Cell chirality in three-dimensional epithelial tissue morphogenesis

Abstract

To investigate inherent cellular chirality in 3D, we developed a dual layered Matrigel system and studied the role of chirality in morphogenesis and tumor development, as well as its mediating factors. Cultivation of Madin-Darby canine kidney (MDCK) epithelial cells embedded within our system assembled into 3D microtissues, which are representative of fundamental acinar and ductal network formations. These primary structures are essential to morphogenesis and the development of human kidneys, lungs and breasts. The microtissues underwent coordinated rotational behavior that was directionally biased and consistent with 2D chiral cultures. The chiral rotational movements were an intrinsic property of the epithelial cells, exhibited by single cells and throughout the progression to multicellular microtissues. Our data suggests an actin-dependent mechanism underlying the chiral behavior of epithelial cells, regulated by alpha-actinin-1 expression. We also utilized our platform to examine the vortical movements of mouse and human mammary epithelial cells, and uncovered an association between chirality and the expression of the H-Ras oncogene. Activation of protein kinase C (PKC), via a cancer promoting drug, enhanced the LR bias of cancer cells and induced invasive behavior with the formation of multicellular extensions. The extensions exhibited chiral twisting while invading the surrounding environment. Overall, these findings highlight the importance of collective cellular chirality in 3D epithelial microtissue self-assembly and tumorigenic tissue development.

Chirality is the innate handedness, or left-right (LR) asymmetry, intrinsic to a variety of structures found throughout nature, ranging from biological molecules to full living organisms. The positioning and orientation of many organs within the human body are asymmetrically biased. Examples of this include the heart, liver, stomach and appendix. The formation of healthy organs requires the development of complex three-dimensional (3D) structures with distinct shapes and physical properties, in order to function properly. Different cell populations need to undergo strategically choreographed migration to reach their proper locations. This orchestrated collective movement is integral to embryogenesis and the development and progression of cancer, as well as numerous other processes. 2D micropatterned cultures revealed that migrating cells exhibit LR biased alignment, and the migrational behavior is dependent on the phenotype of the cell. The LR biases of migrational direction for cancerous cell lines are reversed in comparison to their noncancerous counterparts. To more accurately emulate the morphological and biochemical factors found in native tissue, 3D cultures are often preferable to 2D systems, because they convey more physiologically relevant simulations of in vivo conditions.