Varner, Victor D.

Mechanical forces are increasingly recognized to regulate morphogenesis, but how this is accomplished in the context of the multiple tissue types present within a developing organ remains unclear. Here we use bioengineered “microfluidic chest cavities” to precisely control the mechanical environment of the fetal lung. We show that transmural pressure controls airway branching morphogenesis, the frequency of airway smooth muscle contraction, and the rate of developmental maturation of the lungs, as assessed by transcriptional analyses. Timelapse imaging reveals that branching events are synchronized across distant locations within the lung, and are preceded by long-duration waves of airway smooth muscle contraction. Higher transmural pressure decreases the interval between systemic smooth muscle contractions and increases the rate of morphogenesis of the airway epithelium. These data reveal that the mechanical properties of the microenvironment instruct crosstalk between different tissues to control the development of the embryonic lung.

Abstract The mechanical and chemical properties of the cellular microenvironment can influence the phenotype of cancer cells. The hypoxic conditions within solid breast tumors can contribute to invasiveness and the formation of breast cancer stem cells (CSCs). Integrin-linked kinase (ILK) is a critical mediator for mechanotransduction, a process that converts exogenous mechanical stimuli into biochemical signals. Yet, it is unclear how the physical properties of the tumor microenvironment, including matrix stiffness and hypoxia, integrate to direct the formation of the breast CSC niche. Using an innovative engineered culture model, we found that stiff and hypoxic microenvironments activate integrin signaling and a stem-like gene signature in breast cancer cells, suggesting that integrin signaling, matrix mechanics and oxygen tension cooperatively promote the formation of breast CSCs. Stiff and hypoxic microenvironments promote integrin signaling and the expression of CSC markers including CD44 and Nanog. Knocking down ILK reverts the CSC phenotype of invasive breast cancer cells on stiff matrix, and abrogates their ability to form mammospheres and colonies in soft agar. In contrast, ectopic expression of ILK enhances CSC properties. ILK promotes integrin signaling and the CSC phenotype through the PI3K/Akt pathway. Stiff microenvironments promote tumor formation and metastasis in ovo. ILK depletion significantly abrogates the tumorigenenic and metastatic potential of invasive breast cancer cells in ovo. Importantly, we found that breast cancer cells expressing ILK and the CSC marker CD44 were only present in the regions of tumors predicted to be stiff in breast cancer patient samples. Our data suggest that ILK act as an essential mechanosensor that regulates breast CSC phenotype in response to matrix mechanics and oxygen tension to regulate the formation of breast CSCs. Citation Format: Mei-Fong Pang, Melody Stallings-Mann, Michael J. Siedlik, Victor D. Varner, Siyang Han, Derek C. Radisky, Celeste M. Nelson. Tissue stiffness and hypoxia regulate breast cancer stem cells through ILK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4395.

Significance During branching morphogenesis of the developing lung, it is generally thought that a spatial template of biochemical cues determines the airway branching pattern. Here, however, we demonstrate that physical mechanisms can control the pattern of airway branching. Using a combination of 3D culture experiments and theoretical modeling, we show that a growth-induced physical instability initiates the formation of new epithelial branches. Tuning epithelial growth rate controls the dominant wavelength of the instability, and thereby the branching pattern. These findings emphasize the role of mechanical forces during morphogenesis and indicate that lung development is not a closed genetic system. Physical cues also regulate the spatially patterned cell behaviors that underlie organ assembly in the embryo.