(B) Second round of amplification using the pre-amplified PCR products from (A) as new DNA templates to separately amplify exon 9 (using original primers, 216?bp) and exon 20 (using internal primers, 192?bp). cells from breast cancer patients primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the gene. Captured DTCs grown in cell culture were also sequenced for mutations. Results Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among Cyclosporin H CTCs isolated at different time points. DTCs from this patient propagated contained a mutation, which was maintained despite Cyclosporin H morphological changes during 21?days of cell culture. Conclusions Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies. gene, one of the most frequently mutated genes in breast cancer [22-25]. We demonstrate that this mutation can be detected in single tumor cells isolated from breast cancer patient primary tumor, blood, bone marrow, and metastases, and track mutational status of CTCs over time in a metastatic breast cancer case example and in cultured DTCs from this patient. While we have previously shown that individual CTCs in breast cancer, even from the same blood draw, are transcriptionally heterogeneous [21], here we investigate mutational heterogeneity and concordance among CTCs, DTCs, and single tumor cells from primary tumors and metastases. In particular, for CTCs to be ultimately used to guide drug selection, we hypothesized that CTCs should indeed contain the mutational changes found in metastases. However, our results were surprising and we present here a case that provides a cautionary note that CTCs from any one blood draw alone may not always represent the mutational status of Cyclosporin H tumor cells in bone marrow or distant metastases. Methods Ethics statement This study protocol was approved by Stanfords Human Subjects Research and Institutional Review Board (Protocol 5630). Written informed consent was explained and signed by all participating patients prior to sample collection. Tumor cell isolation, staining, and culture Single cell suspensions used for MagSweeper tumor cell isolation were prepared from primary and metastatic tissue from breast cancer patients. Tumor chunks were finely minced, gently pulled to release single cells or small cell clusters, filtered through a 70 micron mesh followed by centrifugation of the filtrate at 1900?g. The supernatant was discarded and the pellet was resuspended in 1x trypsin (Invitrogen/Life Technologies, Carlsbad, CA, USA) for 5C10 minutes. Rabbit Polyclonal to ATG4D DMEM culture media with 10% FBS (Gibco/Life Technologies, Carlsbad, CA, USA) was added to stop the trypsin reaction. The target single tumor cells Cyclosporin H were labeled with EpCAM-conjugated microbeads and isolated by the MagSweeper as previously described [19,21]. Individual tumor Cyclosporin H cells were aspirated under direct microscopic visualization (Axio Observer A1, Zeiss, Thornwood, NY, USA). Authenticated MCF7 and BT474 human breast cancer cell lines (ATCC, Manassas, VA, USA) were grown in DMEM and trypsinized to release single cells, which were then isolated by the MagSweeper and manually aspirated as single cells. For immunostaining assays, EpCAM-captured cells were treated with DNase I Solution (StemCell Technologies, Vancouver, BC, Canada) to remove the DNA-linker on the magnetic microbeads, and placed on slides. Tumor cells were defined by immunostain assay [26-28] as cells that stained positive for purified anti-cytokeratin (CK+) CAM 5.2 (BD Biosciences, San Jose, CA, USA) and DAPI.