Clinical Applications Of NanoVelcro Rare-Cell Assays For .

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Theranostics 2016, Vol. 6, Issue 9IvyspringInternational Publisher1425Theranostics2016; 6(9): 1425-1439. doi: 10.7150/thno.15359ReviewClinical Applications of NanoVelcro Rare-Cell Assaysfor Detection and Characterization of CirculatingTumor CellsJie-Fu Chen1†, Yazhen Zhu2,3†, Yi-Tsung Lu1†, Elisabeth Hodara1, Shuang Hou3, Vatche G. Agopian4,5,James S. Tomlinson4,6,7, Edwin M. Posadas1 , Hsian-Rong Tseng2 1.2.3.4.5.6.7.†Urologic Oncology Program and Uro-Oncology Research Laboratories, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, LosAngeles, California, USA;Department of Molecular and Medical Pharmacology, California NanoSystems Institute, Crump Institute for Molecular Imaging, University of California,Los Angeles, Los Angeles, California, USA;Department of Pathology, Guangdong Provincial Hospital of TCM, Guangzhou University of Chinese Medicine, Guangzhou, China.Department of Surgery, University of California, Los Angeles, Los Angeles, California, USA;Liver Transplantation and Hepatobiliary Surgery, University of California, Los Angeles, Los Angeles, California, USA;Center for Pancreatic Disease, University of California, Los Angeles, Los Angeles, California, USA;Department of Surgery Greater Los Angeles Veteran’s Affairs Administration, Los Angeles, California, USA.These authors contribute equally to this work. Corresponding authors: Dr. Hsian-Rong Tseng and Edwin M. Posadas. Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Seehttp://ivyspring.com/terms for terms and conditions.Received: 2016.02.23; Accepted: 2016.05.06; Published: 2016.06.15AbstractLiquid biopsy of tumor through isolation of circulating tumor cells (CTCs) allows non-invasive,repetitive, and systemic sampling of disease. Although detecting and enumerating CTCs is ofprognostic significance in metastatic cancer, it is conceivable that performing molecular andfunctional characterization on CTCs will reveal unprecedented insight into the pathogenicmechanisms driving lethal disease. Nanomaterial-embedded cancer diagnostic platforms, i.e.,NanoVelcro CTC Assays represent a unique rare-cell sorting method that enables detectionisolation, and characterization of CTCs in peripheral blood, providing an opportunity tononinvasively monitor disease progression in individual cancer patients. Over the past decade, aseries of NanoVelcro CTC Assays has been demonstrated for exploring the full potential of CTCsas a clinical biomarker, including CTC enumeration, phenotyping, genotyping and expressionprofiling. In this review article, the authors will briefly introduce the development of threegenerations of NanoVelcro CTC Assays, and highlight the clinical applications of each generationfor various types of solid cancers, including prostate cancer, pancreatic cancer, lung cancer, andmelanoma.Key words: Circulating tumor cellCirculating tumor cell (CTC)Pathologic evaluation remains the gold standardfor diagnosis and prognosis in the care of cancerpatients. This approach typically relies on the tissuespecimens obtained by surgical excision orradiographically guided biopsy. While tremendousamounts of information can be obtained from tissues,including histopathology and molecular signatures,this approach has several disadvantages. First, theprocedures to obtain tissues are both invasive andcostly. The risk of morbidity and psychological stresson the patients largely limit the feasibility of invasiveprocedures. Moreover, it has been technicallychallenging to biopsy lesions of certain cancer types orat certain locations, for instance, the osteoblasticmetastasis in prostate cancer. Finally, recent studieshttp://www.thno.org

Theranostics 2016, Vol. 6, Issue 9showing temporospatial heterogeneity [1-7] within atumor raise serious concerns about how accurately asingle biopsy represents a cancer that is spatiallyheterogeneous and evolves over time.As an alternative to solid tumor biopsy, manypropose the use of a “liquid biopsy” based oncirculating tumor cell (CTC) sampling and are activelydeveloping CTC capture techniques.[8] CTCs are raretumor cells shed from all present disease sites thathave active blood perfusion, including primary andmetastatic tumors. Sampled through phlebotomy,CTCs can be obtained easily throughout the course ofcancer; even during the late stages of metastaticdisease without needing invasive and complextraditional biopsy procedures. The ability of serialCTC sampling performed over the course of diseaseoffers the opportunity for real-time, dynamicmonitoring of the disease evolution.[9, 10] Over thepast decade, collaborative and interdisciplinaryresearch groups including chemistry, material science,bioengineering, cancer biology, and oncology havebeen formed to focus their efforts upon CTCdetection, isolation, and characterization.