TEM and Fluorescence Imaging of Human Breast Cancer Cell Cultures

Tissue and organ failure are major health problems in the United States. Tissue engineering is a tool that can assist with identifying treatments for tissue and organ disorders, such as breast cancer, a disease that comprises about a fifth of all cancers in women. Tridimensional (3D) matrix cultures of breast cancer cell lines are more similar to native cancer tissue in terms of morphology and gene expression, as compared to typical monolayer cell cultures. This pilot study was carried out to establish a 3D matrix breast cancer cell culture system, with the goal of comparing its structural characteristics with those of monolayer cultures. To this end, methods for growing the human breast cancer cell line ATCC HCC70 in 3D matrix Geltrex^TM (Invitrogen, 12760-021) were established in parallel with conventional monolayer culture protocols. In order to characterize the general morphology of cells in both conditions, fluorescent probes were used to stain the cytoskeleton (Alexa Fluor® 488; F-actin) and the cell nucleus (Hoechst 33342; DNA). Images were captured using an epifluorescence microscope (Nikon TE2000) and a confocal microscope (Leica TCS SP5 II). Images captured by light and fluorescence microscopy show that the cells in monolayer culture grew in small clusters, adhered to the neighboring cells as well as to the rigid surface, and spread horizontally. In contrast, HCC70 cells maintained in Geltrex^TM grew in multiple layers and formed spheroids. Our optimized histological protocols enabled characterization of the cellular ultrastructure of breast cancer spheroids and monolayer cultures with transmission electron microscopy (Hitachi H-7650). In addition to microvilli, 3D cell surfaces were characterized by membrane protrusions that we speculate may be involved in cell interactions with the matrix. We also observed the presence of intercellular spaces filled with autophagic vacuoles in 3D cells, which were not readily detected in TEM images of 2D-cells. These findings demonstrate that the breast cancer cell line HCC70 responds to different environments with morphological adaptations that may have implications for in vitro studies of cell-cell interactions. Future studies will continue to explore the breast cancer 3D culture system by examining autophagic vacuoles using fluorescent probes. Research supported by P50GM068762.