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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.

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CTC-Enriched Genes Found in Epithelial and Stromal Components of Primary Tumors(A) Expression heatmap of stem cell genes and highly enriched CTC genes in primary tumors and CTC-c cells. Scale in log10(rpm).(B–D) Expression boxplot (left) analysis of (B) Aldh1a2 stem cell and CTC highly enriched genes (C) Klf4 and (D) Igfbp5 genes with RNA-ISH of primary tumors (right). Bar = median, box plot = quartiles, scale in log10(rpm). RNA-ISH color key shown (CK = Krt8+18). Circles indicate a subpopulation of keratin-positive tumor cells with Aldh1a2 marker, and arrowheads identify dual-positive cells at the epithelial-stromal interface (E, epithelial; S, stromal) with DAPI nuclear stain (blue). Low-magnification fluorescent images taken at 100× magnification (scale bar represents 100 μm) and high magnification at 400× (scale bar represents 20 μm).
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Figure 4: CTC-Enriched Genes Found in Epithelial and Stromal Components of Primary Tumors(A) Expression heatmap of stem cell genes and highly enriched CTC genes in primary tumors and CTC-c cells. Scale in log10(rpm).(B–D) Expression boxplot (left) analysis of (B) Aldh1a2 stem cell and CTC highly enriched genes (C) Klf4 and (D) Igfbp5 genes with RNA-ISH of primary tumors (right). Bar = median, box plot = quartiles, scale in log10(rpm). RNA-ISH color key shown (CK = Krt8+18). Circles indicate a subpopulation of keratin-positive tumor cells with Aldh1a2 marker, and arrowheads identify dual-positive cells at the epithelial-stromal interface (E, epithelial; S, stromal) with DAPI nuclear stain (blue). Low-magnification fluorescent images taken at 100× magnification (scale bar represents 100 μm) and high magnification at 400× (scale bar represents 20 μm).

Mentions: CTCs are also likely to be enriched for metastatic precursors capable of initiating metastatic tumor deposits. The relationship between such precursor cells and cancer stem cells is uncertain, as is the relevance of established stem cell markers in identifying these cells. We evaluated putative pancreatic cancer stem cell genes (Rasheed and Matsui, 2012; Rasheed et al., 2010) in the single-cell RNA-seq reads (Figures 4A and S6). Among all candidate markers tested (Aldh1a1, Aldh1a2, Prom1/Cd133, Cd44, Met, EpCAM), only Aldh1a1 and Aldh1a2 were enriched in CTCs. Classical CTCs expressed predominantly the Aldh1a2 isoform, while Aldh1a1 was expressed in a variety of cell types (Figure S6). Within single CTCs, there was no correlation between expression of Aldh1 isoforms and either enrichment for the mesenchymal genes (Cdh11, Vim) or loss of epithelial genes (Cdh1, Muc1), suggesting that stem cell and EMT markers are not intrinsically linked in CTCs. Analysis of primary pancreatic tumors for Aldh1a2 using RNA in situ hybridization (RNA-ISH) identified rare epithelial tumor cells expressing this stem cell marker, but the majority of expression was present within the cancer associated stromal cells (Figure 4B), consistent with immunohistochemistry for ALDH protein in human PDAC (Rasheed et al., 2010).


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)

CTC-Enriched Genes Found in Epithelial and Stromal Components of Primary Tumors(A) Expression heatmap of stem cell genes and highly enriched CTC genes in primary tumors and CTC-c cells. Scale in log10(rpm).(B–D) Expression boxplot (left) analysis of (B) Aldh1a2 stem cell and CTC highly enriched genes (C) Klf4 and (D) Igfbp5 genes with RNA-ISH of primary tumors (right). Bar = median, box plot = quartiles, scale in log10(rpm). RNA-ISH color key shown (CK = Krt8+18). Circles indicate a subpopulation of keratin-positive tumor cells with Aldh1a2 marker, and arrowheads identify dual-positive cells at the epithelial-stromal interface (E, epithelial; S, stromal) with DAPI nuclear stain (blue). Low-magnification fluorescent images taken at 100× magnification (scale bar represents 100 μm) and high magnification at 400× (scale bar represents 20 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: CTC-Enriched Genes Found in Epithelial and Stromal Components of Primary Tumors(A) Expression heatmap of stem cell genes and highly enriched CTC genes in primary tumors and CTC-c cells. Scale in log10(rpm).(B–D) Expression boxplot (left) analysis of (B) Aldh1a2 stem cell and CTC highly enriched genes (C) Klf4 and (D) Igfbp5 genes with RNA-ISH of primary tumors (right). Bar = median, box plot = quartiles, scale in log10(rpm). RNA-ISH color key shown (CK = Krt8+18). Circles indicate a subpopulation of keratin-positive tumor cells with Aldh1a2 marker, and arrowheads identify dual-positive cells at the epithelial-stromal interface (E, epithelial; S, stromal) with DAPI nuclear stain (blue). Low-magnification fluorescent images taken at 100× magnification (scale bar represents 100 μm) and high magnification at 400× (scale bar represents 20 μm).
Mentions: CTCs are also likely to be enriched for metastatic precursors capable of initiating metastatic tumor deposits. The relationship between such precursor cells and cancer stem cells is uncertain, as is the relevance of established stem cell markers in identifying these cells. We evaluated putative pancreatic cancer stem cell genes (Rasheed and Matsui, 2012; Rasheed et al., 2010) in the single-cell RNA-seq reads (Figures 4A and S6). Among all candidate markers tested (Aldh1a1, Aldh1a2, Prom1/Cd133, Cd44, Met, EpCAM), only Aldh1a1 and Aldh1a2 were enriched in CTCs. Classical CTCs expressed predominantly the Aldh1a2 isoform, while Aldh1a1 was expressed in a variety of cell types (Figure S6). Within single CTCs, there was no correlation between expression of Aldh1 isoforms and either enrichment for the mesenchymal genes (Cdh11, Vim) or loss of epithelial genes (Cdh1, Muc1), suggesting that stem cell and EMT markers are not intrinsically linked in CTCs. Analysis of primary pancreatic tumors for Aldh1a2 using RNA in situ hybridization (RNA-ISH) identified rare epithelial tumor cells expressing this stem cell marker, but the majority of expression was present within the cancer associated stromal cells (Figure 4B), consistent with immunohistochemistry for ALDH protein in human PDAC (Rasheed et al., 2010).

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