences were observed in the mean body weight increases of mice from the different experimental diet groups (data not shown).
Mouse mammary tumor line 4526 is a cloned subpopulation from a metastatic nodule that appeared in a mouse growing the 10th in vivo passage of a spontaneous mammary adenocarcinoma in a BALB/cfC3H mouse [ 141. The cell line was obtained from Drs. G. Heppner and A. Fulton. All media and antibiotics were obtained from Whittaker MA Bioproducts, Inc. (Walkersville, MD), with the exception of the calf serum, which was obtained from HyClone (Logan, UT). Tumor cells from frozen stock were thawed and maintained in culture (5% CO,, 37OC) with Eagle’s minimum essential medium with Earle’s salts supplemented with 5% calf serum, 2% essential vitamin mixture, I % L-glutamine, I % nonessential amino acids, 1% sodium pyruvate and 50 pg/ml gentamicin.
13H]thymidine (spec. act., 6.7 Ci/mmol). Mice were injected 3 times at 8 h intervals, and received a total of 45 FCi each. Mice were killed 8 h after the last injection and lungs with metastases prepared for autoradiography. Five mice per dietary group were used for this experiment. Briefly, lungs with metastases were insufflated, using a 21 gauge needle, with 6 ml of 0.2 M glutaraldehyde fixative in phosphate buffer, removed, and placed in fresh fixative for 5 min. Lungs were then diced and placed in fixative overnight at 4OC. After rinsing twice in phosphate buffer for 30 min, the tissues were dehydrated in a graded series of ethanols, infiltrated and embedded in JB-4 embedding medium. Sections were cut at 1 pm, fixed onto glass slides which were then coated with Kodak NTB-3 emulsion (Eastman Kodak Co., Rochester, NY). Slides were placed in light-tight boxes at 4OC for 4 weeks. Slides were then developed, fixed, stained lightly with toluidine blue, and observed using a light microscope equipped with an oilimmersion objective and an intraocular grid. Fields containing metastases were randomly selected from each slide and the number of labeled (4 or more grains above or near the nucleus) and unlabeled cells per field were counted. Values were expressed as percent labeled cells of total cells counted and based on about 100 cells per field.
Tumor transplantation
Tumor cell survival
Tumor cells
and autoradiography
Monolayers of line 4526 in their exponential growth phase and from the same passage were rinsed with calcium- and magnesium-free Hank’s balanced salt solution (HBSS) then treated with a solution of 0.05% trypsin and 0.025% EDTA in the HBSS. The resultant cell suspension was passed through a 21 gauge needle to break up large clumps, washed, counted, and resuspended in HBSS. Mice were injected in the inguinal mammary fat pad with 1 x 104 tumor cells 4 weeks after being fed the experimental diets. Primary tumors were grown in mice until they reached a size of 12 mm x 12 mm (approx. 45 days), at which time mice were injected i.p. with 15 FCi of
Tumor cells were maintained as above, harvested, and passed through a 21 gauge needle to break up large clumps. An aliquot containing 1 x lo5 cells was injected i.v. into the laterai tail vein of mice fed HF diets containing either 1, 2, 4, 8 or 12% 18: 2. Five mice per dietary group were used. After 3 more weeks, mice were killed, their lungs removed, and total metastases quantitated using a stereoscopic microscope. Tumor cell lodgement
To determine whether dietary fat affects the supportive tissue or the tumor cell itself in the process of lodgement, two assays modified
120
from a technique previously described were used [3]. First, to assay for effects on supportive tissue, tumor cells were maintained in culture with complete medium as described above, then grown in medium for 48 h containing serum from mice fed 1% 18 : 2 before intravenous injection into mice fed the different experimental diets. Second, to assess whether 18 : 2 influences the metastatic cell itself, tumor cells were first maintained in culture as described above under “Tumor Cells”, then switched to media containing sera from mice fed each of the experimental diets before intravenous injection into mice fed the 1% 18 : 2 diet. Hence cells, cultured separately, were maintained in medium containing 5% serum from mice fed HF diets that contained either 1, 2, 4, 8, or 12% 18: 2. Although an alternative procedure of growing