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Long-chain omega-3 polyunsaturated fatty acids decrease mammary tumor growth, multiorgan metastasis and enhance survival

  • Saraswoti Khadge
  • Geoffrey M. Thiele
  • John Graham Sharp
  • Timothy R. McGuire
  • Lynell W. Klassen
  • Paul N. Black
  • Concetta C. DiRusso
  • Leah Cook
  • James E. Talmadge
Research Paper

Abstract

Epidemiological studies show a reduced risk of breast cancer (BC) in women consuming high levels of long-chain (LC) omega-3 (ω-3) fatty acids (FAs) compared with women who consumed low levels. However, the regulatory and mechanistic roles of dietary ω-6 and LC-ω-3 FAs on tumor progression, metastasis and survival are poorly understood. Female BALB/c mice (10-week old) were pair-fed with a diet containing ω-3 or an isocaloric, isolipidic ω-6 diet for 16 weeks prior to the orthotopic implantation of 4T1 mammary tumor cells. Major outcomes studied included: mammary tumor growth, survival analysis, and metastases analyses in multiple organs including pulmonary, hepatic, bone, cardiac, renal, ovarian, and contralateral MG (CMG). The dietary regulation of the tumor microenvironment was evaluated in mice autopsied on day-35 post tumor injection. In mice fed the ω-3 containing diet, there was a significant delay in tumor initiation and prolonged survival relative to the ω-6 diet-fed group. The tumor size on day 35 post tumor injection in the ω-3 group was 50% smaller and the frequencies of pulmonary and bone metastases were significantly lower relative to the ω-6 group. Similarly, the incidence/frequencies and/or size of cardiac, renal, ovarian metastases were significantly lower in mice fed the ω-3 diet. The analyses of the tumor microenvironment showed that tumors in the ω-3 group had significantly lower numbers of proliferating tumor cells (Ki67+)/high power field (HPF), and higher numbers of apoptotic tumor cells (TUNEL+)/HPF, lower neo-vascularization (CD31+ vessels/HPF), infiltration by neutrophil elastase+ cells, and macrophages (F4/80+) relative to the tumors from the ω-6 group. Further, in tumors from the ω-3 diet-fed mice, T-cell infiltration was 102% higher resulting in a neutrophil to T-lymphocyte ratio (NLR) that was 76% lower (p < 0.05). Direct correlations were observed between NLR with tumor size and T-cell infiltration with the number of apoptotic tumor cells. qRT-PCR analysis revealed that tumor IL10 mRNA levels were significantly higher (six-fold) in the tumors from mice fed the ω-3 diet and inversely correlated with the tumor size. Our data suggest that dietary LC-ω-3FAs modulates the mammary tumor microenvironment slowing tumor growth, and reducing metastases to both common and less preferential organs resulting in prolonged survival. The surrogate analyses undertaken support a mechanism of action by dietary LC-ω-3FAs that includes, but is not limited to decreased infiltration by myeloid cells (neutrophils and macrophages), an increase in CD3+ lymphocyte infiltration and IL10 associated anti-inflammatory activity.

Keywords

PUFA Omega-3 Mammary tumor Metastasis Survival 

Abbreviations

AA

Arachidonic acid

BC

Breast cancer

CBC

Contralateral breast cancer

CMG

Contralateral mammary gland

DHA

Docosahexaenoic acid

EPA

Eicosapentaenoic acid

FA

Fatty acid

HO

Histologically observed

LC

Long-chain

MFP

Mammary fat pad

MG

Mammary gland

NTB

Non-tumor bearing

NLR

Neutrophil to lymphocyte ratio

PUFA

Polyunsaturated fatty acid

qRT-PCR

Quantitative real-time polymerase chain reaction

TB

Tumor bearing

Introduction

Breast cancer (BC) is one of the leading cause of cancer deaths of women in the United States [1]. However, there is a significant difference in BC incidence between populations consuming Western diets, with those, who consume an Asian diet, typically due to a higher intake of fish, supporting dietary polyunsaturated fatty acid (PUFA) composition as a risk factor for BC [2, 3, 4, 5]. A typical Western diet contains higher levels of omega (ω)-6 FAs relative to ω-3 FAs, mainly long-chain (LC)-ω-3FAs [6]. The LC-ω-3FAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are metabolized to bioactive lipid mediators important in inflammation resolution [7]. In contrast, ω-6 FA metabolites such as arachidonic acid (AA) are pro-inflammatory mediators and chronic production of AA has been associated with inflammation-associated morbidity including BC patients [8]. Thus, the ratio of dietary ω-3 and ω-6 FA has a critical role in determining the inflammatory status of tissue microenvironments, which can modulate tumor growth, metastasis and the efficacy of therapeutic treatments [9, 10].

In murine studies, dietary LC-ω-3FAs early in life results in reduced incidences and growth of mammary tumors compared to the mice fed diets high in ω-6 FA [11, 12]. Further, feeding LC-ω-3FA-enriched diets in adult life results in significantly lower mammary gland (MG) ductal density, proliferation of epithelial cells in the MG, and adipose tissue inflammation, all of which are associated with reduced-risks of BC [13, 14, 15]. However, dietary LC-ω-3FAs regulation of mammary tumor microenvironments, tumor progression, metastasis and survival, has been little studied.

Despite recent advances in the development of molecularly targeted therapies, most deaths due to cancer result from the progressive growth of therapy-resistant metastases [16]. Metastasis is regarded as a highly inefficient process so that < 0.01% of circulating tumor cells eventually succeed in forming secondary tumor growths [17]. The growth of a metastatic lesion in a specific organ is determined by the intrinsic properties of the tumor cells (seed) and the supportive role of the microenvironment of the target organ (soil) so that some cancer cells grow preferentially in one organ while not in other organs [18, 19]. Most invasive BC cells preferentially metastasize to bone, lungs, liver and brain [19] while ovarian [20], cardiac [21] and renal [22] metastases have been less frequently observed. Similarly, 4T1 mammary tumor cells predominantly metastasize to lungs in the spontaneous metastasis model whereas metastasis to liver, heart, kidneys and bone have been less frequently reported and there are no published data on ovarian metastases by 4T1 cells [23, 24, 25, 26]. In animal models and in vitro studies, ω-3 and ω-6 FA have been shown to modulate tissue microenvironments of MG, liver, bone, ovaries, heart and kidneys [13, 27, 28, 29, 30, 31, 32]. However, the role of the modulated microenvironments by pre-exposure to PUFA in the regulation of metastasis to those organs have not been studied. Further, the role of dietary PUFA composition in mammary tumor microenvironments such as inflammation, neo-vascularization and apoptosis have been little studied.

In this study, we hypothesize, that LC-ω-3FA diets can suppress mammary tumor cell proliferation and metastasis by modulating tumor and metastatic microenvironments. By pair-feeding isocaloric and isolipidic diets in an orthotopic model of mammary tumor, we uniquely analyzed the effects of dietary LC-ω-3FA on tumor microenvironments, measured as tumor cell proliferation and apoptosis, neo-vascularization and infiltrating leukocytes with pro-and anti-tumor inflammatory activities. Further, with the injection of low numbers of 4T1 cells, we established a tumor model capable of assessing the differential regulation of dietary PUFA on spontaneous metastasis to different organs including bone, heart, kidneys, ovaries and contralateral mammary glands (CMG), which have not previously been reported.

Materials and methods

Animals, diets and pair-feeding

Female BALB/c mice (6 weeks old) purchased from Charles River Laboratories were housed in groups of five mice per cage, under standard conditions of temperature and humidity, with a 12 h light–dark cycle. The mice were caged in micro-isolators under HEPA filter air supply. Routine health monitoring of control mice was undertaken as part of a protocol to ensure specific pathogen-free conditions. The Institutional Animal Care and Use Committee at the University of Nebraska Medical Center approved the protocol for this study.

Following shipment and acclimatization, mice were segregated at random into the experimental dietary groups; ω-6 and ω-3. All mice were mixed in a single container then randomly assigned into dietary groups. The ω-6 diet consisted of the Lieber-DeCarli control diet (Dyets # 710027) while the ω-3 diet was customized by using the base diet (Dyets # 710166) and adding; 8.4 gm/L olive oil and 20 gm/L of fish oil (NutriGold Triglyceride Omega-3 Gold capsules; Lot# 0081-3180-2) (Table S1). Both liquid diets provided 1.0 Kcal energy per mL of diet and equal calories from macronutrients (35% derived from fat, 47% from carbohydrate, and 18% derived from protein). The ω-6 and ω-3 diets were iso-caloric and iso-lipidic diets, differing primarily by the presence or absence of LC-ω-3FA from fish oil and olive oil respectively. Omega-3 capsules and Lieber-DeCarli powder diets were stored at 4 °C for up to 2 weeks and at − 20 °C for longer times. Diets were prepared and delivered every 24–26 h, and daily consumption monitored. The diets were provided using the pair-fed model as described previously [27]. Briefly, ad libitum access to the liquid diets was provided for the first 5 days for acclimatization. From day six forward, the ω-6 diet group mice were pair-fed based on mean consumption of the ω-3 diet group from the previous day, and this was continued throughout the study. Body weights were determined and recorded every week until mice were used for tumor experiments and twice a week once tumor cells were inoculated. Tumor cells were inoculated 10–16 weeks after the start of liquid diets.