[11] Thesecollaborative scientific endeavors have led to manyimportant studies setting the foundation for therealization of CTCs to function as a liquid biopsy.Initial studies focused on enumeration [12-14] whilerecently, some groups have begun to showgenomic[15-17] and transcriptomic[18, 19] similaritiesbetween CTCs and the traditional tumors biopsies.More and more evidence is supporting the use ofCTCs for investigating the nature of cancer, guidingtherapeutic interventions, and assessing emergingresistance.Conventional CTC assaysThe most widely used CTC detection assaysinclude: (i) Immunomagnetic separation: thesemethods utilize capture agent-labeled magnetic beadsto either positively select [13, 20, 21] CTCs targetingtheir surface markers (e.g., epithelial cell adhesionmolecule [EpCAM]) or negatively deplete [22, 23]white blood cells (WBCs) using anti-CD45. TheCellSearchTM Assay [12-14] is the only FDA-clearedCTC diagnostic technology for metastatic breast,prostate, and colorectal cancers. This assay harvestsCTCs with anti-EpCAM-coated magnetic beads, andthe subsequent immunocytochemistry (ICC) processhelps to identify CTCs (DAPI /cytokeratin,CK /CD45-) from nonspecifically captured WBCs(DAPI /CK-/CD45 ). Recently, several new systems(e.g., MagSweeper[24], IsoFlux[25], naGen/Qiagen[29]) have been developed tofurther improve detection speed and efficiency. (ii)1426Flow cytometry: In conjunction with the use offluorescent markers, flow cytometry [30, 31] is one ofthe most mature technologies for analyzing andsorting subpopulations of cells. However, thisflow-based methodology often has limited detectionpower due to the low abundance of CTCs, and isunable to provide the CTCs’ morphologicalinformation. An improved method, known asensemble-decision aliquot ranking (eDAR),[32, 33]was developed to address this weakness. (iii)Microscopy imaging: Microscopy imaging [34-36] ofICC-treated blood samples allows for highly sensitivedetection of CTCs, accompanied with theirmorphometric characteristics and protein expression.Currently, Epic Sciences is one of the leaders in thecommercial sector, now providing Clinical oratory tests for both CTC enumeration andcharacterization. In contrast to the previous threeapproaches, which require the use of CTC markers,the following two approaches are recognized aslabel-free methods. (iv) CTC filters: Filter-basedapproaches [37-41] have been established to trapCTCs according to their sizes. A wide collection ofcommercial kits/systems from Clearbridge,[39, 40]Rarecells,[42] ScreenCell,[43] and Creatv MicroTechetc. are now available to support research utility.Nevertheless, concerns regarding trophoresis: CTCs can be sorted from WBCs inthe presence of a dielectrophoretic field, since theCTC’s dielectric properties (depending on theirdiameter, membrane area, density, conductivity andvolume) are different from those of WBCs. ApoCell’stechnology[44] leverages these differences in amicrofluidic flow channel to isolate CTCs. SiliconBiosystems’ DEPArray combines the use ofmicroscopy imaging and dielectrophoresis sorting[45]to identify and isolate pre-sorted CTCs, paving theway for downstream single-CTC molecularcharacterizations. (vi) Others: Several reviews [46-48]also summarized a wide collection of CTC detectiontechnologies which may not be included in this article.Microfluidics-enabled CTC assaysThe microfluidic affinity-capture devices [49]developed by Toner et al. sparked the recent logy-enabledCTCassays.This1st-generation (gen) device [49] (i.e., CTC-Chip)featured chemically etched microposts on a siliconsubstrate, on which anti-EpCAM antibodies ts were designed to maximize the contactbetween the device surfaces and the flow throughhttp://www.thno.org

Theranostics 2016, Vol. 6, Issue 9cells. Following CTC capture, ICC was conducted toidentify CTCs. The CTC-Chips erformance than most of the conventional CTCassays. Thereafter, similar device configurations wereadapted to create new microfluidic chips (e.g.,geometrically enhanced differential immunocapture,GEDI [50] approach and Biocept’s CTC assay [51]),and different antibody capture agents wereemployed. Recently, a unique "Ephesia" approach [52]based on microposts of capture agent-coated magneticbeads self-assembled in a microchip demonstratedcombined advantages of both microfluidic andimmunomagnetic cell sorting. The 2nd-gen device [53](i.e., herringbone-chip, HB-Chip) from the e (PDMS) component on a glassslide. Microscale herringbone patterns wereengineered into the PDMS component to introducemicrovortices, leading to enhanced contact betweenthe CTCs and the antibody-coated chip surfaces. Inaddition to the commonly used ICC technique, thetransparent nature of the HB-Chip allowed forimaging of the captured CTCs by standard