Gordon Cann

mRNA-Seq of Single Prostate Cancer Circulating Tumor Cells Reveals Recapitulation of Gene Expression and Pathways Found in Prostate Cancer

Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.

Abstract 4856: Transcriptome sequencing of circulating tumor cells reveals their heterogeneity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL We have developed a novel sample preparation technology for complete purification of circulating tumor cells (CTCs). The target cells are magnetically labeled with anti-EpCAM particles and then immunostained with anti-EpCAM antibodies. CTCs have been selectively captured by applying absorption magnetic forces and desorbing fluidic shear force to the cells (MagSweeper technology). CTCs, defined as EpCAM+ CD45- cells are then picked individually for nucleic acid analysis. We have utilized this platform for isolation of CTCs from prostate cancer patients. From each case, 7.5ml blood sample is processed within 24hr of collection. The purified CTCs have been validated to be EpCAM+ CD45- by immunostaining and qPCR analysis (91%± 6% specificity). So far we have isolated CTCs from 14 out of 35 cases. The CTC counts isolated by MagSweeper have correlated well with the counts obtained from the same cases using FDA-cleared CellSearch system. We are also developing new genomic technologies for whole-genome expression profiling of single CTCs. The purified CTCs are lysed under control conditions, and then processed with our whole transcriptome amplification (WTA) protocol. The amplified cDNA are used for SBS sequencing library preparation. Our WTA protocol has been validated through mRNA-sequencing of cultured single prostate LNCaP and bladder T24 cells. We found that more than 66% of all genes are reproducibly sequenced and the expression profile of single LNCaP and T24 cells correlated well within each cell line (R⁁2=0.75 for LNCaP and R⁁2=0.87 for T24 cells). We have sequenced the whole transcriptome of 7 CTCs and one leukocyte cell isolated from blood sample of a metastatic prostate cancer patient. We found that 7/7 CTC cells express KLK3, AR and EpCAM, 6/7 express CD24 and AMACR, 5/7 express MYC and 0/7 express CD45. On the other hand, the WBC does not express any of these genes except CD45. At present we are analyzing the data for the presence of fusion genes. So far we have not detected the TMPRSS2-ERG fusion gene in any of the CTCs analyzed; however, the search for looking into other known fusion genes is currently under progress. In addition, we clustered CTCs with LNCaP and T24 single cells. We found that the 5/7 CTC cells cluster together, and are different from LNcap or T24 clusters. 2/7 CTCs show different expression profile, demonstrating expected heterogeneity between CTCs. We have introduced a new end-to-end integrated workflow for CTC genomic analysis. The workflow has been successfully tested in isolation and mRNA-seq profiling of CTCs from prostate cancer patient samples. This novel workflow will open up new opportunities in studying CTC biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4856. doi:10.1158/1538-7445.AM2011-4856

Abstract 1144: High-throughput genetic and expression analysis of circulating tumor cells

We are developing novel sample preparation technologies for genomic analysis of circulating tumor cells (CTCs): a new cell-sorting device for purification of single CTCs from a tube of blood; and highly sensitive and quantitative genomic technologies for single cell analysis using Illumina9s microarray and next-generation sequencing platforms. We have prototyped a new ultra-rare cell enrichment device that produces cell samples compatible with Illumina9s nucleic-acid analysis platforms. Sorting is performed on whole blood samples with no pre-fractionation, to minimize the genetic alteration of CTCs. To validate the performance of our cell sorting device, we have monitored the recovery of LNCaP prostate cancer cells spiked into 7.5ml normal blood samples at concentration of 10 cells/7.5ml. For ease of analysis, the cancer cells were labeled with cell tracker dye and leukocyte nuclei in blood were labeled with Hoechst 33342 dye. Cancer cells were immunomagnetically labeled in blood with EpCAM beads and then isolated by our cell sorter. We found that the capturing efficiency of our platform was 60% ± 24% for 10 cells/7.5ml samples. The purity of cancer cells among contaminating white blood cells was 91% ± 6%, after a second round of extraction of individual cancer cells. The entire purification protocol of CTCs from 7.5ml blood samples takes 2 hours. This platform has been earlier validated for CTC isolation from blood samples of breast cancer patients and is currently under evaluation for prostate and ovarian cancer patients. We are also developing new genomic technologies for whole-genome expression profiling, somatic mutation analysis, and transcriptome sequencing of purified CTCs: a) using a Multiple Displacement Amplification (MDA)-based protocol and a 300K-SNP chip readout, we were able to obtain 88.3% and 93.9% call rate, and 97.4% and 99.9% call accuracy with direct cell lysis from 1 and 5 LNCaP cells, respectively. We also detected the chromosomal amplification and loss-of-heterozygosity; b) with our current RNA amplification protocol, we were able to generate reproducible expression profiles, R 2 = 0.37 and 0.75, from 1 and 10 cell inputs, respectively. In addition, the expression profiles correlated well with those obtained with standard 100 ng total RNA input, R 2 = 0.36 and 0.72, respectively; c) we have also developed next-generation sequencing protocols to profile single-cell transciptomes. We have introduced a new platform for isolation of small numbers of CTCs from patient blood samples that are compatible with Illumina genomic assays. Our gDNA and RNA amplification protocols work with direct cell lysates; no need to extract DNA or RNA from single cells. The protocols are compatible with Illumina microarray and next-gen sequencing platforms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1144.

Abstract 1149: Genetic analysis of single cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We are developing highly sensitive and quantitative genomic technologies for genetic and expression analysis of single cells or trace amount of DNA/RNA materials, using microarray and next-generation sequencing platforms. We have tested various Multiple Displacement Amplification (MDA)-based protocols under a variety of reaction conditions. With our current protocol and a 300K-SNP chip readout, we were able to obtain 88.3% and 93.9% call rate, and 97.4% and 99.9% call accuracy with direct cell lysis from 1 cell and 5 cells, respectively. We are also developing RNA amplification methods for high-throughput expression profiling of single cells. With our current protocol, we were able to generate reproducible expression profiles, R2 = 0.73 and 0.77, using 10 pg and 50 pg total RNA input, respectively. In addition, the profiles correlated well with those obtained with standard 100 ng total RNA input, R2 = 0.61 and 0.77, respectively. Our data show that sequencing of single-cell transcriptomes can clearly distinguish embryonic stem cells from embryonic fibroblasts and tumor cells. We are currently using these technologies to study medical specimens such as circulating tumor cells and cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1149.