Culture of 4T1 cells and orthotopic inoculation

Mammary adenocarcinoma 4T1 cells derived from BALB/c mice [33] were used in an orthotopic mammary tumor model. These tumor cells were a generous gift from Dr. Fred R. Miller [33]. Tumor cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Giblco# 11995-065, Grand Island NY) containing 10% fetal bovine serum (#S11150, Atlanta biologics), 2% of 100× MEM vitamins (Gibco # 11120-052), 1% of each of 200 mM l-glutamate (Gibco# 25030-081), 100 mM sodium pyruvate (Gibco# 11360-070), 50× MEM amino acids (Gibco#11130-051), 100× non-essential amino acids (GE healthcare# SH30238.01), and 0.5% of 50 mg/mL gentamycin (Gibco# 15750-060) at 37 °C with 5% CO2.

Cells growing in log phase (40–60% confluent) were treated briefly with 0.05% trypsin–EDTA (Gibco# 25300-054), washed twice at 4 °C and re-suspended in Ca++–Mg++ free (CMF) Hank’s balanced salt solution (HBSS) (Gibco# 14170-112). Live cells were counted using trypan blue (Corning, 25-900CI) and re-suspended in concentrations of 5000 cells/100 μL CMF-HBSS. For tumor inoculation, 100 μL of 4T1 cells were injected in the left inguinal (fifth) mammary fat pad (MFP). The MFPs were palpated daily from day 3 of injection until all animals had palpable tumors. Body weights and tumor volume were measured every 3 days. Tumor volume was calculated using the formula [0.5 (short diameter)2 × long diameter].

Metastasis analysis

Groups of mice (n = 10/group) were autopsied on the day 35 post tumor injection for the single time point metastases analysis in two independent experiments. Overt metastases were analyzed in all animals from both experiments (n = 20/group). Histological analysis of the presence of metastatic foci in cross/longitudinal section of organs was performed in all animals from a single experiment (n = 10/group). The histologically analyzed metastatic foci were abbreviated as histologically observed metastasis (HO-met) in the figures. Lungs, liver, heart and kidneys were fixed in Bouin’s fixative for 24 h prior to counting of the total number of nodules of overt metastases on the surface of the indicated organ using a Stereoscope (Zeiss # Stemi DV4). After overt metastasis analysis, heart and kidneys were longitudinally sectioned (including cephalo-caudal regions of the organ). One-half of the organ was embedded, and cross-sectioned from the deeper tissue side. A piece of liver and spleen were also embedded and sectioned. Both ovaries and a half of the CMGs were collected and fixed in 10% normal buffered formalin (NBF) (Fisher # SF100) for 24 h at RT prior to embedding and cross-sectioning. All the tissues were sectioned at 4 µm thickness, and stained with H & E for the histological analysis of the metastases. The number and size of metastatic foci per cross-section of heart, kidney and ovaries were evaluated using quantitative analyses. The presence of metastases in liver and spleen was confirmed by scanning sections of the tissue at ×200 and ×400. Individual tumor cells were identified based on 4T1 tumor cell morphology.

For skeletal metastasis analysis, both hind limbs were collected and fixed in 10% NBF for 24 h, washed in 1× PBS, and muscles removed to isolate the femurs and tibias. The bones were decalcified by immersing them in decalcification solution (15% EDTA in PBS, pH 7.6) at 4 °C until the bones became soft enough to section. Lateral-longitudinally sectioned femurs–tibias (4 µm) were stained with H & E. Bone metastases were analyzed in medullary spaces of epiphysis/metaphysis and diaphysis of the femurs and the epiphysis/metaphysis areas of the tibia and presented as the number and size of histologically observed metastases per longitudinal section of femurs–tibias.

Immunohistochemistry (IHC)

Tumors and CMGs were collected on day-35 post tumor injection from a single experiment (n = 10/group). The tumors were cross-sectioned and representative sections were fixed in NBF. CMGs were cut in half and representative portions were fixed in NBF, embedded, and sectioned at 4 µm thickness. For IHC analysis, sections were deparaffinized, rehydrated, antigen retrieved, incubated in BLOXALL (Vector labs# SP-6000, Burlingame, CA) and blocked with 2.5% normal horse serum (Vector labs# S-2012) as described previously [13, 27]. The antibodies used in the study were rabbit monoclonal antibodies purchased from Abcam Inc. (Cambridge, MA) including and their concentrations/dilutions used for IHC are as follows: Ki67, 1:100 (ab16667), F4/80, 1:100 (ab111101), CD31, 0.3 µg/ml (ab182981), neutrophil elastase, 1 µg/mL (ab68672) and CD3, 1:100 (ab16669). The sections were incubated overnight at 4 °C with the indicated primary antibodies. For negative controls, serial sections were processed in the absence of primary antibody but with all other reagents employed. The sections were treated with ImmPRESS-AP, anti-rabbit Ig (Vector labs# MP-5401) and the staining was detected using ImmPACT Vector Red alkaline phosphatase reagent (Vector labs# SK-5105) as described previously [13]. Apoptotic nuclei were detected using “In situ Apoptosis Detection Kit” (ab206386, Abcam) as per the kit’s protocol. Images were captured using a Zeiss Axioplan-2 microscope with a HRc camera, and analyzed using Zeiss AxioVision Rel 4.8 software. Two independent investigators examined the sections with “blinded” group assignments and an experienced pathologist validated the results. Subcapsular oxygenated areas of tumor were evaluated for all IHC analyses in tumor tissues. The number of positive cells in ten randomly chosen fields/sample were recorded for a quantitative analysis of Ki67, TUNEL, CD31, Neutrophil-elastase, F4/80 and CD3 positive cells in tumors and Ki67 positive cells in CMGs.

Quantitative real time PCR analysis

Sections of tumors from subcapsular (actively growing oxic areas) of tumors and a section of spleen were collected in a single experiment in which mice were autopsied on day-35 post 4T1 injection (n = 10/group). Freshly collected tissues were fixed in RNA stabilizing solution RNAlater (# 76106, Qiagen) for 24 h at RT, transferred, and stored at − 20 °C until use. RNA extraction, gel electrophoresis, cDNA synthesis and qRT-PCR were performed as described previously [13]. Briefly, total RNA was isolated with TRIzol reagent (15596026, Invitrogen). Complimentary DNA (cDNA) was prepared from RNA using high-capacity cDNA synthesis kit (4374966, Applied Biosystems; AB) as per company protocol. The qRT-PCR master mix was prepared using TaqMan gene expression master mix (4370074, AB), anti-mouse probes and 10 ng of cDNA in Micro Amp fast optical 96 wells plate (4346906, AB). The samples were analyzed using a StepOnePlus qPCR machine (Applied Biosystems). Pre-synthesized mouse probes from Thermofisher were used for the study. The probes used in the study were; GAPDH (Mm99999915_g1), IGF1 (Mm00439560_m1), amphiregulin (AREG; Mm01354339_m1), TNF alpha (TNFα; Mm00443258_m1), CCL2 (Mm00441242_m1), leptin (Lep; Mm00434759_m1), IL6 (Mm00446190_m1), Prostaglandin-endoperoxide synthase (PTGS2; Mm00478374_m1), NFkB1 (Mm00476361), IL10 (Mm01288386), G-CSF (Mm00438334_m1), and GM-CSF (Mm01290062_m1). Negative controls were analyzed for each probe in the plate. Each qRT-PCR experiment was carried out in triplicate (technical replicates), and average cycle thresholds (Cts) were obtained for tumors of mice from each dietary group (n = 10) (biological replicates). The mean Ct values from technical triplicates of each sample was used as the Ct value for the sample. The relative quantity of target mRNA was determined using the Levak method [24]. Briefly, the Ct value of the gene of interest from a sample was normalized to the Ct value of the reference gene for the same sample to obtain the respective threshold cycle difference (dCt). Then the mean dCt values from the biological replicates of each dietary group was used as the dCt of target for the group. Finally, the dCt obtained for the ω-3 group was normalized to the dCt of the target from the ω-6 group to obtain ddCt values and obtained the relative fold change.

Statistical analyses

Results were expressed as a mean ± standard error of the mean (SEM) for animal weights and IHC quantification data and were compared between the dietary groups by Student’s t-test for independent samples. The difference in the incidences of metastases was analyzed by Fisher’s exact test for the differences in the proportions between the experimental groups. Metastases data including organ, weights, metastases number and area of histologically-observed metastases were presented as median values and compared by Mann Whitney’s test. Longitudinal plots for change in body weights and tumor growth and dietary consumption were based on the mean ± SEM value for each time point and the curves between the dietary groups were compared by repeated measures analysis (two-way). Graphs were plotted using Sigma Plot or Graph-pad Prism. Survival analysis was presented as Kaplan–Meier plots and statistical significance was analyzed using the log-rank test. All data were analyzed using SPSS for statistical significance. For qRT-PCR data, dCt values were compared by independent sample t-test. In all instances, differences where p < 0.05 were considered to be statistically significant.

