Phenotypic Profiling of Circulating Tumor Cells in Metastatic Prostate Cancer Patients Using Nanoparticle-Mediated Ranking.

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.

A magneto-activated nanoscale cytometry platform for molecular profiling of small extracellular vesicles.

Exosomal PD-L1 (exoPD-L1) has recently received significant attention as a biomarker predicting immunotherapeutic responses involving the PD1/PD-L1 pathway. However, current technologies for exosomal analysis rely primarily on bulk measurements that do not consider the heterogeneity found within exosomal subpopulations. Here, we present a nanoscale cytometry platform NanoEPIC, enabling phenotypic sorting and exoPD-L1 profiling from blood plasma. We highlight the efficacy of NanoEPIC in monitoring anti-PD-1 immunotherapy through the interrogation of exoPD-L1. NanoEPIC generates signature exoPD-L1 patterns in responders and non-responders. In mice treated with PD1-targeted immunotherapy, exoPD-L1 is correlated with tumor growth, PD-L1 burden in tumors, and the immune suppression of CD8+ tumor-infiltrating lymphocytes. Small extracellular vesicles (sEVs) with different PD-L1 expression levels display distinctive inhibitory effects on CD8 + T cells. NanoEPIC offers robust, high-throughput profiling of exosomal markers, enabling sEV subpopulation analysis. This platform holds the potential for enhanced cancer screening, personalized treatment, and therapeutic response monitoring.

nuPRISM: Microfluidic Genome-Wide Phenotypic Screening Platform for Cellular Nuclei.

Genome-wide loss-of-function screens are critical tools to identify novel genetic regulators of intracellular proteins. However, studying the changes in the organelle-specific expression profile of intracellular proteins can be challenging due to protein localization differences across the whole cell, hindering context-dependent protein expression and activity analyses. Here, we describe nuPRISM, a microfluidics chip specifically designed for large-scale isolated nuclei sorting. The new device enables rapid genome-wide loss-of-function phenotypic CRISPR-Cas9 screens directed at intranuclear targets. We deployed this technology to identify novel genetic regulators of ß-catenin nuclear accumulation, a phenotypic hallmark of APC-mutated colorectal cancer. nuPRISM expands our ability to capture aberrant nuclear morphological and functional traits associated with distinctive signal transduction and subcellular localization-driven functional processes with substantial resolution and high throughput.

Cell invasion in digital microfluidic microgel systems.

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.

A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer related to asbestos exposure. Early diagnosis is challenging due to generic symptoms and a lack of biomarkers. We previously demonstrated that mesothelial precursor cells (MPC) characterized by mesothelin (MSLN)+CD90+CD34+ could be implicated in the development of mesothelioma after asbestos exposure. Here, we aimed to determine the clinical significance of detecting MPC in blood for early-stage diagnosis and prognosis of mesothelioma. METHODS: Due to the rarity of MPC in blood, it is challenging to identify this cell population using conventional techniques. Hence, we have developed a microfluidic liquid biopsy platform called MesoFind that utilizes an immunomagnetic, mesothelin capture strategy coupled with immunofluorescence to identify rare populations of cells at high sensitivity and precision. To validate our technique, we compared this approach to flow cytometry for the detection of MPC in murine blood and lavage samples. Upon successful validation of the murine samples, we then proceeded to examine circulating MPC in 23 patients with MPM, 23 asbestos-exposed individuals (ASB), and 10 healthy donors (HD) to evaluate their prognostic and diagnostic value. FINDING: MPC were successfully detected in the blood of murine samples using MesoFind but were undetectable with flow cytometry. Circulating MPC were significantly higher in patients with epithelioid MPM compared to HD and ASB. The MPC subpopulation, MSLN+ and CD90+, were upregulated in ASB compared to HD suggesting an early role in pleural damage from asbestos. The MPC subpopulation, MSLN+ and CD34+, in contrast, were detected in advanced MPM and associated with markers of poor prognosis, suggesting a predominant role during cancer progression. INTERPRETATION: The identification of circulating MPC presents an attractive solution for screening and early diagnosis of epithelioid mesothelioma. The presence of different subtypes of MPC have a prognostic value that could be of assistance with clinical decisions in patients with MPM. FUNDING: Princess Margaret Hospital Foundation Mesothelioma Research Fund, Toronto General & Western Hospital Foundation.