Microfluidic Isolation of Circulating Tumor Cells and Cancer Stem-Like Cells from Patients with Pancreatic Ductal Adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDAC) requires multimodal therapeutic approaches and disease monitoring for effective treatment. Liquid biopsy biomarkers, including circulating tumor cells (CTCs) and cancer stem-like cells (CSCs), hold promise for evaluating treatment response promptly and guiding therapeutic modifications. Methods: From 24 patients with metastatic PDAC (stage IV, M1) undergoing active systemic treatment, we collected 78 blood samples at different time points for CTC and CSC isolation using a microfluidic platform functionalized with antibodies against a CTC biomarker, epithelial cell adhesion molecule (EpCAM), or a CSC biomarker, CD133. These isolated cells were further verified, via fluorescent staining and imaging, using cytokeratin (CK), CD45, and nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI). Results: The majority (84.4%) of patient blood samples were positive for CTCs (EpCAM+CK+CD45-DAPI+) and 70.8% of patient blood samples were positive for CSCs (CD133+CK+CD45-DAPI+), using the highest baseline value of healthy samples as threshold. The CTC subtypes (EpCAM+CK+CD45-DAPI+CD133+ and EpCAM+CK+CD45-DAPI+CD133-) and CSC subtypes (CD133+CK+CD45-DAPI+EpCAM+ and CD133+CK+CD45-DAPI+EpCAM-) were also analyzed using immunochemical methods. In several cases, CSCs exhibited cytokeratin expression that did not express EpCAM, indicating that they will not be detected using EpCAM-based isolation. Conclusion: The microfluidic platform enabled the reliable isolation of CTCs and CSCs from PDAC patient samples, as well as their subtypes. Complementary assessment of both CTCs and CSCs appears advantageous to assess the profile of tumor progressing in some cases. This research has important implications for the application and interpretation of approved methods to detect CTCs.

[1]  R. Matkowski,et al.  Circulating tumor cells in localized prostate cancer: isolation, cultivation in vitro and relationship to T-stage and Gleason score. , 2014, Anticancer research.

[2]  G. Parmiani,et al.  Immunology of cancer stem cells in solid tumours. A review. , 2014, European journal of cancer.

[3]  前田 真一.  CD133 expression is correlated with lymph node metastasis and vascular endothelial growth factor-C expression in pancreatic cancer , 2008 .

[4]  A. Puisieux,et al.  Generation of Breast Cancer Stem Cells through Epithelial-Mesenchymal Transition , 2008, PloS one.

[5]  R. Henkelman,et al.  Identification of human brain tumour initiating cells , 2004, Nature.

[6]  Laurie E Ailles,et al.  Cancer stem cells in solid tumors. , 2007, Current opinion in biotechnology.

[7]  J. Huober,et al.  Distinct expression of cytokeratin, N-cadherin and CD133 in circulating tumor cells of metastatic breast cancer patients. , 2014, Future oncology.

[8]  K. Pienta,et al.  Cancer stem cells and their role in metastasis. , 2013, Pharmacology & therapeutics.

[9]  M Van Glabbeke,et al.  New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. , 2000, Journal of the National Cancer Institute.

[10]  N. Maitland,et al.  Prospective identification of tumorigenic prostate cancer stem cells. , 2005, Cancer research.

[11]  Shunqiang Wang,et al.  Highly efficient and selective isolation of rare tumor cells using a microfluidic chip with wavy-herringbone micro-patterned surfaces. , 2016, The Analyst.

[12]  W. ElShamy,et al.  Overview: cellular plasticity, cancer stem cells and metastasis. , 2013, Cancer letters.

[13]  Rajan P Kulkarni,et al.  Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology , 2016, Oncotarget.

[14]  Justin C. Grindley,et al.  Tumour-initiating cells: challenges and opportunities for anticancer drug discovery , 2009, Nature Reviews Drug Discovery.

[15]  T. Fehm,et al.  Prognostic Relevance of Circulating Tumor Cells in Molecular Subtypes of Breast Cancer. , 2015, Geburtshilfe und Frauenheilkunde.

[16]  K. Shao,et al.  Progress and challenges of sequencing and analyzing circulating tumor cells , 2017, Cell Biology and Toxicology.

[17]  K. Pantel,et al.  Challenges in circulating tumour cell research , 2014, Nature Reviews Cancer.

[18]  Massimo Cristofanilli,et al.  Considerations in the development of circulating tumor cell technology for clinical use , 2012, Journal of Translational Medicine.

[19]  Joonhee Kim,et al.  Potential mechanisms of CD133 in cancer stem cells , 2017, Life sciences.

[20]  Zongguang Zhou,et al.  Prognostic Value of Cancer Stem Cell Marker CD133 Expression in Gastric Cancer: A Systematic Review , 2013, PloS one.

[21]  Richard J. Jones,et al.  Concise Review: Cancer Stem Cells and Minimal Residual Disease , 2012, Stem cells.

[22]  S. Pandol,et al.  The Potential for Circulating Tumor Cells in Pancreatic Cancer Management , 2017, Front. Physiol..

[23]  D. Roberts,et al.  CD133 Expression Defines a Tumor Initiating Cell Population in Primary Human Ovarian Cancer , 2009, Stem cells.

[24]  M. Beuran,et al.  The epithelial to mesenchymal transition in pancreatic cancer: A systematic review. , 2015, Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.].

[25]  G. Viglietto,et al.  The role of CD133 in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer. , 2009, European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery.

[26]  Robert E. Lewis,et al.  Circulating tumor cells (CTCs) from metastatic breast cancer patients linked to decreased immune function and response to treatment. , 2013, Experimental and molecular pathology.

