RESUMEN
As pancreatic cancer is the third deadliest cancer in the U.S., the ability to study genetic alterations is necessary to provide further insight into potentially targetable regions for cancer treatment. Circulating tumor cells (CTCs) represent an especially aggressive subset of cancer cells, capable of causing metastasis and progressing the disease. Here, we present the Labyrinth-DEPArray pipeline for the isolation and analysis of single CTCs. Established cell lines, patient-derived CTC cell lines and freshly isolated CTCs were recovered and sequenced to reveal single-cell copy number variations (CNVs). The resulting CNV profiles of established cell lines showed concordance with previously reported data and highlight several gains and losses of cancer-related genes such as FGFR3 and GNAS. The novel sequencing of patient-derived CTC cell lines showed gains in chromosome 8q, 10q and 17q across both CTC cell lines. The pipeline was used to process and isolate single cells from a metastatic pancreatic cancer patient revealing a gain of chromosome 1q and a loss of chromosome 5q. Overall, the Labyrinth-DEPArray pipeline offers a validated workflow combining the benefits of antigen-free CTC isolation with single cell genomic analysis.
Asunto(s)
Células Neoplásicas Circulantes , Neoplasias Pancreáticas , Biomarcadores de Tumor , Variaciones en el Número de Copia de ADN , Genómica , Humanos , Células Neoplásicas Circulantes/patología , Neoplasias Pancreáticas/genética , Flujo de Trabajo , Neoplasias PancreáticasRESUMEN
The recent push toward understanding an individual cell's behavior and identifying cellular heterogeneity has created an unmet need for technologies that can probe live cells at the single-cell level. Cells within a population are known to exhibit heterogeneous responses to environmental cues. These differences can lead to varied cellular states, behavior, and responses to therapeutics. Techniques are needed that are not only capable of processing and analyzing cellular populations at the single cell level, but also have the ability to isolate specific cell populations from a complex sample at high throughputs. The new CellMag-Coalesce-Attract-Resegment Wash (CellMag-CARWash) system combines positive magnetic selection with droplet microfluidic devices to isolate cells of interest from a mixture with >93% purity and incorporate treatments within individual droplets to observe single cell biological responses. This workflow is shown to be capable of probing the single cell extracellular vesicle (EV) secretion of MCF7 GFP cells. This article reports the first measurement of ß-Estradiol's effect on EV secretion from MCF7 cells at the single cell level. Single cell processing revealed that MCF7 GFP cells possess a heterogeneous response to ß-Estradiol stimulation with a 1.8-fold increase relative to the control.
Asunto(s)
Separación Celular , Análisis de la Célula Individual , Humanos , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/instrumentación , Células MCF-7 , Separación Celular/métodos , Separación Celular/instrumentación , Dispositivos Laboratorio en un Chip , Vesículas Extracelulares/fisiología , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Estradiol/farmacologíaRESUMEN
Periodontitis affects billions of people worldwide. To address relationships of periodontal niche cell types and microbes in periodontitis, we generated an integrated single-cell RNA sequencing (scRNAseq) atlas of human periodontium (34-sample, 105918-cell), including sulcular and junctional keratinocytes (SK/JKs). SK/JKs displayed altered differentiation states and were enriched for effector cytokines in periodontitis. Single-cell metagenomics revealed 37 bacterial species with cell-specific tropism. Fluorescence in situ hybridization detected intracellular 16 S and mRNA signals of multiple species and correlated with SK/JK proinflammatory phenotypes in situ. Cell-cell communication analysis predicted keratinocyte-specific innate and adaptive immune interactions. Highly multiplexed immunofluorescence (33-antibody) revealed peri-epithelial immune foci, with innate cells often spatially constrained around JKs. Spatial phenotyping revealed immunosuppressed JK-microniches and SK-localized tertiary lymphoid structures in periodontitis. Here, we demonstrate impacts on and predicted interactomics of SK and JK cells in health and periodontitis, which requires further investigation to support precision periodontal interventions in states of chronic inflammation.
Asunto(s)
Comunicación Celular , Queratinocitos , Periodontitis , Análisis de la Célula Individual , Humanos , Queratinocitos/metabolismo , Queratinocitos/inmunología , Periodontitis/microbiología , Periodontitis/metabolismo , Periodontitis/inmunología , Periodontitis/patología , Citocinas/metabolismo , Periodoncio/microbiología , Periodoncio/metabolismo , Periodoncio/patología , Inmunidad Innata , Hibridación Fluorescente in Situ , Masculino , Metagenómica/métodos , Bacterias/metabolismo , Bacterias/genética , Femenino , Adulto , Inmunidad AdaptativaRESUMEN
The mutational and phenotypic landscape of tumors is dynamic, requiring constant monitoring of cancer patients to provide the most up-to-date and effective care. Circulating tumor cells (CTCs) obtained via liquid biopsy can provide tumor DNA, RNA, and protein information that can aid in the diagnosis, prognosis, and treatment of patients. There have been many recent studies and advances in using CTC enumeration, characterization, and expansion to provide personalized cancer treatment, validating the benefit of using CTCs as a biomarker in standard of care procedures. In this paper, we aim to summarize these advances, their limitations, and suggest future areas of study necessary to bring CTC analysis to clinics.