Circulating tumor cell characterization of lung cancer brain metastasis in the cerebrospinal fluid through single-cell transcriptome analysis

AbstractMetastatic lung cancer accounts for about half of the brain metastases (BM). Development of leptomeningeal metastases (LM) are becoming increasingly common, and its prognosis is still poor despite the advances in systemic and local approaches. Cytology analysis in the cerebrospinal fluid (CSF) remains the diagnostic gold standard. Although several previous studies performed in CSF have offered great promise for the diagnostics and therapeutics of LM, a comprehensive characterization of circulating tumor cells (CTCs) in CSF is still lacking. To fill this critical gap of lung adenocarcinoma LM (LUAD-LM), we analyzed the transcriptomes of 1,375 cells from 5 LUAD-LM patient and 3 control samples using single-cell RNA sequencing technology. We defined CSF-CTCs based on abundant expression of epithelial markers and genes with lung origin, as well as the enrichment of metabolic pathway and cell adhesion molecules, which are crucial for the survival and metastases of tumor cells. Elevated expression of CEACAM6 and SCGB3A2 was discovered in CSF-CTCs, which could serve as candidate biomarkers of LUAD-LM. We identified substantial heterogeneity in CSF-CTCs among LUAD-LM patients and within patient among individual cells. Cell-cycle gene expression profiles and the proportion of CTCs displaying mesenchymal and cancer stem cell properties also vary among patients. In addition, CSF-CTC transcriptome profiling identified one LM case as cancer of unknown primary site (CUP). Our results will shed light on the mechanism of LUAD-LM and provide a new direction of diagnostic test of LUAD-LM and CUP cases from CSF samples.

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Single-cell characterization of CRISPR-modified transcript isoforms with nanopore sequencing

Genome Biology ◽

10.1186/s13059-021-02554-1 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Heon Seok Kim ◽

Susan M. Grimes ◽

Anna C. Hooker ◽

Billy T. Lau ◽

Hanlee P. Ji

Keyword(s):

Single Cell ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Splicing Factors ◽

Mrna Isoforms ◽

Structure Detection ◽

Long Read ◽

Cell Gene Expression ◽

Transcript Structure

AbstractWe developed a single-cell approach to detect CRISPR-modified mRNA transcript structures. This method assesses how genetic variants at splicing sites and splicing factors contribute to alternative mRNA isoforms. We determine how alternative splicing is regulated by editing target exon-intron segments or splicing factors by CRISPR-Cas9 and their consequences on transcriptome profile. Our method combines long-read sequencing to characterize the transcript structure and short-read sequencing to match the single-cell gene expression profiles and gRNA sequence and therefore provides targeted genomic edits and transcript isoform structure detection at single-cell resolution.

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Diagnostic Leukapheresis Enables Reliable Transcriptomic Profiling of Single Circulating Tumor Cells to Characterize Inter-Cellular Heterogeneity in Terms of Endocrine Resistance

Cancers ◽

10.3390/cancers11070903 ◽

2019 ◽

Vol 11 (7) ◽

pp. 903 ◽

Cited By ~ 8

Author(s):

Florian Reinhardt ◽

André Franken ◽

Franziska Meier-Stiegen ◽

Christiane Driemel ◽

Nikolas H. Stoecklein ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Single Cell ◽

Endocrine Therapy ◽

Tumor Cells ◽

Expression Profiles ◽

Endocrine Resistance ◽

Gene Expression Profiles ◽

Transcriptome Profiling ◽

Patient Heterogeneity

Circulating tumor cells (CTCs) hold great promise with regard to prognosis, treatment optimization, and monitoring of breast cancer patients. Single CTC transcriptome profiling might help reveal valuable information concerning intra-patient heterogeneity relevant to therapeutic interventions. In this study, we combined Diagnostic Leukapheresis (DLA), which is a microfluidic enrichment using the ParsortixTM system, micromanipulation with CellCelectorTM and subsequent single cell multi-marker transcriptome profiling. First, a PCR panel consisting of 30 different endocrine resistance and phenotypic marker genes was validated for single cell profiling by using different breast cancer cell lines. Second, this panel was applied to characterize uncultured and cultured CTCs, which were enriched from a cryopreserved DLA product obtained from a patient suffering from metastatic breast cancer resistant to endocrine therapy. Gene expression profiles of both CTC populations uncovered inter CTC heterogeneity for transcripts, which are associated with response or resistance to endocrine therapy (e.g., ESR1, HER2, FGFR1). Hierarchical clustering revealed CTC subpopulations with different expressions of transcripts regarding the CTCs’ differential phenotypes (EpCAM, CD44, CD24, MYC, MUC1) and of transcripts involved in endocrine signaling pathways (FOXO, PTEN). Moreover, ER-positive CTCs exhibited significant higher expression of Cyclin D1, which might be relevant for CDK4/6 inhibitor therapies. Overall, gene expression profiles of uncultured and cultured CTCs resulted in a partly combined grouping. Our findings demonstrate that multi-marker RNA profiling of enriched single uncultured CTCs and cultured CTCs form cryopreserved DLA samples may provide important insights into intra-patient heterogeneity relevant for targeted therapies and therapy resistance.

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Circulating tumor cell characterization of lung cancer brain metastases in the cerebrospinal fluid through single‐cell transcriptome analysis

Clinical and Translational Medicine ◽

10.1002/ctm2.246 ◽

2020 ◽

Vol 10 (8) ◽

Author(s):

Haoyu Ruan ◽

Yihang Zhou ◽

Jie Shen ◽

Yue Zhai ◽

Ying Xu ◽

...

