Functional Characterization of Circulating Tumor Cells with a Prostate-Cancer-Specific Microfluidic Device

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.

[1]  Tanja Fehm,et al.  Circulating Tumor Cells in Patients with Breast Cancer Dormancy , 2004, Clinical Cancer Research.

[2]  G. Jenster,et al.  A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. , 1990, Biochemical and biophysical research communications.

[3]  J. Machiels,et al.  Prednisone plus cabazitaxel or mitoxantrone for metastatic castration-resistant prostate cancer progressing after docetaxel treatment: a randomised open-label trial , 2010, The Lancet.

[4]  H. Ananias,et al.  Expression of the gastrin‐releasing peptide receptor, the prostate stem cell antigen and the prostate‐specific membrane antigen in lymph node and bone metastases of prostate cancer , 2009, The Prostate.

[5]  J. Cuzick,et al.  Complex patterns of ETS gene alteration arise during cancer development in the human prostate , 2008, Oncogene.

[6]  J. Bono,et al.  All circulating EpCAM+CK+CD45- objects predict overall survival in castration-resistant prostate cancer. , 2010, Annals of oncology : official journal of the European Society for Medical Oncology.

[7]  R. Weinberg,et al.  Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits , 2009, Nature Reviews Cancer.

[8]  W. Fair,et al.  Expression of the prostate-specific membrane antigen. , 1994, Cancer research.

[9]  N. Bander,et al.  Targeted systemic therapy of prostate cancer with a monoclonal antibody to prostate-specific membrane antigen. , 2003, Seminars in oncology.

[10]  Howard I. Scher,et al.  Circulating Tumor Cells as Biomarkers in Prostate Cancer , 2011, Clinical Cancer Research.

[11]  A. Weiss,et al.  Detection and characterization of carcinoma cells in the blood. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Baselga,et al.  The Evolving War on Cancer , 2011, Cell.

[13]  C Hollmann,et al.  DETECTION OF DISSEMINATED TUMOR CELLS IN PERIPHERAL BLOOD , 2005, Critical reviews in clinical laboratory sciences.

[14]  Mehmet Toner,et al.  Isolation and Characterization of Circulating Tumor Cells from Patients with Localized and Metastatic Prostate Cancer , 2010, Science Translational Medicine.

[15]  B. Vincenzi,et al.  Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal , 2011, Journal of cellular and molecular medicine.

[16]  G. Murphy,et al.  Monoclonal antibodies to a new antigenic marker in epithelial prostatic cells and serum of prostatic cancer patients. , 1987, Anticancer research.

[17]  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.

[18]  Sabine Riethdorf,et al.  Advancing personalized cancer therapy by detection and characterization of circulating carcinoma cells , 2010, Annals of the New York Academy of Sciences.

[19]  N. Bander,et al.  Taxane-induced blockade to nuclear accumulation of the androgen receptor predicts clinical responses in metastatic prostate cancer. , 2011, Cancer research.

[20]  M. Marty,et al.  Phase I and pharmacokinetic study of Taxotere (RP 56976; NSC 628503) given as a short intravenous infusion. , 1993, Cancer research.

[21]  Gerhardt Attard,et al.  Utilizing circulating tumor cells: challenges and pitfalls. , 2011, Current opinion in genetics & development.

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

[23]  R. Bast,et al.  American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. , 2007, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[24]  A. Deblasio,et al.  Sensitive nested reverse transcription polymerase chain reaction detection of circulating prostatic tumor cells: comparison of prostate-specific membrane antigen and prostate-specific antigen-based assays. , 1994, Cancer research.

[25]  Mehmet Toner,et al.  Circulating tumor cells: approaches to isolation and characterization , 2011, The Journal of cell biology.

[26]  D. Dearnaley,et al.  Hormone-sensitive prostate cancer: a case of ETS gene fusion heterogeneity , 2008, Journal of Clinical Pathology.

[27]  V. Reuter,et al.  Five different anti-prostate-specific membrane antigen (PSMA) antibodies confirm PSMA expression in tumor-associated neovasculature. , 1999, Cancer research.

[28]  P. Schellhammer,et al.  Upregulation of prostate-specific membrane antigen after androgen-deprivation therapy. , 1996, Urology.

[29]  Michael Morse,et al.  Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. , 2008, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[30]  Max S Wicha,et al.  Circulating tumor cells: not all detected cells are bad and not all bad cells are detected. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[31]  Glenn Heller,et al.  Circulating Tumor Cell Number and Prognosis in Progressive Castration-Resistant Prostate Cancer , 2007, Clinical Cancer Research.

[32]  Peter Kuhn,et al.  A rare-cell detector for cancer. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[33]  S. Tagawa,et al.  Prostate-Specific Membrane Antigen-Based Therapeutics , 2011, Advances in urology.

[34]  C. Tangen,et al.  Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. , 2004, The New England journal of medicine.

[35]  P. Albers,et al.  Randomized, open-label phase III trial of docetaxel plus high-dose calcitriol versus docetaxel plus prednisone for patients with castration-resistant prostate cancer. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[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]  Ximing J. Yang,et al.  Prostate-specific membrane antigen expression in the neovasculature of gastric and colorectal cancers. , 2009, Human pathology.

[38]  S. Mani,et al.  Validation of the pharmacodynamics of BMS-247550, an analogue of epothilone B, during a phase I clinical study. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[39]  K. Pienta,et al.  Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic Castration-Resistant Prostate Cancer , 2008, Clinical Cancer Research.

[40]  J. Tchinda,et al.  Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. , 2006, Science.

[41]  D. Haber,et al.  Circulating tumor cells: a window into cancer biology and metastasis. , 2010, Current opinion in genetics & development.

[42]  D. Bostwick,et al.  Prostate-specific membrane antigen expression is greatest in prostate adenocarcinoma and lymph node metastases. , 1998, Urology.

[43]  Markus Munz,et al.  The emerging role of EpCAM in cancer and stem cell signaling. , 2009, Cancer research.

[44]  Paraskevi Giannakakou,et al.  Mitotic Kinesin Inhibitors Induce Mitotic Arrest and Cell Death in Taxol-resistant and -sensitive Cancer Cells* , 2005, Journal of Biological Chemistry.

[45]  I. Tannock,et al.  Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. , 2004, The New England journal of medicine.

[46]  S. Krobitsch,et al.  Effects of EpCAM overexpression on human breast cancer cell lines , 2011, BMC Cancer.

[47]  C. Cordon-Cardo,et al.  Prostate-specific membrane antigen expression in normal and malignant human tissues. , 1997, Clinical cancer research : an official journal of the American Association for Cancer Research.

[48]  Xiaoyu Jia,et al.  Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data. , 2009, The Lancet. Oncology.

[49]  Ruud H. Brakenhoff,et al.  Detection, clinical relevance and specific biological properties of disseminating tumour cells , 2008, Nature Reviews Cancer.

[50]  M. Jordan,et al.  Microtubules as a target for anticancer drugs , 2004, Nature Reviews Cancer.

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

[52]  S. Varambally,et al.  Antibody-based detection of ERG rearrangement-positive prostate cancer. , 2010, Neoplasia.

[53]  T. Fojo,et al.  Mitosis is not a key target of microtubule agents in patient tumors , 2011, Nature Reviews Clinical Oncology.

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