Abstract 1073: Breast cancer intravasation signature from patient fine-needle aspirates

A key step in the progression of metastatic breast cancer is the process of blood vessel intravasation. Understanding the molecular mechanisms that underlie this crucial step is essential in designing strategies to block the same with an aim to prevent metastatic disease. Recently our group has developed and published an in vitro intravasation assay (1) that recapitulates the process of intravasation and allows the use of patient derived breast cancer cells. Previously our group had identified a breast cancer invasion signature that helps facilitate clinical decision making in breast cancer patients (2). Here using fine needle aspirates from breast cancer resections we collected viable cancer cells which were labeled green using vital dyes and mixed with a macrophage cell line labeled red and layered on the top compartment of a transwell. The bottom of the transwell membrane contains a layer of extracellular matrix and a tight layer of HUVECs. Green cancer cells were separated one at a time using a florescent microscope and a micromanipulator. The cancer cells capable of intravasation are divided into two groups, one group that has crossed the entire layer and fallen to the bottom of the well, and a second group that crossed the endothelial cells but are still bound to the bottom of the endothelial layer. Cancer cells from top of the transwell which did not intravasate were also collected. RNA was extracted from the few cells collected by micromanipulation from all the three groups, amplified and converted to Cy3 labeled cDNA. The labeled cDNA was hybridized to whole genome microarrays. Gene expression was compared between the three groups of cells. Both unsupervised and supervised data analysis was performed on the three groups of cells from five patients. Significance Analysis of Microarrays, Ingenuity Pathway Analysis and Gene Set Enrichment Analysis was performed on these datasets. Supervised analysis and validation of the altered genes was performed using qRT-PCR. Preliminary data analysis indicates the upregulation of the genes associated with Breast Cancer Stem cells and DNA repair. This is the first report of whole genome gene expression analysis from cells collected by FNA from patients, separated in vitro into intravasation competent and intravasation incompetent and unsupervised whole genome analysis performed to identify intravasation specific genes and pathways. Once unique targets and pathways are identified in intrvasation competent cells, a