PROJECT SUMMARY/ABSTRACT This R21 proposal specifically responds to RFA-CA-18-002 ? Innovative Molecular and Cellular Analysis Technologies for Basic and Clinical Cancer Research. The long-term goal is to develop and validate a novel click chemistry-mediated cell sorting method (i.e., Click Chips) for enumeration and quantitative molecular characterization of circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). We envision that Click Chips can be seamlessly coupled with a multi-marker sorting strategy to capture and purify HCC CTCs from blood 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 the disease. CTCs can be recovered and analyzed repeatedly over the disease course, providing potential insights into the molecular mechanisms governing disease progression, while averting the need for numerous invasive biopsy. HCC, the 2nd most common cause of cancer-related deaths worldwide, is in dire need of prognostic biomarkers. 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 patients receiving chemotherapy. Our joint research team at UCLA has recently developed a multi-marker capture cocktail that allows for detection of HCC CTCs across all disease stages. Developing new liquid biopsy diagnostics, capable of conducting both HCC CTC enumeration and molecular analysis, holds great promise to significantly augment the ability of current staging criteria to realize longitudinal monitoring of disease progression and treatment responses. Recognizing the limitations associated with the conventional antibody-mediated CTC sorting, our team aims to develop a new class of CTC assays based on click chemistry-mediated cell capture. In contrast to antibody-mediated CTC sorting methods, a pair of highly reactive click chemistry motifs (i.e., tetrazine, Tz, and trans-cyclooctene, TCO) were grafted onto cell-capture substrates and CTCs, respectively. When TCO-grafted CTCs 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, by incorporating a disulfide bond into the surface linker that tethers Tz onto the substrate, the CTCs captured on the 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) increased sensitivity and specificity of CTC enrichment by replacing antibody-mediated capture with click chemistry, and ii) highly effective CTC purification due to the disulfide-cleavage driven CTC release mechanism. The proposal will be implemented via Specific Aim 1: to conduct exploratory development of Click Chips for HCC CTCs, and Specific Aim 2: to conduct initial clinical validation of Click Chips using HCC blood samples.