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Article: RNA-Seq Library Generation from Rare Human Cells Isolated by FACS.

Gkountela, Sofia / Clark, Amander T

2016 Volume 3, Issue 12

Abstract: High throughput RNA Sequencing has revolutionized transcriptome analyses. However, most available protocols require micrograms of RNA rendering this technique not feasible for analyzing small numbers of cells, including precious rare cell types isolated ... ...

Abstract	High throughput RNA Sequencing has revolutionized transcriptome analyses. However, most available protocols require micrograms of RNA rendering this technique not feasible for analyzing small numbers of cells, including precious rare cell types isolated from human tissues or organs. Here, we used an RNA Amplification System and describe a method for preparing RNA sense-strand cDNA libraries compatible with an Illumina sequencing platform starting from limited numbers of human fetal germ cells as well as human embryonic stem cells (hESCs) isolated using Fluorescence Activated Cell Sorting (FACS). With this protocol we generated seven RNA-Seq libraries starting from 4,000 germ cells sorted from fetal ovaries (n = 2) and fetal testes (n = 2) at 16-16.5 weeks of development and 4,000 sorted hESCs (n = 3). We predict that multiplexed libraries can also be generated by replacing the single-plex 3' adapter used here with a multiplexing compatible 3' adapter and indexed PCR primers.
Language	English
Publishing date	2016-07-19
Publishing country	United States
Document type	Journal Article
ZDB-ID	2833269-6
ISSN	2331-8325
ISSN	2331-8325
DOI	10.21769/bioprotoc.791
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Article ; Online: Stem-like features of cancer cells on their way to metastasis.

Gkountela, Sofia / Aceto, Nicola

Biology direct

2016 Volume 11, Page(s) 33

Abstract: Unlabelled: More than 90 % of cancer-related deaths are due to the development of a systemic metastatic disease. Clearly, much remains to be understood about the biological principles that govern human cancer metastasis, aiming at the ambitious ... ...

Abstract	Unlabelled: More than 90 % of cancer-related deaths are due to the development of a systemic metastatic disease. Clearly, much remains to be understood about the biological principles that govern human cancer metastasis, aiming at the ambitious objective to decrease metastasis-related mortality in patients. For many years, research on metastasis has been conducted in great part on experimental mouse models, mainly because of the difficulties in sampling, longitudinal studies, and molecular interrogation of a human metastatic disease. However, recently, extraordinary advances in microfluidic technologies are allowing the isolation and characterization of human circulating tumor cells (CTCs) that escaped a primary tumor mass and are in the process of seeding a distant metastasis. Analysis of human CTCs has now revealed important features of cancer metastasis, such as the high metastatic potential of CTC-clusters compared to single CTCs, the dynamic expression of epithelial and mesenchymal markers on CTCs during treatment, and the possibility to culture CTCs from patients for a real-time and individualized testing of drug susceptibility. Nevertheless, several aspects of CTC biology remain unsolved, such as the characterization of the stem-like cell population among human CTCs. Here, we focus on describing the latest findings in the CTC field, and discuss them in the context of cancer stem cell biology. Defining the molecular features of those few metastasis-initiating, stem-like CTCs holds the exceptional promise to develop metastasis-tailored therapies for patients with cancer. Reviewers: This article was reviewed by Elisa Cimetta, Luca Pellegrini and Sirio Dupont (nominated by LP).
MeSH term(s)	Animals ; Biomarkers, Tumor/analysis ; Humans ; Mice ; Neoplastic Cells, Circulating/metabolism ; Neoplastic Stem Cells/metabolism
Chemical Substances	Biomarkers, Tumor
Language	English
Publishing date	2016-07-26
Publishing country	England
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ISSN	1745-6150
ISSN (online)	1745-6150
DOI	10.1186/s13062-016-0135-4
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Article ; Online: A big surprise in the little zygote: the curious business of losing methylated cytosines.

Gkountela, Sofia / Clark, Amander T

Cell stem cell

2014 Volume 15, Issue 4, Page(s) 393–394

Abstract: In stem cell biology, the dynamic addition and removal of 5-methylcytosines (5mCs) are necessary for lineage differentiation, nuclear reprogramming, and embryonic development. Recent investigations have sought to understand the mechanisms of how 5mCs are ...

