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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 Single-Cell Isolation(A) Schematic of the CTC-iChip-negative inertial focusing device system.(B) Mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion is shown in log10.(C) CTC enumeration by immunofluorescent staining (CK+/CD45−/DAPI+) from normal and cancer mice. Bar represents mean.(D) Representative image of CK-positive CTCs. DAPI (blue), CK (red), and CD45 (green). Scale bar, 20 μm. Bright-field image highlighting lack of immunomagnetic anti-CD45 beads on CK+ CTCs (white circle).
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Figure 1: CTC Single-Cell Isolation(A) Schematic of the CTC-iChip-negative inertial focusing device system.(B) Mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion is shown in log10.(C) CTC enumeration by immunofluorescent staining (CK+/CD45−/DAPI+) from normal and cancer mice. Bar represents mean.(D) Representative image of CK-positive CTCs. DAPI (blue), CK (red), and CD45 (green). Scale bar, 20 μm. Bright-field image highlighting lack of immunomagnetic anti-CD45 beads on CK+ CTCs (white circle).

Mentions: The CTC-iChip combines initial hydrodynamic size-based separation of all nucleated cells (leukocytes [WBCs] and CTCs) away from red blood cells, platelets, and plasma, with subsequent inertial focusing of the nucleated cells into a single streamline to achieve high-efficiency in-line magnetic sorting. While tumor epitopes are highly variable, WBC cell-surface markers are well established; applying magnetic-conjugated anti-WBC to this very high-throughput microfluidic cell-separation device can thus exclude the vast majority of WBCs to reveal a small number of untagged CTCs (Figure 1A). Whole-blood labeling using 100 anti-CD45 beads per WBC achieved >103 depletion in normal mice, mice bearing orthotopic tumors, and the KPC mice (Figure 1B).


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 Single-Cell Isolation(A) Schematic of the CTC-iChip-negative inertial focusing device system.(B) Mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion is shown in log10.(C) CTC enumeration by immunofluorescent staining (CK+/CD45−/DAPI+) from normal and cancer mice. Bar represents mean.(D) Representative image of CK-positive CTCs. DAPI (blue), CK (red), and CD45 (green). Scale bar, 20 μm. Bright-field image highlighting lack of immunomagnetic anti-CD45 beads on CK+ CTCs (white circle).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: CTC Single-Cell Isolation(A) Schematic of the CTC-iChip-negative inertial focusing device system.(B) Mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion is shown in log10.(C) CTC enumeration by immunofluorescent staining (CK+/CD45−/DAPI+) from normal and cancer mice. Bar represents mean.(D) Representative image of CK-positive CTCs. DAPI (blue), CK (red), and CD45 (green). Scale bar, 20 μm. Bright-field image highlighting lack of immunomagnetic anti-CD45 beads on CK+ CTCs (white circle).
Mentions: The CTC-iChip combines initial hydrodynamic size-based separation of all nucleated cells (leukocytes [WBCs] and CTCs) away from red blood cells, platelets, and plasma, with subsequent inertial focusing of the nucleated cells into a single streamline to achieve high-efficiency in-line magnetic sorting. While tumor epitopes are highly variable, WBC cell-surface markers are well established; applying magnetic-conjugated anti-WBC to this very high-throughput microfluidic cell-separation device can thus exclude the vast majority of WBCs to reveal a small number of untagged CTCs (Figure 1A). Whole-blood labeling using 100 anti-CD45 beads per WBC achieved >103 depletion in normal mice, mice bearing orthotopic tumors, and the KPC mice (Figure 1B).

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