Limits...
Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.

Ting DT, Wittner BS, Ligorio M, Vincent Jordan N, Shah AM, Miyamoto DT, Aceto N, Bersani F, Brannigan BW, Xega K, Ciciliano JC, Zhu H, MacKenzie OC, Trautwein J, Arora KS, Shahid M, Ellis HL, Qu N, Bardeesy N, Rivera MN, Deshpande V, Ferrone CR, Kapur R, Ramaswamy S, Shioda T, Toner M, Maheswaran S, Haber DA - Cell Rep (2014)

Bottom Line: To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing.Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness.The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

View Article: PubMed Central - PubMed

Affiliation: Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA; Department of Medicine, Harvard Medical School, Boston, MA 02114, USA.

Show MeSH

Related in: MedlinePlus

Single-Cell RNA-Seq Global Analysis(A) Unsupervised hierarchical clustering of candidate single CTCs (1, 3, and 7), single WBCs (2), single MEFs (5), single NB508 cancer cell line (6), and bulk primary tumors diluted to 10 or 100 pg of RNA (4). CTC-c, classical CTCs; CTC-plt, platelet-adhered CTCs; CTC-pro, proliferative CTCs. Data shown log transformed and median polished.(B) Principal component analysis of single-cell samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230325&req=5

Figure 2: Single-Cell RNA-Seq Global Analysis(A) Unsupervised hierarchical clustering of candidate single CTCs (1, 3, and 7), single WBCs (2), single MEFs (5), single NB508 cancer cell line (6), and bulk primary tumors diluted to 10 or 100 pg of RNA (4). CTC-c, classical CTCs; CTC-plt, platelet-adhered CTCs; CTC-pro, proliferative CTCs. Data shown log transformed and median polished.(B) Principal component analysis of single-cell samples.

Mentions: Single-cell RNA-seq performance was comparable for all samples analyzed, with a mean 4.4–8.5 million reads, of which a mean 46%–61% uniquely aligned to the mouse genome (Figure S1). Genome-aligned reads were annotated and counted using UCSC Known Gene transcriptome reference and normalized in reads per million (rpm). Normalized reads were then analyzed by unsupervised hierarchical clustering (Figure 2A). Single-cell transcriptomes from MEFs, the NB508 pancreatic cancer cell line, and normal WBCs clustered tightly, supporting the analytic reliability of the RNA-seq strategy. Three distinct clustering patterns of candidate CTCs were identified, all of which were distinct from matched primary tumor sequences and cancer cell lines. Principal component analysis shows the clustering and interrelationships of these different groups (Figure 2B).


Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.

Ting DT, Wittner BS, Ligorio M, Vincent Jordan N, Shah AM, Miyamoto DT, Aceto N, Bersani F, Brannigan BW, Xega K, Ciciliano JC, Zhu H, MacKenzie OC, Trautwein J, Arora KS, Shahid M, Ellis HL, Qu N, Bardeesy N, Rivera MN, Deshpande V, Ferrone CR, Kapur R, Ramaswamy S, Shioda T, Toner M, Maheswaran S, Haber DA - Cell Rep (2014)

Single-Cell RNA-Seq Global Analysis(A) Unsupervised hierarchical clustering of candidate single CTCs (1, 3, and 7), single WBCs (2), single MEFs (5), single NB508 cancer cell line (6), and bulk primary tumors diluted to 10 or 100 pg of RNA (4). CTC-c, classical CTCs; CTC-plt, platelet-adhered CTCs; CTC-pro, proliferative CTCs. Data shown log transformed and median polished.(B) Principal component analysis of single-cell samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230325&req=5

Figure 2: Single-Cell RNA-Seq Global Analysis(A) Unsupervised hierarchical clustering of candidate single CTCs (1, 3, and 7), single WBCs (2), single MEFs (5), single NB508 cancer cell line (6), and bulk primary tumors diluted to 10 or 100 pg of RNA (4). CTC-c, classical CTCs; CTC-plt, platelet-adhered CTCs; CTC-pro, proliferative CTCs. Data shown log transformed and median polished.(B) Principal component analysis of single-cell samples.
Mentions: Single-cell RNA-seq performance was comparable for all samples analyzed, with a mean 4.4–8.5 million reads, of which a mean 46%–61% uniquely aligned to the mouse genome (Figure S1). Genome-aligned reads were annotated and counted using UCSC Known Gene transcriptome reference and normalized in reads per million (rpm). Normalized reads were then analyzed by unsupervised hierarchical clustering (Figure 2A). Single-cell transcriptomes from MEFs, the NB508 pancreatic cancer cell line, and normal WBCs clustered tightly, supporting the analytic reliability of the RNA-seq strategy. Three distinct clustering patterns of candidate CTCs were identified, all of which were distinct from matched primary tumor sequences and cancer cell lines. Principal component analysis shows the clustering and interrelationships of these different groups (Figure 2B).

Bottom Line: To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing.Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness.The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

View Article: PubMed Central - PubMed

Affiliation: Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA; Department of Medicine, Harvard Medical School, Boston, MA 02114, USA.

Show MeSH
Related in: MedlinePlus