Abstract A19: Label-free analysis of tissue polarity in live three-dimensional culture of breast epithelium by Raman imaging of lipid phase

The formation of the basoapical polarity axis in epithelia is critical for maintaining the homeostasis of differentiated tissues. Factors that influence cancer development notoriously affect tissue organization. Apical polarity appears as a specific tissue feature that, once disrupted, would facilitate the onset of mammary tumors. Thus, developing means to rapidly measure apical polarity alterations would greatly favor screening for factors that endanger or protect the breast epithelium. A Raman scattering based platform was used for label-free determination of apical polarity in live breast epithelial structures (acini) produced in three-dimensional cell culture. The coherent anti-Stokes Raman scattering signal permitted the visualization of the apical and basal surfaces in the equatorial plane of an acinus. Raman microspectroscopy subsequently revealed that in polarized acini lipids were more ordered at the apical membranes compared to basal membranes, and that an inverse situation occurred in acini that lost apical polarity upon treatment with Ca²⁺-chelator EGTA. This method overcame variation between different cultures by tracking the status of apical polarity longitudinally for the same acini. Therefore the disruption of apical polarity by dietary breast cancer risk factor, 6 fatty acid, could be observed significantly with this method, even when the effect was too moderate to permit a conclusive assessment by visually scoring the acinus population for apical location of tight junction marker ZO-1. Analysis of apical polarity markers and cytoskeleton by fluorescence microcopy revealed that the change of lipid ordering upon apical polarity loss corresponded to actual disruption of the apical polarity complex. Our label-free platform will enable rapid identification of environmental factors that either initiate or prevent the very early stage of breast neoplasia. Identifying risk and protective factors will facilitate the design of primary prevention strategies to reduce breast cancer incidence. Furthermore, the use of the membrane lipid phase as readout to assess tissue polarity should be applicable to other types of epithelia where over 90% of cancers originate.

Citation Information: Cancer Prev Res 2011;4(10 Suppl):A19.