Abstract	In stem cell biology, the dynamic addition and removal of 5-methylcytosines (5mCs) are necessary for lineage differentiation, nuclear reprogramming, and embryonic development. Recent investigations have sought to understand the mechanisms of how 5mCs are added and in particular how 5mCs are removed from DNA during embryogenesis.
MeSH term(s)	Animals ; Cell Nucleus/metabolism ; DNA Methylation/genetics ; Female ; Male ; Mammals/genetics
Language	English
Publishing date	2014-10-03
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Comment
ZDB-ID	2375354-7
ISSN	1875-9777 ; 1934-5909
ISSN (online)	1875-9777
ISSN	1934-5909
DOI	10.1016/j.stem.2014.09.005
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Article: Recent advances in the biology of human circulating tumour cells and metastasis.

Gkountela, Sofia / Szczerba, Barbara / Donato, Cinzia / Aceto, Nicola

ESMO open

2016 Volume 1, Issue 4, Page(s) e000078

Abstract: The development of a metastatic disease is recognised as the cause of death of over 90% of patients diagnosed with cancer. Understanding the biological features of metastasis has been hampered for a long time by the difficulties to study widespread ... ...

Abstract	The development of a metastatic disease is recognised as the cause of death of over 90% of patients diagnosed with cancer. Understanding the biological features of metastasis has been hampered for a long time by the difficulties to study widespread cancerous lesions in patients, and by the absence of reliable methods to isolate viable metastatic cells during disease progression. These difficulties negatively impact on our ability to develop new agents that are tailored to block the spread of cancer. Yet, recent advances in specialised devices for the isolation of circulating tumour cells (CTCs), hand-in-hand with technologies that enable single cell resolution interrogation of their genome and transcriptome, are now paving the way to understanding those molecular mechanisms that drive the formation of metastasis. In this review, we aim to summarise some of the latest discoveries in CTC biology in the context of several types of cancer, and to highlight those findings that have a potential to improve the clinical management of patients with metastatic cancer.
Language	English
Publishing date	2016-08-03
Publishing country	England
Document type	Review ; Journal Article
ISSN	2059-7029
ISSN	2059-7029
DOI	10.1136/esmoopen-2016-000078
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Article: Cancer Diagnosis Using a Liquid Biopsy: Challenges and Expectations.

Castro-Giner, Francesc / Gkountela, Sofia / Donato, Cinzia / Alborelli, Ilaria / Quagliata, Luca / Ng, Charlotte K Y / Piscuoglio, Salvatore / Aceto, Nicola

Diagnostics (Basel, Switzerland)

2018 Volume 8, Issue 2

Abstract: The field of cancer diagnostics has recently been impacted by new and exciting developments in the area of liquid biopsy. A liquid biopsy is a minimally invasive alternative to surgical biopsies of solid tissues, typically achieved through the withdrawal ...

Abstract	The field of cancer diagnostics has recently been impacted by new and exciting developments in the area of liquid biopsy. A liquid biopsy is a minimally invasive alternative to surgical biopsies of solid tissues, typically achieved through the withdrawal of a blood sample or other body fluids, allowing the interrogation of tumor-derived material including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) fragments that are present at a given time point. In this short review, we discuss a few studies that summarize the state-of-the-art in the liquid biopsy field from a diagnostic perspective, and speculate on current challenges and expectations of implementing liquid biopsy testing for cancer diagnosis and monitoring in the clinical setting.
Language	English
Publishing date	2018-05-09
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2662336-5
ISSN	2075-4418
ISSN	2075-4418
DOI	10.3390/diagnostics8020031
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Article ; Online: Primate Primordial Germ Cells Acquire Transplantation Potential by Carnegie Stage 23.