primary tumors in dietary-fat manipulated mice and preparing single cell suspensions for injection was tried, it proved to be less satisfactory than the procedure described above. The reasons for that are: first, contamination of the primary tumor digests with other cell types (the primary consisted of 70% tumor cells, 15% inflammatory cells, and 15% other types including fibroblasts; unpublished observations) ; and second, lipids from cells of dietaryfat manipulated mice can flux during the 24 h labeling period and cultures need to be supplemented with autologous mouse serum. When monolayers were 50% confluent, they were rinsed and then incubated in fresh medium that contained 0.5 pCi/ml, 5[lz51]iodo-2’-deoxyuridine (spec. act. 5 pCi/ mg) . In pilot studies, the optimal conditions for labeling without reduction in proliferation were determined. After 24 h, cells were rinsed 3 times using HBSS, harvested, passed through a 21 gauge needle to break up large clumps and aliquots containing 1 x lo5 cells were injected i.v. into the lateral tail vein of mice. Aliquots identical to injected tumor cell suspensions were collected at the same time. After 12 and 24 h post-injection mice were killed, their kidneys, lungs, spleen, liver and heart excised, rinsed in 3 changes of 70% ethanol
for 48 h to dissociate membrane bound label [3], and individually counted in a gamma radiation counter. Radioactivity was calculated on a per cell basis, correcting for counter and isotope background [2]. Five mice per group were utilized for this experiment which was repeated twice. Fatty acid analysis To assay fatty acid composition of tumor cells, line 4526 was grown in medium supplemented with 5 % serum from mice fed either 1, 2,4, 8, or 12% 18 : 2 diets. To assay fatty acid composition of the lungs and in order not to include any micrometastasis, a parallel group of mice fed the experimental diets but not injected with tumor cells were used. Fatty acid composition of cells and tissues was analysed as previously described [8]. Briefly, cell monolayers or lung tissues were homogenized in 1.2 ml of 0.1 M KC1 to which was added 6 ml of chloroform/methanol (2 : 1, v/v). The homogenate was transferred to a glass centrifuge tube, vortexed for 60 s, then centrifuged. The chloroform phase with extracted lipids was run through Na,SO, into a glass tube and the solvent evaporated under a continuous stream of nitrogen. The resultant lipid residues were dissolved in chloroform and stored under nitrogen at - 70°C until further use. Fatty acid methyl esters of samples were dissolved in methylene chloride and analyzed using a Hewlett-Packard 5880A gas-liquid chromatograph. The retention times were compared with those of known standards, and the relative levels of fatty acid in the lipid calculated by dividing the integrated areas of each peak by total area occupied by all peaks on the chromatogram. Statistical analysis Differences in body weight and fatty acid composition among the different groups were tested for statistical significance using one-way analysis of variance. Differences in metastasis were tested for statistical significance using the Wilcoxon rank-sum test for two groups or the Kruskal-Wallis one-way analysis of variance
121 Table 2.
Effect of dietary 18 : 2 level on lodgement
of line 4526 tumor cells=.
% 18:2in diet
12h
1 2 4 8 12
2840 3860 4140 3930 4310
Totalb
Spleen
Lung
24h
f f f f f
195’ 275 385 370 430
1000 1040 1520 1340 1450
24h
12 h
f llOd f 9Od + 85 + 100 f 105
400 350 290 300 450
* f + f +
30 25 20 50 80
100 120 140 250 220
24 h
12h
-c + f + +
15d lo’ 5’ 25 20
3900 4800 5130 4930 5290
+ + + + +
300” 400 450 425 415
1190 1280 1800 1690 1820
f: f f f +
150d 125d 195 165 155
‘Labeled tumor cells were grown in medium supplemented with serum from mice fed the 1% 18 : 2 diet and injected into mice fed HF diets that contained either 1, 2, 4, 8, or 12% 18 : 2. Values represent the mean number of cells lodged f S.E. for n = 75. bSum of the tumor cells distributed to 5 major sites: the heart, liver, kidneys, spleen and lungs, in the experimental metastasis assays. Significantly (P< 0.05) less than other dietary groups for this time point. dSignificantly (P< 0.05) less than 4.8 and 12% 18 : 2 dietary groups for this time point. “Significantly (P< 0.05) less than 8 and 12% 18 : 2 dietary groups for this time point.