Results

Dietary LC-ω3FA regulate mammary tumor induction, growth and mouse survival

Mice (n = 20/group) were fed the experimental diets for 10–16 weeks before use in two independent tumor experiments. The supplemental figure (Fig. S1) shows the data from pair-fed groups of mice in a single experiment (n = 20/group). There was no difference in the average dietary consumption between the groups during the non-tumor bearing (NTB) period; (Fig. S1A) but the dietary consumption levels significantly decreased in the ω-6 group during the tumor bearing (TB) phase of the experiments (Fig. S1B).

The effects of dietary LC-ω-3FAs on the regulation of mammary tumor initiation, growth and survival from pooled data of two independent studies are shown in (Fig. 1). Out of n = 40 mice/group used in the study, 100% (40/40) of the mice from the ω-6 group and 95% (38/40) of mice from the ω-3 group developed palpable tumors within the experimental period (89 days). Among the mice which developed tumors, the median time to a palpable tumor were significantly delayed for the ω-3 group (10 days, range 6–32 days) compared to a median of 7 days, range 5–22 days for the ω-6 diet group (Fig. 1A). Similarly, mice fed the ω-3 diet had a significantly slower mammary tumor growth compared to the ω-6 diet-fed mice (Fig. 1B). These data are supported by a 50.2% smaller tumor volume (446.3 ± 52.3 mm3) and 48.8% lower tumor weight (0.6 ± 0.1 g) in mice fed the ω-3 diet compared to the tumor volume (888.5 ± 115.2 mm3) and weight (1.2 ± 0.2 g) in the mice fed the ω-6 diet on the day-35 post 4T1-tumor injection autopsy (n = 20/group, p < 0.05) (Fig. 1C, D). The body weights of the mice were evaluated at different times during the experiment to analyze the effects of dietary PUFA composition on body mass before and during tumor growth (Fig. 1E). There was no difference in body weights when the experimental diets were started at the age of 10 weeks. However, after feeding the diets for 16 weeks, mice given the ω-3 diet had significantly lower body weights compared to the mice on ω-6 diet, resulting in a significant difference in the body weights on the day of tumor injection. When the tumors were palpable, changes in body weights may be influenced by multiple factors in addition to diet, including tumor growth and weight, and tumor-associated metabolic changes (cachexia). Thus, we analyzed the body weights at autopsy [day 35 post tumor injection Fig. 1E(c)] and after subtracting the weight of the tumor on that day [Fig. 1E(d)]. Both dietary groups of mice lost significant body weight by the day of autopsy compared to their weights at tumor injection. The ω-3 diet mice had significantly lower body weights (with or without tumor) on day 35 post tumor injection; although, the % change in body weights (-tumor weights) relative to their weights on tumor injection day were not different between the groups [Fig. 1E(e)].

Fig. 1

Dietary PUFA regulation of 4T1 tumor induction, growth and survival. Mice were provided an isocaloric, isolipidic pair fed ω-3 (W3 in figure) or ω-6 (W6 in figure) diet for 10 weeks prior to orthotopic injection of 4T1 cells in the left rear mammary gland (MG). Pooled data from two experiments were analyzed as shown. A Kaplan–Meier plot of the time to development of a palpable tumor (log-rank test). B Longitudinal changes in tumor volume (repeated measures test). C Tumor weight and D tumor volume were compared from mice autopsied on day 35 post-tumor injection. Horizontal line represents median values and Mann–Whitney test was used to analyze the difference between the dietary groups. E Body weights of the mice were compared at key study landmarks: body weights at the start of the experimental diets, at the time of tumor injection, at autopsy on day-35 post tumor injection, host weight at autopsy (body weight minus tumor weight) and percent body weight loss at autopsy relative to the day of tumor injection. $,#p < 0.05 for the change in host body weight at autopsy in ω-3 and ω-6 groups respectively, compared to the respective weights on tumor injection day (t-test). F Kaplan–Meier survival plot of mice on ω-3 and ω-6 diets (n = 20mice/group) (log-rank test). *p < 0.05 compared to mice fed the ω-6 diet

Among the mice used in the study, 20/group that were pooled from two studies were autopsied on day 35 post injection and the extent and sites of metastases assessed and the survival of the remaining 20 mice/group was monitored. During the study period of 89 days, two mice from the ω-3 group did not develop tumors, and one mouse had a palpable tumor, which did not grown to a measurable size by 89 days post tumor injection, when the study terminated. The survival analysis showed that a mice fed the LC-ω3FA diet for 16 weeks of adult life prior to tumor challenge significantly prolonged overall survival (a median survival time (MST) of 46.5 days) compared to the MST of 35.5 days for the ω-6 diet-fed mouse group (p < 0.05) (Fig. 1F).

Effect of dietary PUFA in 4T1 tumor metastasis to lung, liver and bone

Metastasis analysis was performed on the day 35 autopsied mice from two independent experiments (n = 10/group/experiment). The extent of metastasis was determined by counting overt metastases on the organ surfaces [34] and by evaluating the weights of organs autopsied on the same day. Further, smaller and deep tissue metastases were evaluated by analyzing metastatic lesions in H & E stained tissue sections. Among the organs investigated, the lungs had the highest number of metastases in both dietary groups and metastases were detected in all animals (Table 1). However, the ω-3 diet group mice had a 64% lower number of pulmonary overt metastases (median = 28; range 2–187) compared to the ω-6 group (Median = 78; range 8–312) (p < 0.05) (Fig. 2b). This observation was supported by the lower lung weights in mice fed the ω-3 diet that were 36% lighter (median = 0.27 g; range 0.16–0.74 g) relative to the lungs of the ω-6 fed group (median = 0.47 g; range 0.16–0.96 g) (p < 0.05) (Fig. 2c). Histopathological analysis of lung metastases showed that the majority of metastatic pulmonary foci were localized superficially on the lungs while metastatic foci in the lungs from mice given the ω-6 diet were localized throughout the pulmonary parenchyma (Fig. 2a). An analysis of hepatic metastases (Table 1) (Fig. 2d–f) showed that there was a 47% lower incidence of mice with hepatic overt metastases in the mice that received the ω-3 diet (8/20 mice) compared to the incidence in the ω-6 group (15/20 mice) (p = 0.05). However, there were no significant difference in the median number of hepatic overt metastases nor the weights of livers between the dietary groups (Fig. 2e, f). Histopathological analysis showed that the livers from ω-3 diet-fed mice had fewer and smaller metastatic foci deeper in the hepatic parenchymal tissue compared to mice in the ω-6 fed group (Fig. 2d). There were individual tumor cells and foci of few tumor cells in the livers from the ω-3 fed group, while the histologically observed metastases in the livers from the ω-6 group were macrometastases (≥ 2 mm in size). Quantitative analysis of the hepatic metastasis size was challenging due to extensive extramedullary myelopoiesis (EMM) within the metastatic foci preventing reliable analyses of the metastasis area. The tumor weights were directly correlated with the weights of lungs, the number of overt metastases in lungs and the weights of liver suggesting a direct relationship to tumor growth with metastases to these organs (Fig. S2A, C).

Table 1

Dietary Lc-ω-3FA regulation of incidences of metastasis

Metastases incidence

Organ

Mice/group (n)

Mice with overt-met [n (%)]

Mice/group (n)

Mice with HO-met [n (%)]

W3

W6

W3

W6

Lung

20

20 (100%)

20 (100%)

n/a

n/a

n/a

Liver

20

8 (40%)

15 (75%)

n/a

n/a

n/a

Heart

20

2 (10%)*

12 (60%)

10

2 (20%)

6 (60%)

Kidney (at least one)

20

2 (10%)*

15 (75%)

10

1 (10%)

6 (60%)

Kidneys (both)

20

2 (10%)

7 (35%)

10

0 (0%)

2 (20%)

Ovary (at least one)

 

n/a

n/a

10

1 (10%)*

9 (90%)

Ovary (both)

 

n/a

n/a

10

0 (0%)*

6 (60%)

Bone (one leg)

 

n/a

n/a

10

1 (10%)

5 (50%)

Bone (both leg)

 

n/a

n/a

10

0 (0%)

3 (30%)

Mice autopsied on day 35 post tumor injection were analyzed for the incidence of overt-metastasis (overt-met) (n = 20 mice/group) and histologically-observed metastasis HO-met (n = 10 mice/group). The results are presented as number of mice with at least one metastasis in the indicated organ (n) and percent representation per total animal analyzed in the group (%)

n/a indicates either the analysis was not applicable or not done

*p < 0.05 ω-3 versus ω-6 diet-fed mice. Fisher exact test for the difference in proportions was used to analyze statistical differences