Clark, Amander T / Gkountela, Sofia / Chen, Di / Liu, Wanlu / Sosa, Enrique / Sukhwani, Meena / Hennebold, Jon D / Orwig, Kyle E

Stem cell reports

2017 Volume 9, Issue 1, Page(s) 329–341

Abstract: Primordial germ cells (PGCs) are the earliest embryonic progenitors in the germline. Correct formation of PGCs is critical to reproductive health as an adult. Recent work has shown that primate PGCs can be differentiated from pluripotent stem cells; ... ...

Abstract	Primordial germ cells (PGCs) are the earliest embryonic progenitors in the germline. Correct formation of PGCs is critical to reproductive health as an adult. Recent work has shown that primate PGCs can be differentiated from pluripotent stem cells; however, a bioassay that supports their identity as transplantable germ cells has not been reported. Here, we adopted a xenotransplantation assay by transplanting single-cell suspensions of human and nonhuman primate embryonic Macaca mulatta (rhesus macaque) testes containing PGCs into the seminiferous tubules of adult busulfan-treated nude mice. We discovered that both human and nonhuman primate embryonic testis are xenotransplantable, generating colonies while not generating tumors. Taken together, this work provides two critical references (molecular and functional) for defining transplantable primate PGCs. These results provide a blueprint for differentiating pluripotent stem cells to transplantable PGC-like cells in a species that is amenable to transplantation and fertility studies.
Language	English
Publishing date	2017-07-11
Publishing country	United States
Document type	Journal Article
ZDB-ID	2720528-9
ISSN	2213-6711 ; 2213-6711
ISSN (online)	2213-6711
ISSN	2213-6711
DOI	10.1016/j.stemcr.2017.05.002
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Article ; Online: Cancer Diagnosis Using a Liquid Biopsy

Francesc Castro-Giner / Sofia Gkountela / Cinzia Donato / Ilaria Alborelli / Luca Quagliata / Charlotte K. Y. Ng / Salvatore Piscuoglio / Nicola Aceto

Diagnostics, Vol 8, Iss 2, p

Challenges and Expectations

2018 Volume 31

Abstract	The field of cancer diagnostics has recently been impacted by new and exciting developments in the area of liquid biopsy. A liquid biopsy is a minimally invasive alternative to surgical biopsies of solid tissues, typically achieved through the withdrawal of a blood sample or other body fluids, allowing the interrogation of tumor-derived material including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) fragments that are present at a given time point. In this short review, we discuss a few studies that summarize the state-of-the-art in the liquid biopsy field from a diagnostic perspective, and speculate on current challenges and expectations of implementing liquid biopsy testing for cancer diagnosis and monitoring in the clinical setting.
Keywords	circulating tumor cells ; single cell analysis ; deep-sequencing ; liquid biopsy ; cancer diagnosis ; cancer treatment ; Medicine (General) ; R5-920
Language	English
Publishing date	2018-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Circulating Tumor Cell Clustering Shapes DNA Methylation to Enable Metastasis Seeding

Gkountela, Sofia / Castro-Giner, Francesc / Szczerba, Barbara Maria / Vetter, Marcus / Landin, Julia / Scherrer, Ramona / Krol, Ilona / Scheidmann, Manuel C / Beisel, Christian / Stirnimann, Christian U / Kurzeder, Christian / Heinzelmann-Schwarz, Viola / Rochlitz, Christoph / Weber, Walter Paul / Aceto, Nicola

Cell. 2019 Jan. 10, v. 176, no. 1-2

2019

Abstract: The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single ...

Abstract	The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer.
Keywords	DNA methylation ; animal models ; binding sites ; breast neoplasms ; dissociation ; embryonic stem cells ; enzyme inhibitors ; metastasis ; neoplasm cells ; patients ; sodium-potassium-exchanging ATPase ; transcription factors
Language	English
Dates of publication	2019-0110
Size	p. 98-112.e14.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	187009-9
ISSN	1097-4172 ; 0092-8674
ISSN (online)	1097-4172
ISSN	0092-8674
DOI	10.1016/j.cell.2018.11.046
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: PRMT5 is required for human embryonic stem cell proliferation but not pluripotency.