clinicalhistopathological stains (i.e., haematoxylin and eosinstain). Although the microfluidic setting improvesCTC-capture performance, the majority of themicrofluidic CTC assays suffers from depth of fieldissues when performing microscopy imaging due tothe vertical depths of 3-dimensional ectional imaging scans that generate largeimage files are required in order to avoid out-of-focusor superimposed micrographs. By coupling a pair ofmicroelectrodes at the terminal of a plasticmicrofluidic chip[54], enzymatic release of thecaptured CTCs can be electrically counted without theissue of microscopy imaging. In contrast to their 1stand 2nd-gen devices, their 3rd-gen iChip[55] representsa groundbreaking label-free approach, whichcombines negative immunomagnetic depletionprocesses with an inertial focusing setting in anintegrated microchip. Most importantly, thisapproach allows for the recovery of unmanipulatedCTCs with desired molecular integrity and viability,allowing for downstream expressional profiling[18],as well as ex vivo culture and drug susceptibilitytesting[56]. The sorting mechanism of iChip, however,was recently reported to compromise the isolation ofCTC clusters, which potentially contain CTCs withhigh metastatic potential.[57] “Cluster-Chip”, amicrochip that can be used individually or inconjunction with CTC-iChips to isolate CTC clusters,was developed to address this issue.[58] Othermicrofluidic CTC assays based on unique principles,1427including micro-nuclear magnetic resonance (μNMR)platform[59], cell rolling[60], supported lipid bilayer(SLB)-coated microfluidic devices[61], and Vortextechnology[62, 63] have also been developed anddemonstrated. In addition to the microfluidic rization, and ex vivo expansion of CTCs, asectioned microfluidic device (known as the VelocityValley Chip) that selectively captures CTCs in amanner dependent on the number of magnetic beadsgrafted on the surface of a given CTC [64] has beendesigned. This device was employed to separate CTCsinto subpopulations by EpCAM expression ofindividual CTCs. Overall, microfluidic technology hasshown its potential in enriching and isolating CTCsamenable for subsequent molecular and functionalcharacterizations.NanoVelcro CTC Assays: Threegenerations of developmentRecent advances in the field of nanotechnologyoffer powerful solutions [65-67] resulting in a widerange of in-depth characterizations of CTCs whiledrastically reducing costs. Ultimately, deployment ofthese emerging advances will bring oncology closer tothe goal of personalized care. It has long beenrecognized that there are nanoscale componentspresent in the tissue microenvironment, including theextracellular matrix, and the cellular membrane.These provide structural and biochemical supportthat regulate cellular behavior and fate. Inspired bythe nanoscale interactions observed in the tissuemicroenvironment, Dr. Tseng’s research team atUCLA pioneered the development of “NanoVelcro”cell-affinity substrates [68, 69].In this uniqueapproach, capture agent-coated nanostructuredsubstrates are utilized to immobilize CTCs with highefficiency. The working mechanism of NanoVelcrocell-affinity substrates mimics that of VelcroTM – whenthe two fabric strips of a Velcro fastener are pressedtogether, interactions between the hairy surfaces ontwo strips leads to strong affinity between cells andnanosubstrates. In addition to the silicon nanowiresubstrate (SiNS)[68], the general applicability of theNanoVelcro cell-affinity assay is supported byextensive research endeavors devoted to exploitingdifferent nanomaterials, e.g., polymer 74], gold clusters on silicon nanowires[75],Fe3O4 nanoparticles[76], DNA networks[77], andgraphene oxide nanosheets[78] to achieve highaffinity capture of CTCs and other types of rare cells.In parallel, the team has also established a 3-color ICCprotocol[79] using DAPI, anti-CD45, and anti-CK tohttp://www.thno.org

Theranostics 2016, Vol. 6, Issue 9identify nanosubstrate-immobilized CTCs. Single-cellimage cytometry data covering DAPI staining,CK/CD45 expression and object size can be used todistinguish CTCs (DAPI /CK /CD45-, sizes 6 µm)from nonspecifically captured WBCs (DAPI /CK-/CD45 , sizes 12 µm), and cellular debris. With theinitial proof-of-concept demonstration of theNanoVelcro substrates and ICC protocol in place,three generations of NanoVelcro CTC Chips havebeen established[69] (Figure 1) to achieve differentclinical utilities.The 1st-gen NanoVelcro Chip [9, 80], composedof a SiNS and an overlaid microfluidic chaotic mixer,was created for CTC enumeration. The performance( 85% of CTC capture efficiency) of these NanoVelcroChips was measured using artificial CTC samples.1428Side-by-side analytical validation studies usingclinical blood samples show that the sensitivity of the1st-gen NanoVelcro Chip exceeds [9] that of theFDA-approved CellSearchTM Assay. Notably, theNanoVelcro-like approach allows immobilization ofCTCs onto a flat and small surface, thus facilitatingthe implementation of subsequent high-resolutionimmunofluorescence microscopy imaging of CTCswithout multiple cross-sectional imaging scansrequired for the majority of the existing microfluidicCTC assays. Moving beyond CTC enumeration, the2nd-gen NanoVelcro Chips[16, 81, 82], known asNanoVelcro-LCM approach were developed byreplacing SiNS with a transparent substrate o-glycolic acid). The transparent PLGAFigure 1. Conceptual illustration of three generations of the NanoVelcro CTC Assays developed by the UCLA team to achieve different clinicalutilities. 