Fig. 2

Dietary PUFA regulation of 4T1 metastasis to the lungs, liver, spleen and femur. Mice from two experiments were autopsied on day 35 post tumor injection and analyzed for overt metastases (overt-met) (n = 20/group) and histologically-observed metastases (HO-mets) (n = 10mice/group). a Hematoxylin and eosin (H & E) stained section of lungs with HO-mets. b Median number of gross-mets and c median lung weights at autopsy. d H & E liver sections with HO-mets. e Median number of overt-met on the liver surface and f median liver weights at autopsy. g H & E stained sections from spleens showing foci of myeloid cells. h Spleen weights. i Linear regression analysis of tumor weight. j H & E stained sections of decalcified femurs that had HO-met. k One mouse from the ω-3 group has one smaller HO-met while five mice in the ω-6 group had small to large sized HO-mets. a, d, j, k Were taken at ×100. g, j Were taken at ×25 and ×400 magnification respectively. *p < 0.05 compared to the mice fed ω-6 diet (Mann–Whitney test)

The effects of dietary PUFA on the spleen was evaluated based on weights and histological analyses. There were significantly lower spleen weights in the ω-3 dietary groups compared to the ω-6 group (Fig. 2h). Further, the spleen weights directly correlated with tumor weights in both groups of mice (Fig. 2i). Histological analyses of spleen sections showed splenomegaly was predominantly due to increases in EMM in the sub-capsular and red pulp areas, as previously reported [35]. Individual tumor cells and/or clusters of a few tumor cells were observed in all spleens independent of the dietary group (Fig. 2g). However, the incidence of distinct histologically observed metastases (micro and macro metastases) was non-significantly (33%) lower in the spleens from ω-3 diet-fed mice (4/10) compared to the ω-6 group (6/10) (p = 0.6).

Bone metastases by 4T1 tumors have been rarely reported, with the exception of sub-clone 4T1.2 tumor cells which were selected for their propensity to metastasize to bone [36]. In the current study, we report dietary PUFA regulation of bone metastases in this 4T1 orthotopic primary tumor bearing mice model. The presence of osseous metastases was suggested by the observation of hind-limb paralysis in the ω-6 group of mice. We analyzed metastases in the longitudinal sections of femurs and tibias from both hind limbs of mice autopsied on day 35 post tumor injection to evaluate the effects of dietary PUFAs on bone metastases (n = 10 mice/group). Mice fed the ω-3 diet had an 80% lower incidence of bone metastases in at least one leg (1/10 mice) relative to the incidence in the ω-6 group (5/10 mice) (p = 0.1). Similarly, (3/10) mice on ω-6 diet had metastases in the femur and/or tibias from both hind limbs, which was not observed in mice fed the ω-3 diet (0/10) (p = 0.08) (Table 1). Further, there were significantly fewer (median = 0; range 0–1) and smaller (median = 0; range 0–0.01 mm2) metastases per femurs/tibias in ω-3 diet-fed mice, compared to the retrospective analyses of number (median = 0; range 0–6) and size (median = 0; size: 0–0.35 mm2) of metastases in the ω-6 diet-fed mice (Fig. 2j, k). Histopathological analyses showed the larger metastases of the ω-6 group were localized to the femur metaphyses, specifically around the growth plate, while smaller metastases were present throughout the distal and proximal epiphyses and diaphyses of the femurs and epiphyses of the tibias. The only metastasis detected in an ω-3 diet-fed mouse was localized in the femur diaphysis medullary space (Fig. 2j). Despite variations in the location of metastases, all of the detected metastases in long bones were adjacent to trabecular bone, indicating potential interactions of tumor cells and sites of active hematopoiesis/osteogenesis in bone microenvironments.

Effect of dietary PUFAs in 4T1 tumor metastasis to heart, kidneys and ovaries

Heart, kidneys, and ovaries are not primary targets of 4T1 mammary tumor metastasis [24, 37, 38] and therefore are infrequently reported. In this study, we observed an 83% lower incidence of cardiac metastasis in mice receiving dietary LC-ω-3FAs (2/20 mice) compared to the incidence in mice on the ω-6 diet (12/20 mice) (p < 0.05) (Table 1). Mice from the ω-3 group with cardiac metastases had a single nodule of overt metastasis (median = 0; range 0–1) on the pericardium, which was a significantly lower number compared to the frequency found on the pericardium from the mice fed ω-6 diet (median = 1; range 0–5). Histological analysis of myocardial metastases deeper in the cardiac tissue was performed by evaluating the frequency and size of histologically observed metastases per cross section of cardiac tissue (n = 10/group) (Fig. 3a, b). The results showed the incidence of histologically observed metastases in cardiac tissue was 67% lower in mice fed the ω-3 diet (2/10) relative to the incidence in the ω-6 group (6/10) (p = 0.16). Both of the mice from the ω-3 group with myocardial metastases had a single metastatic foci (median = 0; range 0–1) while mice on the ω-6 diet had a median = 1; range 0–5 of histologically observed metastases in heart (p = 0.07). The median size of histologically observed cardiac metastases in the ω-3 group was significantly smaller (median = 0; range 0–1.73) mm2 compared to the size of metastases in ω-6 group (median = 0.49; range 0–7.69) mm2 (p < 0.05). The cardiac metastases in mice fed the ω-3 diet were limited to the pericardium; while the metastatic foci were present in pericardium, as well as deeper in the cardiac tissue of the mice from the ω-6 group.

Fig. 3

Dietary PUFA regulation of 4T1 metastasis to the heart, kidneys and ovaries. Mice (n = 10/group) autopsied on day 35 post tumor injection were analyzed for histologically observed metastases (HO-mets). a Hematoxylin & eosin (H & E) stained cardiac cross section showing myocardial HO-mets. b Two of the ten mice fed the ω-3 diet had small cardiac HO-mets and six of the ten mice on the ω-6 diet had larger HO-mets. c H & E stained renal cross-sections showing HO-mets in each dietary group. d One of the ten mice fed the ω-3 diet had one small renal HO-mets in one kidney, while six of the ten mice on the ω-6 diet had larger HO-mets. e H & E stained ovarian cross-sections showing HO-mets in each dietary group. f One of the ten mice fed the ω-3 diet had small ovarian HO-mets, while eight of the nine mice on the ω-6 diet had larger HO-mets. Scale bars in images a, c and e represent 200 µm length. Horizontal line in b, d and f represents median. *p < 0.05 mice fed ω-3 versus ω-6 diet

Renal metastases were analyzed by counting the number of overt metastatic nodules associated with the renal capsule, and evaluating the number and size of histologically observed metastases per renal cross-section (Fig. 3c, d). We analyzed metastases in both kidneys of the mice and expressed our data as the incidence of metastases in one kidney or both kidneys per mouse. The incidence of having overt metastasis in at least one kidney of the mice fed ω-3 diet (2/20) was significantly (87%) lower relative to the incidence in the ω-6 group (15/20). Similarly, there was a 72% lower incidence of overt metastases in both kidneys of mice fed ω-3 diet (2/20) relative to the ω-6 group (7/20) (p = 0.4) (Table 1). Further, the median number of overt metastases per kidney for the ω-3 group (median: 0 range 0–2) was significantly lower from that of the ω-6 group (median:1, range 0–4). Histological analysis (Fig. 3c, d) confirmed that the incidence of histologically observed metastases in at least one kidney was 83% lower in the mice fed ω-3 diet (1/10 mice) relative to the ω-6 group (6/10 mice). None of mice from the ω-3 group had histologically observed metastases on both kidneys; while, (2/10) of mice on the ω-6 diet had metastases in both kidneys. The single metastatic lesion in the renal cross-section of a mouse from the ω-3 group was found attached to the peri-capsular area, but did not invade into the renal parenchyma. In contrast, amongst the 20 metastatic lesions analyzed in the kidneys from mice fed the ω-6 diet, 35% were located in the peri-capsular fat and attached to the renal capsule, 45% were located on the adrenal gland and 20% were located in the deeper area renal parenchyma. The median size of histologically observed renal metastases in a ω-3 diet-fed mice (median = 0; range 0–0.22 mm2) was significantly lower compared to renal metastases in the ω-6 diet-fed mice (median = 0; range 0–3.55 mm2). Overall, the results suggest that the incidence of kidney metastases (both overt and histologically observed) were significantly lower in the mice fed the ω-3 diet compared to the mice fed the ω-6 diet.

