Isolation and Characterization of Tumor Cells from the Ascites of Ovarian Cancer Patients: Molecular Phenotype of Chemoresistant Ovarian Tumors

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Abstract

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12–14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.

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Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer.

Sharmila Bapat, Avinash Mali, Chaitanyananda Koppikar … (2005)

The cellular mechanisms underlying the increasing aggressiveness associated with ovarian cancer progression are poorly understood. Coupled with a lack of identification of specific markers that could aid early diagnoses, the disease becomes a major cause of cancer-related mortality in women. Here we present direct evidence that the aggressiveness of human ovarian cancer may be a result of transformation and dysfunction of stem cells in the ovary. A single tumorigenic clone was isolated among a mixed population of cells derived from the ascites of a patient with advanced ovarian cancer. During the course of the study, yet another clone underwent spontaneous transformation in culture, providing a model of disease progression. Both the transformed clones possess stem cell-like characteristics and differentiate to grow in an anchorage-independent manner in vitro as spheroids, although further maturation and tissue-specific differentiation was arrested. Significantly, tumors established from these clones in animal models are similar to those in the human disease in their histopathology and cell architecture. Furthermore, the tumorigenic clones, even on serial transplantation continue to establish tumors, thereby confirming their identity as tumor stem cells. These findings suggest that: (a) stem cell transformation can be the underlying cause of ovarian cancer and (b) continuing stochastic events of stem and progenitor cell transformation define the increasing aggression that is characteristically associated with the disease.

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Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway.

Zhiwei Wang, Yiwei Li, Dejuan Kong … (2009)

Despite rapid advances in many fronts, pancreatic cancer (PC) remains one of the most difficult human malignancies to treat due, in part, to de novo and acquired chemoresistance and radioresistance. Gemcitabine alone or in combination with other conventional therapeutics is the standard of care for the treatment of advanced PC without any significant improvement in the overall survival of patients diagnosed with this deadly disease. Previous studies have shown that PC cells that are gemcitabine-resistant (GR) acquired epithelial-mesenchymal transition (EMT) phenotype, which is reminiscent of "cancer stem-like cells"; however, the molecular mechanism that led to EMT phenotype has not been fully investigated. The present study shows that Notch-2 and its ligand, Jagged-1, are highly up-regulated in GR cells, which is consistent with the role of the Notch signaling pathway in the acquisition of EMT and cancer stem-like cell phenotype. We also found that the down-regulation of Notch signaling was associated with decreased invasive behavior of GR cells. Moreover, down-regulation of Notch signaling by siRNA approach led to partial reversal of the EMT phenotype, resulting in the mesenchymal-epithelial transition, which was associated with decreased expression of vimentin, ZEB1, Slug, Snail, and nuclear factor-kappaB. These results provide molecular evidence showing that the activation of Notch signaling is mechanistically linked with chemoresistance phenotype (EMT phenotype) of PC cells, suggesting that the inactivation of Notch signaling by novel strategies could be a potential targeted therapeutic approach for overcoming chemoresistance toward the prevention of tumor progression and/or treatment of metastatic PC.

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Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells.

Toni Celià-Terrassa, Óscar Meca-Cortés, Francesca Mateo … (2012)

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs.

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Author and article information

Contributors

Shannon M. Hawkins: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2012

Publication date (Electronic): 8 October 2012

Volume: 7

Issue: 10

Electronic Location Identifier: e46858

Affiliations

[1 ]Women’s Cancer Research Centre, Royal Women’s Hospital, Victoria, Australia

[2 ]Department of Surgery, University of Melbourne, St Vincent Hospital, Victoria, Australia

[3 ]Department of Surgery, University of Melbourne, Royal Melbourne Hospital, Victoria, Australia

[4 ]Prince Henry’s Institute for Medical Research, Victoria, Australia

[5 ]Department of Anatomical Pathology, Royal Women’s Hospital, Victoria, Australia

[6 ]St Vincent Institute, Victoria, Australia

[7 ]Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia

Baylor College of Medicine, United States of America

Author notes

* E-mail: Nuzhat.Ahmed@ 123456thewomens.org.au

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: AL NA. Performed the experiments: AL RL MB. Analyzed the data: AL MB SN KS JP JKF NA. Contributed reagents/materials/analysis tools: HZ MAQ. Wrote the paper: AL NA. Edited the manuscript: AL MB KS EWT MAQ JKF NA.

Article

Publisher ID: PONE-D-12-20118

DOI: 10.1371/journal.pone.0046858

PMC ID: 3466197

PubMed ID: 23056490

SO-VID: f74ea374-1a43-484a-903e-1811b2382328

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 11 July 2012

Date accepted : 10 September 2012

Page count

Pages: 20

Funding

This was supported by Women’s Cancer Foundation, National Health and Medical Research Council of Australia (JKF, RegKey#441101), the National Breast Cancer Foundation (EWT; EMPathy Breast Cancer Network, Australia) and the Victorian Government’s Operational Infrastructure Support Program (Australia). The funders had no role in study desugn, data collection and analysis, decision to publish or preparation of the manuscript.

Isolation and Characterization of Tumor Cells from the Ascites of Ovarian Cancer Patients: Molecular Phenotype of Chemoresistant Ovarian Tumors

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Most cited references 32

Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer.

Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway.

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells.

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