by ranks. Differences were expressed when the level of significance was 0.05 or less. Statistical analyses were carried out using the Microsoft computer program (Ecosoft, Indianapolis, IN). Results Tumor cell lodgement
To test the influence of 18 : 2 on the tumor cell itself, cells were cultured in media supplemented with serum from mice that were fed various levels of 18 : 2 and injected into host mice fed 1% 18 : 2. No significant (P > 0.05) differences among the groups were observed in the lodgement of tumor cells in the body organs after 12 or 24 h (data not shown). However, in the reverse experiment to determine the influence of 18 : 2 on the supportive tissue, where tumor cells cultured in medium supplemented with serum from mice fed 1% 18 : 2 were injected into host mice that were fed diets contaiing various levels of 18 : 2, significant (P < 0.05) differences were observed (Table 2). To determine the nonorgan-specific effects of 18 : 2 on metastasis, the total number of tumor cells contained in
$
500
z z” P 3
450
2
400
r cz 6
350
z -!? z’
300
c r”
250 I 0
I
I
I
I
I
I
2
4
6
8
10
12
%18:2 Fig. 1.
14
in Diet
Effect of dietary 18 : 2 level on the survival of line 4526 mammary tumor cells. Mice were fed the experimental diets for 4 weeks then injected i.v. with 1 x lo5 unlabeled cells. After 21 days, mice were killed and metastases quantitated. The number of surface lung nodules of mice fed 8 and 12% 18 : 2 diets was significantly (P < 0.05) increased when compared to the number found in the lung of mice fed 1, 2, or 4% 18 : 2. Brackets are S.E. for n = 75.
122
the major sites, heart, liver, kidneys, spleen and lungs, was examined. After 12 h, the total number of cells lodged was significantly less (P < 0.05) in mice fed 1% 18 : 2 compared to mice fed the other diets. As expected, fewer cells were lodged in the organs after 24 h when compared to 12 h. After 24 h, the total number of cells lodged in organs of mice fed 1 or 2% 18 : 2 was significantly (P < 0.05) less than the number of cells lodged in mice fed 4, 8, or 12% 18: 2. At the 12 and 24 h time points, significantly (P < 0.05) fewer cells were lodged in the lungs of mice fed 1% or 1% and 2% 18 : 2, respectively, when compared to mice fed 4, 8 and 12% 18 : 2 diets. With respect to the spleen, no differences were observed in tumor cell lodgment after 12 h, but after 24 h, significantly (P < 0.05) fewer cells were lodged in the spleens of mice fed HF diets that contained 1, 2, or 4% 18 : 2 when compared to groups fed 8 or 12% 18 : 2. No significant (P > 0.05) differences in the number of tumor cells lodged in the heart, liver, or kidney, were observed among the dietary groups (data not shown).
cells within metastases of lungs of mice fed HF diets containing 1, 2, 4, 8, or 12% 18 : 2 was 18.8 f 2,4, 19.3 & 1.9, 22.3 f 3.4, 21.3 f 1.5 and 19.0 f 2.7, respectively. No significant (P > 0.05) differences were observed in the percentage of labeled cells when metastases of lungs from groups of mice fed various levels of 18 : 2 were compared. Tumor cell survival The effect of dietary 18 : 2 level on tumor cell survival in lungs of mice fed the various diets is shown in Fig. 1. There was a linear increase in the number of surface lung nodules with increasing dietary 18 : 2 level. However, the only statistically significant differences (P < 0.05) were the increased number of nodules in the 8 and 12% dietary groups compared to micefed1,2,or4% 18:2. Fatty acid composition
The total fatty acid composition of cells cultured in media supplemented with serum from mice fed various levels of dietary 18 : 2 was significantly (P < 0.05) altered (Table 3). Thus, cells injected i.v. for the lodgement studies differed in their fatty acid composition, depending upon the serum. The relative levels of oleic acid (18 : 1) were significantly (P < 0.05) decreased and the relative levels of
Proliferation
Autoradiography of the lung was performed to study proliferation of tumor cells within the metastatic nodule. The percentage of labeled
Table3. Fatty acid composition Fatty acid
% of total fatty acids with varying dietary 18 : 2 levels 1%
SFAb 16 : 1 18: 1 18 : 2 20:4
of line 4526 cells grown in vitro”.