[27]  S. Capitani,et al.  Up-modulation of PLC-β2 reduces the number and malignancy of triple-negative breast tumor cells with a CD133+/EpCAM+ phenotype: a promising target for preventing progression of TNBC , 2017, BMC Cancer.

[28]  M. Clarke,et al.  Identification of pancreatic cancer stem cells. , 2007, Cancer research.

[29]  Mehmet Toner,et al.  Inertial Focusing for Tumor Antigen–Dependent and –Independent Sorting of Rare Circulating Tumor Cells , 2013, Science Translational Medicine.

[30]  Douglas B. Evans,et al.  ALDH Activity Selectively Defines an Enhanced Tumor-Initiating Cell Population Relative to CD133 Expression in Human Pancreatic Adenocarcinoma , 2011, PloS one.

[31]  M. Margelí,et al.  Detection of disseminated tumor cells in locally advanced breast cancer patients before primary systemic therapy. , 2013, Breast.

[32]  C. Heeschen,et al.  Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. , 2007, Cell stem cell.

[33]  Thomas J George,et al.  Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip. , 2014, Lab on a chip.

[34]  Wenjun Guo,et al.  The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells , 2008, Cell.

[35]  S. Kelley,et al.  Nanoparticle-mediated binning and profiling of heterogeneous circulating tumor cell subpopulations. , 2015, Angewandte Chemie.

[36]  K. Isselbacher,et al.  Isolation of circulating tumor cells using a microvortex-generating herringbone-chip , 2010, Proceedings of the National Academy of Sciences.

[37]  W. Huttner,et al.  Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[38]  S. Natsugoe,et al.  Establishment of a highly migratory subclone reveals that CD133 contributes to migration and invasion through epithelial–mesenchymal transition in pancreatic cancer , 2011, Human Cell.

[39]  N. Navin.  Cancer genomics: one cell at a time , 2014, Genome Biology.

[40]  T. Price,et al.  Circulating tumour cells: the evolving concept and the inadequacy of their enrichment by EpCAM-based methodology for basic and clinical cancer research. , 2014, Annals of oncology : official journal of the European Society for Medical Oncology.

[41]  H. Tseng,et al.  Improving pancreatic cancer diagnosis using circulating tumor cells: prospects for staging and single-cell analysis , 2015, Expert review of molecular diagnostics.

[42]  C. Perou,et al.  Discrete microfluidics for the isolation of circulating tumor cell subpopulations targeting fibroblast activation protein alpha and epithelial cell adhesion molecule , 2017, npj Precision Oncology.

[43]  S. Digumarthy,et al.  Isolation of rare circulating tumour cells in cancer patients by microchip technology , 2007, Nature.

[44]  M. van Glabbeke,et al.  New guidelines to evaluate the response to treatment in solid tumors , 2000, Journal of the National Cancer Institute.

[45]  B. Monk,et al.  Circulating tumor cells predict progression free survival and overall survival in patients with relapsed/recurrent advanced ovarian cancer. , 2011, Gynecologic oncology.

[46]  Yutaka Naito,et al.  Expression of cancer stem cell markers ALDH1, CD44 and CD133 in primary tumor and lymph node metastasis of gastric cancer , 2012, Pathology international.

[47]  L. Ricci-Vitiani,et al.  Colon cancer stem cells , 2007, Gut.

[48]  Peter Dirks,et al.  Cancer stem cells: an evolving concept , 2012, Nature Reviews Cancer.

[49]  Jason P. Gleghorn,et al.  Capture of circulating tumor cells from whole blood of prostate cancer patients using geometrically enhanced differential immunocapture (GEDI) and a prostate-specific antibody. , 2010, Lab on a chip.

[50]  P. Marcato,et al.  Targeting cancer-initiating cells with oncolytic viruses. , 2009, Molecular therapy : the journal of the American Society of Gene Therapy.

[51]  Alexander Stojadinovic,et al.  Current Approaches and Challenges in Monitoring Treatment Responses in Breast Cancer , 2014, Journal of Cancer.

[52]  C. Lim,et al.  Isolation and retrieval of circulating tumor cells using centrifugal forces , 2013, Scientific Reports.

[53]  Paul A. Wiggins,et al.  Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state , 2011, Proceedings of the National Academy of Sciences.

[54]  Lie Yao,et al.  Circulating tumor cells in pancreatic cancer patients: efficacy in diagnosis and value in prognosis. , 2016, Discover medicine.

[55]  K. Pantel,et al.  Real-time liquid biopsy: circulating tumor cells versus circulating tumor DNA. , 2013, Annals of translational medicine.

[56]  D. Heymann,et al.  Circulating Tumor Cells: A Review of Non-EpCAM-Based Approaches for Cell Enrichment and Isolation. , 2016, Clinical chemistry.

[57]  Pu Xia,et al.  Prognostic value of circulating CD133+ cells in patients with gastric cancer , 2015, Cell proliferation.

[58]  I. Malanchi,et al.  Neutrophils support lung colonization of metastasis-initiating breast cancer cells , 2015, Nature.

[59]  M. Okada,et al.  [New response evaluation criteria in solid tumours-revised RECIST guideline (version 1.1)]. , 2009, Gan to kagaku ryoho. Cancer & chemotherapy.

[60]  Kangfu Chen,et al.  A Novel Microfluidic Device for Isolation of Circulating Tumor Cells from Pancreatic Cancer Blood Samples. , 2017, Methods in molecular biology.

[61]  Alison Stopeck,et al.  Circulating tumor cells, disease progression, and survival in metastatic breast cancer. , 2004, The New England journal of medicine.

[62]  Shicai Chen,et al.  CD133 Expression and the Prognosis of Colorectal Cancer: A Systematic Review and Meta-Analysis , 2013, PloS one.