Beyond Single-Cell Analysis of Metallodrugs by ICP-MS: Targeting Cellular Substructures

Platinum compounds such as cisplatin (cisPt) embody the backbone of combination chemotherapy protocols against advanced lung cancer. However, their efficacy is primarily limited by inherent or acquired platinum resistance, the origin of which has not been fully elucidated yet, although of paramount interest. Using single cell inductively coupled plasma mass spectrometry (SC-ICP-MS), this study quantifies cisPt in single cancer cells and for the first time in isolated nuclei. A comparison of cisPt uptake was performed between a wild type (wt) cancer cell line and related resistant sublines. In both, resistant cells, wt cells, and their nuclei, cisPt uptake was measured at different incubation times. A lower amount of cisPt was found in resistant cell lines and their nuclei compared to wt cells. Moreover, the abundance of internalized cisPt decreased with increasing resistance. Interestingly, concentrations of cisPt found within the nuclei were higher than compared to cellular concentrations. Here, we show, that SC-ICP-MS allows precise and accurate quantification of metallodrugs in both single cells and cell organelles such as nuclei. These findings pave the way for future applications investigating the potency and efficacy of novel metallodrugs developed for cancer treatment.

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Quantification of single-cell nanoparticle concentrations and the distribution of these concentrations in cell population

Journal of The Royal Society Interface ◽

10.1098/rsif.2013.1152 ◽

2014 ◽

Vol 11 (94) ◽

pp. 20131152 ◽

Cited By ~ 21

Author(s):

Jason T. Rashkow ◽

Sunny C. Patel ◽

Ryan Tappero ◽

Balaji Sitharaman

Keyword(s):

Single Cell ◽

Inductively Coupled Plasma ◽

Single Cell Analysis ◽

Single Cells ◽

Large Population ◽

Cellular Heterogeneity ◽

Physical Parameters ◽

Animal Cells ◽

Promising Tool ◽

Icp Ms

Quantification of nanoparticle uptake into cells is necessary for numerous applications in cellular imaging and therapy. Herein, synchrotron X-ray fluorescence (SXRF) microscopy, a promising tool to quantify elements in plant and animal cells, was employed to quantify and characterize the distribution of titanium dioxide (TiO 2 ) nanosphere uptake in a population of single cells. These results were compared with average nanoparticle concentrations per cell obtained by widely used inductively coupled plasma mass spectrometry (ICP-MS). The results show that nanoparticle concentrations per cell quantified by SXRF were of one to two orders of magnitude greater compared with ICP-MS. The SXRF results also indicate a Gaussian distribution of the nanoparticle concentration per cell. The results suggest that issues relevant to the field of single-cell analysis, the limitation of methods to determine physical parameters from large population averages leading to potentially misleading information and the lack of any information about the cellular heterogeneity are equally relevant for quantification of nanoparticles in cell populations.

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Highly efficient single-cell analysis of microbial cells by time-resolved inductively coupled plasma mass spectrometry

Journal of Analytical Atomic Spectrometry ◽

10.1039/c4ja00040d ◽

2014 ◽

Vol 29 (9) ◽

pp. 1598-1606 ◽

Cited By ~ 35

Author(s):

Shin-ichi Miyashita ◽

Alexander S. Groombridge ◽

Shin-ichiro Fujii ◽

Ayumi Minoda ◽

Akiko Takatsu ◽

...

Keyword(s):

Single Cell ◽

Inductively Coupled Plasma ◽

Single Cell Analysis ◽

High Time Resolution ◽

Time Resolved ◽

Microbial Cells ◽

Inductively Coupled ◽

Highly Efficient ◽

Icp Ms ◽

Plasma Mass Spectrometry

Highly efficient single-cell elemental analysis of microbial cells was achieved using a developed ICP-MS system with approximately 100% cell introduction efficiency and high time resolution.

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Single-cell analysis by use of ICP-MS

Journal of Analytical Atomic Spectrometry ◽

10.1039/d0ja00194e ◽

2020 ◽

Vol 35 (9) ◽

pp. 1784-1813 ◽

Cited By ~ 2

Author(s):

Sarah Theiner ◽

Konrad Loehr ◽

Gunda Koellensperger ◽

Larissa Mueller ◽

Norbert Jakubowski

Keyword(s):

Mass Spectrometry ◽

Single Cell ◽

Inductively Coupled Plasma ◽

Single Cell Analysis ◽

Review Article ◽

Cell Analysis ◽

Inductively Coupled ◽

Icp Ms ◽

Plasma Mass Spectrometry ◽

Recent Trends

This tutorial review article is highlighting the fundamentals, instrumentation, and most recent trends of single-cell analysis by use of inductively coupled plasma-mass spectrometry (ICP-MS).

