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Advances in single-cell level profiling of the proteome require quantitative and versatile platforms, especially for rare cell analyses such as circulating tumor cell (CTC) profiling. Here we demonstrate an integrated microfluidic chip that uses magnetic nanoparticles to capture single tumor cells with high efficiency, permits on-chip incubation, and facilitates in situ cell-surface protein expression analysis. Combined with phage-based barcoding and next-generation sequencing technology, we were able to monitor changes in the expression of multiple surface markers stimulated in response to CTC adherence. Interestingly, we found fluctuations in the expression of Frizzled2 (FZD2) that reflected the microenvironment of the single cells. This platform has a high potential for in-depth screening of multiple surface antigens simultaneously in rare cells with single-cell resolution, which will provide further insights regarding biological heterogeneity and human disease.

Circulating tumor cells (CTCs) break free from primary tumors and travel through the circulation system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe an in vivo genome-wide CRISPR knockout screen using CTCs directly isolated from a mouse xenograft. This screen elucidated

Microenvironmental factors play critical roles in regulating stem cell fate, providing a rationale to engineer biomimetic microenvironments that facilitate rapid and effective stem cell differentiation. Three-dimensional (3D) hierarchical microarchitectures have been developed to enable rapid neural differentiation of multipotent human mesenchymal stromal cells (HMSCs) via mechanotransduction. However, low cell viability during long-term culture and poor cell recovery efficiency from the architectures were also observed. Such problems hinder further applications of the architectures in stem cell differentiation. Here, we present improved 3D nanostructured microarchitectures functionalized with cell-adhesion-promoting arginylglycylaspartic acid (RGD) peptides. These RGD-functionalized architectures significantly upregulated long-term cell viability and facilitated effective recovery of differentiated cells from the architectures while maintaining high differentiation efficiency. Efficient recovery of highly viable differentiated cells enabled the downstream analysis of morphology and protein expression to be performed. Remarkably, even after the removal of the mechanical stimulus provided by the 3D microarchitectures, the recovered HMSCs showed a neuron-like elongated morphology for 10 days and consistently expressed microtubule-associated protein 2, a mature neural marker. RGD-functionalized nanostructured microarchitectures hold great potential to guide effective differentiation of highly viable stem cells.

The analysis of circulating tumor cells (CTCs) provides a means to collect information about the evolving properties of a tumor during cancer progression and treatment. For patients with metastatic prostate cancer, noninvasive serial measurements of bloodborne cells may provide a means to tailor therapeutic decisions based on an individual patient's response. Here, we used a high-sensitivity profiling approach to monitor CTCs in patients with metastatic castrate-resistant prostate cancer (mCRPC) undergoing treatment with abiraterone and enzalutamide, two drugs used to treat advanced prostate cancer. The capture and profiling approach uses antibody-functionalized magnetic nanoparticles to sort cells according to protein expression levels. CTCs are tagged with magnetic nanoparticles conjugated to an antibody specific for the epithelial cell adhesion molecule (EpCAM) and sorted into four zones of a microfluidic device based on EpCAM expression levels. Our approach was compared to the FDA-cleared CellSearch method, and we demonstrate significantly higher capture efficiency of low-EpCAM cells compared to the commercial method. The nanoparticle-based approach detected CTCs from 86% of patients at baseline, compared to CellSearch which only detected CTCs from 60% of patients. Patients were stratified as prostate specific antigen (PSA) progressive versus responsive based on clinically acceptable definitions, and it was observed that patients with a limited response to therapy had elevated levels of androgen receptor variant 7 (ARV7) and the mesenchymal marker, N-cadherin, expressed on their CTCs. In addition, these CTCs exhibited lower EpCAM expression. The results highlight features of CTCs associated with disease progression on abiraterone or enzalutamide, including mesenchymal phenotypes and increased expression levels of ARV7. The use of a high-sensitivity method to capture and profile CTCs provides more informative data concerning the phenotypic properties of these cells as patients undergo treatment relative to an FDA-cleared method.

Circulating tumor cells (CTCs) break free from primary tumors and travel through the bloodstream and lymphatic system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe the first in vivo genome-wide CRISPR KO screen using CTCs directly isolated from a mouse xenograft. This screen elucidated SLIT2 – a gene encoding a secreted protein acting as a cellular migration cue – as the most significantly represented gene knockout in the CTC population. SLIT2 knockout cells are highly metastatic with hypermigratory and mesenchymal phenotype. Reduced expression of SLIT2 is observed in human tumors, indicating its role as a negative modulator of tumor progression and metastasis.

A digital microfluidic technique allows for analysis of sub-populations of invading cells by transcriptomics., Microfluidic methods for studying cell invasion can be subdivided into those in which cells invade into free space and those in which cells invade into hydrogels. The former techniques allow straightforward extraction of subpopulations of cells for RNA sequencing, while the latter preserve key aspects of cell interactions with the extracellular matrix (ECM). Here, we introduce “cell invasion in digital microfluidic microgel systems” (CIMMS), which bridges the gap between them, allowing the stratification of cells on the basis of their invasiveness into hydrogels for RNA sequencing. In initial studies with a breast cancer model, 244 genes were found to be differentially expressed between invading and noninvading cells, including genes correlating with ECM-remodeling, chemokine/cytokine receptors, and G protein transducers. These results suggest that CIMMS will be a valuable tool for probing metastasis as well as the many physiological processes that rely on invasion, such as tissue development, repair, and protection.

Molecular-level features of tumours can be tracked via single-cell analyses of circulating tumour cells (CTCs). Yet single-cell measurements of protein expression for rare CTCs are hampered by the presence of a large number of non-target cells. Here, we show that the antibody-mediated labelling of intracellular proteins in the nucleus, mitochondria, and cytoplasm of human cells with magnetic nanoparticles enables the analysis of target proteins at the single-cell level by sorting the cells according to their nanoparticle content in a microfluidic device with cell-capture zones sandwiched between arrays of magnets. We used the magnetic labelling and cell-sorting approach to track the expression of therapeutic protein targets in CTCs isolated from blood samples of mice with orthotopic prostate xenografts and from patients with metastatic castration-resistant prostate cancer. We also show that mutated proteins that are drug targets or markers of therapeutic response can be directly identified in CTCs and analysed at the single-cell level, and used to predict how mice with drug-susceptible and drug-resistant pancreatic tumour xenografts respond to therapy.