GRF 2023 9043345 PI: Chia-Hung CHEN

Droplet Functional Barcoding Technology for Single-Cell Multidimensional Profiling Applied to Precision Immunotherapy

Researcher(s)

• Chia-Hung CHEN (Principal Investigator / Project Coordinator)Department of Biomedical Engineering
• Kwan Ting CHOW (Co-Investigator)Department of Biomedical Sciences

Description

Immunotherapy, such as adoptive cell therapy (ACT) harnessing the patient’s immune system, heralds a new era of personalized medicine, offering hope for curative responses in patients with serious illnesses. Despite the demonstrated success, there are large variations in patient responses, including non-healing effects and unpredictable toxicity, which could be attributed in part to cell functional heterogeneity, transcriptomic difference, and interpatient heterogeneity of cell infusion products. This makes it imperative to characterize the multidimensional profile of single cells at the systems level, while there are currently no available platforms to approach that at scale.Here, we will develop a novel droplet functional barcoding technology for single cell phenotype sequencing that will integrate functional analysis and transcriptomic profiling in the same single cell, in a highly multiplexed, and high throughput manner. Single-cell secretome (~20-102 cytokines), cytotoxic effector function (by assaying ~5 cancer intracellular proteins), and transcriptomics (~105 RNAs) will be simultaneously measured in a throughput ~104 cells via a single sequencing experiment. Together, single-cell functional assays and transcriptomic profiles allow us to reveal novel insights into the functional consequences of molecular heterogeneity in immune systems. We can therefore exploit drugs and genetic tools to manipulate specific cells for disease treatments.This research involves three parts. First, the cholesterol-linked capture antibodies and DNA barcoded antibodies (DNA tags) will be synthesized, forming a cell secretomic barcode library. Cholesterol-linked capture antibodies will anchor to the cell surface to capture the target secreted proteins. The captured proteins will then be labeled by DNA tags for single-cell multiplexed secretomic analysis. Second, a microfluidic droplet injector cell loading device will be developed to coencapsulate single immune cells and single cancer cells in the droplets. In a killing process, the cancer cell membrane will be degraded to release intracellular cancer proteins. By capturing these proteins from dead cancer cells on immune cell surface for barcoding, the cytotoxicity can be measured. Notably, after labeling the captured proteins by DNA tags, the phenotypes of barcoded cells can be effectively analyzed by sequencing (Phenotype-seq) for multidimensional profiling (secretomics + cytotoxicity + transcriptomics). Third, two immunotherapy applications will be demonstrated: TCR-T cell analysis to guide the cell manufacturing workflow and tumor-specific TCR screening for immunotherapy against hepatocellular carcinoma (HCC). The successful realization of proposed technology for high throughput single-cell multidimensional analysis will allow an effective assay for the determination of the most optimal TCR for patients, as a major advancement toward precision medicine. 

Detail(s)