Self-organization to Morphogenesis

We developed 3D culture models of the breast to achieve the objectives of observing cancer in statu nascendi (in the nascent state), an event that has been beyond reach until now. This allows for the study of the reciprocal cell-cell and cell-matrix interactions that result in normal tissue structure and their breakdown during carcinogenesis, as well as the role of mammotropic hormones in normal morphogenesis and neoplasia. In particular, we are studying the role of mechanical properties of the matrix and of physical forces as determinants of form and function. For this purpose, we are collaborating with Professor Vyvyan Howard, Dr. Kurt Saetzler (University of Ulster at Coleraine) and Professor Helen Byrne (University of Oxford). Using a Systems Biology approach we will develop a multi-scale computer model. Using this model, “predictive” in silico experimentswill be conducted with the purpose of informing the design of experiments in the in vitro 3D-culture model.

Hormone-Sensitive 3D model for studying mammary gland morphogenesis:

We have developed a novel, 3D culture model to study the influence of specific reproductive hormones on the various morphogenetic processes that take place during mammary gland development. As the gland goes through the different stages of development, the mammary epithelial tree invade the mammary fat pad, branches out and during pregnancy, forms alveoli which then secrete milk. All these changes in the epithelium are influences by estrogen, progesterone and prolactin. We have used an estrogen-sensitive and ER+, PR+ and Prl+ human breast cancer cell line, T47D cells, embedded in type I collagen gels to show the particular effect each hormone has on the epithelial phenotype. As shown in the figure below, T47D cells elongate in the presence of estrogen, elongate and form protrusions (structures similar to branching) in presence of estrogen and progesterone, and form alveoli in presence of prolactin and estrogen. This model can now be further used to study these specific processes and how other cell types, particularly stromal cells, can affect these processes.

T47D cells 3D model soto sonnenschein
T47D breast cancer cells embedded in collagen gels stained with carmine alum. (Top) Representative structures formed by T47D cells by hormone treatment. (Bottom) Reconstructed 3D projections of epithelial structures from z-stack images using Volocity software. (Speroni et al 2014)

 

Biomechanical Factors in mammary gland morphogenesis

The main epithelial structures in the mammary gland are ducts. However, during pregnancy and lactation, lobular epithelial structures called acini are formed at the end of the ducts. These acini produce and secrete milk and the ducts act as conduits through which the milk is delivered. The development of the mammary gland is largely dictated by stromal-epithelial interactions. Biomechanical factors, such as matrix stiffness, have been shown to be key players in such interactions. For example, epithelial cells cannot form acini in 3D cultures when the stiffness of the matrix is greater than that of a normal mammary gland. We are interested how such biomechanical forces can influence mammary epithelial morphogenesis.

Type I collagen is the predominant collagen type present in the mammary gland. It has been suggested that increase in collagen I fiber density results in a stiffer matrix and a stiffer matrix is correlated with malignancy in breast neoplasia. Collagen fiber organization is also important for normal mammary gland development. In our 3D in vitro cultures, we have shown that collagen fiber alignment begins before epithelial cells form ducts or acini (see videos below).

Collagen fiber (in green) organization during acinus formation 

formation of an acinus

 

Collagen fiber (in green) organization during duct formation

Formation of a duct

 

Collagen fiber organization around MCF10A acinus and duct in 3D gels soto sonnenschein
Collagen fiber organization around MCF10A acinus and duct in 3D gels. Fiber alignment was visualized using reflectance confocal microscopy (RCM) (Barnes et al 2014)

 

Collagen fiber organization is also influenced by hormonal treatment during morphogenesis of T47D cells, as shown below. Fiber aligment was visualized using second harmonic generation imaging in this case.

collagen organization T47D hormones soto sonnenschein
Collagen fiber organization around breast epithelial structures derived from T47D cells in 3D collagen gels. Fiber organization analyzed using second harmonic generation imaging. (Speroni et al 2014)

 

 

 

 

 

 

 

 

 

 

Recommended Reading – 

Barnes C, Speroni L, Quinn K, Montevil M, Saetzler K, Bode-Animashaun G, McKerr G, Georgakoudi I, Downes S, Sonnenschein C, Howard CV, Soto AM: From single cells to tissues: interactions between the matrix and human breast cells in real time. PLoS ONE 2014; 9: e93325

Speroni L, Whitt GS, Xylas J, Quinn KP, Jondeau-Cabaton A, Georgakoudi I, Sonnenschein C, Soto AM: Hormonal regulation of epithelial organization in a 3D breast tissue culture model. Tissue Engineering Part C Methods 2014; 20: 42-51