SpiDe-Sr: blind super-resolution network for precise cell segmentation and clustering in spatial proteomics imaging.

Spatial proteomics elucidates cellular biochemical changes with unprecedented topological level. Imaging mass cytometry (IMC) is a high-dimensional single-cell resolution platform for targeted spatial proteomics. However, the precision of subsequent clinical analysis is constrained by imaging noise and resolution. Here, we propose SpiDe-Sr, a super-resolution network embedded with a denoising module for IMC spatial resolution enhancement. SpiDe-Sr effectively resists noise and improves resolution by 4 times. We demonstrate SpiDe-Sr respectively with cells, mouse and human tissues, resulting 18.95%/27.27%/21.16% increase in peak signal-to-noise ratio and 15.95%/31.63%/15.52% increase in cell extraction accuracy. We further apply SpiDe-Sr to study the tumor microenvironment of a 20-patient clinical breast cancer cohort with 269,556 single cells, and discover the invasion of Gram-negative bacteria is positively correlated with carcinogenesis markers and negatively correlated with immunological markers. Additionally, SpiDe-Sr is also compatible with fluorescence microscopy imaging, suggesting SpiDe-Sr an alternative tool for microscopy image super-resolution.

Proteome signatures of joint toxicity to arsenic (As) and lead (Pb) in human brain organoids with optic vesicles.

Arsenic (As) and lead (Pb) are toxins found in the natural surroundings, and the harmful health outcomes caused by the co-exposure of such toxins have become a considerable problem. However, the joint neurotoxicity of As and Pb to neurodevelopment and the underlying mechanisms remain unclear. Pluripotent stem cell-derived human brain organoids are emerging animal model alternatives for understanding neurological-related diseases. Therefore, we utilized brain organoids with optic vesicles (OVB-organoids) to systematically analyze the neurotoxicity of As and Pb. After 24 h of As and/or Pb exposure, hematoxylin-eosin staining revealed that As and Pb exposure could cause disorders in the structure of the ventricular zone and general cell disarrangement in OVB-organoids. Immunostaining displayed that OVB-organoids are more susceptible to As and Pb co-exposure than independent exposure in apoptosis, proliferation, and cell differentiation. Meanwhile, even though As and Pb could both hinder cell proliferation, contrary to Pb, As could induce an increasing proportion of mitotic (G2/M) cells. The proteome landscape of OVB-organoids illustrated that Pb synergized with As in G2/M arrest and the common role of As and Pb in carcinogenesis. Besides, proteomics analyses suggested the consequential role of autophagy and Wnt pathway in the neurotoxicity of As and Pb co-exposure. Overall, our findings provide penetrating insights into the cell cycle, carcinogenesis, autophagy, and Wnt pathway underlying the As and Pb binary exposure scenarios, which could enhance our understanding of the mixture neurotoxicity mechanisms.

Three-dimensional microfluidics with dynamic fluidic perturbation promotes viability and uniformity of human cerebral organoids.

Human cerebral organoids (COs), generated from stem cells, are emerging animal alternatives for understanding brain development and neurodegeneration diseases. Long-term growth of COs is currently hindered by the limitation of efficient oxygen infiltration and continuous nutrient supply, leading to general inner hypoxia and cell death at the core region of the organoids. Here, we developed a three-dimensional (3D) microfluidic platform with dynamic fluidic perturbation and oxygen supply. We demonstrated COs cultured in the 3D microfluidic system grew continuously for over 50 days without cell death at the core region. Increased cell proliferation and enhanced cell differentiation were also observed and verified with immunofluorescence staining, proteomics and metabolomics. Time-lapse proteomics from 7 consecutive acquisitions between day 4 and day 30 identified 546 proteins differently expressed accompanying COs growth, which were mainly relevant to nervous system development, in utero embryonic development, brain development and neuron migration. Our 3D microfluidic platform provides potential utility for culturing high-homogeneous human organoids.

SAIF: Label-Free Separation of Circulating Tumor Cells Using a Self-Amplified Inertial Focusing Microfluidic Chip.

Circulating tumor cells (CTCs) are rare cells existing in the bloodstream with a relatively low number, which facilitate as a predictor of cancer progression. However, it is difficult to obtain highly purified intact CTCs with desired viability due to their low percentage among blood cells. In this chapter, we demonstrate the detailed steps for the fabrication and application of the novel self-amplified inertial-focused (SAIF) microfluidic chip that enables size-based, high-throughput, label-free separation of CTCs from the patient blood. The SAIF chip introduced in this chapter demonstrates the feasibility of an extremely narrow zigzag channel (with 40 µm channel width) connected with two expansion regions to effectively separate different-sized cells with amplified separation distance.