Representative images of a ovarian cross-section with histologically observed metastases and variations in their size between the dietary groups are presented in (Fig. 3e, f) (n = 10/group). Among the ovarian cross-sections examined, the ovaries with an intact capsule, cortex, medulla and follicles were included in the quantitative analysis histologically observed metastases. The incidence of having metastasis in at least one ovary was significantly lower (89%) in mice fed a ω-3 diet (1/10 mice) relative to the incidence in the ω-6 diet-fed group (8/9 mice). Similarly, in the ω-3 diet-fed group, none of mice had metastases in both ovaries (0/9 mice) while 86% (6/7 mice) had metastases in both ovaries in mice on the ω-6 diet. There was significantly lower number of histologically observed metastases (median = 0; range 0–3) and they were significantly smaller in size (median = 0; range 0–0.01 mm2) in the ovaries from mice fed the ω-3 diet, compared to the frequency of (median = 3; range 0–8) and size (median = 0.09; range 0–0.94 mm2) of metastases in ovarian cross-section in the mice fed the ω-6 diet. Collectively, these results suggested that mice on the ω-3 diet had a significantly lower incidence, frequency and smaller size of ovarian metastases compared to the observations in the mice fed the ω-6 diet.

Metastases in the CMG

The fifth left MG was injected with tumor cells and the fourth right MG was assessed for the presence of metastasis in a CMG that is distant to the primary tumor. Out of n = 10 mice/group in which histologically observed metastases were examined in CMGs, metastases were observed in three mice in the ω-3 group (range 0–1 metastatic lesions/CMG) and four mice in ω-6 group (range 0–6 metastatic lesions/CMG) respectively (p = 0.4). In addition to studies of metastases to the CMG, we also examined metastasis to the associated MG lymph node (Fig. 4a, b). Among the analyzed lymph nodes in the CMG sections, metastases were observed in 100% of lymph nodes from both dietary groups. We did not find any lymph node in the tumor injected MGs, which might be due to the aggressive growth of the tumor throughout the MG.

Fig. 4

Dietary PUFA regulation of contralateral mammary gland metastasis. Mice (n = 10/group) autopsied on day-35 post tumor injection were analyzed for histologically-observed metastases (HO-mets) in contralateral mammary gland (CMG). a H & E stained CMG sections showing tumor HO-mets arrested in the CMG (b). Incidences of HO-mets in the CMG lymph node (LN) and the CMGs. Note, that a CMG LN was sectioned in only six of the ten CMG. c Proliferating tumor cells in CMG HO-mets as shown by Ki67 staining. d Quantitative analysis of proliferating tumor cell frequency in the HO-met of the CMGs. e Ki67 staining for proliferating cells in the ductal wall of CMGs and f their frequency. g F4/80+ macrophages in the ductal wall of CMGs and h their frequency. i and j F4/80+ macrophages in adipose tissue of CMGs. Images of a were taken at 100x and those of c, e, g and i were taken at ×200. *p < 0.05 mice fed ω-3 versus ω-6 diet and were analyzed using t-test

In a histological analysis, tumor cell morphology of the CMG metastases from the ω-6 diet appeared more proliferative, compared to metastases in CMGs from ω-3 mice, an observation confirmed by Ki67 staining. In the Ki67 IHC studies, the frequency of proliferating tumor cells in the CMGs from ω-3 diet-fed mice were 74% lower compared to tumor cells from the CMG from the ω-6 group (n = 3, p < 0.05) (Fig. 4c, d). In addition to the CMG metastases, the mammary ducts in the CMG from ω-6 fed mice had a significantly higher number of proliferating cells (Ki67+), which was rarely observed in the mammary ducts from the ω-3 group. Our analysis of 10 fields/sample for n = 10 mice/group showed that ω-3 diet-fed mice had 97% fewer proliferating cells in the ductal epithelial layer (Fig. 4e, f) relative to mice on the ω-6 diets (p < 0.05). We previously showed that NTB mice on the ω-3 diet had thinner epithelia, ductal stroma, and fewer proliferating epithelial cells and macrophages in adipose tissue of MFPs compared to mice in the ω-6 fed group [13]. The current analysis of CMGs from TB mice support these previous observations; but the extent of effects may be influenced by the presence of the primary tumor. In the current study, the 4T1 TB mice fed the ω-3 diet had 59% fewer F4/80+ macrophages in the periductal area and 52% fewer in the adipose tissues of the CMG, compared to the respective analysis in CMG from TB mice fed ω-6 diet (n = 10, p < 0.05) (Fig. 4g, j). The number of infiltrating macrophages in the CMG from 4T1 TB mice were increased relative to the number in NTB mice (data not shown). Further, the morphology of macrophages in the CMGs from TB mice ppeared to be larger and morphologically activated, with multiple cytoplasmic granules, compared to those observed in the MGs from NTB mice independent of the dietary group (Fig. S3A, B). These results suggest, dietary PUFA might influence the number of infiltrating macrophages in both NTB MGs and TB CMGs but the presence of primary tumor further enhance the number and activity of infiltrating macrophages. Additionally, individual tumor cells were observed around the ducts in association with an increased number of infiltrating macrophages, notably in the CMGs from the ω-6 group. Collectively, our data document that prior exposure to dietary PUFAs modulate the CMGs microenvironments to be tumor promoting or suppressing depending on the type of PUFA and might further regulate the proliferation of tumor cells in the CMG metastases.

Dietary PUFA regulation of mammary tumor cell proliferation, apoptosis and neo-vascularization

Mammary tumors from mice (n = 10 mice/group) were collected on day 35 post tumor injection for the analysis of dietary PUFA regulation of the tumor microenvironment. We obtained representative cross-sections from these tumors containing oxic, hypoxic and necrotic tumor zones. The sub-capsular, oxic areas of tumors were used for all IHC evaluations. Our data showed that a diet high in LC-ω3FA, fed for 16 weeks, prior to tumor challenge decreased the number of proliferating (Ki67+) primary tumor cells by 44% (49 ± 7 cells/HPF) compared to the tumors from the mice fed the ω-6 diet (87 ± 7 cells/HPF) (p < 0.05) (Fig. 5a, b). In contrast, the tumors in the ω-3 fed mice had a 50% higher frequency of apoptotic tumor cells (8 ± 1 cells/HPF) relative to the tumors from the mice fed ω-6 diet (4 ± 1 cells/HPF) (p < 0.05) (Fig. 5c, d).

Fig. 5

Dietary PUFA regulation of 4T1 tumor proliferation, apoptosis and neo-angiogenesis. Mice (n = 10/group) were autopsied on day 35 post tumor injection and well oxygenated, peripheral tumor sections analyzed by immunohistochemistry (IHC). a Representative images of tumor tissue stained with Ki67 staining. b Frequency of proliferating tumor cells. c Tumor tissue showing TUNEL+ nuclei. d Differences in TUNEL positive apoptotic cells between the dietary groups. e Intra-tumoral neo-angiogenesis as shown by CD31+ vessels in actively growing areas of tumor. f Differences in tumor associated neo-angiogenesis between the dietary groups. Correlation analysis between tumor weights and g Ki67+ cells. h TUNEL+ nuclei. i CD31+ vessels per high power field (HPF). Relationship between the number of CD31+ vessels in tumor with j the number of F4/80+ cells and k the number of CD3+ cells in tumor. Pearson’s correlation analysis was used for statistical test. Images a, c and e were taken at ×400. *p < 0.05 compared to the mice fed ω-6 diet

PUFA regulation of intra-tumoral neo-vascularization was also evaluated as CD31+ vessels in oxic areas of the tumors. These studies showed that mice fed an ω-3 diet had 34% fewer neo-vascular vessels (14 ± 1 vessels/HPF) relative to the counts in the ω-6 diet-fed mice (22 ± 2 vessels/HPF) (p < 0.05) (Fig. 5e, f). Morphologically, the vessels in the tumors from the ω-3 group were smaller and of uniform size; while the vessels in ω-6 fed mice were larger, connecting with an irregular structure. The number of CD31 + vessels, and the number of proliferating tumor cells were positively correlated with the size of the tumors, while an inverse correlation was observed between the number of apoptotic cells in the tumors and the tumor size (Fig. 5g, i). We note that the tumor sizes of the mice on ω-3 diet were significantly smaller relative to tumors of mice on the ω-6 diets (Fig. 1c, d). Further, a direct correlation was found between the CD31+ vessels and the number of F4/80+ cells in tumors (Fig. 5j) indicating a potential role of tumor infiltrating macrophages in enhancing neo-vascularization.

