Sickle-like Inertial Microfluidic System for Online Rare Cell Separation and Tandem Label-Free Quantitative Proteomics (Orcs-Proteomics).

Label-free proteomics with trace clinical samples provides a wealth of actionable insights for personalized medicine. Clinically acquired primary cells, such as circulating tumor cells (CTCs), are usually with low abundance that is prohibitive for conventional label-free proteomics analysis. Here, we present a sickle-like inertial microfluidic system for online rare cell separation and tandem label-free proteomics (namely, Orcs-proteomics). Orcs-proteomics adopts a buffer system with 0.1% N-dodecyl ß-d-maltoside (DDM), 1 mM Tris (2-carboxyethyl) phosphine (TCEP), and 2 mM 2-chloroacetamide (CAA) for cell lysis and reductive alkylation. We demonstrate the application of Orcs-proteomics with 293T cells and manage to identify 913, 1563, 2271, and 2770 protein groups with 4, 13, 68, and 119 cells, respectively. We then spike MCF7 cells with white blood cells (WBCs) to simulate the patient's blood sample. Orcs-proteomics identifies more than 2000 protein groups with an average of 61 MCF7 cells. We further recruit two advanced breast cancer patients and collect 5 and 7 CTCs from each patient through minimally invasive blood drawing. Orcs-proteomics manages to identify 973 and 1135 protein groups for each patient. Therefore, Orcs-proteomics empowers rare cells simultaneously to be separated and counted for proteomics and provides technical support for personalized treatment decision making with rare primary patient samples.

Integrated microfluidic single-cell immunoblotting chip enables high-throughput isolation, enrichment and direct protein analysis of circulating tumor cells.

Effective capture and analysis of a single circulating tumor cell (CTC) is instrumental for early diagnosis and personalized therapy of tumors. However, due to their extremely low abundance and susceptibility to interference from other cells, high-throughput isolation, enrichment, and single-cell-level functional protein analysis of CTCs within one integrated system remains a major challenge. Herein, we present an integrated multifunctional microfluidic system for highly efficient and label-free CTC isolation, CTC enrichment, and single-cell immunoblotting (ieSCI). The ieSCI-chip is a multilayer microfluidic system that combines an inertia force-based cell sorter with a membrane filter for label-free CTC separation and enrichment and a thin layer of a photoactive polyacrylamide gel with microwell arrays at the bottom of the chamber for single-cell immunoblotting. The ieSCI-chip successfully identified a subgroup of apoptosis-negative (Bax-negative) cells, which traditional bulk analysis did not detect, from cisplatin-treated cells. Furthermore, we demonstrated the clinical application of the ieSCI-chip with blood samples from breast cancer patients for personalized CTC epithelial-to-mesenchymal transition (EMT) analysis. The expression level of a tumor cell marker (EpCAM) can be directly determined in isolated CTCs at the single-cell level, and the therapeutic response to anticancer drugs can be simultaneously monitored. Therefore, the ieSCI-chip provides a promising clinical translational tool for clinical drug response monitoring and personalized regimen development.

Rapid and efficient capturing of circulating tumor cells from breast cancer Patient's whole blood via the antibody functionalized microfluidic (AFM) chip.

Accurate enumeration of circulating tumor cells (CTCs) in cancer patient's blood functions as a form of "liquid biopsy", which is pivotal for cancer screening, prognosis, and diagnosis. Herein, we demonstrate a novel antibody functionalized microfluidic (AFM) chip that rapidly and accurately qualifies CTCs from breast cancer patient's whole blood. The AFM chip consists of three buffering zones, and four main capturing zones filled with equilateral triangular pillars and periodically distributed obstacles. We validate the AFM chip with three Epithelial cell adhesion molecule (EpCAM) positive cancer cell lines, including breast (MCF-7), prostate (PC3), and lung cancer cell lines (A549), achieving capture efficiencies of 99.5%, 98.5%, and 96.72%, respectively, at a flow rate of 0.6 mL/hour. We further confirm the efficacy of the AFM chip with five advanced breast cancer patients' whole blood to capture EpCAM+/CK19+/CD45-/DAPI + CTCs. Interestingly, high number of CTCs were identified from each patient's 1 mL whole blood (595-2270), The AFM chip is highly efficient at rapidly capturing CTCs from cancer patients' whole blood without requiring extra equipment, which is critically beneficial for clinical application.

Highly sensitive and portable mRNA detection platform for early cancer detection.

