In Vivo Flow Cytometry of Circulating Tumor-Associated Exosomes

Circulating tumor cells (CTCs) demonstrated the potential as prognostic markers of metastatic development. However, the incurable metastasis can already be developed at the time of initial diagnosis with the existing CTC assays. Alternatively, tumor-associated particles (CTPs) including exosomes can be a more valuable prognostic marker because they can be released from the primary tumor long before CTCs and in larger amount. However, little progress has been made in high sensitivity detection of CTPs, especially in vivo. We show here that in vivo integrated photoacoustic (PA) and fluorescence flow cytometry (PAFFC) platform can provide the detection of melanoma and breast-cancer-associated single CTPs with endogenously expressed melanin and genetically engineered proteins or exogenous dyes as PA and fluorescent contrast agents. The two-beam, time-of-light PAFFC can measure the sizes of CTCs and CTPs and identify bulk and rolling CTCs and CTC clusters, with no influence on blood flow instability. This technique revealed a higher concentration of CTPs than CTCs at an early cancer stage. Because a single tumor cell can release many CTPs and in vivo PAFFC can examine the whole blood volume, PAFFC diagnostic platform has the potential to dramatically improve (up to 105-fold) the sensitivity of cancer diagnosis.

[1]  Paula D. Bos,et al.  Metastasis: from dissemination to organ-specific colonization , 2009, Nature Reviews Cancer.

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

[3]  B. Ferry,et al.  Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay. , 2011, Thrombosis research.

[4]  Sridhar Ramaswamy,et al.  Circulating Breast Tumor Cells Exhibit Dynamic Changes in Epithelial and Mesenchymal Composition , 2013, Science.

[5]  Andrew F. Hill,et al.  Exosomes: Vehicles for the Transfer of Toxic Proteins Associated with Neurodegenerative Diseases? , 2012, Front. Physio..

[6]  Laurence Zitvogel,et al.  Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming , 2001, Nature Medicine.

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

[8]  Mustafa Sarimollaoglu,et al.  Synergy of photoacoustic and fluorescence flow cytometry of circulating cells with negative and positive contrasts , 2013, Journal of biophotonics.

[9]  V. Zharov,et al.  In vivo photoacoustic time-of-flight velocity measurement of single cells and nanoparticles. , 2011, Optics letters.

[10]  Attila Tárnok,et al.  In vivo flow cytometry: A horizon of opportunities , 2011, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[11]  S. Wickline,et al.  Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis. , 2011, Cancer research.

[12]  Nada Jabado,et al.  New technologies for the detection of circulating tumour cells. , 2010, British medical bulletin.

[13]  Vladimir P Zharov,et al.  In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser. , 2009, Cancer research.

[14]  Cesar M. Castro,et al.  Magnetic nanosensor for detection and profiling of erythrocyte-derived microvesicles. , 2013, ACS nano.

[15]  Thomas Kelly,et al.  In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells. , 2009, Nature nanotechnology.

[16]  F. Nicolantonio,et al.  Liquid biopsy: monitoring cancer-genetics in the blood , 2013, Nature Reviews Clinical Oncology.

[17]  V. Zharov,et al.  In vivo photoswitchable flow cytometry for direct tracking of single circulating tumor cells. , 2014, Chemistry & biology.

[18]  J. Rak,et al.  Extracellular Vesicles – Biomarkers and Effectors of the Cellular Interactome in Cancer , 2013, Front. Pharmacol..

[19]  Claudia Mongini,et al.  Emerging Roles of Exosomes in Normal and Pathological Conditions: New Insights for Diagnosis and Therapeutic Applications , 2015, Front. Immunol..

[20]  Youli Zu,et al.  Detecting Circulating Tumor Cells: Current Challenges and New Trends , 2013, Theranostics.

[21]  Mustafa Sarimollaoglu,et al.  Photoacoustic and photothermal detection of circulating tumor cells, bacteria and nanoparticles in cerebrospinal fluid in vivo and ex vivo , 2013, Journal of biophotonics.

[22]  I. Fidler,et al.  AACR centennial series: the biology of cancer metastasis: historical perspective. , 2010, Cancer research.

[23]  Mustafa Sarimollaoglu,et al.  In vivo flow cytometry of circulating clots using negative photothermal and photoacoustic contrasts , 2011, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[24]  C. Théry,et al.  Membrane vesicles as conveyors of immune responses , 2009, Nature Reviews Immunology.

[25]  J. Rak,et al.  Extracellular vesicles as prospective carriers of oncogenic protein signatures in adult and paediatric brain tumours , 2013, Proteomics.

[26]  Gema Moreno-Bueno,et al.  Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET , 2012, Nature Medicine.

[27]  G. Camussi,et al.  Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche. , 2011, Cancer research.

[28]  F. Reyal,et al.  Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression. , 2012, Cancer research.

[29]  Cesar M. Castro,et al.  Nano-plasmonic exosome diagnostics , 2015, Expert review of molecular diagnostics.

[30]  Hakho Lee,et al.  Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor , 2014, Nature Biotechnology.

[31]  Hongtao Zhang,et al.  Non-invasive, ultra-sensitive, high-throughput assays to quantify rare biomarkers in the blood. , 2008, Methods.

[32]  Vladimir P Zharov,et al.  Photoacoustic flow cytometry. , 2012, Methods.

[33]  E. Lianidou.  Circulating tumor cells--new challenges ahead. , 2012, Clinical chemistry.

[34]  I. Fidler,et al.  The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited , 2003, Nature Reviews Cancer.

[35]  I. Bauerfeind,et al.  Circulating microparticles in breast cancer patients: a comparative analysis with established biomarkers. , 2008, Anticancer research.

[36]  John W. Park,et al.  Circulating Tumor Cells , 2017, Methods in Molecular Biology.

[37]  Jacob G. Scott,et al.  Unifying metastasis — integrating intravasation, circulation and end-organ colonization , 2012, Nature Reviews Cancer.

[38]  Graça Raposo,et al.  Extracellular vesicles: Exosomes, microvesicles, and friends , 2013, The Journal of cell biology.

[39]  Yuki Takahashi,et al.  Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection. , 2013, Journal of biotechnology.

[40]  Larry Norton,et al.  Tumor Self-Seeding by Circulating Cancer Cells , 2009, Cell.

[41]  M. Kesimer,et al.  Nanoparticle analysis of circulating cell-derived vesicles in ovarian cancer patients. , 2012, Analytical biochemistry.

[42]  Klaus Pantel,et al.  [The circulating tumor cells: liquid biopsy of cancer]. , 2014, Klinicheskaia laboratornaia diagnostika.