Wnt and Neuregulin1/ErbB signalling extends 3D culture of hormone responsive mammary organoids

The development of in vitro culture systems quantitatively and qualitatively recapitulating normal breast biology is key to the understanding of mammary gland biology. Current three-dimensional mammary culture systems have not demonstrated concurrent proliferation and functional differentiation ex vivo in any system for longer than 2 weeks. Here, we identify conditions including Neuregulin1 and R-spondin 1, allowing maintenance and expansion of mammary organoids for 2.5 months in culture. The organoids comprise distinct basal and luminal compartments complete with functional steroid receptors and stem/progenitor cells able to reconstitute a complete mammary gland in vivo. Alternative conditions are also described that promote enrichment of basal cells organized into multiple layers surrounding a keratinous core, reminiscent of structures observed in MMTV-Wnt1 tumours. These conditions comprise a unique tool that should further understanding of normal mammary gland development, the molecular mechanism of hormone action and signalling events whose deregulation leads to breast tumourigenesis

Influence of adypocyte secretions on angiogenic process and lesser therapeutic response

L'obésité, en constance augmentation, est un facteur de risque établi de cancer mammaire chez les femmes en post-ménopause associé à un mauvais pronostic favorisant la survenue de métastases et la moindre réponse thérapeutique chez ces patientes. Parmi les différentes hypothèses émises expliquant ce lien entre obésité et cancer, plusieurs arguments bibliographiques suggèrent une implication des sécrétions adipocytaires, dont les concentrations plasmiques sont modulées en situation d'obésité, dans la tumorogenèse mammaire. Les objectifs de ce travail étaient d'analyser le rôle des sécrétions adipocytaires, reflétant une situation d'obésité, dans le processus d'angiogenèse ainsi que dans la moindre réponse thérapeuthique aux traitements anti-cancéreux, notamment d'hormonothérapie.(...)Nos résultats suggèrent que les sécrétions adipocytaires sont impliquées dans la régulation de la tumorogenèse mammaire ce qui ouvre des perspectives préventives et/ou thérapeutiques prometteuses, ciblant les adipokines, pour les femmes en surpoids particulièrement à risque.Obesity, constantly incrasing, is an established risk factor for breast cancer in post-menopausal women associed with a pour prognosis favoring the occurence of metastases and lower therapeutic response in these patient. Among the various hypotheses explaining the link between obesity and breast cancer, multiple bibliographic arguments suggest the involvement of adipocyte secretions, whose plasma concentrations are modulated in case of obesity, in mammary tumorogenesis. The objectives of thesis were to highlight the implication of adipocyte secretions, in a context of obesity, in the angiogenic process and therapeutic response to hormonal cancer traetments. (...) Adipocyte secretions are involved in the regulation of mammary tumorigenesis which opens up promising preventive or therapeutic perspectives targeting adipokines in situation of overweight

Adipocyte/breast cancer cell crosstalk in obesity interferes with the anti-proliferative efficacy of tamoxifen.

Obesity is a well-known risk factor of breast cancer in post-menopausal women that also correlates with a diminished therapeutic response. The influence of adipocytes and their secretome, i.e. adipokines, on the efficacy of hormone therapy has yet to be elucidated.We investigated, ex vivo, whether mature adipocytes, differentiated from adipose stem cells of normal-weight (MA20) or obese (MA30) women, and their secretions, were able to counteract the effects of tamoxifen (Tx) which is known to decrease neoplastic cell proliferation.In a tridimensional model and in a model of co-culture, the anti-proliferative effect of Tx on MCF-7 cancer cells was counteracted by MA30. These two models highlighted two different specific gene expression profiles for genes encoding cytokines or involved in angiogenesis based on the adipocyte microenvironment and the treatment. Thus it notably showed altered expression of genes such as TNFα that correlated with IL-6. In addition, leptin, IL-6 and TNFα, at concentrations reflecting plasma concentrations in obese patients, decreased the anti-proliferative efficacy of 4-hydroxytamoxifen (a major active metabolite of Tx).These findings bring insights on adipocytes and mammary cancer cell interactions in Tx therapy, particularly in overweight/obese people. Indeed, patient' adipokine status would give valuable information for developing individual strategies and avoid resistance to treatment

Tridimensional adipose mammary equivalent model.

<p>(A) Hematoxylin Phloxin Safran staining showed a pluristratified and differentiated epithelium on a connective underlying tissue made of fibroblasts surrounded by their ECM coloured in yellow in the porous scaffold. (B) Mature adipocytes were visible after Oil Red O staining coloring their vesicles in pink. (C) Ki67 immunohistochemical staining of the tridimensional adipose equivalent model using affinity-purified polyclonal biotinylated antibodies raised against Ki67 (Magnification: ×400). Positive staining appeared in brown. (D) Ki67 immunofluorescent staining of the tridimensional adipose equivalent model. Positive staining appeared in red. (E) Comparison of cellular proliferation in tridimensional adipose equivalent model with or without mature adipocytes and tamoxifen treatment.</p

qRT-PCR assays on 3D adipose equivalent model.

<p>qRT-PCR assays on 3D adipose equivalent model.</p

Effect of mature adipocytes, their secretions and Tamoxifen (Tx) on cell proliferation.

<p>(A) MCF-7 cells were co-cultured with MA obtained from women of normal weight (MA20), overweight (MA27) or obese (MA30) women and treated with Tx. (B) 184B5 cells were co-cultured with MA27 with or without Tx. For A) and B), the proliferation was quantified after 72h using a resazurine test (Fluoroskan Ascent^® FL). (C) MCF-7 and (D) 184B5 cells were also cultured with conditioned media from the culture of MA20 (CM20) and AM30 (CM30) with or without Tx treatment. Results were expressed as Mean ± SEM, ¤ MA <i>vs</i> Control, * Tx <i>vs</i> Control, § MA+Tx <i>vs</i> Tx; $ CM30 <i>vs</i> CM20, £ CM+Tx <i>vs</i> CM, # CM30+Tx <i>vs</i> CM20+Tx (n = 3, Student's t test).</p

Effects of Leptin, IL-6 and TNFα on MCF-7 cell proliferation after 4-OH-tamoxifen treatment.

<p>After adhesion, MCF-7 were treated with leptin (0; 10; 100 ng/mL) (A), IL-6 (0, 2.3; 83 pg/mL) (B), TNFα (0; 0.7; 12.5 pg/mL) (C) and with 12.5 μM of 4-OH-Tx. After 72h, cell proliferation was quantified using a resazurine test (Fluoroskan Ascent^® FL). Mean ± SEM, $ 4-OH-Tx +/- adipokine <i>vs</i> Control, ¤ 4-OH-Tx +/- adipokine <i>vs</i> 4-OH-Tx (n = 3, Student's t test).</p

Principal component analysis (PCA) to explore the relationships among genes on 3D adipose skin equivalent model.

<p>The expression of genes in MCF-7 mammary cell line co-cultured or not with mature adipocytes from women with a normal weight (MA20) or obese women (MA30) with or without tamoxifen treatment were analyzed by PCA. A first global PCA was carried out (A) followed by a second one taking into account the biological function of the different studied genes (B, C, D, E).</p