45.0 3.6 27.2 12.3 6.4
2% f -t f -c f
1.2 0.5 1.2 0.6 0.9
47.7 2.7 24.0 12.9 4.7
4% * k f f f
1.1 0.5 0.9 0.5 0.7
48.4 2.5 25.8 15.8 4.5
12%
8% + f f f +
2.1 0.3 1.1 0.7d 0.6
45.9 3.0 21.8 20.2 5.1
f f + -c f
1.5 0.6 0.5 0.9 0.5
47.5 3.2 20.9 21.3 4.8
* f f + *
2.1 0.4 0.9 0.9 0.4
Walues represent the mean relative percent f S.E. for n = 15. Cells were grown in medium supplemented with 5% serum from mice fed either 1, 2, 4, 8, or 12% 18 : 2 diets, then extracted, derivatized, and analyzed by gas-liquid chromatography. bSaturated fatty acids (SFA) include 12 : 0, 14 : 0, 16 : 0 and 18 : 0. cSignificantly (P < 0.05) decreased when compared with 1, 2 and 4% 18 : 2 dietary groups for this fatty acid. dSignificantly (P< 0.05) greater than 1 and 2% 18 : 2 dietary groups for this fatty acid. -Significantly (P < 0.05) greater than 1, 2 and 4% dietary 18 : 2 groups for this fatty acid.
123
Table 4.
Fatty acid composition
Fatty acid
46 of total fatty acids with varying dietary 18 : 2 levels 1%
SFAb 18 : 1 18 : 2 20 : 4 Others
of lung tissue”.
51.4 22.6 3.5 10.3 4.7
4%
2% f 0.1 f 1.1 f 0.2 f 0.5 f 1.1
52.3 20.7 6.1 10.0 6.6
f f f f +
0.6 0.7 0.2 0.1 1.2
8%
50.4 17.4 10.4 10.8 6.8
-c f * f f
0.7 1.2d 0.4’ 0.4 1.2
45.8 17.2 18.7 9.2 5.1
12% f f f -c k
0.7= l.ld 1.4’ 1.1 1.6
44.1 14.1 24.3 9.3 4.3
+ f k -c f
0.4 0.3 0.5’ 0.7 0.9
*Values represent the mean relative percent of total f SE. for n = 30. Lungs were obtained from mice fed the experimental diets but not injected with tumor cells. bSaturatedfatty acids (SFA) include 12 : 0,14 : 0, 16 : 0 and 18 : 0. cSignificantly (P < 0.05) decreased when compared with 1, 2 and 4% dietary groups for this fatty acid. dSignificantly (P < 0.05) decreased when compared with 1 and 2% dietary groups and significantly (P < 0.05) increased when compared with 12% dietary group for this fatty acid. Significantly (P < 0.05) decreased when compared with other dietary groups for this fatty acid. Significantly (P < 0.05) increased when compared with group(s) containing less dietary 18 : 2.
