Imagine beyond: recent breakthroughs and next challenges in mammary gland biology and breast cancer research

On 8 December 2022 the organizing committee of the European Network for Breast Development and Cancer labs (ENBDC) held its fifth annual Think Tank meeting in Amsterdam, the Netherlands. Here, we embraced the opportunity to look back to identify the most prominent breakthroughs of the past ten years and to reflect on the main challenges that lie ahead for our field in the years to come. The outcomes of these discussions are presented in this position paper, in the hope that it will serve as a summary of the current state of affairs in mammary gland biology and breast cancer research for early career researchers and other newcomers in the field, and as inspiration for scientists and clinicians to move the field forward

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

<i>Myh9</i> knock-out does not impede fibroblast motility.

(A) Detailed time-lapse snapshots of fibroblast-organoid contact establishment in dispersed co-cultures with control or Myh9-KO fibroblasts and tdTomato+ organoids. Scale bar: 50 μm. (B) Quantification of fibroblast-organoid contacts established in the first 3 days of co-culture, comparing GFP+ and GFP- fibroblasts (GFP is a marker of adenoviral transduction). The plot shows mean ± SD. Statistical analysis: two-tailored t test; n = 3 independent biological replicates, N = 20 organoids per experiment. The data underlying the graphs shown in the figure can be found in S1 Data. (TIFF)</p

The list of detection agents used in this study.

Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. Here, we employed fibroblast-organoid or fibroblast-spheroid co-culture systems and time-lapse imaging to reveal that physical contact between fibroblasts and epithelial cells and fibroblast contractility are required to induce mammary epithelial branching. Pharmacological inhibition of ROCK or non-muscle myosin II, or fibroblast-specific knock-out of Myh9 abrogate fibroblast-induced epithelial branching. The process of fibroblast-induced branching requires epithelial proliferation and is associated with distinctive epithelial patterning of yes associated protein (YAP) activity along organoid branches, which is dependent on fibroblast contractility. Moreover, we provide evidence for the in vivo existence of contractile fibroblasts specifically surrounding terminal end buds (TEBs) of pubertal murine mammary glands, advocating for an important role of fibroblast contractility in branching in vivo. Together, we identify fibroblast contractility as a novel stromal factor driving mammary epithelial morphogenesis. Our study contributes to comprehensive understanding of overlapping but divergent employment of mechanically active fibroblasts in developmental versus tumorigenic programs.</div

Fibroblasts organization around bifurcating TEB.

Z-stack scroll-through of mammary gland whole-organ imaging, showing a bifurcating TEB. DAPI in blue, vimentin in white, tdTomato in red. MIP and appropriate scale bar are depicted in Fig 6. (AVI)</p

MCF7-ras spheroid budding in co-cultures requires cell contractility.

(A, C) Photographs of spheroids on day 4 of dispersed co-culture with fibroblasts upon treatment with no inhibitor (mock), with blebbistatin (Bleb, A) or with Y27632 (C). Top gray and red bars indicate proportion of branched spheroids out of all spheroids per condition. Scale bar: 100 μm. (B, D) Quantification of number of branches/buds per branched spheroid in conditions from (A). The plot shows mean ± SD, each lined dot shows mean from each experiment, each faint dot shows single spheroid measurement, n = 4 (B) or 5 (D) biologically independent experiments, N = 20 spheroids per experiment. Statistical analysis: two-tailored t test. The data underlying the graphs shown in the figure can be found in S1 Data. (TIFF)</p

Mammary epithelial branching morphogenesis upon FGF2 treatment or fibroblast co-culture.

The video is composed of time-lapse videos capturing 5 days of epithelial morphogenesis in 3D organoid culture with no growth factor in the basal organoid medium (left), with FGF2 in the basal organoid medium (middle), or in fibroblast-organoid co-culture without addition of any growth factors to the basal organoid medium. Snapshots from the videos are depicted in Fig 1A. (AVI)</p

Quantification of fibroblast loops.

(A) A representative confocal image of a dispersed co-culture on day 4. Scale bar: 20 μm, scale bar in detail: 10 μm. (B) A representative confocal image of a dispersed organoid-fibroblast co-culture on day 3. The arrowhead indicates the fibroblast loop at the branch neck. Scale bar: 50 μm. (C) Quantification of the presence of fibroblast loops around organoid branches in dispersed co-cultures. The plot shows mean ± SD. Statistical analysis: two-tailored t test; n = 3 independent biological replicates, N = 5–12 organoids per experiment; 56 branches in total. The data underlying the graphs shown in the figure can be found in S1 Data. (TIFF)</p

Combination of fibroblasts and FGF2-STAB induces TEB-like phenotype of organoids.

(A) Time-lapse snap-shots of organoids grown in basal organoid medium with no exogenous growth factors (basal M), with FGF2-STAB, co-cultured with fibroblasts or co-cultured with fibroblasts with FGF2-STAB. Scale bar: 100 μm. (B) Quantification of organoid branching. The plot shows mean ± SD. n = 2 independent biological replicates, N = 20 organoids per experiment. (C) Quantification of number of branches per branched organoid. The plot shows mean ± SD. n = 2 independent biological replicates, N = 12–19 branching organoids per experiment. (D) Examples of luminized and full branch on bright-field imaging and quantification of the branch phenotypes. n = 2 independent biological replicates, N = 12–19 branching organoids per experiment. (E) Representative confocal images of organoids on day 5 of culture with FGF2-STAB or fibroblasts. Scale bar: 100 μm. (F) Quantification of maximum number of cell layers in a branch in confocal images. The plot shows mean ± SD. The dots represent averages from individual experiments. Statistical analysis: two-tailored t test; n = 3 independent biological replicates, N = 9–13 organoids per experiment. (G) Quantification of the percentage of organoids with KRT5+ cells present within the layers of KRT5-cells (basal-in-luminal, BIL cells) in confocal images. The plot shows mean ± SD. Statistical analysis: two-tailored t test; n = 3 independent biological replicates, N = 9–13 organoids per experiment. (H) A schematic representation of uncoupling fibroblast contraction and growth factor signaling in organoids. The data underlying the graphs shown in the figure can be found in S1 Data. FGF2, fibroblast growth factor 2; TEB, terminal end bud.</p