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Recent studies have linked conjugated linoleic acid (CLA) to altered tumorigenesis of several sites. We showed recently that a mixture of CLA isomers was able to significantly decrease mammary tumor metastasis in mice. That effect was seen with as little as 0.1% CLA in the diet. Other studies with dietary CLA have shown that various isomers may have differential effects. The purpose of this work was to assess which individual CLA isomers had similar effects in alteration of mouse mammary tumor metastasis. For that, we fed six 20% (w/w) total fat diets which contained either no CLA, low (0.1%, w/w) or high (0.25%, w/w) levels of cis9,trans11-CLA (c9,t11), trans10,cis12-CLA (t10,c12) or a mixture of the 2 isomers (0.125% of each, w/w) as free fatty acids. Neither the separate isomers nor the mixture had an effect on the latency or growth of primary line 4526 tumors when compared to the group without CLA. However, all diets containing CLA significantly decreased the total tumor burden (volume of tumor, mm(3)) in lungs of mice from both spontaneous metastasis (reduced by 42-73%) as well as implantation and survival of the metastatic cell (reduced by 46-61%) when compared with diets containing no CLA. Diets containing a greater concentration of either c9,t11 or t10,c12 had a significantly greater effect compared to the lower concentrations of the respective isomers when metastatic nodule size and total tumor burden were assessed. The diet containing both isomers decreased total tumor burden similarly to the diets containing the lower concentration of each of the isomers. Thus, the effects of c9,t11 and t10,c12 may not be additive and possibly share similar mechanisms for decreasing metastatic tumor burden in mice transplanted with mammary tumor cells.

Recent studies have shown that conjugated linoleic acid (CLA) can inhibit the initiation and thus, incidence of mammary tumors in rodents. The concentration of CLA required for these effects was as low as 0.1% of the diet, with no increased effects above 1%. To date, there is little evidence that CLA has any effect on growth or metastasis of mammary tumors. In this report, we demonstrate that CLA, at the concentrations used in previous studies, had a significant effect on the latency, metastasis, and pulmonary tumor burden of transplantable murine mammary tumors grown in mice fed 20% fat diets. The latency of tumors from mice fed CLA was significantly increased when compared with the 0% CLA control diet. The volume of pulmonary tumor burden, as a result of spontaneous metastasis, decreased proportionately with increasing concentrations of dietary CLA. With 0.5 and 1% CLA, pulmonary tumor burden was significantly decreased compared to mice treated with the eicosanoid inhibitor, indomethacin and fed diets containing no CLA. Tumors of mice fed as little as 0.1% CLA and as much as 1% had significantly decreased numbers of pulmonary nodules when compared with diets containing no CLA. The decrease in the number of pulmonary nodules by CLA was nearly as effective as indomethacin, a known suppressor of tumor growth and metastasis in this malignant model. These data suggest that effects of CLA on mammary tumorigenesis may go beyond the reported alterations in tumor incidence and effect later stages, especially metastasis.

We have previously shown that mice fed a high (n-3) fatty acid-containing diet with 20% (w/w) total fat had significantly slower mammary tumor growth, decreased numbers of metastatic pulmonary nodules, and decreased total metastatic load. In this study we sought to determine whether tumor vascularization was altered in mice fed diets varying in concentrations of (n-3) and (n-6) fatty acids. Several direct or indirect parameters of vascularization were tested. With 20% dietary fat, fish oil (FO) or a mixture of FO and safflower oil (FS) significantly reduced blood vascular area, mast cell number and macrophage infiltration in solid mammary tumors compared to tumors grown in mice fed safflower oil (SO). A decreasing trend was seen in the percent area of vessels positive for CD31 and vascular endothelial growth factor (VEGF) in the 20% FO and 20% FS compared to the 20% SO dietary groups. VEGF concentrations were twice as high in smaller tumors (100 mm3) from all dietary groups as compared to larger tumors (500 mm3). A two-fold increase in VEGF levels was found in the 20% SO dietary group compared to the 20% FO group in 100-mm3 but not larger tumors. We conclude that at 20% total fat, the n-3 fatty acids found in fish oil may inhibit primary mammary tumor growth through modulation of select determinants of vascularization.

