Inflammation of mammary adipose tissue occurs in overweight and obese patients exhibiting early-stage breast cancer
Charlotte Vaysse Jon Lømo Øystein Garred Frøydis Fjeldheim Trygve Lofteroed Ellen Schlichting Anne McTiernan Hanne Frydenberg Anders Husøy Steinar Lundgren
Growing evidence indicates that adiposity is associated with breast cancer risk and negatively affects breast cancer recurrence and survival, a paracrine role of mammary adipose tissue being very likely in this process. In contrast to other adipose depots, ...
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PubMed Central (PMC)
Introduction

Growing evidence from both clinical and preclinical studies indicates that adiposity is associated with breast cancer risk,1, 2 and may act as a negative prognostic factor influencing breast cancer recurrence and survival.1–3 These observations have recently been supported in mechanistic studies, observing that adiposity-associated factors, such as hormones, lipids, adipokines, and pro-inflammatory mediators are associated with breast cancer development and progression.4, 5

Accumulating studies point to a role of mammary adipose tissue (MAT) adjacent to the tumors in breast cancer development and progression, as adipose tissue (AT) represents a major component of the breast tumor microenvironment.6, 7 We and others have demonstrated that a bidirectional crosstalk takes place between breast cancer cells and tumor-surrounding AT.8–13 Tumor-surrounding adipocytes may stimulate the aggressiveness of cancer cells by secreting extracellular matrix such as collagen VI and its fragment endotrophin,11, 12 matrix metalloproteases,13 chemokines,10 and pro-inflammatory cytokines such as Interleukin-6 (IL6).14 IL-6 is an important growth factor for estrogen receptor-α (ERα)-positive breast cancer, and elevated serum IL-6 is associated with poor prognosis.15, 16 These results strongly support the innovative concept that adipocytes may participate in a highly complex cycle orchestrated by cancer cells to support tumor initiation, growth, and metastasis, possibly amplified in overweight and obese breast cancer patients.

In obesity, both the cellar composition and the secretion of various bioactive substances of AT is deregulated, generating a low-grade, chronic inflammatory state.4, 5, 17 This inflammatory state is due to the recruitment of macrophages.18 During weight gain, there is a shift in macrophage sub-type to a pro-inflammatory, M1 polarization,18, 19 leading to chronic inflammation of the AT with pro-inflammatory release of TNF-α, IL-6, IL-1β, PAI-1 (reviewed in ref. 18). In a very elegant study (after in vivo labeling of macrophages), Lumeng et al. demonstrated that the obesity-induced switch in macrophages activation state is due to the recruitment of additional macrophages from the circulation with high M1 gene expression, that then forms clusters surrounding necrotic adipocytes.20 It is therefore now recognized that these clusters (also called crown-like structure or CLS) represent inflammation foci.20 Interestingly, the critical size of adipocyte-triggering death is dependent on the site of the adipose depot, in line with the view that adipocytes found in different depots have different properties.21 Independently of a cancer context, it has been shown that obesity-induced macrophage infiltration occurs also in MAT and this was, as expected, associated with an increase in pro-inflammatory gene expression.22, 23

Existence of such an inflammatory state in MAT is likely to explain an amplification of its paracrine effect on tumor development and progression among overweight and obese women. In contrast to sub-cutaneous adipose tissue (SAT) and visceral adipose tissue (VAT),21, 24 less is known about CLS accumulation in MAT in obese, but also in overweight women. The pioneering work of Dannenberg and collaborators has demonstrated that CLS occurs in the MAT of obese subjects bearing breast cancers.23, 25 However, several questions remain unclear concerning the link between CLS accumulation in MAT and overweight/obesity in a cancer context. Previous studies were performed in mastectomy specimens, including a vast majority of patients with aggressive and high-grade tumors and positive lymph nodes involvement.25–27 Thus, it remains undetermined if CLS also occurs in localized early-stage breast tumors. Another major issue is whether body mass index (BMI), common clinical measure of body composition, is the most appropriate measure of adiposity to reflect the presence of CLS in MAT, or whether other anthropometric measures focused on abdominal obesity such as waist to hip ratio (WHR) or truncal fat measured with Dual X-ray Absorptiometry scan (DXA) are more appropriate. Finally, the associations among CLS, metabolic dysfunction and low grade inflammation (such as glucose/HbA1c levels, dyslipidemia and level of C-Reactive protein, CRP) have been focused on only in one study25 and need to be further investigated.