Pancreatic cancer, at unresectable advanced stages, presents poor prognoses, which could be prevented by early pancreatic cancer diagnosis methods. Recently, a promising early-stage pancreatic cancer biomarker, extracellular vesicles (EVs) related glypican-1 (GPC1) mRNA, is found to overexpress in pancreatic cancer cells. Current mRNA detection methods usually require expensive machinery, strict preservation environments, and time-consuming processes to guarantee detection sensitivity, specificity, and stability. Herein, we propose a novel two-step amplification method (CHAGE) via the target triggered Catalytic Hairpin Assembly strategy combined with Gold-Enhanced point-of-care-testing (POCT) technology for sensitive visual detection of pancreatic cancer biomarker. First, utilizing the catalyzed hairpin DNA circuit, low expression of the GPC1 mRNA was changed into amplification product 1 (AP1, a DNA duplex) as the next detection targets of the paper strips. Second, the AP1 was loaded onto a lateral flow assay and captured with the gold signal nanoparticles to visualize results. Finally, the detected results can be further enhanced by depositing gold to re-enlarge the sizes of gold nanoparticles in detection zones. As a result, the CHAGE methodology lowers the detection limit of mRNA to 100 fM and provides results within 2 h at 37 °C. Furthermore, we demonstrate the successful application in discriminating pancreatic cancer cells by analyzing EVs' GPC1 mRNA expression levels. Hence, the CHAGE methodology proposed here provides a rapid and convenient POCT platform for sensitive detection of mRNAs through unique probes designs (COVID, HPV, etc.).

Single-Cell Immunoblotting based on a Photoclick Hydrogel Enables High-Throughput Screening and Accurate Profiling of Exogenous Gene Expression.

Fast and accurate profiling of exogenous gene expression in host cells is crucial for studying gene function in cellular and molecular biology, but still faces the challenge of incomplete co-expression of reporter genes and target genes. Here, a single-cell transfection analysis chip (scTAC) is presented, which is based on the in situ microchip immunoblotting method, for rapid and accurate analysis of exogenous gene expression in thousands of individual host cells. scTAC not only can assign information of exogenous gene activity to specific transfected cells, but enables the acquisition of continuous protein expression even in low co-expression scenarios. It is demonstrated that scTAC can reveal the relationship of expression level between reporter genes and target genes, which is helpful for evaluating transient transfection strategy efficiency. The advantages of this method for the study of fusion protein expression and downstream protein expression in signaling pathway in rare cells are shown. Empirically, an EGFP-TSPAN8 fusion plasmid is transfected into MCF-7 breast cancer cells and the expressions of two cancer stemness biomarkers (ALDHA1 and SOX2) are analyzed. The scTAC method clearly reveals an interesting phenomenon that transfected adherent MCF-7 cells exhibit some stem cell characteristics, but they do not have stem cell appearance.

Immunocyte Profiling Using Single-Cell Mass Cytometry Reveals EpCAM+ CD4+ T Cells Abnormal in Colon Cancer.

Colon cancer (CC) is one of the leading causes of cancer related mortality. Research over past decades have profoundly enhanced our understanding of immunotherapy, a major clinical accomplishment, and its potential role toward treating CC. However, studies investigating the expression of these immune checkpoints, such as epithelial cell adhesion molecule (EpCAM), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1), by peripheral blood mononuclear cells (PBMCs) is lacking. Here, high-dimensional mass cytometry (CyTOF) is used to investigate immune alterations and promising immunotherapeutic targets expression by PBMCs of CC patients. Results reveal that expression of EpCAM and PD-L1 on CD4+ T cells significantly increased in patients with CC, compared with age- and sex- matching healthy controls and patients with colonic polyps. These differences are also validated in an independent patient cohort using flow cytometry. Further analysis revealed that EpCAM+ CD4+ T cells are PD-L1+ CCR5+ CCR6+. Immunofluorescence staining results demonstrate that the increase of EpCAM+ CD4+ T cells is also observed in tumor tissues, rather than para-cancerous tissues. To ascertain the functional disorders of the identified cell subset, phosphorylated signaling protein levels are assessed using imaging mass cytometry. Increases in pp38 MAPK and pMAPKAPK2 are observable, indicating abnormal activation of pp38 MAPK-pMAPKAPK2 signaling pathway. Results in this study indicate that EpCAM+ CD4+ T cells may play a role in CC development. Detailed knowledge on the functionality of EpCAM+ CD4+ T cells is of high translational relevance.