We and others have previously shown that dietary fat can alter the growth and metastasis of rodent mammary tumors. Few transplantable tumor models have been used to study the effects of dietary n-6 versus n-3 fatty acids on mammary tumorigenesis. Here we study the effects of fish oil and safflower oil on the growth and metastasis of an animal model that in several ways parallels the human disease. Tumor latency, growth and metastasis were studied in mice fed diets that contained either 10 or 20% total fat which was varied in the type of fat with either menhaden fish oil (FO), safflower oil (SO) or a 50/50 mixture of the two. Tumor latency was significantly longer and tumor growth was significantly slower in mice fed the 20% FO diet. When spontaneous metastasis was assessed, mice fed diets containing FO had significantly decreased numbers of pulmonary nodules and total metastatic load. Likewise, mice fed FO diets had a lower level of implantation and survival of pulmonary metastases. Thus, in our animal model, diets containing n-3 fatty acids in fish oil significantly decrease primary breast tumor growth and its metastasis.

We have previously shown that a high fat diet (20% w/w) containing 12% linoleic acid (18:2) can significantly increase the metastasis of mammary tumor cells when compared with high fat diets that contain 8% or less 18:2 with a constant level of oleic acid (18:1). This effect may have been due to an alteration of eicosanoid metabolism because the cyclooxygenase inhibitor, indomethacin, abolished the increase. Because 18:1 may interfere with the metabolism of 18:2 to 20:4, we have now tested whether the 18:1 that supplements the 18:2 diet can have an effect on spontaneous or experimental metastasis of the line 4526 murine mammary tumor. For this, six 20% fat diets were formulated with 1%, 6%, and 12% 18:2 and either high or low levels of 18:1. Our results indicate that the amount of select fatty acids other than 18:2 at 12% has no significant effect on mouse growth, tumor growth, or tumor latency. When spontaneous metastatic burden was calculated, no significant differences between mice fed diets containing 1% and 6% 18:2 were observed. However, 4 to 5 times more of a metastatic burden was observed in mice fed diets containing 12% 18:2. No significant differences were observed between high and low 18:1 diets when the 18:2 content was 1 or 12%. However, at 6% 18:2, 18:1 significantly decreased metastatic burden. When experimental metastasis was assessed, relatively low levels of surface lung nodules were observed at 1% and 6% 18:2, but significantly higher levels were observed at 12% 18:2.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of the cyclo-oxygenase inhibitor, indomethacin (IM), on the metastasis, development and prostaglandin E (PGE) levels of line 4526 mammary tumors grown in mice fed high fat (HF, 20%, w/w) diets containing various levels of linoleic acid (18:2) was investigated. Control mice that grew primary tumors and were fed HF diets containing 12% 18:2 (w/w) had 2-3 times the number of lung metastases than mice fed 1%, 4%, or 8% 18:2. Chronic treatment of mice with 10 micrograms/ml IM in drinking water reduced metastasis in 1% and 4% 18:2-fed mice compared to controls and completely inhibited the increased metastasis of mice fed the 12% 18:2 diet. Treatment with IM also increased the latency and decreased the growth rates of primary 4526 tumors of all dietary groups. Treatment of mice with a higher dosage of IM (20 micrograms/ml), decreased tumor metastasis even further compared to controls, but did not decrease tumor growth rate compared to the lower dosage of IM (10 micrograms/ml). Tumor PGE levels, measured by radioimmunoassay (RIA), were decreased by IM treatment. These data provide evidence that arachidonic acid metabolites such as PGE may be involved in the metastasis of 4526 mammary tumors.

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, 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 [3H]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.

The effect of dietary fat concentration and saturation on cell composition and structure of line 168 mouse mammary tumors in vivo was studied using morphometry and electron microscopy. Both the concentration and saturation of fat fed to mice had a significant influence on the volume ratio of mast cells infiltrating line 168 tumors. Tumors of mice fed diets containing a high concentration (20%) of either safflower oil (SO) or palm oil (PO) had 2-3 times the volume ratio of mast cells than mice fed diets containing a low concentration (5%) of either fat. There were no significant differences among diets with respect to other inflammatory cell populations. Mice fed either one of the high fat diets had tumor cells with inclusions that ultrastructurally, appeared to consist of lipid. Dietary fat, however, had no observable affect on cell junctions or other morphological characteristics. Greater infiltration of mast cells in tumors of mice fed high fat diets and the eventual formation of new blood capillaries may explain the decreased latency of tumor onset and enhanced growth of tumors in mice fed diets with high concentrations of fat.