Abstract:Breast cancer (BC) affects 1 in every 8 women in the United States and is currently the most prevalent cancer worldwide. Precise staging at diagnosis and prognosis are essential components for the clinical management of BC patients. In this study, we set out to evaluate the feasibility of the high-definition single cell (HDSCA) liquid biopsy (LBx) platform to stratify late-stage BC, early-stage BC, and normal donors using peripheral blood samples. Utilizing 5 biomarkers, we identified rare circulating events w… Show more
“…Figure 1 shows a schematic of the overall data science pipeline. It is important to note that NDs often exhibit small numbers of rare cells across the range of channel types 18 , making a rigorous statistical treatment necessary to distinguish SCLC from ND. Figure 1 HDSCA3.0 Workflow.…”
Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with low five-year survival rates. Recently described molecular phenotypes of SCLC exhibit differential vulnerabilities heralding potential for stratified treatment. Whilst tumor biopsy in SCLC is challenging, circulating tumor cells in the liquid biopsy are prevalent and can be repeatedly sampled accommodating the dynamic plasticity of SCLC phenotypes. The aim of this study was to characterize the heterogeneity of rare circulating cells with confirmed tumor origin and to explore a liquid biopsy approach for future clinical trials of targeted therapies. This study applied the 3rd generation of a previously validated direct imaging platform to 14 chemo-naive SCLC patients and 10 non-cancerous normal donor (ND) samples. Phenotypic heterogeneity of circulating rare cells in SCLC was observed and a patient-level classification model was established to stratify SCLC patients from non-cancerous donors. Eight rare cell groups, with combinations of epithelial, endothelial, and mesenchymal biomarker expression patterns, were phenotypically characterized. The single-cell genomic analysis confirmed the cancer cell plasticity in every rare cell group harboring clonal genomic alterations. This study shows rare cell heterogeneity and confirms cellular plasticity in SCLC providing a valuable resource for better opportunities to discover novel therapeutic targets in SCLC.
“…Figure 1 shows a schematic of the overall data science pipeline. It is important to note that NDs often exhibit small numbers of rare cells across the range of channel types 18 , making a rigorous statistical treatment necessary to distinguish SCLC from ND. Figure 1 HDSCA3.0 Workflow.…”
Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with low five-year survival rates. Recently described molecular phenotypes of SCLC exhibit differential vulnerabilities heralding potential for stratified treatment. Whilst tumor biopsy in SCLC is challenging, circulating tumor cells in the liquid biopsy are prevalent and can be repeatedly sampled accommodating the dynamic plasticity of SCLC phenotypes. The aim of this study was to characterize the heterogeneity of rare circulating cells with confirmed tumor origin and to explore a liquid biopsy approach for future clinical trials of targeted therapies. This study applied the 3rd generation of a previously validated direct imaging platform to 14 chemo-naive SCLC patients and 10 non-cancerous normal donor (ND) samples. Phenotypic heterogeneity of circulating rare cells in SCLC was observed and a patient-level classification model was established to stratify SCLC patients from non-cancerous donors. Eight rare cell groups, with combinations of epithelial, endothelial, and mesenchymal biomarker expression patterns, were phenotypically characterized. The single-cell genomic analysis confirmed the cancer cell plasticity in every rare cell group harboring clonal genomic alterations. This study shows rare cell heterogeneity and confirms cellular plasticity in SCLC providing a valuable resource for better opportunities to discover novel therapeutic targets in SCLC.
“…After washing in TBS for 5 min, the cells were stained with 0.1 μg/mL DAPI (Life Technologies Corp, CA, USA) in TBS for 10 min, washed in TBS for 5 min, dipped in distilled water, mounted with a glycerol-based media, and coverslipped. Slides were then imaged as described below. − …”
With their intricate design, nanoparticles (NPs) have become indispensable tools in the quest for precise cellular targeting. Among various NPs, gold NPs stand out with unique features such as chemical stability, biocompatibility, adjustable shape, and size-dependent optical properties, making them particularly promising for molecular detection by leveraging the surface-enhanced Raman scattering (SERS) effect. Their multiplexing abilities for the simultaneous identification of multiple biomarkers are important in the rapidly evolving landscape of diverse cellular phenotypes and biomolecular profiling. However, the challenge is ensuring that SERS NPs can effectively target specific cells and biomarkers among intricate cell types and biomolecules with high specificity. In this study, we improve the functionalization of SERS NPs, optimizing their targeting efficiency in cellular applications for ca. 160 nm NP-based probes. Spherical SERS NPs, conjugated with antibodies targeting epidermal growth factor receptor and human epidermal growth factor receptor 2, were incubated with cells overexpressing these proteins, and their specific binding potential was quantified at each stage by using flow cytometry to achieve optimal targeting efficiency. We determined that maintaining an average of 3.5 × 10 5 thiols per NP, 300 antibodies per NP, 18,000 NPs per cell, conducting a 15 min staining incubation at 4 °C in a shaker, and using SM(PEG) 12 as a cross-linker for the NP conjugation were crucial to achieve the highest targeting efficiency. Fluorescence and Raman imaging were used with these parameters to observe the maximum ability of these NPs to efficiently target suspended cells. These highly sensitive contrast agents demonstrate their pivotal role in effective active targeting, making them invaluable for multiplexing applications across diverse biological environments.
“…Among these, the third-generation high-definition single-cell assay (HDSCA3.0) workflow provides the ability to identify and characterize epithelial, mesenchymal, endothelial, and hematopoietic cells, as well as large EVs. A study demonstrated that, beyond CTCs, assessing a wider range of circulating events enhanced the ability to categorize normal donors, EBC, and MBC patients into distinct groups ( 133 ).…”
Section: Challenges Of a Multiparametric Approachmentioning
Liquid biopsy has emerged as a crucial tool in managing breast cancer (BC) patients, offering a minimally invasive approach to detect circulating tumor biomarkers. Until recently, the majority of the studies in BC focused on evaluating a single liquid biopsy analyte, primarily circulating tumor DNA and circulating tumor cells (CTCs). Despite the proven prognostic and predictive value of CTCs, their low abundance when detected using enrichment methods, especially in the early stages, poses a significant challenge. It is becoming evident that combining diverse circulating biomarkers, each representing different facets of tumor biology, has the potential to enhance the management of patients with BC. This article emphasizes the importance of considering these biomarkers as complementary/synergistic rather than competitive, recognizing their ability to contribute to a comprehensive disease profile. The review provides an overview of the clinical significance of simultaneously analyzing CTCs and other biomarkers, including cell-free circulating DNA, extracellular vesicles, non-canonical CTCs, cell-free RNAs, and non-malignant cells. Such a comprehensive liquid biopsy approach holds promise not only in BC but also in other cancer types, offering opportunities for early detection, prognostication, and therapy monitoring. However, addressing associated challenges, such as refining detection methods and establishing standardized protocols, is crucial for realizing the full potential of liquid biopsy in transforming our understanding and approach to BC. As the field evolves, collaborative efforts will be instrumental in unlocking the revolutionary impact of liquid biopsy in BC research and management.
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