Long-Term High-Fat Diet Limits the Protective Effect of Spontaneous Physical Activity on Mammary Carcinogenesis.

Breast cancer is influenced by factors such as diet, a sedentary lifestyle, obesity, and postmenopausal status, which are all linked to prolonged hormonal and inflammatory exposure. Physical activity offers protection against breast cancer by modulating hormones, immune responses, and oxidative defenses. This study aimed to assess how a prolonged high-fat diet (HFD) affects the effectiveness of physical activity in preventing and managing mammary tumorigenesis. Ovariectomised C57BL/6 mice were provided with an enriched environment to induce spontaneous physical activity while being fed HFD. After 44 days (short-term, ST HFD) or 88 days (long-term, LT HFD), syngenic EO771 cells were implanted into mammary glands, and tumour growth was monitored until sacrifice. Despite similar physical activity and food intake, the LT HFD group exhibited higher visceral adipose tissue mass and reduced skeletal muscle mass. In the tumour microenvironment, the LT HFD group showed decreased NK cells and TCD8+ cells, with a trend toward increased T regulatory cells, leading to a collapse of the T8/Treg ratio. Additionally, the LT HFD group displayed decreased tumour triglyceride content and altered enzyme activities indicative of oxidative stress. Prolonged exposure to HFD was associated with tumour growth despite elevated physical activity, promoting a tolerogenic tumour microenvironment. Future studies should explore inter-organ exchanges between tumour and tissues.

Spontaneous Physical Activity in Obese Condition Favours Antitumour Immunity Leading to Decreased Tumour Growth in a Syngeneic Mouse Model of Carcinogenesis.

Our goal was to evaluate the effect of spontaneous physical activity on tumour immunity during aging. Elderly (n = 10/group, 33 weeks) ovariectomized C57BL/6J mice fed a hyperlipidic diet were housed in standard (SE) or enriched (EE) environments. After 4 weeks, orthotopic implantation of syngeneic mammary cancer EO771 cells was performed to explore the immune phenotyping in the immune organs and the tumours, as well as the cytokines in the tumour and the plasma. EE lowered circulating myostatin, IL-6 and slowed down tumour growth. Spleen and inguinal lymph node weights reduced in relation to SE. Within the tumours, EE induced a lower content of lymphoid cells with a decrease in Th2, Treg and MDCS; and, conversely, a greater quantity of Tc and TAMs. While no change in tumour NKs cells occurred, granzyme A and B expression increased as did that of perforin 1. Spontaneous physical activity in obese conditions slowed tumour growth by decreasing low-grade inflammation, modulating immune recruitment and efficacy within the tumour.

Vitamin D supplementation associated with physical exercise promotes a tolerogenic immune environment without effect on mammary tumour growth in C57BL/6 mice.

PURPOSE: High plasma vitamin D (VitD) level and regular exercise (Ex) are known to have anti-cancer and immunomodulatory effects. This study aimed to evaluate the impact of VitD supplementation and imposed physical Ex on mammary tumour growth and immune response in ovariectomised mice fed high-fat (HF) diet. METHODS: Ovariectomised 33-week-old mice C57BL/6 (n = 60), housed in enriched environment (EE), were fed HF diet (450 kcal/100 g) supplemented or not with VitD (HF/HF + D: 125/1225 IU/100 g) for 12 weeks and submitted or not to Ex (HF + Ex; HF + D + Ex) on treadmill (45 min/day, 5 days/week). At w8, syngeneic tumour cells EO771 were orthotopically injected into the 4th mammary gland. Spontaneous activity (SPA), maximal speed (MS) and forelimb grip strength (GS) were measured. Tumour immune cells infiltrate was phenotyped by FACS. Data (mean ± SEM) were analysed by two-way ANOVA + Tukey post-test. RESULTS: Ex (p = 0.01) and VitD (p = 0.05) reduced body weight gain. Exercise decreased visceral fat mass [g: 1.5 ± 0.8 (HF); 1.2 ± 0.65 (HF + Ex); 0.9 ± 0.6 (HF + D + Ex); p = 0.03]. SPA (p < 0.0001) and GS (p = 0.01) were higher in HF + D + Ex mice vs others. No effect of Ex or VitD on tumour growth was detected. In tumour, VitD decreased the proportion of NK (p = 0.03), while Ex increased it (p = 0.03). The Th1/Th2 ratio is lowered by VitD (p = 0.05), while Tc/Treg ratio was not affected either by Exercise or VitD. CONCLUSION: In our experimental conditions, VitD supplementation and physical exercise have synergetic effects reducing the weight gain under HF diet and improving the physical capacities of mice. VitD coupled with exercise induces an immunosuppressive response without effect on tumour growth.

Cell Cycle Synchronization of the Murine EO771 Cell Line Using Double Thymidine Block Treatment.

This study shows that double thymidine block treatment efficiently arrests the EO771 cells in the S-phase without altering cell growth or survival. A long-term analysis of cell behavior, using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) staining, show synchronization to be stable and consistent over time. The EO771 cell line is a medullary breast-adenocarcinoma cell line isolated from a spontaneous murine mammary tumor, and can be used to generate murine tumor implantation models. Different biological (serum or amino acid deprivation), physical (elutriation, mitotic shake-off), or chemical (colchicine, nocodazole, thymidine) treatments are widely used for cell synchronization. Of the different methods tested, the double thymidine block is the most efficient for synchronization of murine EO771 cells if a large quantity of highly synchronized cells is recommended to study functional and biochemical events occurring in specific points of cell cycle progression.

