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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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SPARC Expression in Human PDAC Enhances Invasion and Metastasis(A–C) Relative SPARC expression in (A) patient-derived human PDAC cell lines and in (B) PDAC3 cell line with shRNA against SPARC and nontarget (NT). Error bars represent range. (C) Proliferation of PDAC3 cell lines determined by MTT.(D) Tumor spheres in PDAC3 shNT versus shSPARC counted per 4× field (error bars represent SD).(E) Boyden migration chamber assay stained with crystal violet and imaged.(F) Scratch assay of shSPARC and shNT cell lines at 24 hr.(G) Invasion of shSPARC and shNT cell lines quantitated by number of nuclei/20× field. p value < 0.01 (**), 0.001 (***), 0.0001 (****). Error bars represent SD.(H) Percentage of detectable lung metastases by in vivo luciferase imaging after 3 weeks after tail vein inoculation of PDAC3 cell lines. Fisher’s exact test p value is shown.(I) Normalized metastasis burden in mice with orthotopic pancreatic tumors from PDAC3 cell lines. Error bars represent SD (*p < 0.05).
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Figure 6: SPARC Expression in Human PDAC Enhances Invasion and Metastasis(A–C) Relative SPARC expression in (A) patient-derived human PDAC cell lines and in (B) PDAC3 cell line with shRNA against SPARC and nontarget (NT). Error bars represent range. (C) Proliferation of PDAC3 cell lines determined by MTT.(D) Tumor spheres in PDAC3 shNT versus shSPARC counted per 4× field (error bars represent SD).(E) Boyden migration chamber assay stained with crystal violet and imaged.(F) Scratch assay of shSPARC and shNT cell lines at 24 hr.(G) Invasion of shSPARC and shNT cell lines quantitated by number of nuclei/20× field. p value < 0.01 (**), 0.001 (***), 0.0001 (****). Error bars represent SD.(H) Percentage of detectable lung metastases by in vivo luciferase imaging after 3 weeks after tail vein inoculation of PDAC3 cell lines. Fisher’s exact test p value is shown.(I) Normalized metastasis burden in mice with orthotopic pancreatic tumors from PDAC3 cell lines. Error bars represent SD (*p < 0.05).

Mentions: In order to define the functional consequences of SPARC expression in pancreatic cancer cells, we screened a panel of patient-derived, low-passage PDAC cell lines for expression. Two human PDAC cell lines with relatively high SPARC expression were identified (PDAC2 and PDAC3), making it possible to test the consequences of small hairpin RNA (shRNA)-mediated knockdown (Figures 6A, 6B, and S7). Suppression of endogenous SPARC expression in both PDAC2 and PDAC3 cell lines using two independent shRNA constructs did not affect proliferation in 2D cultures or anchorage-independent tumor sphere formation (Figures 6C, 6D, and S7). However, SPARC knockdown by both shRNAs significantly reduced pancreatic cancer cell migration in wound scratch assays and their invasive properties, as measured by in vitro Boyden assays (Figures 6E–6G and S7). Tail vein injection of SPARC-suppressed PDAC3 cells using both shRNA constructs generated significantly fewer lung metastases than cells expressing nontargeting hairpin (shNT) controls (Figure 6H). Metastases generated from orthotopic pancreatic xenografts were also significantly reduced for SPARC-suppressed PDAC3 cells, as measured by luciferase imaging and normalized for primary tumor size (Figure 6I). Thus, SPARC expression by pancreatic cancer cells appears to selectively enhance their invasive and migratory properties to augment metastatic virulence. The high levels of SPARC expression evident in virtually all pancreatic CTCs thus raises the possibility that it contributes significantly to the metastatic spread of pancreatic cancer.


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)

SPARC Expression in Human PDAC Enhances Invasion and Metastasis(A–C) Relative SPARC expression in (A) patient-derived human PDAC cell lines and in (B) PDAC3 cell line with shRNA against SPARC and nontarget (NT). Error bars represent range. (C) Proliferation of PDAC3 cell lines determined by MTT.(D) Tumor spheres in PDAC3 shNT versus shSPARC counted per 4× field (error bars represent SD).(E) Boyden migration chamber assay stained with crystal violet and imaged.(F) Scratch assay of shSPARC and shNT cell lines at 24 hr.(G) Invasion of shSPARC and shNT cell lines quantitated by number of nuclei/20× field. p value < 0.01 (**), 0.001 (***), 0.0001 (****). Error bars represent SD.(H) Percentage of detectable lung metastases by in vivo luciferase imaging after 3 weeks after tail vein inoculation of PDAC3 cell lines. Fisher’s exact test p value is shown.(I) Normalized metastasis burden in mice with orthotopic pancreatic tumors from PDAC3 cell lines. Error bars represent SD (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: SPARC Expression in Human PDAC Enhances Invasion and Metastasis(A–C) Relative SPARC expression in (A) patient-derived human PDAC cell lines and in (B) PDAC3 cell line with shRNA against SPARC and nontarget (NT). Error bars represent range. (C) Proliferation of PDAC3 cell lines determined by MTT.(D) Tumor spheres in PDAC3 shNT versus shSPARC counted per 4× field (error bars represent SD).(E) Boyden migration chamber assay stained with crystal violet and imaged.(F) Scratch assay of shSPARC and shNT cell lines at 24 hr.(G) Invasion of shSPARC and shNT cell lines quantitated by number of nuclei/20× field. p value < 0.01 (**), 0.001 (***), 0.0001 (****). Error bars represent SD.(H) Percentage of detectable lung metastases by in vivo luciferase imaging after 3 weeks after tail vein inoculation of PDAC3 cell lines. Fisher’s exact test p value is shown.(I) Normalized metastasis burden in mice with orthotopic pancreatic tumors from PDAC3 cell lines. Error bars represent SD (*p < 0.05).
Mentions: In order to define the functional consequences of SPARC expression in pancreatic cancer cells, we screened a panel of patient-derived, low-passage PDAC cell lines for expression. Two human PDAC cell lines with relatively high SPARC expression were identified (PDAC2 and PDAC3), making it possible to test the consequences of small hairpin RNA (shRNA)-mediated knockdown (Figures 6A, 6B, and S7). Suppression of endogenous SPARC expression in both PDAC2 and PDAC3 cell lines using two independent shRNA constructs did not affect proliferation in 2D cultures or anchorage-independent tumor sphere formation (Figures 6C, 6D, and S7). However, SPARC knockdown by both shRNAs significantly reduced pancreatic cancer cell migration in wound scratch assays and their invasive properties, as measured by in vitro Boyden assays (Figures 6E–6G and S7). Tail vein injection of SPARC-suppressed PDAC3 cells using both shRNA constructs generated significantly fewer lung metastases than cells expressing nontargeting hairpin (shNT) controls (Figure 6H). Metastases generated from orthotopic pancreatic xenografts were also significantly reduced for SPARC-suppressed PDAC3 cells, as measured by luciferase imaging and normalized for primary tumor size (Figure 6I). Thus, SPARC expression by pancreatic cancer cells appears to selectively enhance their invasive and migratory properties to augment metastatic virulence. The high levels of SPARC expression evident in virtually all pancreatic CTCs thus raises the possibility that it contributes significantly to the metastatic spread of pancreatic cancer.

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