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| Status |
Public on Sep 17, 2015 |
| Title |
RNA-Seq of Single Prostate CTCs Implicates Non-Canonical Wnt Signaling in Antiandrogen Resistance |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Prostate 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.
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| Overall design |
A total of 221 single candidate prostate CTCs were isolated from 18 patients with metastatic prostate cancer with bone metastases 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.
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| Contributor(s) |
Miyamoto DT, Zheng Y, Wittner BS, Lee RJ, Zhu H, Broderick KT, Desai R, Fox DB, Brannigan BW, Trautwein J, Arora KS, Dahl DM, Sequist LV, Smith MR, Kapur R, Wu C, Shioda T, Ramaswamy S, Ting DT, Toner M, Maheswaran S, Haber DA |
| Citation(s) |
26383955 |
| Submission date |
Apr 16, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ben S. Wittner |
| E-mail(s) |
wittner.ben@mgh.harvard.edu
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| Organization name |
Massachusetts General Hospital
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| Department |
Center for Cancer Research
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| Lab |
Lawrence
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| Street address |
149 13th Street
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
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| Platforms (1) |
| GPL16288 |
AB 5500xl Genetic Analyzer (Homo sapiens) |
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| Samples (169)
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| Relations |
| BioProject |
PRJNA281419 |
| SRA |
SRP057275 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE67980_readCounts.txt.gz |
2.2 Mb |
(ftp)(http) |
TXT |
| GSE67980_sampleProperties.txt.gz |
1.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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