title: Molecular Genetic Characterization of Individual Cancer Cells Isolated via Single-Cell Printing.
abstract: Intratumoral genetic heterogeneity may impact disease outcome. Gold standard for dissecting clonal heterogeneity are single-cell analyses. Here, we present an efficient workflow based on an advanced Single-Cell Printer (SCP) device for the study of gene variants in single cancer cells. To allow for precise cell deposition into microwells the SCP was equipped with an automatic dispenser offset compensation, and the 384-microwell plates were electrostatically neutralized. The ejection efficiency was 99.7% for fluorescent beads (n = 2304) and 98.7% for human cells (U-2 OS or Kasumi-1 cancer cell line, acute myeloid leukemia [AML] patient; n = 150). Per fluorescence microscopy, 98.8% of beads were correctly delivered into the wells. A subset of single cells (n = 81) was subjected to whole genome amplification (WGA), which was successful in all cells. On empty droplets, a PCR on LINE1 retrotransposons yielded no product after WGA, verifying the absence of free-floating DNA in SCP-generated droplets. Representative gene variants identified in bulk specimens were sequenced in single-cell WGA DNA. In U-2 OS, 22 of 25 cells yielded results for both an SLC34A2 and TET2 mutation site, including cells harboring the SLC34A2 but not the TET2 mutation. In one cell, the TET2 mutation analysis was inconclusive due to allelic dropout, as assessed via polymorphisms located close to the mutation. Of Kasumi-1, 23 of 33 cells with data on both the KIT and TP53 mutation site harbored both mutations. In the AML patient, 21 of 23 cells were informative for a TP53 polymorphism; the identified alleles matched the loss of chromosome arm 17p. The advanced SCP allows efficient, precise and gentle isolation of individual cells for subsequent WGA and routine PCR/sequencing-based analyses of gene variants. This makes single-cell information readily accessible to a wide range of applications and can provide insights into clonal heterogeneity that were indeterminable solely by analyses of bulk specimens.
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Freiburg, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Riba
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Renz
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Niemöller
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Bleul
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Pfeifer
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Juliane M
authors.initials: JM
authors.lastname: Stosch
authors.affiliation: Department of Internal Medicine III, University of Munich, Munich, Germany.
authors.forename: Klaus H
authors.initials: KH
authors.lastname: Metzeler
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Hackanson
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Lübbert
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Justus
authors.initials: J
authors.lastname: Duyster
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Freiburg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Koltay
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Freiburg, Germany.|Hahn-Schickard Society for Applied Research, Freiburg, Germany.|BIOSS - Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Zengerle
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Claus
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Freiburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Zimmermann
authors.affiliation: Department of Medicine I, Medical Center - University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Heiko
authors.initials: H
authors.lastname: Becker