| Circulating tumor cells (CTCs) are tumor cells that are disseminated from the local site into the vasculature and thereby can be obtained by a simple blood draw from the patients as a biopsy of the tumor. The technology of using CTCs as a source to understand the current state of the tumor has been advancing and there are many studies that have demonstrated the importance of characterizing these cells. Gene expression profiling of CTCs has been a challenge since RNA degrades faster when compared to genomic DNA. Even though there are studies that have demonstrated single cell RNA-seq, the methods used are not robust and reproducible and were also expensive for the large cohort study. Hence we decided to use Parsortix (Angle. Inc, UK) which is a simple, cheap and robust technology to enrich CTCs. One of the challenges that remained even after using Parsrotix was the number of background WBCs that were along with the enriched fraction. Therefore, we performed different methods to optimize the enrichment fraction obtained using this technology and determined an efficient method by using Rosette Sep Depletion followed by Parsortix. Therefore, we hypothesized that using this technology in our enrichment process would enhance the ratio of CTCs to WBC we obtain in the fraction and enable more reliable and high-throughput sample analysis for detection of prostate cancer specific transcripts. To test this hypothesis, we decided to use a specific panel of genes that are prostate specific. Before using the method to identify signatures from patient samples, the method optimized were tested using LnCap cells. Further, this assay was validated using patient sample who were diagnosed with prostate cancer. |
