Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing

Lee, Mei-Chong Wendy; López-Díaz, Fernando J.; Khan, Shahid Y.; Tariq, Muhammad; Dayn, Yelena; Vaske, Charles J.; Radenbaugh, Amie; Kim, Hyunsung John; Emerson, Beverly M.; Pourmand, Nader

doi:10.1073/pnas.1404656111

Cited by 171 publications

(159 citation statements)

References 59 publications

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“…In this study, we confirmed that TSA alone could induce DNA damage in ECSS cell lines by detecting of γH2AX and single-cell gel electrophoresis (Fig. 1) When cells encounter environmental stresses, the cellular transcriptional profiles will be altered to adapt and overcome the stresses [35]. The stress-responsive genes benefit the cell for better survival.…”

Section: Discussionsupporting

confidence: 67%

Knockdown of Rad9A enhanced DNA damage induced by trichostatin A in esophageal cancer cells

Pang

Luo

et al. 2015

Tumor Biol.

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Histone deacetylase (HDAC) inhibitors have recently emerged as a new class of anticancer agents. As a classical HDAC inhibitor, trichostatin A (TSA) has been shown to possess many anticancer activities such as induction of cell cycle arrest, promotion of cell death, and enhancement of radiosensitity. In our previous work, we found that TSA treatment induced Rad9 gene expression, which suggested that Rad9 might play a role in TSA-induced biological effects. As Rad9 is involved in maintaining genomic integrity, we further analyzed the DNA damage induced by TSA and combined with Rad9 knockdown in esophageal cancer cells (ESCCs). Our results showed that TSA treatment alone induced significantly DNA damage in ESCC cells. Simultaneously, TSA also induced Rad9 gene expression both at transcriptional and translational levels in EC109 cells, but not in KYSE150 cells. Further, the induction of Rad9 by TSA was accompanied with increased level of histone H3K9 acetylation in Rad9 promoter region. To understand the role of Rad9 in TSA-induced DNA damage, Rad9 gene expression was efficiently knocked down by small interfering RNA (siRNA), which led to enhanced DNA damage and cell death induced by TSA. Our data suggested that Rad9 plays an important role in DNA damage, which is related to the biological effects of TSA.

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Section: Discussionsupporting

confidence: 67%

Knockdown of Rad9A enhanced DNA damage induced by trichostatin A in esophageal cancer cells

Pang

Luo

et al. 2015

Tumor Biol.

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“…4 In addition to basic functional studies of healthy tissue, single cell transcriptomics also makes it possible to study the states of diseased cells both before and after drug treatment, and has been employed to profile mutations that drive drug resistance in tumors. 5 We recently introduced a photochemical method for more precisely isolating mRNA from single cells within living tissue using Transcriptome In Vivo Analysis (TIVA). 6 …”

Section: Introductionmentioning

confidence: 99%

Oligonucleotide modifications enhance probe stability for single cell transcriptomein vivoanalysis (TIVA)

2017

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Single cell transcriptomics provides a powerful discovery tool for identifying new cell types and functions as well as a means to probe molecular features of the etiology and treatment of human diseases, including cancer. However, such analyses are limited by the difficulty of isolating mRNA from single cells within biological samples. We recently introduced a photochemical method for isolating mRNA from single living cells, Transcriptome In Vivo Analysis (TIVA). The TIVA probe is a “caged” polyU:polyA oligonucleotide hairpin designed to enter live tissue, where site-specific activation with 405 nm laser reveals the polyU-biotin strand to bind mRNA in a target cell, enabling subsequent mRNA isolation and sequencing. The TIVA method is well suited for analysis of living cells in resected tissue, but has not yet been applied to living cells in whole organisms. Adapting TIVA to this more challenging environment requires a probe with higher thermal stability, more robust caging, and greater nuclease resistance. In this paper we present modifications to the original TIVA probe with multiple aspects of enhanced stability. These newer probes utilize an extended 22mer polyU capture strand with two 9mer polyA blocking strands (“22/9/9”) for higher thermal stability pre-photolysis and improved mRNA capture affinity post-photolysis. The “22/9/9 GC” probe features a terminal GC pair to reduce pre-photolysis interactions with mRNA by more than half. The “PS-22/9/9” probe features a phosphorothioated backbone, which extends serum stability from <1 h to at least 48 h, and also mediates uptake into cultured human fibroblasts.

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“…Genetic mutations and functional changes at the proteomic level alter the cellular composition and thus might change inherent density signatures. Further biological changes during many physiological events, such as differentiation (1,2), cell death (3,4), aging (5), immune response (6, 7), or drug resistance (8) are accompanied by transient changes in cellular magnetic signatures, predominantly owing to the formation of intracellular paramagnetic reactive oxygen species. Characterizing these dramatic changes in fundamental cellular properties down to the individual cell level can reveal subpopulations in seemingly homogenous populations that cannot be seen using conventional assays, which average out or dilute changes in these cellular subsets (9).…”

mentioning

confidence: 99%

Magnetic levitation of single cells

Durmus

Tekin

Güven

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

205

297

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Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10. We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine.

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Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing

Cited by 171 publications

References 59 publications

Knockdown of Rad9A enhanced DNA damage induced by trichostatin A in esophageal cancer cells

Knockdown of Rad9A enhanced DNA damage induced by trichostatin A in esophageal cancer cells

Oligonucleotide modifications enhance probe stability for single cell transcriptomein vivoanalysis (TIVA)

Magnetic levitation of single cells

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