Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing


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Jonas Demeulemeester1,Parveen Kumar2,Elen K. Møller3,Silje Nord3,David C. Wedge4,April Peterson5,Randi R. Mathiesen6,Renathe Fjelldal7,Masoud Zamani Esteki8,A. Jason Grundstad9,Elin Borgen7,Lars O. Baumbusch10,Anne-Lise Børresen-Dale3,Kevin P. White9,Bjørn Naume11,Vessela N. Kristensen3,Peter Van Loo1,Thierry Voet2
1The Francis Crick Institute, London, UK / Human Genome Laboratory, Department of Human Genetics, University of Leuven, Leuven, Belgium,2Laboratory of Reproductive Genomics, Department of Human Genetics, University of Leuven, Leuven, Belgium / Single-cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, UK,3Department of Genetics, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Oslo, Norway / K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway,4Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, UK,5Institute for Genomics & Systems Biology and Department of Human Genetics, University of Chicago, Chicago, Illinois, USA / (Present address) Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin, USA,6Department of Genetics, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Oslo, Norway / Division of Surgery and Cancer Medicine, Department of Oncology, Oslo University Hospital, Radiumhospitalet, Oslo, Norway / (Present address) Department of Oncology, Akershus University Hospital, Lørenskog, Norway,7Department of Pathology, Oslo University Hospital, Radiumhospitalet, Oslo, Norway,8Laboratory of Reproductive Genomics, Department of Human Genetics, University of Leuven, Leuven, Belgium,9Institute for Genomics & Systems Biology and Department of Human Genetics, University of Chicago, Chicago, Illinois, USA,10Department of Genetics, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Oslo, Norway / (Present address) Department of Pediatric Research, Women and Children's Division, Oslo University Hospital, Rikshospitalet, Oslo, Norway,11K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway / Division of Surgery and Cancer Medicine, Department of Oncology, Oslo University Hospital, Radiumhospitalet, Oslo, Norway

Abstract

Background

Single-cell micro-metastases of solid tumours often occur in bone marrow. These disseminated tumour cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. Unfortunately, the molecular nature of DTCs remains elusive, as well as when and from where in the tumour they originate. Here, we apply single-cell sequencing and subclonal reconstruction to identify and trace the origin of DTCs in breast cancer.

Method

We sequenced the genomes of 63 single cells isolated by micromanipulation from the bone marrow of six patients using established immunocytochemical markers and morphologic characteristics for epithelial tumour cells. We compared the cells’ DNA copy number aberration (CNA) landscapes with those of the primary tumours and lymph node metastasis, and genotyped somatic mutations called on bulk tumour exomes in the single-cell sequences. Evolutionary reconstruction analysis of bulk tumour and DTC genomes enabled ordering of CNA events in molecular pseudo-time.

Results

CNA landscape analysis revealed that almost half of the cells classified as tumor cells are indeed DTCs disseminating from the observed tumor. The remaining cells represented non-aberrant ‘normal’ cells and ‘aberrant cells of unknown origin’ that have CNA profiles discordant from the tumour. Probing somatic mutations confirmed that these cells did not derive from the same lineages as the observed breast cancers. Intriguingly, their prevalence tends to increase with patient age. Evolutionary reconstruction pinpointed the origin of the DTCs to either the main tumour clone, primary tumour subclones, or subclones in an axillary lymph node metastasis.

Conclusion

Single-cell sequencing of bone-marrow epithelial-like cells, in parallel with intratumour genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. Metastatic potential is acquired relatively late during breast cancer evolution. A heterogeneous population of CNA-positive cells of unknown origin is prominent in bone marrow.