Dietary PUFA regulation of mammary tumor inflammation

Tumor infiltrating neutrophils were assessed as the frequency of elastase positive (NE+) cells, as were the frequency of F4/80+ macrophages and CD3+ T-cells to characterize the tumor microenvironment between the dietary groups (n = 10). There were 52% fewer neutrophils infiltrating the tumors from mice fed the ω-3 diet (10 ± 2 cells/HPF) compared to tumors from mice fed the ω-6 diet (22 ± 4 cells/HPF) (p < 0.05) (Fig. 6a, b). Similarly, infiltrating F4/80 positive macrophages were 49% lower in mice fed the ω-3 diet (5 ± 1 cells/HPF) compared to the mice on the ω-6 diet (10 ± 1cells/HPF) (p < 0.05) (Fig. 6c, d). In contrast, mice on the ω-3 diet had a 102% more infiltrating T-cells (23 ± 3 cells/HPF) compared to tumors from mice on the ω-6 diet (11 ± 1 cells/HPF) (p < 0.05) (Fig. 6e, f). Based on clinical studies that have shown that the neutrophil to lymphocyte ratio (NLR) can be used as an independent prognostic factor and that a high NLR is associated with shorter survival of patients [39] we assessed these ratios as well. Feeding a LC-ω3FA diet for 16 weeks prior to tumor challenge significantly lowered the NLR by 76% in 4T1 mammary tumors compared to the mice fed the isocaloric, isolipidic ω-6 diet. Further, the NLR directly correlated with tumor size (Fig. 6j). In addition, a direct correlation between tumor size and infiltration by neutrophil-elastase positive (NE+) myeloid cells and macrophages, but an inverse correlation with infiltrated T-cells (Fig. 6g, i) was noted. Further, the number of T-cell in tumors was inversely correlated with the intra-tumoral neo-vascularization (Fig. 5k) and directly correlated with the number of apoptotic tumor cells (Fig. 6k) Thus, the differences in the tumor microenvironments may be associated with both tumor size and indirectly or directly associated with diet. This suggests a critical role for dietary PUFA composition in the regulation of the inflammatory tumor microenvironment and that an increase in a dietary LC-ω3FA may regulate tumor growth by decreasing inflammatory cells and increasing lymphocyte infiltration.

Fig. 6

Dietary PUFA regulation of 4T1 tumor inflammatory cell infiltration. Mice (n = 10/group) autopsied on day 35 post tumor injection were assessed by IHC for leukocyte infiltration of well oxygenated, peripheral tumor sections. a Infiltration of neutrophil-elastase positive (NE+) myeloid cells in tumor. b Quantitative differences in the number of infiltrated cells per high power field (HPF) between the groups. c Infiltration by F4/80+ macrophages in tumors. d Differences in the number of invading macrophages per HPF between the groups. e T-cell infiltration assessed as CD3+ cells. f Plot comparing the frequency of T-cells per HPF infiltrating the tumors. Correlation analysis of tumor weights. g Neutrophil-elastase+ cells. h F4/80+ nuclei and i CD3+ cells per HPF (Pearson’s correlation analysis). j Relationship between neutrophil to lymphocyte ratio (NLR) (evaluated by NE+ cell number/T-cell number) and tumor weight (Pearson correlation analysis). k Correlation analysis of CD3+ T-cells with apoptotic cells in tumor. Images a, c and e were taken at ×400. *p < 0.05 fed ω-3 versus ω-6 diet (independent sample t-test for IHC data and one sample t-test for qPCR data)

Cytokines and chemokines are mediators of inflammation, including myeloid cell proliferation and infiltration and can have a critical role in tumor growth and progression, depending on the type and function of the cellular mediator. The mRNA expression of inflammatory and immune regulatory mediators in tumors and spleens was analyzed from mice autopsied on day-35 post tumor injection (n = 10/group). Sections of tumors from subcapsular, actively growing tumor areas were collected for the evaluation of mRNA expression to avoid the influence of hypoxic condition on the mRNA expression of the target genes. Among the analyzed target genes, there was no difference in mRNA expression levels of NFkB, G-CSF, GM-CSF, TNFα, CCL2, PTGS2, LEP, IGF1, CXCL5, CXCL1 and AREG in tumors between the dietary groups. Unexpectedly, we observed a significant (sixfold) higher expression level of IL10 mRNA in the tumors from mice fed ω-3 diets relative to the expression in tumors from ω-6 diet-fed mice (Fig. 7a). We compared the expression levels of the indicated genes in tumors of both groups with the respective gene expression levels in NTB spleens to evaluate if mRNA expressions was directed by tumor growth and/or dietary PUFA differences. Our data showed that the tumor mRNA levels of PTGS2, CCL2 and G-CSF from both dietary groups were significantly higher relative to the respective mRNA levels in NTB spleens. In contrast, IL10 mRNA level was not different in the tumors of the ω-3 group but significantly lower in the tumors from ω-6 group relative to the level in NTB spleens (Fig. 7b). Further, there was a indirect correlation between the mRNA levels of IL10 with tumor size (Fig. 7c) but no relationship was observed in the levels of G-CSF, PTGS2 and CCL2 with tumor size (Fig. 7d–f). Collectively our data showed that the mRNA expression of PTGS2, CCL2 and G-CSF were associated with the presence of 4T1 tumors and that diets may have minimal effects on their expression; however, the IL10 expression in the tumors might have been modulated by dietary PUFA composition.

Fig. 7

Dietary PUFA regulation of inflammatory mediators. Tumor tissue from peripheral tumor sections, and sections of spleen were collected from mice autopsied on day-35 post tumor injection for evaluation of mRNA expression by quantitative real time polymerase chain reaction (qRT-PCR) (n = 10/group). a qPCR analysis of the mRNA levels of the indicated genes in the tumors from ω-3 (W3) diet-fed mice. Data are expressed as the fold change (FC) relative to the corresponding mRNA levels in the tumors from ω-6 group. b mRNA levels as a FC of the indicated genes in tumors from ω-3 and ω-6 diet-fed mice relative to the expression levels in the spleen from non-tumor bearing (NTB) control mice. c Correlation analysis comparing FC of IL10 mRNA in tumors. d G-CSF. e PTGS2. f CCL2 relative to the levels in non tumor-bearing (NTB) spleen versus tumor weight d. For statistical analysis, dCts were compared by Student’s t-test in a and b. *p < 0.05 versus ω-6 diet group and # p < 0.05 versus NTB spleen. Pearson’s correlation analysis was performed for (cf)

Discussion

In this study, we present evidence that LC-ω-3FAs containing diets in adult life significantly decreased 4T1 mammary tumor induction, growth, metastases to multiple organs and enhanced survival of mice compared to mice pair-fed with an iso-caloric, iso-lipidic ω-6FA diet. The outcomes were consistent with the observed differences in tumor microenvironments including tumor cell proliferation, apoptosis, neo-vascularization and infiltrating immune cells between the two dietary groups. We have previously shown that feeding a LC-ω-3FAs diet for 16 weeks of adult life resulted in decreased MG density, ductal epithelial cell proliferation and decreased expression of inflammatory mediators in MGs [13] and liver [27]. In our current study, we extended these observations to dietary PUFA mediated alterations in mammary tumor microenvironments that regulate tumor growth and metastasis.

The observations of significantly lower body weight gains in mice fed a LC-ω-3FA diet compared to the mice fed an ω-6FA diet during a NTB phase is consistent with our previous studies using the same dietary model [13, 27]. BALB/c mice are refractory to obesity [40], such that their body weight did not increase significantly following consumption of a high fat diet (60% calories from fat) for 16 weeks [40]. Thus our observation of significant differences in body weights, on diets with 36% calories from fat after a 16 week diet feeding period may be due to lack of body fat deposits on the mice fed LC-ω-3FAs diets, rather than a significant increase in body weights of the ω-6 diet group [13]. The potential mechanisms behind the difference in body fat depositions are more likely by differential regulation of metabolism and adipogenesis by dietary PUFAs [41, 42]. During the tumor bearing phase, mice fed the ω-6 diet lost more weight compared to their body weights prior to tumor injection. During tumor growth, body weight is more influenced by tumor associated metabolic disorders including cachexia and adipose tissue atrophy, than the effects related to dietary calories/composition [43, 44]. Further, when the energy generated from dietary calories is not sufficient to fulfil the requirement for hyper-proliferative tumor cells, beta-oxidation of body fats occur to generate energy endogenously, resulting in the weight loss [45]. In these studies, tumors were initiated more rapidly and grew faster resulting in tumors twice as large on day 35 and there were more metastases in the mice fed an ω-6 diet compared to the mice from the ω-3 group, so it is likely that they lost more weight due to tumor growth associated factors as discussed previously.

Whether tumor growth and metastasis are regulated by PUFA composition or specific PUFA metabolism is not directly addressed in our studies. However, we have separated PUFA composition from caloric composition with the use of an isocaloric diet. This provides a model to address the role of ω-3 versus ω-6FA composition on tumor growth and metastasis. Further, the timing (in-utero, pre/puberty or adult) of dietary PUFA consumption may also have a critical role in these parameters as well as mammary tumorigenesis. The results from the studies based on in-utero/pre-pubertal dietary exposure helps to understand the role of PUFAs in mammary tumorigenesis during MG development. Studies with autochthonous mammary tumors have shown that fish oil based diets markedly suppress mammary tumor incidence, multiplicity and MG hyperplasia in murine mammary tumor virus (MMTV)-HER-2/neu transgenic mice [46, 47]. Similarly, pre-pubertal consumption of a low-fat (16% calories from fat) ω-3 diet but not a high-fat (39% calories) ω-3 diet induced mammary epithelial differentiation by reducing terminal end buds, increasing apoptosis and reducing cell proliferation, as well as, decreasing mammary tumor incidence, compared to a low-fat ω-6FA diet [11, 48]. These results emphasized the critical role of calories from FAs as well as dietary composition in mammary tumorigenesis.