18 : 2 were significantly (P< 0.05) increased in the 8 and 12% dietary groups when compared with cells grown in medium supplemented with serum from mice fed HF diets containing 1, 2, or 4% 18 : 2. Cells cultured in medium containing serum from mice fed 4% 18 : 2 had significantly (P < 0.05) greater levels of 18 : 2 than the 1 and 2% groups and significantly (P < 0.05) lower levels when compared to the 8 and 12% groups. To assess whether the fatty acids of the lungs, the major site of metastasis, changed as the level of dietary 18 : 2 varied, composition was assessed. Lung tissues of mice fed various levels of dietary 18 : 2 were also significantly altered (Table 4). The levels of saturated fatty acids (12: 0, 14: 0, 16: 0 and 18 :0)were decreased (P < 0.05) in animals fed 8 and 12% 18 : 2 when compared with the 1, 2 and 4% 18 : 2 dietary groups. Oleic acid was significantly (P < 0.05) decreased in mice fed 4, 8 and 12% 18 : 2 when compared with the 1 and 2% 18 : 2 groups and the relative levels of 18 : 2 significantly (P< 0.05) increased with increases in dietary availability of 18 : 2. Discussion
Metastasis is a complex multistep event in
the progression of malignant tumors that may be influenced by many systemic and local host factors as well as the properties of the tumor cell. We have previously shown that dietary 18 : 2 influences spontaneous metastasis in general. We now wish to determine whether 18 : 2 influences some of these specific events such as implantation, proliferation and survival. We have observed a modulation in the lodgement and survival of line 4526 mammary tumor cells by changing dietary 18 : 2 concentrations. When dietary 18 : 2 has an effect on the lodgement and implantation of the tumor cell it was on the host tissues rather than on the tumor cells themselves, because we observed significant increases in the lodgement of similar cells all cultured in 1% mouse sera from dietmanipulated mice, and no differences in lodgement of lipid-manipulated cells injected into similar mice all fed 1% 18 : 2 diets. Lack of an effect when the fatty acid composition of the sera changed was not due to lack of incorporation because our analysis indicated an increase in 18 : 2 levels and a decrease in oleic acid levels in the tumor cells as dietary 18 : 2 levels increased. The level of initial tumor cell lodgment in the lung appears to be directly related to the level of 18 : 2 found in that tissue. In our longer term assay using
124
autoradiograpy, we observed no difference in the rate of proliferation with the established metastatic nodule. Although we would also like to determine whether dietary fat has a direct influence on only the tumor cell, the microenvironment, or both, in the process of cell proliferation at a metastatic site, that could not be assessed because the tumor cell would change its fatty acid composition once placed in a different lipid environment, e.g. in dietaryfat manipulated mice. In a short period of time, tumor cell fatty acid composition would be similar to the fatty acid levels in the diet and the serum. For that reason, only short term experimental metastasis assays were used and only effects observed with a short term assay may be associated with changes in the tumor cell or the microenvironment. Fatty acid analysis of tumor cells injected into mice showed that changes in the serum were associated with changes in the lipid composition of the cells. Results of the tumor cell survival study indicate that 18 : 2 increased survival of tumor cells that lodged, since there were more surface lung metastases 21 days postinjection in mice that were fed diets containing higher levels of 18 : 2 (8 and 12%). Moreover, the increase in the number of surface lung nodules observed here and in other studies, was probably not due to an increase in the proliferation of tumor cells within the nodule, since we observed no differences in the percentage of radiolabeled cells among the dietary groups. The mechanism(s) involved in the 18 : 2mediated increases in lodgement and survival of line 4526 mammary tumor cells is not known. However, there are a few possible explanations. Increasing the level of 18 : 2 in the diet may serve to alter the synthesis of eicosanoids [5,11]. Eicosanoids can effectively modulate mechanisms of the immune system [9,10,13] and alter events in hemostasis [6,7,12]. Lymphocytes and macrophages could be suppressed by dietary fat and prostaglandins [ 1,9,13] resulting in an increase in the number of tumor cells that survive to form secondary foci.
