Ultrafast Single-Cell Level Enzymatic Tumor Profiling

Abstract

In the context of tumor analysis, the implementation of precision medicine requires on-time clinical measurements, which requires rapid large-scale single-cell screening that obtains cell population distributions and functions in tumors to determine disease progression for therapeutics. In this study, a high-throughput screening (HTS) platform integrating optical fluorescence detectors and a computational method was developed as a droplet-based microfluidic flow cytometer (Droplet-μFC) to comprehensively analyze multiple proteolytic activities of a patient-derived tumor (with ∼0.5−2 M cells) at single-cell resolution within 2 h. The data-driven analytical method identified distinct cell types and status through protease profiling with high precision. Multiple protease activities of single cells harvested from a tumor were thus determined with a throughput of ∼100 cells per second. This platform was used to screen protease activities of a wide range of cell types, forming a library. With the development of advanced computational clustering and cell mapping, rapid quantitative tumor profiling with a comprehensive description of cell population distributions and functions could be obtained for clinical treatments.

Droplet-based microfluidics in biomedical applications

“…MMP9 activity was quantified in the encapsulated lymphoma cells (U937), invasive breast ductal carcinoma cells (MCF-7) cells, and mouse embryonic fibroblast cells (NIH3T3) as the negative control, to differentiate cancer and normal cells as well as different cancer cell lines by monitoring their enzymatic fingerprints. To study cancer cell heterogeneity, the patient-derived glioblastoma samples were introduced to a droplet-based microfluidic system, where encapsulation of the cells within droplets was followed by screening and computational analysis of single-cell protease profiles [199]. A library of cell types including HUVECs, astrocytes (NHAs), fibroblasts (NHDFs), white blood cells (WBCs), and cell lines associated with brain tumors were generated based on their distinct MMP/ADAM activity profiles within 2 h with the throughput of ∼100 cells s −1 .…”

Label-free Mass Cytometry for Unveiling Cellular Metabolic Heterogeneity

“…The metabolite coverages of CyESI-MS were assessed in both positive and negative ion modes. In the range of m/z 100−1000, 421, and 425, ion signals related to cellular metabolites were picked out in positive and negative modes, respectively (Figure 4A, Figure S8), of which 174 and 117 cellular metabolites were assigned based on accurate mass measurements as well as MS 2 information obtained from population cells (Table S8− 9). Abundant metabolites assigned in positive mode were labeled on the mass spectra of selected m/z regions (Figure 4B−D).…”

“…Different types of cells play specific roles in the life activities of a complex living organism . Unveiling cell heterogeneity provides deep insights of types, cycle stages, degrees of differentiation, , and fates , of cells, which is expected to contribute to the prognosis, diagnosis, and therapy of metabolism-related diseases. , Methods for the characterization of phenotypes and functions of single cells, such as fluidic platforms derived from flow cytometry, single-cell sequencing, − and RNA fluorescence in situ hybridization, have revealed heterogeneity of cell populations which were once considered homogeneous. Among these methods, fluorescence-based flow cytometry and its derivatives have been widely used in analyzing immunophenotypes of heterogeneous cell populations, − owing to its advantages of high-throughput detection of single cells.…”

Functional Stem Cell Sorting via Integrative Droplet Synchronization

“…Droplet microfluidics has emerged as an important platform that provides new experimental possibilities for high-throughput screening. , The continuous generation of large amounts of monodispersed droplets containing a single cell enables effective compartmentalization for secretion analysis. For example, cell-secreted enzymes have been investigated by inserting fluorescence resonance energy transfer (FRET)-based sensors within droplets for high-throughput biological sample profiling . However, for droplet-based single-cell assays, it is essential to encapsulate toxic chemical sensors and cells within the droplets together for incubation, which is harmful to the cells.…”

“…For example, cell-secreted enzymes have been investigated by inserting fluorescence resonance energy transfer (FRET)-based sensors within droplets for highthroughput biological sample profiling. 33 However, for droplet-based single-cell assays, it is essential to encapsulate toxic chemical sensors and cells within the droplets together for incubation, which is harmful to the cells. In fact, there are only a few specific biocompatible chemical sensors available for cell encapsulation for secretion analysis.…”