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.

Application of Microfluidics in Single-cell Manipulation, Omics and Drug Development.

BACKGROUND: Cell heterogeneity exists among different tissues, even in the same type of cells. Cell heterogeneity leads to a difference in cell size, functions, biological activity, and for cancer cells it causes different drug responses and resistance. Meanwhile, microfluidics is a promising tool for single-cell research to reveal cell heterogeneity. METHODS: Through literature research conducted over the past ten years on microfluidics, we summarize and introduce the application of microfluidics in single-cell separation and manipulation, featuring techniques, such as acoustic manipulation, optical manipulation, single-cell trapping, and patterning, as well as single-cell omics including singlecell genomics, single-cell transcriptomics, single-cell proteome, single-cell metabolome, and drug development. RESULTS: Microfluidics is a flexible, precise tool, and it is easy to integrate with different functions. Firstly, it can be used as an important tool to separate rare but important cells according to the cell`s biological or physical properties. Secondly, microfluidics can provide the possibility of single-cell omics. Thirdly, microfluidics can be used in drug development, specifically in drug delivery and drug combination. Meanwhile, droplet microfluidics has gradually become the most powerful tool to encapsulate single-cells with other reagents for DNA, RNA, or protein analysis. CONCLUSION: Microfluidics is a robust platform technology that is able to accomplish rare cell separation, efficient single-cell omics analysis and provide a platform for drug development and drug delivery.

Simplified ARCHITECT microfluidic chip through a dual-flip strategy enables stable and versatile tumoroid formation combined with label-free quantitative proteomic analysis.

Recent years, microfluidic three-dimensional (3D) tumor culture technique has made great progress in tumor microenvironment simulation and drug screening. Meanwhile, as their functionality and complexity increase, it is more difficult for current chip models to selectively collect specific-layer cells from tumoroids for further analysis. Moreover, a simplified and robust method for tumoroid formation with highly consistent size and repeatable 3D morphology is relatively ncessary. Here, we report an ARCHITECT (ARtificial CHIp for Tumor Enables Confocal Topography observation) chip, through a dual-flip strategy to implement straightforward tumoroid establishment. This platform guarantees stable batch-to-batch tumoroids formation and allows high resolution confocal imaging. Moreover, an initial cell density as low as 65 cells per chamber is efficient to deliver a tumoroid. With this ARCHITECT chip, different-layer cells of interest could be collected from tumoroid for label-free quantitative (LFQ) proteomic analysis. For application demonstration, we mainly verified this platform for lung carcinoma (A549) tumoroid construction and proteomic analysis at out layer. Our data indicate that the out-layer cells of A549 tumoroid show extensively distinct proteomic expressions compared to two-dimensional cultured A549 cells. The up-regulated proteins are mainly related to tumorigenicity, proliferation and metastasis. And the differentially expressed proteins are mainly relevant to lipid metabolism pathway which is essential to tumor progression and proliferation. This platform provides a simplified yet robust technique to connect microfluidic tumoroid construction and LFQ proteomic analysis. The simplicity of this technique should open the way to numerous applications such as discovering the innovative targets for cancer treatment, and studying the mophological and proteomic heterogeneity of different-layer cells across the tumoroid.

Label-free Separation of Circulating Tumor Cells Using a Self-Amplified Inertial Focusing (SAIF) Microfluidic Chip.

Circulating tumor cells (CTCs) are rare cells existing in the bloodstream with a relatively low number, which facilitate as a predictor of cancer progress. However, it is difficult to obtain highly purified intact CTCs with desired viability due to the low percentage of CTCs among blood cells. In this work, we demonstrate a novel self-amplified inertial focused (SAIF) microfluidic chip that enables size-based, high-throughput, label-free separation of CTCs from a patient's blood. The SAIF chip introduced in this study demonstrated the feasibility of an extremely narrow zigzag channel (with 40 µm channel width) connected with two expansion regions to effectively separate different-sized cells with amplified separation distance. The chip performance was optimized with different-sized polystyrene (PS) particles and blood cells spiked with three different types of cancer cells. The separation efficiencies for blood cells and spiked cancer cells are higher than 80%. Recovery rates of cancer cells were tested by spiking 1500 lung cancer cells (A549), breast cancer cells (MCF-7), and cervical cancer cells (HeLa) separately to 3 mL 0.09% saline with 3 × 106 white blood cells (WBCs). The recovery rates for larger cells (MCF-7 and HeLa) were 79.1 and 85.4%, respectively. Viabilities of the cells harvested from outlets were all higher than 97% after culturing for 24, 48, and 72 h. The SAIF chip performance was further confirmed using the real clinical patient blood samples from four lung cancer patients. Theoretical force balance analysis in physics, computational simulations, and experimental observations indicate that the SAIF chip is simple but effective, and high-throughput separation CTCs can be readily achieved without complex structures.

