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Genetic profiling of putative breast cancer stem cells from malignant pleural effusions.

Abstract A common symptom during late stage breast cancer disease is pleural effusion, which is related to poor prognosis. Malignant cells can be detected in pleural effusions indicating metastatic spread from the primary tumor site. Pleural effusions have been shown to be a useful source for studying metastasis and for isolating cells with putative cancer stem cell (CSC) properties. For the present study, pleural effusion aspirates from 17 metastatic breast cancer patients were processed to propagate CSCs in vitro. Patient-derived aspirates were cultured under sphere forming conditions and isolated primary cultures were further sorted for cancer stem cell subpopulations ALDH1+ and CD44+CD24-/low. Additionally, sphere forming efficiency of CSC and non-CSC subpopulations was determined. In order to genetically characterize the different tumor subpopulations, DNA was isolated from pleural effusions before and after cell sorting, and compared with corresponding DNA copy number profiles from primary tumors or bone metastasis using low-coverage whole genome sequencing (SCNA-seq). In general, unsorted cells had a higher potential to form spheres when compared to CSC subpopulations. In most cases, cell sorting did not yield sufficient cells for copy number analysis. A total of five from nine analyzed unsorted pleura samples (55%) showed aberrant copy number profiles similar to the respective primary tumor. However, most sorted subpopulations showed a balanced profile indicating an insufficient amount of tumor cells and low sensitivity of the sequencing method. Finally, we were able to establish a long term cell culture from one pleural effusion sample, which was characterized in detail. In conclusion, we confirm that pleural effusions are a suitable source for enrichment of putative CSC. However, sequencing based molecular characterization is impeded due to insufficient sensitivity along with a high number of normal contaminating cells, which are masking genetic alterations of rare cancer (stem) cells.
PMID
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Authors

Mayor MeshTerms
Keywords
Journal Title plos one
Publication Year Start




PMID- 28423035
OWN - NLM
STAT- In-Process
DA  - 20170419
LR  - 20170419
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 12
IP  - 4
DP  - 2017
TI  - Genetic profiling of putative breast cancer stem cells from malignant pleural
      effusions.
PG  - e0175223
LID - 10.1371/journal.pone.0175223 [doi]
AB  - A common symptom during late stage breast cancer disease is pleural effusion,
      which is related to poor prognosis. Malignant cells can be detected in pleural
      effusions indicating metastatic spread from the primary tumor site. Pleural
      effusions have been shown to be a useful source for studying metastasis and for
      isolating cells with putative cancer stem cell (CSC) properties. For the present 
      study, pleural effusion aspirates from 17 metastatic breast cancer patients were 
      processed to propagate CSCs in vitro. Patient-derived aspirates were cultured
      under sphere forming conditions and isolated primary cultures were further sorted
      for cancer stem cell subpopulations ALDH1+ and CD44+CD24-/low. Additionally,
      sphere forming efficiency of CSC and non-CSC subpopulations was determined. In
      order to genetically characterize the different tumor subpopulations, DNA was
      isolated from pleural effusions before and after cell sorting, and compared with 
      corresponding DNA copy number profiles from primary tumors or bone metastasis
      using low-coverage whole genome sequencing (SCNA-seq). In general, unsorted cells
      had a higher potential to form spheres when compared to CSC subpopulations. In
      most cases, cell sorting did not yield sufficient cells for copy number analysis.
      A total of five from nine analyzed unsorted pleura samples (55%) showed aberrant 
      copy number profiles similar to the respective primary tumor. However, most
      sorted subpopulations showed a balanced profile indicating an insufficient amount
      of tumor cells and low sensitivity of the sequencing method. Finally, we were
      able to establish a long term cell culture from one pleural effusion sample,
      which was characterized in detail. In conclusion, we confirm that pleural
      effusions are a suitable source for enrichment of putative CSC. However,
      sequencing based molecular characterization is impeded due to insufficient
      sensitivity along with a high number of normal contaminating cells, which are
      masking genetic alterations of rare cancer (stem) cells.
FAU - Tiran, Verena
AU  - Tiran V
AD  - Department of Internal Medicine, Division of Oncology, Medical University of
      Graz, Graz, Austria.
FAU - Stanzer, Stefanie
AU  - Stanzer S
AD  - Department of Internal Medicine, Division of Oncology, Medical University of
      Graz, Graz, Austria.
FAU - Heitzer, Ellen
AU  - Heitzer E
AD  - Institute of Human Genetics, Medical University of Graz, Graz, Austria.
FAU - Meilinger, Michael
AU  - Meilinger M
AD  - Department of Internal Medicine, Division of Pulmonology, Medical University of
      Graz, Graz, Austria.
AD  - Second Internal Division of Pulmonology, Otto Wagner Spital, Vienna, Austria.
FAU - Rossmann, Christopher
AU  - Rossmann C
AD  - Department of Internal Medicine, Division of Oncology, Medical University of
      Graz, Graz, Austria.
FAU - Lax, Sigurd
AU  - Lax S
AD  - Institute of Pathology, LKH Graz West, Graz, Austria.
FAU - Tsybrovskyy, Oleksiy
AU  - Tsybrovskyy O
AD  - Institute of Pathology, Medical University of Graz, Graz, Austria.
FAU - Dandachi, Nadia
AU  - Dandachi N
AD  - Department of Internal Medicine, Division of Oncology, Medical University of
      Graz, Graz, Austria.
AD  - Research Unit Epigenetic and Genetic Cancer Biomarkers, Medical University of
      Graz, Graz, Austria.
FAU - Balic, Marija
AU  - Balic M
AD  - Department of Internal Medicine, Division of Oncology, Medical University of
      Graz, Graz, Austria.
AD  - Research Unit Circulating Tumor Cells and Cancer Stem Cells, Medical University
      of Graz, Graz, Austria.
LA  - eng
PT  - Journal Article
DEP - 20170419
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
EDAT- 2017/04/20 06:00
MHDA- 2017/04/20 06:00
CRDT- 2017/04/20 06:00
PHST- 2017/01/25 [received]
PHST- 2017/03/22 [accepted]
AID - 10.1371/journal.pone.0175223 [doi]
AID - PONE-D-17-03292 [pii]
PST - epublish
SO  - PLoS One. 2017 Apr 19;12(4):e0175223. doi: 10.1371/journal.pone.0175223.
      eCollection 2017.

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