EO771, the first luminal B mammary cancer cell line from C57BL/6 mice.

BACKGROUND: Despite decades of therapeutic trials, effective diagnosis, many drugs available and numerous studies on breast cancer, it remains the deadliest cancer in women. In order to choose the most appropriate treatment and to understand the prognosis of the patients, breast cancer is divided into different subtypes using a molecular classification. Just as there remains a need to discover new effective therapies, models to test them are also required. METHODS: The EO771 (also named E0771 or EO 771) murine mammary cancer cell line was originally isolated from a spontaneous tumour in C57BL/6 mouse. Although frequently used, this cell line remains poorly characterized. Therefore, the EO771 phenotype was investigated. The phenotype was compared to that of MCF-7 cells, known to be of luminal A subtype and to express estrogen receptors, as well as MDA-MB-231 cells, which are triple negative. Their sensitivity to hormonal treatment was evaluated by viability tests. RESULTS: The EO771 were estrogen receptor α negative, estrogen receptor ß positive, progesterone receptor positive and ErbB2 positive. This phenotype was associated with a sensitivity to anti-estrogen treatments such as tamoxifen, 4-hydroxy-tamoxifen, endoxifen and fulvestrant. CONCLUSIONS: On account of the numerous results published with the EO771 cell line, it is important to know its classification, to facilitate comparisons with corresponding types of tumours in patients. Transcriptomic and protein analysis of the EO771 cell line classified it within the luminal B subtype. Luminal B cancers correspond to one of the subtypes most frequently encountered in patients and associated with a poor prognosis.

Modulation of inter-organ signalling in obese mice by spontaneous physical activity during mammary cancer development.

Accumulative evidence links breast cancer development to excess weight and obesity. During obesity, dysregulations of adipose tissue induce an increase in pro-inflammatory adipokine secretions, such as leptin and oestrogen secretions. Furthermore, a raise in oxidative stress, along with a decrease in antioxidant capacity, induces and maintains chronic inflammation, which creates a permissive environment for cancer development. Physical activity is recommended as a non-pharmacological therapy in both obese and cancer situations. Physical activity is associated with a moderation of acute inflammation, higher antioxidant defences and adipokine regulation, linked to a decrease of tumour-cell proliferation. However, the biological mechanisms underlying the relationship between oxidative stress, low-grade inflammation, carcinogenesis, obesity and physical activity are poorly understood. Our study is based on old, ovariectomised mice (C57BL/6J mice, 33 weeks old), fed with a high fat diet which increases adipose tissue favouring overweight and obesity, and housed in either an enriched environment, promoting physical activity and social interactions, or a standard environment constituting close to sedentary conditions. Our model of mammary carcinogenesis allowed for the exploration of tissue secretions and signalling pathway activation as well as the oxidative status in tumours to clarify the mechanisms involved in a multiple factorial analysis of the data set. The multiple factorial analysis demonstrated that the most important variables linked to moderate, spontaneous physical activity were the increase in growth factor (epithelial growth factor (EGF), hepatocyte growth factor (HGF)) and the activation of the signalling pathways (STAT3, c-jun n-terminal kinases (JNK), EKR1/2, nuclear factor-kappa B (NF-κB)) in the gastrocnemius (G). In inguinal adipose tissue, the NF-κB inflammation pathway was activated, increasing the IL-6 content. The adiponectin plasma (P) level increased and presented an inverse correlation with tumour oxidative status. Altogether, these results demonstrated that spontaneous physical activity in obesity conditions could slow down tumour growth through crosstalk between muscle, adipose tissue and tumour. A spontaneous moderate physical activity was able to modify the inter-organ exchange in a paracrine manner. The different tissues changed their signalling pathways and adipokine/cytokine secretions, such as adiponectin and leptin, resulting in a decrease in anti-oxidative response and inflammation in the tumour environment. This model showed that moderate, spontaneous physical activity suppresses tumour growth via a dialogue between the organs close to the tumour.

The interrelationship between physical activity and metabolic regulation of breast cancer progression in obesity via cytokine control.

Regular physical activity is known to protect against the development of breast cancer and mediate direct anti-inflammatory effects on adipose tissue. While direct relationships between muscle activity, adipose tissue and breast tissue have been highlighted in recent years, few studies have focused on the effects of obesity and physical activity during the development of breast cancer, particularly at the level of cell signaling. Skeletal muscle and adipose tissue modulate the cell metabolism by secreting myokines and adipokines. These secreted cytokines belong to a crosstalk network via cell signaling pathway modulation. The understanding of the tissue crosstalk is fundamental to the management of physical activity in the care of obese breast cancer patients. Therefore, this review focuses on the effects of obesity and physical activity during the development of breast cancer, particularly at the level of cell signaling. We focuse on the main mediators, secreted by both adipose and muscle tissue, which are implicated in breast cancer development. We presente the variation of these mediators in the physiopathological context of their secreted tissue. Then, we open the discussion on the crosstalk of these tissues in breast carcinogenesis.