Thus, the main aim of the present study was to elaborate on whether MAT inflammation in lumpectomy specimens, as reflected by CLS, is associated with mammary adipocyte size, body composition assessed by various methods of assessments, and serum biomarkers, in patients with the most common types of breast tumors in routine clinical practice.

Discussion

In this sample of breast cancer patients, 71% of whom underwent breast-conserving surgery, we observed a strong positive correlation between patient’s body composition, breast adipocytes size, and the presence of CLS in MAT close to the tumor, likely reflecting local inflammation. Our results further highlight that BMI is an adequate predictor of the presence of CLS in MAT among postmenopausal women, whereas the measure of truncal fat percentage might be more predictive in premenopausal women. Our study also underlines that CLS are present in overweight patients in accordance with the fact that a relatively small increase in adipocyte size is observed between CLS positive and negative patients, suggesting that this tissue might be prone to inflammation during hypertrophy. Finally, the presence of CLS was associated with systemic markers such as TG/HDL-cholesterol ratio, TG (in the postmenopausal population), glucose/HbA1C whereas serum CRP was associated with the presence of CLS in premenopausal women. These compelling results demonstrate that excess adiposity is associated with MAT inflammation, a condition that could contribute to breast cancer development and progression in a paracrine manner.

Our results extend previous observations suggesting that MAT inflammation occurs with excess adiposity as previously observed for others fat depots such as SAT and VAT. In fact, only a limited number of studies have reported the presence of CLS in MAT of overweight/obese patients with breast cancers, and all emanated from the same research group.26–28 Our study extends previous results, as we also demonstrate that the presence of CLS occurs in a series of overweight/obese patients that mostly exhibited low-grade tumors treated by breast tumor resection. This is in contrast to previous studies that were performed on mastectomy specimens including a majority of patients with aggressive, high grade tumors and positive lymph node involvement.26–28 Importantly, in our study, the analyses of CLS were performed in fat tissue in the same block as the tumors, and therefore were in close proximity to the tumor mass. Moreover, by including both breast conservative and radical surgeries, our study reflects the current clinical practices on management of breast cancer stage I and II with the evolution of techniques and therapeutics.29 Our findings therefore highlight that MAT inflammation associated with excess weight might have important implications in clinical practice since it concerns a large majority of patients treated for breast cancer.

All three methods of measuring body composition in this study showed a significant correlation between CLS density and adipocyte size in breast tissue (Fig. ). Importantly, this correlation was found for BMI, which is considered a rough measurement of body composition but is still commonly used in clinical practice.1, 30 WHR and truncal fat percentage31 were also strongly correlated with adipocyte size and CLS density. When OR were calculated, all the three markers BMI, WHR and truncal fat percentage assessed by DXA predict the occurrence of CLS in the overall population. However, while BMI predicted presence of CLS in postmenopausal women, truncal fat was a better predictor in premenopausal women. After menopause, increase in BMI is associated with increased amounts of visceral adiposity (to the same levels of male subjects) whereas a lower incidence of visceral obesity according to BMI is observed in premenopausal women.32 Accordingly, after menopause the BMI reflects abdominal distribution of fat whereas in premenopausal women, these two variables could be dissociated.32 In obesity, VAT is more inflammatory than SAT and the accumulation of VAT is strongly associated with obesity-related complications like Type 2 diabetes and coronary artery disease.33 Interestingly, a higher odds of CLS presence for HbA1c levels (that indirectly reflect insulin resistance) was seen in premenopausal (7.96) than in postmenopausal women (1.75). Therefore, our results suggest that MAT inflammation is more likely to occur in patients exhibiting visceral adiposity (and potentially a metabolic syndrome). This link deserves further investigations since it could have important consequences at clinical levels.

Our study shows that the occurrence of CLS in MAT is not limited to obese patients since overweight patients had a 3-fold higher risk of CLS presence compared with lean/normal-weight patients. These results indicate that MAT inflammation might occur even with moderate excess of AT. In regression analysis, a linear association between size of mammary adipocytes and CLS-density was observed suggesting that when breast adipocytes reach a critical size they die, triggering macrophage infiltration, as observed also in AT in other locations.21, 34, 35 Interestingly, the increase in adipocyte size in samples that contained CLS was rather modest (1.08-fold, mean size 69.7 µm in CLS positive vs. 64.3 μm in CLS negative samples) in accordance with a previous study also performed in MAT.36 These results are in contrast with the 2-fold increase previously reported in both SAT and VAT.34 Taken together, these results suggest that the adipocyte size limit triggering cell death in MAT may be smaller than the one observed in other adipose depots, therefore explaining the occurrence of CLS in overweight patients found in our study. Additional studies directly measuring the number of dead adipocytes in AT (as determined by the rate of adipocyte death and the rate of dead adipocyte clearance by macrophages) are needed to confirm this hypothesis.