Project IDentif.AI: Harnessing Artificial Intelligence to Rapidly Optimize Combination Therapy Development for Infectious Disease Intervention.

In 2019/2020, the emergence of coronavirus disease 2019 (COVID-19) resulted in rapid increases in infection rates as well as patient mortality. Treatment options addressing COVID-19 included drug repurposing, investigational therapies such as remdesivir, and vaccine development. Combination therapy based on drug repurposing is among the most widely pursued of these efforts. Multi-drug regimens are traditionally designed by selecting drugs based on their mechanism of action. This is followed by dose-finding to achieve drug synergy. This approach is widely-used for drug development and repurposing. Realizing synergistic combinations, however, is a substantially different outcome compared to globally optimizing combination therapy, which realizes the best possible treatment outcome by a set of candidate therapies and doses toward a disease indication. To address this challenge, the results of Project IDentif.AI (Identifying Infectious Disease Combination Therapy with Artificial Intelligence) are reported. An AI-based platform is used to interrogate a massive 12 drug/dose parameter space, rapidly identifying actionable combination therapies that optimally inhibit A549 lung cell infection by vesicular stomatitis virus within three days of project start. Importantly, a sevenfold difference in efficacy is observed between the top-ranked combination being optimally and sub-optimally dosed, demonstrating the critical importance of ideal drug and dose identification. This platform is disease indication and disease mechanism-agnostic, and potentially applicable to the systematic N-of-1 and population-wide design of highly efficacious and tolerable clinical regimens. This work also discusses key factors ranging from healthcare economics to global health policy that may serve to drive the broader deployment of this platform to address COVID-19 and future pandemics.

Microdroplets-on-chip: A review.

Single-cell analysis serves as an important approach to study cell functions and interactions. Catering to the demand of Big Data Era, fast reactions for single cells and paralleled high-throughput analysis have become an urgent need. Microdroplet in microfluidics has advantages of modularity and integrity, as well as high throughput and sensitivity, which present great potential in the field of single-cell analysis. This review is carried out on three aspects to introduce microdroplet chips for single-cell analysis: droplet formation, droplet detection and practical functions. Structures of droplet formation are categorized into three types, including T-shaped channel, flow-involved channel and three-dimensional micro-vortice. The detection methods, including fluorescence, Raman spectroscopy, mass spectroscopy and electrochemical detection, are summarized from applications. Both pros and cons for existing techniques are reviewed and discussed. The functions of microdroplets-on-chip cover cell culture, nucleic acid test and cell identification. For each field, principles/mechanisms and/or schematic images are laconically introduced. Microdroplet in microfluidics has become a major research direction in single-cell analysis. With updated methods of droplet formation such as inertial ordering and micro-vortice, microdroplets-based biochips will expect high throughput detection and high-accuracy trace detection for clinical diagnosis in the near future.

High-Throughput Isolation of Circulating Tumor Cells Using Cascaded Inertial Focusing Microfluidic Channel.

Circulating tumor cells (CTCs) are rare cells that detach from a primary or metastasis tumor and flow into the bloodstream. Intact and viable tumor cells are needed for genetic characterization of CTCs, new drug development, and other research. Although separation of CTCs using spiral channel with two outlets has been reported, few literature demonstrated simultaneous isolation of different types of CTCs from human blood using cascaded inertial focusing microfluidic channel. Herein, we introduce a cascaded microfluidic device consisting of two spiral channels and one zigzag channel designed with different fluid fields, including lift force, Dean drag force, and centrifugal force. Both red blood cells (RBCs)-lysed human blood spiked with CTCs and 1:50 diluted human whole blood spiked with CTCs were tested on the presented chip. This chip successfully separated RBCs, white blood cells (WBCs), and two different types of tumor cells (human lung cancer cells (A549) and human breast cancer cells (MCF-7)) simultaneously based on their physical properties. A total of 80.75% of A549 and 73.75% of MCF-7 were faithfully separated from human whole blood. Furthermore, CTCs gathered from outlets could propagate and remained intact. The cell viability of A549 and MCF-7 were 95% and 98%, respectively. The entire separating process for CTCs from blood cells could be finished within 20 min. The cascaded microfluidic device introduced in this study serves as a novel platform for simultaneous isolation of multiple types of CTCs from patient blood.