Gkountela, Sofia / Li, Ziwei / Chin, Chee Jia / Lee, Serena A / Clark, Amander T

Stem cell reviews and reports

2014 Volume 10, Issue 2, Page(s) 230–239

Abstract: Human pluripotent stem cells (PSCs) are critical in vitro tools for understanding mechanisms that regulate lineage differentiation in the human embryo as well as a potentially unlimited supply of stem cells for regenerative medicine. Pluripotent human ... ...

Abstract	Human pluripotent stem cells (PSCs) are critical in vitro tools for understanding mechanisms that regulate lineage differentiation in the human embryo as well as a potentially unlimited supply of stem cells for regenerative medicine. Pluripotent human and mouse embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts share a similar transcription factor network to maintain pluripotency and self-renewal, yet there are considerable molecular differences reflecting the diverse environments in which mouse and human ESCs are derived. In the current study we evaluated the role of Protein arginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewal and pluripotency given its critical role in safeguarding mouse ESC pluripotency. Unlike the mouse, we discovered that PRMT5 has no role in hESC pluripotency. Using microarray analysis we discovered that a significant depletion in PRMT5 RNA and protein from hESCs changed the expression of only 78 genes, with the majority being repressed. Functionally, we discovered that depletion of PRMT5 had no effect on expression of OCT4, NANOG or SOX2, and did not prevent teratoma formation. Instead, we show that PRMT5 functions in hESCs to regulate proliferation in the self-renewing state by regulating the fraction of cells in Gap 1 (G1) of the cell cycle and increasing expression of the G1 cell cycle inhibitor P57. Taken together our data unveils a distinct role for PRMT5 in hESCs and identifies P57 as new target.
MeSH term(s)	Animals ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p57/genetics ; Cyclin-Dependent Kinase Inhibitor p57/metabolism ; Embryonic Stem Cells/physiology ; Embryonic Stem Cells/transplantation ; G1 Phase Cell Cycle Checkpoints ; Gene Expression ; Humans ; Mice ; Mice, SCID ; Protein-Arginine N-Methyltransferases/physiology ; Teratoma/pathology
Chemical Substances	CDKN1C protein, human ; Cyclin-Dependent Kinase Inhibitor p57 ; PRMT5 protein, human (EC 2.1.1.319) ; Protein-Arginine N-Methyltransferases (EC 2.1.1.319)
Language	English
Publishing date	2014-01-30
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2495577-2
ISSN	2629-3277 ; 1558-6804 ; 1550-8943
ISSN (online)	2629-3277 ; 1558-6804
ISSN	1550-8943
DOI	10.1007/s12015-013-9490-z
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Article ; Online: An In Vivo CRISPR Screen Identifies Stepwise Genetic Dependencies of Metastatic Progression.

Cancer research

2021 Volume 82, Issue 4, Page(s) 681–694

Abstract: Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes ...

Abstract	Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches. Significance: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.
MeSH term(s)	Animals ; Biomarkers, Tumor/genetics ; Biomarkers, Tumor/metabolism ; Breast Neoplasms/blood ; Breast Neoplasms/genetics ; Breast Neoplasms/pathology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics ; Cell Cycle Proteins/metabolism ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Neoplasm Metastasis ; Neoplastic Cells, Circulating/metabolism ; Neoplastic Cells, Circulating/pathology ; Protein Serine-Threonine Kinases/genetics ; Protein Serine-Threonine Kinases/metabolism ; Proto-Oncogene Proteins/genetics ; Proto-Oncogene Proteins/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA-Seq/methods ; Survival Analysis ; Xenograft Model Antitumor Assays/methods ; Polo-Like Kinase 1 ; Mice
Chemical Substances	Biomarkers, Tumor ; Cell Cycle Proteins ; Proto-Oncogene Proteins ; RNA, Guide, CRISPR-Cas Systems ; Protein Serine-Threonine Kinases (EC 2.7.11.1)
Language	English
Publishing date	2021-12-13
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1432-1
ISSN	1538-7445 ; 0008-5472
ISSN (online)	1538-7445
ISSN	0008-5472
DOI	10.1158/0008-5472.CAN-21-3908
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