The current study is based on dietary consumption during adult life using iso-caloric and isolipidic diets to assess the role of PUFA composition on the growth of orthotopically implanted mammary tumors. As we started the diets earlier than tumor injections, our study emphasizes the potential prophylactic role of dietary LC-ω-3FAs in the control of tumor growth and metastasis by modulating microenvironments by prior exposure of the diets. Among the 40 mice/group in two studies, two mice from the ω-3 group did not develop tumors. Potential reasons for this might be due to technical issues related to the low number of tumor cells injected (5000 4T1 cells), compared to most prior reports [40, 49, 50]. Alternatively, the delay in tumor development in the ω-3 group may be due to ω-3FAs mediated microenviromental changes. We report that mice fed the ω-3 diet had a significant delay in tumor induction resulting in 50% smaller tumors at autopsy on the day 35 post tumor injection, compared to the mice fed the ω-6 diet. These findings are consistent with previous studies using autochthonous mammary tumors in rodents fed ω-3 FA enriched diets [46, 47] or studies with the fat-1 transgenic mice capable of endogenously producing ω-3 FA [12] and orthotopic mammary tumor models [51, 52]. However, this is the first study using rodents receiving isocaloric and isolipidic diets and pair feeding in the analysis of tumor growth and metastasis to multiple organs.

4T1 mammary carcinoma tumors spontaneously metastasize to multiple organs [53] in a process involving both soil and seed properties [18, 19]. BC usually metastasize to lymph nodes, bone, lungs and liver [19, 54] and less frequently to the ovaries [20] heart and kidneys [21, 22]. The 4T1 mammary tumor, is an aggressive tumor; spontaneously metastasizing to the lungs [33] and less frequently to other organs including the liver, brain, bone, [23, 24, 25, 26, 37, 40] heart and kidney [23, 26] while, no report, published to our knowledge, has described metastasis to ovaries and CMG by the parental 4T1 tumor cells. In our present study, we report dietary PUFA regulation of a frequency and number of spontaneous metastases to both common and infrequent organs. The presence of pulmonary metastasis was anticipated as lungs are preferential site of metastasis in most murine mammary tumor models. Further, the direct correlation of lung weights and pulmonary overt metastases with the tumor size indicates the potential role of slower tumor growth in ω-3 diet-fed mice in having less burden of pulmonary metastases compared to the ω-6 group is a novel observation.

Although, the incidence of hepatic metastasis was lowered by 35% in the ω-3 group, we did not observe significant differences in either hepatic overt metastasis counts or liver weights between the dietary groups. We previously reported that the liver weights of NTB mice fed an ω-6 diet were significantly lower compared to the livers from mice fed the ω-3 diet. The differences in liver weights were accompanied by differences in hepatic storage of fat versus glycogen in mice given ω-6 or ω-3 diets respectively [27]. Thus, our observation of similar liver weights in TB mice between the dietary groups may be due to an increase in liver weights of TB mice on the ω-6 diet, potentially by the presence of a greater number of metastases in the hepatic parenchyma (Fig. 2d). Spontaneous bone metastases are infrequently reported by primary 4T1 tumors, compared to their occurrence following intra-cardiac or intra-tibial inoculation of 4T1 cells or the bone trophic variant that favors colonization of the femurs/tibias (4T1.2) [36, 55, 56]. In the current study, using a low number of 4T1 parent cells, we observed dietary PUFA regulation of bone metastases and an 80% lower incidence of spontaneous femur/tibia metastases in mice fed an ω-3 diet relative to mice given an ω-6 diet. Previous studies have shown that dietary fish oil prevents osteolytic lesions; following intra-cardiac injection of MDA-MB-231 breast cancer cells and bone METs [57]. These data were supported by the findings that DHA and EPA significantly attenuated the migration/invasion of MDA-MB-231 BC cells in vitro as well as reduced cell migration to bone [57, 58]. Further, other studies have shown a beneficial role of fish oil in bone health, including inhibitory activities of DHA for osteoclastogensis [29] and calcium bioavailabity [59], and a negative correlation of the dietary ω-6: ω-3 ratio with bone formation [28, 60]. Thus, it is not clear, if a dietary PUFA modulation of osteolytic pre-metastatic niches in bone acts as a chemoattractant for metastases to bone, or PUFA modulated bone metastases regulate osteolysis suggesting that further mechanistic studies of bone metastases are needed.

Cardiac and renal MET are rarely reported metastatic sites for any tumor type. The lower incidence may be due to the lymphatic network in these organs, composition of the tissue microenvironments or physiological functions of the organ (for example, myocardial contractions) [21, 61]. However, it is also possible that metastases to these sites may have been overlooked when focusing on the burden of metastases in the more common metastatic sites. This is supported by the frequent findings of cardiac metastases in post-mortem examinations [62, 63, 64]. In this study, a diet enriched in ω-3 FA was associated with a lower incidences of cardiac and renal metastases relative to the respective incidences in mice fed an ω-6 diet. Further, the observation of a significantly lower number and size of histologically observed in the heart and kidney of mice given the ω-3 diet suggests a role of the dietary LC-ω-3FAs on the establishment and growth of metastatic cells in those tissues, potentially due to a modulated tissue microenvironments. However, our study is the first reporting dietary PUFA regulation on cardiac and renal metastasis of a murine carcinoma, thus further mechanistic studies are warranted.

Although the ovary is not a preferential site for BC metastasis, ovarian metastasis have been detected in autopsies from young women with BC [20, 65]. Differential diagnosis of ovarian metastasis by BC are challenging because ovarian cancer is frequently diagnosed as a primary tumor instead of as a metastatic lesion [66]. We did not find any previous reports of spontaneous 4T1 ovarian metastasis or for any other mammary tumors. Interestingly, we observed a significant decrease in number of mice with ovarian metastasis that were fed the LC-ω-3FA diet relative to mice in the ω-6 group. Further, metastases were found extensively in the follicular regions in mice fed an ω-6 diet, while the growth was limited to medullary region of ovaries in mice fed the ω-3FA diet, indicating the critical role of PUFAs in growth of tumor cells in the ovarian microenvironment. Potential mechanisms are the regulation of prostaglandin E2 and estrogen levels by LC-ω-3FA, as reported with ovarian cancer growth [67, 68, 69]. However, further mechanistic studies in animal models of mammary tumors are needed to evaluate the specific roles of LC-ω-3FAs in BC ovarian metastases.

Contralateral BC (CBC) metastasis have been diagnosed in 11% of BC survivors [70]. CBC can be a new cancer or invasion of metastatic tumor cells in the second breast, but due to challenges in differential diagnosis of the clonal origins, most CBC are treated as a second BC [70, 71]. A recent study reported a direct relationship between breast density and incidence of CBC in women with a primary BC [72]. Recently, we showed that the MGs of NTB mice fed a LC-ω-3FA diet had significantly lower ductal densities and macrophage infiltration, relative to the MGs from NTB mice given an ω-6 diet [73]. However, there are no reports on the association of LC-ω-3FA in the induction of CBC, nor murine studies on the role of ω-3FA, on metastasis to CMG. In the current study, we did not find significant differences in the incidence of CMG metastasis between the dietary groups. Nevertheless, there were significantly fewer proliferating tumor cells in the metastasis of CMG of mice fed an ω-3 diet, which was accompanied by fewer proliferating epithelial cells in the ductal epithelium and infiltrated macrophages relative to the respective findings in mice given an ω-6 diet (Fig. 4c–h). Additionally, the macrophages infiltrated in tumor tissue were larger and granulated compared to the macrophages we observed in MGs from NTB mice (Fig. S3). These data suggest that tumors growth might regulate the activation of macrophages in CMGs from TB mice however further studies are warranted to evaluate the macrophage phenotypes. Collectively, our previous data from NTB mice [73] and our current analyses of CMG metastasis in 4T1 TB mice, suggests that the consumption of LC-ω-3FAs may suppress pro-inflammatory mediators and growth factors, thus modulating the microenvironment inhibiting successful 4T1 metastasis in the CMG of mice fed the ω-3 diet. These observation are supported by previous findings of the ability of ω-3FA to inhibit mammary tumor growth in vitro [74] and in vivo [12, 75].

Splenic BC METs are a rare event in humans [76] and have been infrequently reported in mice bearing 4T1 murine carcinomas [23, 77]. Splenomegaly in 4T1 tumor bearing mouse is strongly associated with extramedullary hematopoiesis, driven by myeloid growth factor secretion by 4T1 cells [78, 79, 80]. In these studies, as we expected, we observed splenomegaly in TB mice from both dietary groups compared to a normal mouse spleen; however, mice from the ω-3FA group had significantly smaller spleens relative to those from ω-6FA diet-fed mice. Spleen weights were directly correlated with tumor size in both groups and the histology demonstrated sub-capsular myeloid hyperplasia. Thus, the observation of smaller spleens in the ω-3 groups may be associated with the decreased tumor size.