In this study, we observed decreased tumor cell lodgement cells after 12 h of mice fed a low level of 18 : 2 (1%). After 24 h, we observed decreased lodgement in mice fed 1% and 2% 18 : 2. After 21 days, we observed a decrease in the number of experimental lung nodules in mice fed 1, 2 and 4%. In other studies [8] we observed increased spontaneous metastasis of primary line 4526 tumors from the mammary fat pad of mice fed 12% 18 : 2. These results suggest that increasing the level of 18 : 2 in the diet, first affects the number of cells that lodge. This may occur either by suppressing the immune system such that fewer cells are removed from the circulation, or by increasing the probability that cells will lodge by increasing platelet aggregation. Second, the level of dietary 18 : 2 can affect the number of cells or clumps that ultimately become secondary tumor foci. This may occur by modulating mechanisms of immune response. Acknowledgements
The authors wish to thank Dr. Steve Lanning of Capitol City Products for providing the coconut oil. This work was supported in part by grants from the Gustavus and Louise Pfeiffer Research Foundation and the National Dairy Board. Administered by the National Dairy Council. References Erickson, K.L. (1986) Mechanisms of dietary fat modulation of tumorigenesis: changes in immune response. In: Progress in Clinical and Biological Research, pp. 555586, Editors: C. Ip, D.F. Bitt, A.E. Rogers and C. Mettlin. Alan R. Liss, New York. Erickson, K.L., Schlanger, D.S., Adams, D.A., Fregeau, D.R. and Stern, J.S. (1984) Influence of dietary fatty acid concentration and geometric configuration on murine mammary tumorigenesis and experimental metastasis. J. Nutr., 114,18341842. Fidler, I.J. (1970) Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with i251-5iodo-2’-deoxyuridine. J. Natl. Cancer Inst., 45, 778782. Fidler, I.J. (1978) Tumor heterogeneity and the bilogy of cancer invasion and metastasis. Cancer Res., 38, 26512660.
125 5
6
7
8
9
10
Galli, C. (1980) Dietary influences on prostaglandin synthesis. In: Advances in Nutritional Research, pp. 95126. Editors: H.H. Draper. Plenum Press, New York. Gorelik, E., Wiltrout, R.H., Okumura, K., Habu, S. and Herberman, R.B. (1982) Role of NK cells in the control of metastatic spread and growth of tumor cells in mice. Int. J. Cancer, 30, 107-112. Honn, K.V., Bockman, R.S. and Marnett, L.J. (1981) Prostaglandins and cancer: a review of tumor initiation through tumor metastasis. Prostaglandins, 21.833-864. Hubbard, N.E. and Erickson, K.L. (1987) Enhancement of metastasis from a transplantable mouse mammary tumor by dietary linoleic acid. Cancer Res., 47, 6171-6175. Johnston, P.V. and Marshall, L.A. (1985) Dietary fat, prostaglandins and the immune response. Prog. Food Nutr. Sci., 8.3-25. Lala, P.L., Parhar, R.S. and Singh, P. (1986) Indomethatin therapy abrogates the prostaglandin-mediated supression of natural killer activity in tumor-bearing mice and prevents tumor metastasis. Cellular Immunol., 99, 108118.
11
12
13
14
15
Mathias, M.M. and DuPont, J. (1985) Quantitative relationships between dietary linoleate and prostaglandin (eicosanoid) biosynthesis. Lipids, 20, 791-801. Menter, D.G., Hatfield, J.S., Harkins, C., Sloane, B.F., Taylor, J.D., Crtssman, J.D. and Honn, K.V. (1987) Tumor cell-platelet interactions in vitro and their relationship to in uiuo arrest of hematogenously circulating tumor cells. Clin. Exp. Metastasis, 5, 65-78. Merttn, J., Stackpoole, A. and Shumway, S. (1985) Nutrition and immunity: the immunoregulatory effect of n-6 essential fatty acids is mediated through prostaglandin E. Int. Archs. Allergy Appl. Immunol., 77,390-395. Miller, F.R., Miller, D.E. and Heppner, G.H. (1983) Characterization of metastatic heterogeneity among subpopulations of a single mouse mammary tumor: heterogeneity in phenotypic stability. Invasion Metastasis, 3, 2231. National Research Council (1978) Nutrient Requirements of Laboratory Animals, 3rd edn. National Academy of Sciences, Washington, DC.