Click Chemistry-Mediated Microfluidic Sorting for HCC CTCs

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Molecular & Cellular Analysis Technologies
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PROJECT SUMMARY/ABSTRACTThis R21 proposal specifically responds to RFA-CA-18-002 ? Innovative Molecular and Cellular AnalysisTechnologies for Basic and Clinical Cancer Research. The long-term goal is to develop and validate a novelclick chemistry-mediated cell sorting method (i.e., Click Chips) for enumeration and quantitative molecularcharacterization of circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). We envision that ClickChips can be seamlessly coupled with a multi-marker sorting strategy to capture and purify HCC CTCs fromblood samples to expedite the detection and characterization of HCC CTCs. CTCs are regarded as a liquid biopsy of tumors, allowing non-invasive and systemic sampling of thedisease. CTCs can be recovered and analyzed repeatedly over the disease course, providing potential insightsinto the molecular mechanisms governing disease progression, while averting the need for numerous invasivebiopsy. HCC, the 2nd most common cause of cancer-related deaths worldwide, is in dire need of prognosticbiomarkers. Current clinicopathologic and radiographic staging systems, and serum biomarkers (e.g., AFP)poorly discriminate between early-stage patients amenable to surgical therapy and advanced-stage patientsreceiving chemotherapy. Our joint research team at UCLA has recently developed a multi-marker capturecocktail that allows for detection of HCC CTCs across all disease stages. Developing new liquid biopsydiagnostics, capable of conducting both HCC CTC enumeration and molecular analysis, holds great promise tosignificantly augment the ability of current staging criteria to realize longitudinal monitoring of diseaseprogression and treatment responses. Recognizing the limitations associated with the conventional antibody-mediated CTC sorting, our teamaims to develop a new class of CTC assays based on click chemistry-mediated cell capture. In contrast toantibody-mediated CTC sorting methods, a pair of highly reactive click chemistry motifs (i.e., tetrazine, Tz, andtrans-cyclooctene, TCO) were grafted onto cell-capture substrates and CTCs, respectively. When TCO-graftedCTCs flow through the integrated device, a click reaction (between TCO on CTCs and Tz on the substrate)leads to irreversible immobilization of CTCs with dramatically improved sensitivity and specificity. Further, byincorporating a disulfide bond into the surface linker that tethers Tz onto the substrate, the CTCs captured onthe substrate can be released/recovered upon exposure to a mild disulfide cleavage agent (i.e.,dithiobutylamine), allowing for effective CTC purification. The innovation of Click Chips includes i) increasedsensitivity and specificity of CTC enrichment by replacing antibody-mediated capture with click chemistry, andii) highly effective CTC purification due to the disulfide-cleavage driven CTC release mechanism. The proposalwill be implemented via Specific Aim 1: to conduct exploratory development of Click Chips for HCC CTCs, andSpecific Aim 2: to conduct initial clinical validation of Click Chips using HCC blood samples.