The tumor microenvironment has a crucial role in tumor progression. In these studies, we observed a significantly lower number of proliferating tumor cells, a greater number of apoptotic tumor cells and less neo-vascularization in the sub-capsular oxic areas of tumors from mice fed an ω-3 diet relative to the same analyses in the ω-6 fed groups. DHA has been shown to induce tumor cell apoptosis and reduce proliferation in vitro and in vivo, potentially by increasing lipid peroxidation [81], tumor cell cytotoxicity [10], inhibition of pro-inflammatory eicosanoids [82] and cell cycle arrest [83, 84]. The induction of tumor cell apoptosis has an important role in cancer therapy and represents a target for many treatment strategies. As LC-ω-3 FAs appear to cause selective cytotoxicity towards cancer cells with little or no toxicity to normal cells, many studies have assessed the role of ω-3 PUFAs as a therapeutic adjuvant, to improve the efficacy and tolerability of traditional anticancer therapies [10, 85, 86, 87].

Neo-vascularization is essential for tumor metastases, thus it is an important target in the treatment of solid tumors, including BC [88, 89]. Our observation of lower numbers of CD31+ vessels in tumor tissue from an ω-3 diet-fed mice suggests a decrease in tumor neo-vascularization by dietary LC-ω-FAs. In addition to a direct effect of active metabolites of LC-ω-3FAs on mediators of neo-vascularization, such as vascular endothelial growth factor (VEGF) [90], modulation of tumor infiltrating inflammatory cells might also indirectly regulate neo-vascularization. Recent evidence indicates that tumor-associated immune cells, including macrophages, neutrophils, and mast cells can stimulate tumor angiogenesis [91, 92, 93, 94]. We found that infiltration of macrophages and neutrophils in tumor oxic areas from mice fed ω-3 diets was significantly lower, relative to their infiltration in the tumors from ω-6 diet-fed mice. Further, the number of neutrophils and macrophages in tumor tissue directly correlated with tumor size and a direct correlation was observed between the numbers of macrophage with the extent of neo-vascularization in tumors. These results suggests that macrophages mediating downregulation of neovascularization might be one of the potential mechanisms of tumor growth suppression by dietary LC-ω-3FA. Our results are supported by previous findings on the effects of LC-ω-3FA metabolites in the suppression of tumor-associated macrophage infiltration and the association of macrophages with tumor angiogenesis [95, 96, 97, 98].

The presence of tumor infiltrating T-cells has been associated with a better prognosis and response to cytotoxic treatments in BC patients [99]. In contrast, a neutrophilic response can inhibit immunity by suppressing the cytotoxic activity of T-cells and is associated with a poor prognosis in cancer [100]. The NLR of blood is indicative of systemic inflammation and clinical studies have demonstrated the prognostic value of a systemic NLR in multiple cancers, including BC [39, 101]. We evaluated the NLR in the tumor microenvironment as an index of pro-tumorigenic immune response. Our finding of a greater number of T-cells and fewer neutrophils in tumors from mice fed a diet rich in LC-ω-FAs, relative to the numbers in the ω-6 diet group and a direct correlation of NLR with tumor size, indicates the potential of dietary LC-ω-3FAs in tumor growth suppression by decreasing tumor supporting inflammation. Further, our results also support a potential role of LC-ω-FA as an adjuvant to enhance tumor therapies [102, 103, 104].

In an analysis of potential mediators of inflammation and/or immune-regulation in the tumor microenvironment, the expression of IL10 was significantly higher in tumors and spleens of ω-3 diet-fed mice relative to the ω-6 group and IL10 expression in tumors was negatively correlated with tumor size. These data suggest a potential role for dietary LC-ω-3FAs in the regulation of tumor and systemic inflammation via IL10 mediated pathways. In tumor immunology, IL10 has multifaceted roles and has been associated with both poor and good cancer prognoses [105, 106, 107]. It has been suggested that IL10 contributes to an immune suppressive tumor microenvironment, however, recent studies have shown a role for IL10 in stimulating antitumor activity of CD8+ T-cells [108] suggesting IL10 as a potential therapeutic target for cancer treatment [109, 110]. The ω-3FAs have been associated with increased IL10 production in in vitro and in vivo studies, [111, 112] but the role of LC-ω-3FA mediated IL10 in tumor microenvironment has not been fully elucidated. In the present study, since increased IL10 expression in tumors was associated with tumor size, our data indicates a potential novel mechanism of dietary ω-3FA mediated tumor growth suppression; however, future mechanistic studies are warranted.

In our studies, we did not observe any significant difference in the expression of the cytokines and growth factors we analyzed in tumors between the dietary groups, although the expression of PTGS2, CCL2 and G-CSF were significantly higher in the tumors from both groups relative to the respective levels in NTB spleens. Studies have shown 4T1 tumor message level of G-CSF [113, 114], PTGS2 (COX-II) [115] and CCL2 [116] were associated with tumor growth and METs. It is noted that in our prior study [113], the G-CSF message was associated with tumor cells and not infiltrating leukocytes, consistent with in vitro studies showing high levels of G-CSF message and protein with 4T1 tumor cells [114, 117]. Thus, our data indicate that the increase in intra-tumoral expression of G-CSF, COX-II and CCL2 might be tumor cells dependent phenomenon and the dietary PUFA may not significantly regulate those inflammatory mediators.

In this study, we reported the effects of dietary PUFAs in mammary tumors using the 4T1 murine cell line and BALB/c mice. To validate that these effects are not limited to a single tumor and mouse strain, further studies are warranted using different mammary tumor cell lines and mouse strains. We confirmed the activity of LC-ω-3FAs and ω-6FAs on the tumor growth and metastasis of RENCA tumors in syngeneic BALB/c mice. Our findings from the current studies on mammary tumor suggest a potential prophylactic role of dietary LC-ω-3FAs in breast cancer, and also emphasize for the need for future studies (mechanistic/translational/clinical) to evaluate the therapeutic role of LC-ω-3FAs in breast cancer growth, metastasis and in clinical management of co-morbidities in survivors. There are ongoing clinical trials on the role of ω-3FAs in breast cancer prevention (https://clinicaltrials.gov/ct2/show/NCT02295059) and a recent clinical study suggested beneficial effects of ω-3FAs. They are able to reduce arthralgia associated with aromatase inhibitor treatment in obese breast cancer patients. Moreover, clinical studies focusing on the role of LC-ω-3FAs as an adjuvant therapy and translational studies in understanding the modulation of primary tumor and metastatic sites microenvironments would provide an insight on targeting LC-ω-3FAs associated pathways for an advancement of anti-cancer therapies.

In summary, using isocaloric, isolipidic diets in a pair-fed model, we conclude that dietary LC-ω-3FAs suppress mammary tumor growth resulting in lower frequency of metastasis to the preferential metastatic sites such as lungs and prolonged survival of the mice. Moreover, considering the differences in metastatic burden (incidence/frequency and size) in multiple organs (specifically low preferential sites; heart, kidney, ovaries, bone and CMGs) between the ω-3 and ω-6 dietary groups, out data indicate dietary PUFA composition has additive effects in metastasis, in addition to the regulation of metastasis by primary tumor growth. Further, analyses of the tumor microenvironments showed significantly lower neo-vascularization, macrophage infiltration, and NLR and higher T-cell infiltration, tumor cell apoptosis and increased IL10 expression in the ω-3 diet-fed mice. Together, these factors might contribute the suppressive effects on to the tumor growth by dietary ω-3 PUFAs.

Notes

Acknowledgements

We gratefully acknowledge funding from the Fred & Pamela Buffett Cancer Center’s NIH Cancer Center Support Grant No. (P30CA036727) and the Nebraska Center for Integrated Biomolecular Communications (Grant No. P20GM113126) for this project. Also, funding from the UNMC College of Medicine (LWK) Endowed Chair.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

10585_2018_9941_MOESM1_ESM.docx (2.6 mb)
Supplementary material 1 (DOCX 2659 KB)

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Saraswoti Khadge
    • 1
  • Geoffrey M. Thiele
    • 1
    • 3
    • 4
  • John Graham Sharp
    • 2
  • Timothy R. McGuire
    • 5
  • Lynell W. Klassen
    • 3
    • 4
  • Paul N. Black
    • 6
  • Concetta C. DiRusso
    • 6
  • Leah Cook
    • 1
  • James E. Talmadge
    • 1
    • 3
  1. 1.Department of Pathology and MicrobiologyUniversity of Nebraska Medical CenterOmahaUSA
  2. 2.Department of Genetics, Cell Biology and AnatomyUniversity of Nebraska Medical CenterOmahaUSA
  3. 3.Department of Internal MedicineUniversity of Nebraska Medical CenterOmahaUSA
  4. 4.Veteran Affairs Nebraska-Western Iowa Health Care SystemOmahaUSA
  5. 5.Department of Pharmacy PracticeUniversity of Nebraska Medical CenterOmahaUSA
  6. 6.Department of BiochemistryUniversity of Nebraska-LincolnLincolnUSA

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