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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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Human and Mouse CTCs across Different Epithelial Cancer Express High Levels of ECM Protein Genes(A) Expression heatmap of mouse single primary tumor cells, bulk tumor, and CTCs for ECM protein genes. Scale in log10(rpm).(B) RNA-ISH of ECM protein genes Dcn and Sparc with CK (Krt8+18) in mouse primary PDAC tumors.(C) RNA-ISH of SPARC with CK (KRT7,8,18,+19) in human primary PDAC tumors. Arrowheads identify dual-positive cells at the epithelial-stromal interface (E, epithelial; S, stromal) with DAPI nuclear stain (blue). Images taken at 400× magnification (scale bar represents 20 μm).(D) Expression boxplot of highly expressed ECM genes in human PDAC, breast (BR), and prostate (PR) CTCs. Bar, median; boxplot, quartiles; scale in log10(rpm). Holm-adjusted p value < 0.05 (*), 0.01 (**), 0.001 (***).
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Figure 5: Human and Mouse CTCs across Different Epithelial Cancer Express High Levels of ECM Protein Genes(A) Expression heatmap of mouse single primary tumor cells, bulk tumor, and CTCs for ECM protein genes. Scale in log10(rpm).(B) RNA-ISH of ECM protein genes Dcn and Sparc with CK (Krt8+18) in mouse primary PDAC tumors.(C) RNA-ISH of SPARC with CK (KRT7,8,18,+19) in human primary PDAC tumors. Arrowheads identify dual-positive cells at the epithelial-stromal interface (E, epithelial; S, stromal) with DAPI nuclear stain (blue). Images taken at 400× magnification (scale bar represents 20 μm).(D) Expression boxplot of highly expressed ECM genes in human PDAC, breast (BR), and prostate (PR) CTCs. Bar, median; boxplot, quartiles; scale in log10(rpm). Holm-adjusted p value < 0.05 (*), 0.01 (**), 0.001 (***).

Mentions: In addition to highly expressing Dcn, CTCs consistently had high levels of multiple ECM gene transcripts. GO analysis of all CTC-enriched genes (Table S2) identified 32 proteinaceous ECM genes (GO:0005578, OR 2.4, q-value 4.8 × 10−3). These genes are normally expressed in reactive stromal cells, rather than in epithelial cancer cells, and while recent studies have highlighted the importance of the stroma in supporting pancreatic cancer pathogenesis and metastasis (Feig et al., 2012; Neesse et al., 2011, 2013; Olive et al., 2009; Provenzano et al., 2012), the expression of these stroma-associated ECM genes within tumor cells in circulation was unexpected. Using RP differential expression analysis, we compared CTCs with purified EGFP-tagged primary tumor single cells (TuGMP3) and bulk tumor samples (tumor cells admixed with reactive stromal cells). Six proteinaceous ECM genes were highly expressed by CTCs and by stromal component, but not by epithelial cells within primary tumors: Dcn, Sparc, Ccdc80, Col1a2, Col3a1, and Timp2 (Figure 5A). RNA-ISH analysis of both Dcn and Sparc confirmed diffuse expression in stromal elements of mouse primary tumors, with rare areas where these transcripts are colocalized with keratin-expressing cells at the epithelial-stromal border (Figure 5B). SPARC is a well-known ECM protein gene found in stroma of human primary PDAC (Infante et al., 2007; Neuzillet et al., 2013; Sato et al., 2003). Indeed, RNA-ISH analysis of 198 primary human PDACs demonstrates abundant stromal cell expression of SPARC transcripts in 99% of cases, with up to a third of tumors with rare epithelial cells expressing this ECM gene product (Figure 5C). Consistent with these observations, RNA-seq of EGFP-tagged single primary tumor cells (Figure 5A) identified only 1 of 20 cells (5%) with coexpression of high levels (>100 rpm) of Sparc and Krt19. In summary, abundant expression of ECM genes is a common feature of all keratin-rich classical CTCs. This is in marked contrast to the primary tumor, where these gene products are secreted by supporting stromal cells and not by the epithelial cancer cells. However, rare cells at the epithelial-stromal interface of primary tumors do appear to express both keratins and ECM genes, consistent with the pattern observed in CTCs themselves.