1st-Gen NanoVelcro Chip [9, 80], composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, was created for CTC enumeration. Inconjunction with the use of the laser capture microdissection (LCM) technique, 2nd-gen NanoVelcro-LMD technology [16, 81, 82], was developed for single-CTC isolation. Theindividually isolated CTCs can be subjected to single-CTC genotyping. By grafting thermoresponsive polymer brushes onto SiNS, 3rd-gen Thermoresponsive NanoVelcro CTCChips [83, 84] were developed for purification of CTCs via capture and release of CTCs at 37 C and 4 C, respectively. The surface-grafted polymer brushes were responsiblefor altering the accessibility of the capture agent on NanoVelcro substrates, allowing for rapid CTC purification with desired viability and molecular integrity. (Reprinted withpermission from Tseng 2014, Copyright, American Chemical Society). We compare the performance and differences of the three generations of NanoVelcro CTC Assays ina table.http://www.thno.org

Theranostics 2016, Vol. 6, Issue 9NanoVelcro substrate retains the desired CTC captureperformance, and allows for seamless integrationwith a laser capture microdissection (LCM) techniqueto isolate immobilized CTCs with single-cellresolution. The individually isolated CTCs can besubjected to single-CTC genotyping (both Sangersequencing [82] and next-generation sequencing[NGS][16, 81]) to serve as liquid biopsies. In order toincrease throughput, lower labor, and address theneed for viable/unfixed CTCs, the UCLA teamdeveloped the 3rd-gen Thermoresponsive NanoVelcroChips [83, 84] and demonstrated the ability to captureand release viable CTCs at 37 C and 4 C, respectively.By grafting thermoresponsive polymer brushes[83](poly(N-isopropylacrylamide, PIPAAm) onto SiNSvia atom transfer radical polymerization, thetemperature-dependent conformational changes ofpolymer brushes can effectively alter the accessibilityof capture agents on SiNS, allowing for rapid CTCpurification with desired viability and molecularintegrity. The advent of the 3rd-gen ThermoresponsiveNanoVelcro Chips is expected to open up newopportunities to connect with a wider range ofmolecular and functional assays. The continuousresearch endeavors put together by the UCLA teamand its clinical collaborators have demonstrated theuse of NanoVelcro CTC assays in clinical settings tofacilitate the concept of CTC-based liquid biopsy.These results are briefly summarized in this reviewarticle.Enumerating CTCs using 1st-genNanoVelcro CTC AssayGiven the CTC detection performance observedfor the 1st-gen NanoVelcro CTC Assay [80],continuous efforts were devoted to test its utility forCTC detection in different solid tumors in conjunctionwith the use of combined capture and ICC antibodies.The initial clinical studies focused on prostate cancerwith the intention to address the issue thatCellSearchTM assay is unable to detect CTCs in a largeportion of late stage prostate cancer patients [14].These clinical validation studies [9] were jointlyconducted by Urologic Oncology teams at RonaldReagan UCLA Medical Center and Cedars-SinaiMedical Center (CSMC). Forty prostate cancerpatients (32 with metastatic disease and 8 withlocalized disease) were recruited. CTCs wereidentified in all 40 patients, indicating a consistentefficiency of 1st-gen NanoVelcro Assay for CTCenumeration in prostate cancer patients acrossdifferent stages of disease. The team also performedserial enumeration allowing the comparison of CTCnumber changes after 4-10 weeks of therapy, andobserved a statistically significant reduction in CTC1429counts in the clinical responders. Further, long-termfollow ups were also performed for 460 days withserial CTC collection and enumeration. In this case,CTC numbers faithfully represented the initialresponse and subsequent failures during nilutamideand sipuleucel-t treatment. This study demonstratesthe consistency of the 1st-gen NanoVelcro CTC Assayover time for CTC enumeration, and shows thatcontinuous monitoring of CTC numbers can beemployed to follow responses to differen

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