It is now clearly established that the presence of CLS is associated with a pro-inflammatory response in the affected tissue.21, 24 In fact, we demonstrated here that macrophages forming CLS were positive for IL6 expression in all the studied samples. This pro-inflammatory environment is likely to favor the progression of estrogen receptor (ER) + positive samples that represents the vast majority of our samples. Indeed, several of the proinflammatory mediators associated with CLS, including TNFα, IL-1β, IL-6, and Prostaglandin E2 (PGE2) are known to up regulate aromatase expression (reviewed in36). Very interestingly, it has been demonstrated that cyclooxygenase (COX)-2-derived PGE(2) stimulates a transduction pathway contributing to enhance expression of aromatase (the rate-limiting enzyme for estrogen biosynthesis), and elevated progesterone receptor (PgR) levels in breast tissues from overweight and obese women.28 This deciphered pathway may contribute to initiation, progression as well as resistance to anti-aromatase therapies of ER + breast cancers in overweight and obese patients.

Inflammation of AT is a key component of the occurrence of insulin resistance and the metabolic syndrome. In our study, an association between systemic markers (CRP, TG/HDL cholesterol ratio, glucose, and HbA1c levels) and the presence of CLS was observed (see Table ). These systemic markers could also contribute in addition to local inflammation to cancer progression. For example, elevated CRP levels are associated with shortened disease-specific and overall survival in breast cancer patients.5, 37–39 Similarly, insulin resistance could contribute directly (through elevated insulin levels) or indirectly (through its effect on the bioavailability of insulin-like growth factor I) to breast cancer occurrence and progression.40 Finally, TG/HDL-cholesterol ratio, that may function as a surrogate for insulin resistance,41 is also associated with breast cancer occurrence42 and prognosis.43 Given the increase in unfavorable metabolic profiles worldwide, and their observed negative effects on breast cancer development and prognosis, there is a need of further studies to improve our knowledge regarding the association between components of metabolic syndrome and MAT inflammation.

In conclusion, our findings support that the presence of CLS in MAT is increased with overweight and obesity. Our findings were obtained in a series of patients representing those commonly seen in clinical practice. In addition, we demonstrate that BMI is an adequate measure to predict the presence of CLS in MAT among postmenopausal women, whereas in premenopausal women the measure of truncal fat percentage might be more predictive, highlighting a potential link between visceral adiposity and the presence of CLS in MAT. The presence of CLS in MAT close to the tumors would contribute to generate a pro-inflammatory environment favorable to breast cancer occurrence and progression. In addition to the local modification of AT, changes in metabolic parameters may also contribute to this deleterious environment. Interestingly, two very recent studies, although performed in small number of patients, suggest that the presence of CLS might be associated to breast cancer prognosis with a decrease in progression-free survival44 and distant recurrence-free survival.25

Patients included in this study are part of a clinical protocol (with a final enrollment of 600 patients) and we thus expect to evaluate the impact of CLS presence in MAT on response to treatment, relapse, survival, with regards to different cancer sub-types. In fact, it will be highly interesting to investigate the occurrence of CLS in sub-groups (i.e., HER2 over-expressing as well as triple negative compared to ER + tumors) according to overweight/obesity that was not possible in the current study due to the low numbers of non-ER + patients. This large prospective longitudinal study will comprehensively investigate the prognostic role of MAT inflammation with its associated circulating abnormalities. If studies confirm the link between CLS and breast cancer prognosis, this could have therapeutic impact.

Several strategies have been developed to target inflammation in cancer patients including for example the use of anti-inflammatory drugs (COX2 inhibitors, aspirin, and anti-inflammatory steroids) or anti-cytokines drugs (anti-IL-6 and anti-TNF-α).45 It will therefore be important to design specific clinical trials to evaluate the interest of such anti-inflammatory strategies in the sub-population of CLS positive patients. Additional and larger studies are clearly needed to make these findings of importance in daily clinical practice.