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)

Human and Mouse CTCs across Different Epithelial Cancer Express High Levels of ECM Protein Genes(A) Expression heatmap of mouse single primary tumor cells, bulk tumor, and CTCs for ECM protein genes. Scale in log10(rpm).(B) RNA-ISH of ECM protein genes Dcn and Sparc with CK (Krt8+18) in mouse primary PDAC tumors.(C) RNA-ISH of SPARC with CK (KRT7,8,18,+19) in human primary PDAC tumors. Arrowheads identify dual-positive cells at the epithelial-stromal interface (E, epithelial; S, stromal) with DAPI nuclear stain (blue). Images taken at 400× magnification (scale bar represents 20 μm).(D) Expression boxplot of highly expressed ECM genes in human PDAC, breast (BR), and prostate (PR) CTCs. Bar, median; boxplot, quartiles; scale in log10(rpm). Holm-adjusted p value < 0.05 (*), 0.01 (**), 0.001 (***).
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Related In: Results  -  Collection

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Figure 5: Human and Mouse CTCs across Different Epithelial Cancer Express High Levels of ECM Protein Genes(A) Expression heatmap of mouse single primary tumor cells, bulk tumor, and CTCs for ECM protein genes. Scale in log10(rpm).(B) RNA-ISH of ECM protein genes Dcn and Sparc with CK (Krt8+18) in mouse primary PDAC tumors.(C) RNA-ISH of SPARC with CK (KRT7,8,18,+19) in human primary PDAC tumors. Arrowheads identify dual-positive cells at the epithelial-stromal interface (E, epithelial; S, stromal) with DAPI nuclear stain (blue). Images taken at 400× magnification (scale bar represents 20 μm).(D) Expression boxplot of highly expressed ECM genes in human PDAC, breast (BR), and prostate (PR) CTCs. Bar, median; boxplot, quartiles; scale in log10(rpm). Holm-adjusted p value < 0.05 (*), 0.01 (**), 0.001 (***).
Mentions: In addition to highly expressing Dcn, CTCs consistently had high levels of multiple ECM gene transcripts. GO analysis of all CTC-enriched genes (Table S2) identified 32 proteinaceous ECM genes (GO:0005578, OR 2.4, q-value 4.8 × 10−3). These genes are normally expressed in reactive stromal cells, rather than in epithelial cancer cells, and while recent studies have highlighted the importance of the stroma in supporting pancreatic cancer pathogenesis and metastasis (Feig et al., 2012; Neesse et al., 2011, 2013; Olive et al., 2009; Provenzano et al., 2012), the expression of these stroma-associated ECM genes within tumor cells in circulation was unexpected. Using RP differential expression analysis, we compared CTCs with purified EGFP-tagged primary tumor single cells (TuGMP3) and bulk tumor samples (tumor cells admixed with reactive stromal cells). Six proteinaceous ECM genes were highly expressed by CTCs and by stromal component, but not by epithelial cells within primary tumors: Dcn, Sparc, Ccdc80, Col1a2, Col3a1, and Timp2 (Figure 5A). RNA-ISH analysis of both Dcn and Sparc confirmed diffuse expression in stromal elements of mouse primary tumors, with rare areas where these transcripts are colocalized with keratin-expressing cells at the epithelial-stromal border (Figure 5B). SPARC is a well-known ECM protein gene found in stroma of human primary PDAC (Infante et al., 2007; Neuzillet et al., 2013; Sato et al., 2003). Indeed, RNA-ISH analysis of 198 primary human PDACs demonstrates abundant stromal cell expression of SPARC transcripts in 99% of cases, with up to a third of tumors with rare epithelial cells expressing this ECM gene product (Figure 5C). Consistent with these observations, RNA-seq of EGFP-tagged single primary tumor cells (Figure 5A) identified only 1 of 20 cells (5%) with coexpression of high levels (>100 rpm) of Sparc and Krt19. In summary, abundant expression of ECM genes is a common feature of all keratin-rich classical CTCs. This is in marked contrast to the primary tumor, where these gene products are secreted by supporting stromal cells and not by the epithelial cancer cells. However, rare cells at the epithelial-stromal interface of primary tumors do appear to express both keratins and ECM genes, consistent with the pattern observed in CTCs themselves.

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