| Abstract | Background: The most common methods for CTC isolation from peripheral blood depend on positive selection based on tumor cells’ surface EpCAM expression. However, EpCAM expression may vary between CTCs and may be downregulated in cells undergoing epithelial-to-mesenchymal transition, potentially resulting in the failure to capture CTC subsets having a critical role as intermediates in cancer progression.
Methods: We previously reported on a feasibility study using an unbiased approach with three different enrichment methods, involving both positive and negative selection, and obtained consistently high recovery of small numbers of MDA-MB-231 cancer cells spiked into donor human peripheral blood using the RosetteSep Human Circulating Epithelial Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Vancouver, BC Canada). Cytospins prepared from recovered cells were used for identification and enumeration by immunocytochemistry using fluorescently laballed anti-CD45 and anti-CK19 antibodies. We optimized RNA amplification from the equivalent of one MDA-MB-231 cell (~10 pg) and used the resulting amplified RNA in microarray experiments. Peripheral blood from MBC patients was enriched for CTC, RNA was isolated and amplified, and microarray and qPCR analysis was used to investigate global and gene-specific transcription changes, respectively.
Results: Numbers of CTC enriched from eight MBC patients ranged from 1 to 125 per 5 mL blood. EpCAM, CK19, vimentin, and CD45 expression by CTC was determined by qPCR. Preliminary results from microarray analysis of enriched CTC from four of six initial patients showed ~ 130 common genes upregulated more than twofold, including genes involved in tumorigenesis. Two of six patients had insufficiently amplified RNA. Gene enrichment analysis showed an abundance of genes involved in translation.
Conclusions: Although preliminary, these methods may lead to a more thorough characterization of micrometastases in advanced breast cancer and potentially early stage disease. Further microarray analysis and optimization methods are ongoing in MBC patients. |
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