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.
Highlights
Intratumoral clonal heterogeneity may impact treatment response to chemotherapy or targeted therapies and the outcome of cancer patients [1,2]
As we and others have shown for acute myeloid leukemia (AML), single-cell sequencing is useful for verifying the clonal architecture concluded from next generation sequencing (NGS) data and for resolving the clonal assignment of mutations when NGS provides ambiguous or complex clonal architectures [6,7,8,9]
We study gene mutations and polymorphisms in cancer cells using routine PCR and Sanger sequencing after whole genome amplification (WGA) in order to evaluate the co-occurrence of mutations in individual cells and the clonal genetic architecture
Summary
Intratumoral clonal heterogeneity may impact treatment response to chemotherapy or targeted therapies and the outcome of cancer patients [1,2]. Information on gene mutations derived from generation sequencing (NGS) of bulk cell populations has been increasingly used to gain insights into the clonal heterogeneity of malignancies. This bioinformatically inferred data may only give an approximation of the definite clonal architecture. Genetic information on the single-cell level has become more accessible in the recent years This led to several studies which revealed deeper insights into the clonal architecture and evolution of various types of solid cancers and leukemias, all of which highlighted the importance of single-cell analyses [3,4,5,6,7,8,9,10]. As we and others have shown for acute myeloid leukemia (AML), single-cell sequencing is useful for verifying the clonal architecture concluded from NGS data and for resolving the clonal assignment of mutations when NGS provides ambiguous or complex clonal architectures [6,7,8,9]
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