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Microfluidic platform accelerates tissue processing into single cells for molecular analysis and primary culture models

Nature Communications ◽

10.1038/s41467-021-23238-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jeremy A. Lombardo ◽

Marzieh Aliaghaei ◽

Quy H. Nguyen ◽

Kai Kessenbrock ◽

Jered B. Haun

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Single Cells ◽

Cell Analysis ◽

Processing Technologies ◽

Microfluidic Platform ◽

Tissue Processing ◽

Single Cell Rna Sequencing ◽

Culture Models ◽

Tissue Digestion

AbstractTissues are complex mixtures of different cell subtypes, and this diversity is increasingly characterized using high-throughput single cell analysis methods. However, these efforts are hindered, as tissues must first be dissociated into single cell suspensions using methods that are often inefficient, labor-intensive, highly variable, and potentially biased towards certain cell subtypes. Here, we present a microfluidic platform consisting of three tissue processing technologies that combine tissue digestion, disaggregation, and filtration. The platform is evaluated using a diverse array of tissues. For kidney and mammary tumor, microfluidic processing produces 2.5-fold more single cells. Single cell RNA sequencing further reveals that endothelial cells, fibroblasts, and basal epithelium are enriched without affecting stress response. For liver and heart, processing time is dramatically reduced. We also demonstrate that recovery of cells from the system at periodic intervals during processing increases hepatocyte and cardiomyocyte numbers, as well as increases reproducibility from batch-to-batch for all tissues.

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Multiplexed profiling of single-cell extracellular vesicles secretion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814348116 ◽

2019 ◽

Vol 116 (13) ◽

pp. 5979-5984 ◽

Cited By ~ 22

Author(s):

Yahui Ji ◽

Dongyuan Qi ◽

Linmei Li ◽

Haoran Su ◽

Xiaojie Li ◽

...

Keyword(s):

Single Cell ◽

Cell Lines ◽

Extracellular Vesicles ◽

Single Cell Analysis ◽

Comprehensive Evaluation ◽

Single Cells ◽

Deep Understanding ◽

Principal Component ◽

Cell Heterogeneity ◽

Indispensable Tool

Extracellular vesicles (EVs) are important intercellular mediators regulating health and diseases. Conventional methods for EV surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EV secretion. Herein, by using spatially patterned antibody barcodes, we realized multiplexed profiling of single-cell EV secretion from more than 1,000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to a deep understanding of previously undifferentiated single-cell heterogeneity underlying EV secretion. Notably, we observed that the decrement of certain EV phenotypes (e.g.,CD63+EV) was associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EV secretion and cytokines secretion simultaneously from the same single cells to investigate the multidimensional spectrum of cellular communications, from which we resolved tiered functional subgroups with distinct secretion profiles by visualized clustering and principal component analysis. In particular, we found that different cell subgroups dominated EV secretion and cytokine secretion. The technology introduced here enables a comprehensive evaluation of EV secretion heterogeneity at single-cell level, which may become an indispensable tool to complement current single-cell analysis and EV research.

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A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS

The Analyst ◽

10.1039/c6an02638a ◽

2017 ◽

Vol 142 (10) ◽

pp. 1703-1710 ◽

Cited By ~ 23

Author(s):

A. J. Herrmann ◽

S. Techritz ◽

N. Jakubowski ◽

A. Haase ◽

A. Luch ◽

...

Keyword(s):

Mass Spectrometry ◽

Inductively Coupled Plasma ◽

Single Cells ◽

Staining Procedure ◽

Simple Metal ◽

Inductively Coupled ◽

Icp Ms ◽

High Lateral Resolution ◽

Plasma Mass Spectrometry ◽

Staining Methods

High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods.

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Direct observation of frequency modulated transcription in single cells using light activation

eLife ◽

10.7554/elife.00750 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 90

Author(s):

Daniel R Larson ◽

Christoph Fritzsch ◽

Liang Sun ◽

Xiuhau Meng ◽

David S Lawrence ◽

...

Keyword(s):

Single Cell ◽

Single Molecule ◽

Single Cell Analysis ◽

Cell Model ◽

Single Cells ◽

Single Gene ◽

Gene Activation ◽

Light Activation ◽

Dynamic Synthesis ◽

Single Cell Model

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus.

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More than efficacy revealed by single-cell analysis of antiviral therapeutics

10.1101/606715 ◽

2019 ◽

Author(s):

Wu Liu ◽

Mehmet U. Caglar ◽

Zhangming Mao ◽

Andrew Woodman ◽

Jamie J. Arnold ◽

...

