Dataset View [GSE67980]

TitleRNA-Seq of Single Prostate CTCs Implicates Non-Canonical Wnt Signaling in Antiandrogen Resistance
ArticleHaber DA,Maheswaran S,Toner M,Ting DT,Ramaswamy S,Shioda T,Wu CL,Kapur R,Smith MR,Sequist LV,Dahl DM,Desai N,Arora KS,Trautwein J,Brannigan BW,Fox DB,Desai R,Broderick KT,Zhu H,Lee RJ,Wittner BS,Zheng Y,Miyamoto DT.RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance.Science (New York, N.Y.).2015 Sep 18
Bio ProjectBioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA281419
SraSRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP057275
Overall DesginA total of 221 single candidate prostate CTCs were isolated from 18 patients with metastatic prostate cancer and 4 patients with localized prostate cancer. Of these, 133 cells (60%) had RNA of sufficient quality for amplification and next generation RNA sequencing, and 122 (55%) had >100,000 uniquely aligned sequencing reads. In addition to candidate CTCs, we also obtained comprehensive transcriptomes for 12 bulk primary prostate cancers (macrodissected for >70% tumor content), 30 single cells derived from four different prostate cancer cell lines, and 5 patient-derived leukocyte controls. The leukocytes were readily distinguished by their expression of hematopoietic lineage markers and served to exclude any CTCs with potentially contaminating signals. Strict expression thresholds were used to define lineage-confirmed CTCs, scored by prostate lineage-specific genes (PSA, PSMA, AMACR, AR) and standard epithelial markers (KRT7, KRT8, KRT18, KRT19, EpCAM). 28 cells were excluded given the presence of leukocyte transcripts suggestive of cellular contamination or misidentification during selection, and 17 cells were excluded given low expression of both prostate lineage-specific genes and 5 standard epithelial markers. The remaining 77 cells, defined as lineage-confirmed CTCs, displayed expression of either prostate lineage-specific or epithelial genes, and low expression of leukocyte-specific genes, consistent with their tumor of origin.
SummaryProstate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging, hence sampling circulating tumor cells (CTCs) may reveal drug resistance mechanisms. We established single cell RNA-sequencing profiles of 77 intact CTCs isolated from 13 patients (mean 6 CTCs/patient) using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing on AR inhibitor, compared with untreated cases indicates activation of noncanonical Wnt signaling (P=0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, while its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.
Experimental ProtocolSamples that have “source name” above set to “candidate PCa CTC” or “lineage-confirmed PCa CTC” were extracted as follows. We applied the CTC-iChip (PMID: 23552373) to efficiently deplete normal hematopoietic cells from whole blood specimens. Cell surface staining of remaining unfixed cells for epithelial (EpCAM) and mesenchymal (CDH11) markers (PMID: 23845299), combined with absent staining for the common leukocyte marker CD45, was used to select candidate tumor cells for micromanipulation. Samples that have “source name” set to “primary PCa tumor” were frozen primary prostate cancer tissue samples from patients with localized prostate cancer who underwent prostatectomy, that were then sectioned and microdissected for >70% tumor content. Samples that have “source name” above set to “single cell from PCa cell line” were trypsinized into single-cell suspension and then micromanipulated. Only samples for which amplification was successful were submitted for sequencing. White blood cell samples from donor Pr23 were processed using the CTC-iChip and micromanipulated after staining for CD45. White blood cells from healthy donor HD1 were isolated from his blood using a micromanipulator, after enrichment of the mononuclear fraction using a BD Vacutainer CPT Cell Preparation Tube with Sodium Citrate and staining for CD45. For all samples RNA was extracted and amplified as in Tang, et. al. (PMID: 20203668).; Libraries were constructed as previously described (Tang, F., Barbacioru, C., Nordman, E., Li, B., Xu, N., Bashkirov, V.I., Lao, K., and Surani, M.A. (2010). RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat Protoc 5, 516-535). Briefly, to generate cDNA, samples were treated with reverse transcription master mix (0.05 uL RNase inhibitor, 0.07uL T4 gene 32 protein, and 0.33uL SuperScript III Reverse Transcriptase per 1X volume) and incubated on thermocycler at 50C for 30 minutes and 70C for 15 minutes. To remove free primer, 1.0uL of EXOSAP mix was added to each sample, which was incubated at 37C for 30 minutes and inactivated at 80C for 25 minutes. Next, a 3'-poly-A tail was added to the cDNA in each sample by incubating in master mix (0.6uL 10X PCR Buffer II, 0.36uL 25mM MgCl2, 0.18uL 100mM dATP, 0.3uL Terminal Transferase, 0.3uL RNase H, and 4.26uL H2O per 1X volume) at 37C for 15 minutes and inactivated at 70C for 10 minutes. A second strand cDNA was synthesis by dividing each sample into 4 and incubating in master mix (2.2uL 10X High Fidelity PCR Buffer, 1.76uL 2.5mM each dNTP, 0.066uL UP2 Primer at 100uM, 0.88uL 50mM MgSO4, 0.44uL Platinum Taq DNA Polymerase, and 13.654uL H2O per 1X volume) at 95C for 3 minutes, 50C for 2 minutes, and 72C for 10 minutes. DNA was sheared using a Covaris S2 system and then prepared for ABI 5500XL library construction with end polishing, size selection of 200-500 bp using AMPure XP, ABI barcode adaptor ligation, amplification and purification with AMPure XP, and then pooling of barcoded samples for emulsion PCR wiht template beads preparation. Samples were then loaded per protocol on the ABI 5500XL.; RNA-Seq using oligo-dT cDNA synthesis and amplification of cDNA libraries using custom universal PCR primers
Data processingColor space reads were aligned using tophat version 2.0.4 and bowtie1 version 0.12.7 with the no-novel-juncs argument set with human genome version hg19 and transcriptome defined by the hg19 knownGene table from genome.ucsc.edu.; Reads that did not align or aligned to multiple locations in the genome were discarded.; The hg19 table knownToLocusLink from genome.ucsc.edu was used to map, if possible, each aligned read to the gene who's exons the read had aligned to. The reads count for each gene was the number of reads that were so mapped to that gene.; The read count was divided by the total number of reads that were mapped to any gene and multiplied by one million to form the reads-per-million (rpm) count.; Genome_build: hg19; Supplementary_files_format_and_content: The readCounts.txt file gives the read counts described above for each sample (columns) and gene (rows).
Public OnPublic on Sep 17 2015

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