NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells
Advanced Drug Delivery Reviews
Circulating tumor cells (CTCs) are cancer cells shredded from either a primary tumor or a metastatic site and circulate in the blood as the potential cellular origin of metastasis. By detecting and analyzing CTCs, we will be able to noninvasively monitor disease progression in individual cancer patients and obtain insightful information for assessing disease status, thus realizing the concept of "tumor liquid biopsy". However, it is technically challenging to identify CTCs in patient blood
... es because of the extremely low abundance of CTCs among a large number of hematologic cells. In order to address this challenge, our research team at UCLA pioneered a † These authors contribute equally to this work. unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with remarkable efficiency. Four generations of NanoVelcro CTC assays have been developed over the past decade for a variety of clinical utilities. The 1 st -gen NanoVelcro chips, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, were created for CTC enumeration. The 2 nd -gen NanoVelcro chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation in conjunction with the use of the laser microdissection (LMD) technique. By grafting thermoresponsive polymer brushes onto SiNS, the 3 rd -gen Thermoresponsive NanoVelcro chips have demonstrated the capture and release of CTCs at 37 and 4 °C respectively, thereby allowing for rapid CTC purification while maintaining cell viability and molecular integrity. Fabricated with boronic acid-grafted conducting polymer-based nanomaterial on chip surface, the 4 th -gen NanoVelcro Chips (Sweet chip) were able to purify CTCs with well-preserved RNA transcripts, which could be used for downstream analysis of several cancer specific RNA biomarkers. In this review article, we will summarize the development of the four generations of NanoVelcro CTC Assays, and the clinical applications of each generation of devices. Graphical abstract Jan et al. represents a disease whose biological and molecular nature varies from site to site and changes over time in the course of treatment interventions. Despite its difficulty, a re-biopsy procedure is often recommended to detect a possible new biology profile of cancer cells during the clinical treatment course in some solid tumors (e.g. lung cancer). As a non-invasive alternative to tumor biopsy, researchers have been exploring the use of circulating tumor cells (CTCs) as "liquid biopsies" of solid tumors. CTCs are blood borne tumor cells shed from either primary or metastatic sites. Through a simple blood draw, CTCs can be detected and recovered throughout the course of disease development without needing invasive and painful biopsy procedures. In addition to conventional diagnostic imaging and serum marker detection, detecting and characterizing CTCs in patient blood provides an opportunity for early diagnosis of cancer metastasis. Further, serial CTC tests can be performed over the disease progression with relatively high frequency, creating an opportunity to perform real-time, dynamic monitoring of an evolving and adapting malignant process [8, 9] . To address this unmet need, there have been significant research endeavors, especially in the fields of chemistry, materials science, and bioengineering, devoted to developing CTC detection, isolation, and characterization technologies  . However, identifying CTCs in blood samples has been technically challenging due to the extremely low abundance (a few to hundreds per milliliter) of CTCs among a large number (10 9 mL −1 ) of hematologic cells in the blood. Initial CTC studies focused on enumeration and protein expression analysis    . More recent research efforts have demonstrated that CTCs and their matching tumor tissues share significant similarities at the genomic    and transcriptomic [18, 19] levels. Mounting evidence has consistently shown CTCs to be a powerful tool with which we can study the underlying biology of cancer, guide therapeutic interventions, and monitor the progression of disease. Existing CTC assays In order to effectively conduct detection and analysis of CTCs, a variety of methodologies have been developed. (i) Immunomagnetic separation: Positive selection/enrichment of CTCs[13, 20, 21] can be achieved using capture agent-labeled magnetic beads. For epithelial-origin solid tumors, anti-EpCAM  is the most widely used capture agent. Alternatively, negative depletion [23, 24] of white blood cells (WBCs) using magnetic beads tagged by anti-CD45 can result in purified CTCs. CellSearch™    is the only FDAapproved CTC assay with prognostic utility in metastatic breast, colorectal, and prostate cancers. CellSearch™ Assay enriches CTCs with magnetic beads tagged with anti-EpCAM, followed by immunocytochemistry (ICC) treatment to distinguish CTCs (DAPI+/cytokeratin −CK+/CD45−) from WBCs (DAPI+/CK−/CD45+) and cell debris in the background. New immunomagnetic technologies including MagSweeper, magnetic sifters, AdnaGen/ Qiagen, IsoFlux, VerIFAST and Cynvenio, were developed to improve the speed, efficiency, and cost of CTC detection and characterization. (ii) Flow cytometry: Although one of the most commonly used cell-sorting technologies for the analysis of subpopulations of fluorescently labeled cells [31, 32], flow cytometry lacked the ability to reveal sufficient morphological information to satisfy the standards set by pathologists for CTCs. The development of ensemble decision aliquot ranking (eDAR) [33, 34] addressed this drawback. (iii) Microfluidics-enabled CTC assays: Massachusetts General Hospital Jan et al.