Keyword(s):

Single Cell ◽

Drug Targets ◽

Single Cell Analysis ◽

Single Cells ◽

Principal Component ◽

Viral Population ◽

Cell Analysis ◽

Toxic Dose ◽

Time Required

SUMMARYDevelopment of antiviral therapeutics emphasizes minimization of the effective dose and maximization of the toxic dose, first in cell culture and later in animal models. Long-term success of an antiviral therapeutic is determined not only by its efficacy but also by the duration of time required for drug-resistance to evolve. We have developed a microfluidic device comprised of ~6000 wells, with each well containing a microstructure to capture single cells. We have used this device to characterize enterovirus inhibitors with distinct mechanisms of action. In contrast to population methods, single-cell analysis reveals that each class of inhibitor interferes with the viral infection cycle in a manner that can be distinguished by principal component analysis. Single-cell analysis of antiviral candidates reveals not only efficacy but also properties of the members of the viral population most sensitive to the drug, the stage of the lifecycle most affected by the drug, and perhaps even if the drug targets an interaction of the virus with its host.

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μCB-seq: Microfluidic cell barcoding and sequencing for high-resolution imaging and sequencing of single cells

10.1101/2020.02.18.954974 ◽

2020 ◽

Author(s):

Tyler N. Chen ◽

Anushka Gupta ◽

Mansi Zalavadia ◽

Aaron M. Streets

Keyword(s):

High Resolution ◽

Single Cell ◽

Single Cell Analysis ◽

Protein Localization ◽

Single Cells ◽

Optical Measurements ◽

Detection Sensitivity ◽

High Resolution Imaging ◽

Cell Analysis ◽

Resolution Imaging

AbstractSingle-cell RNA sequencing (scRNA-seq) enables the investigation of complex biological processes in multicellular organisms with high resolution. However, many phenotypic features that are critical to understanding the functional role of cells in a heterogeneous tissue or organ are not directly encoded in the genome and therefore cannot be profiled with scRNA-seq. Quantitative optical microscopy has long been a powerful approach for characterizing diverse cellular phenotypes including cell morphology, protein localization, and chemical composition. Combining scRNA-seq with optical imaging has the potential to provide comprehensive single-cell analysis, allowing for functional integration of gene expression profiling and cell-state characterization. However, it is difficult to track single cells through both measurements; therefore, coupling current scRNA-seq protocols with optical measurements remains a challenge. Here, we report Microfluidic Cell Barcoding and Sequencing (μCB-seq), a microfluidic platform that combines high-resolution imaging and sequencing of single cells. μCB-seq is enabled by a novel fabrication method that preloads primers with known barcode sequences inside addressable reaction chambers of a microfluidic device. In addition to enabling multi-modal single-cell analysis, μCB-seq improves gene detection sensitivity, providing a scalable and accurate method for information-rich characterization of single cells.

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MBRS-59. SINGLE-CELL WHOLE-GENOME SEQUENCING DISSECTS INTRA-TUMOURAL GENOMIC HETEROGENEITY AND CLONAL EVOLUTION IN CHILDHOOD MEDULLOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.563 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii408-iii408

Author(s):

Marina Danilenko ◽

Masood Zaka ◽

Claire Keeling ◽

Stephen Crosier ◽

Rafiqul Hussain ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Single Cell ◽

Genome Sequencing ◽

Copy Number ◽

Single Cell Analysis ◽

Mutational Analysis ◽

Single Cells ◽

Clonal Evolution ◽

Whole Genome ◽

Clinically Significant

Abstract Medulloblastomas harbor clinically-significant intra-tumoral heterogeneity for key biomarkers (e.g. MYC/MYCN, β-catenin). Recent studies have characterized transcriptional heterogeneity at the single-cell level, however the underlying genomic copy number and mutational architecture remains to be resolved. We therefore sought to establish the intra-tumoural genomic heterogeneity of medulloblastoma at single-cell resolution. Copy number patterns were dissected by whole-genome sequencing in 1024 single cells isolated from multiple distinct tumour regions within 16 snap-frozen medulloblastomas, representing the major molecular subgroups (WNT, SHH, Group3, Group4) and genotypes (i.e. MYC amplification, TP53 mutation). Common copy number driver and subclonal events were identified, providing clear evidence of copy number evolution in medulloblastoma development. Moreover, subclonal whole-arm and focal copy number alterations covering important genomic loci (e.g. on chr10 of SHH patients) were detected in single tumour cells, yet undetectable at the bulk-tumor level. Spatial copy number heterogeneity was also common, with differences between clonal and subclonal events detected in distinct regions of individual tumours. Mutational analysis of the cells allowed dissection of spatial and clonal heterogeneity patterns for key medulloblastoma mutations (e.g. CTNNB1, TP53, SMARCA4, PTCH1) within our cohort. Integrated copy number and mutational analysis is underway to establish their inter-relationships and relative contributions to clonal evolution during tumourigenesis. In summary, single-cell analysis has enabled the resolution of common mutational and copy number drivers, alongside sub-clonal events and distinct patterns of clonal and spatial evolution, in medulloblastoma development. We anticipate these findings will provide a critical foundation for future improved biomarker selection, and the development of targeted therapies.

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