Keyword(s):

Lung Cancer ◽

Cerebrospinal Fluid ◽

Brain Metastases ◽

Tumor Cell ◽

Single Cell ◽

Transcriptome Analysis ◽

Circulating Tumor Cell ◽

Cell Transcriptome ◽

Single Cell Transcriptome

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Stromal Cells Derived from Non-Small Cell Lung Cancer and Normal Lung Tissue Display Mesenchymal Stem Cell Characteristics and Differ in Their Gene Expression Profiles and Functional Behaviour

Pneumologie ◽

10.1055/s-0029-1213954 ◽

2009 ◽

Vol 63 (S 01) ◽

Author(s):

S Gottschling ◽

A Jauch ◽

M Granzow ◽

R Kuner ◽

T Muley ◽

...

Keyword(s):

Gene Expression ◽

Lung Cancer ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Stromal Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Small Cell ◽

Normal Lung ◽

Small Cell Lung

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The Jekyll and Hyde of Cellular Senescence in Cancer

Cells ◽

10.3390/cells10020208 ◽

2021 ◽

Vol 10 (2) ◽

pp. 208

Author(s):

Dilara Demirci ◽

Bengisu Dayanc ◽

Fatma Aybuke Mazi ◽

Serif Senturk

Keyword(s):

Cancer Cell ◽

Cellular Senescence ◽

Expression Profiles ◽

Treatment Strategies ◽

Gene Expression Profiles ◽

Therapy Resistance ◽

Disease Recurrence ◽

Adverse Outcomes ◽

Cell Senescence ◽

Great Promise

Cellular senescence is a state of stable cell cycle arrest that can be triggered in response to various insults and is characterized by distinct morphological hallmarks, gene expression profiles, and the senescence-associated secretory phenotype (SASP). Importantly, cellular senescence is a key component of normal physiology with tumor suppressive functions. In the last few decades, novel cancer treatment strategies exploiting pro-senescence therapies have attracted considerable interest. Recent insight, however, suggests that therapy-induced senescence (TIS) elicits cell-autonomous and non-cell-autonomous implications that potentially entail detrimental consequences, reflecting the Jekyll and Hyde nature of cancer cell senescence. In essence, the undesirable manifestations that generally culminate in inflammation, cancer stemness, senescence reversal, therapy resistance, and disease recurrence are dictated by the persistent accumulation of senescent cells and the SASP. Thus, mitigating these pro-tumorigenic effects by eliminating these cells or inhibiting their SASP production holds great promise for developing innovative therapeutic strategies. In this review, we describe the fundamental aspects and dynamics of cancer cell senescence and summarize the comprehensive research on the adverse outcomes of TIS. Furthermore, we underline the rationale and motivation of emerging senotherapeutic modalities surrounding the removal of senescent cells and the SASP to help maximize the overall efficacy of cancer therapies.

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4 Molecularly guided multiplexed digital spatial analysis reveals differential gene expression profiles in the WNT-β-catenin pathway between melanoma and prostate tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0004 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A4-A4

Author(s):

Anushka Dikshit ◽

Dan Zollinger ◽

Karen Nguyen ◽

Jill McKay-Fleisch ◽

Kit Fuhrman ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Single Molecule ◽

Spatial Information ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Fluorescence Assay ◽

Differentially Expressed ◽

Tumor Type ◽

Prostate Tumors

BackgroundThe canonical WNT-β-catenin signaling pathway is vital for development and tissue homeostasis but becomes strongly tumorigenic when dysregulated. and alter the transcriptional signature of a cell to promote malignant transformation. However, thorough characterization of these transcriptomic signatures has been challenging because traditional methods lack either spatial information, multiplexing, or sensitivity/specificity. To overcome these challenges, we developed a novel workflow combining the single molecule and single cell visualization capabilities of the RNAscope in situ hybridization (ISH) assay with the highly multiplexed spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler (DSP) RNA assays. Using these methods, we sought to spatially profile and compare gene expression signatures of tumor niches with high and low CTNNB1 expression.MethodsAfter screening 120 tumor cores from multiple tumors for CTNNB1 expression by the RNAscope assay, we identified melanoma as the tumor type with the highest CTNNB1 expression while prostate tumors had the lowest expression. Using the RNAscope Multiplex Fluorescence assay we selected regions of high CTNNB1 expression within 3 melanoma tumors as well as regions with low CTNNB1 expression within 3 prostate tumors. These selected regions of interest (ROIs) were then transcriptionally profiled using the GeoMx DSP RNA assay for a set of 78 genes relevant in immuno-oncology. Target genes that were differentially expressed were further visualized and spatially assessed using the RNAscope Multiplex Fluorescence assay to confirm GeoMx DSP data with single cell resolution.ResultsThe GeoMx DSP analysis comparing the melanoma and prostate tumors revealed that they had significantly different gene expression profiles and many of these genes showed concordance with CTNNB1 expression. Furthermore, immunoregulatory targets such as ICOSLG, CTLA4, PDCD1 and ARG1, also demonstrated significant correlation with CTNNB1 expression. On validating selected targets using the RNAscope assay, we could distinctly visualize that they were not only highly expressed in melanoma compared to the prostate tumor, but their expression levels changed proportionally to that of CTNNB1 within the same tumors suggesting that these differentially expressed genes may be regulated by the WNT-β-catenin pathway.ConclusionsIn summary, by combining the RNAscope ISH assay and the GeoMx DSP RNA assay into one joint workflow we transcriptionally profiled regions of high and low CTNNB1 expression within melanoma and prostate tumors and identified genes potentially regulated by the WNT- β-catenin pathway. This novel workflow can be fully automated and is well suited for interrogating the tumor and stroma and their interactions.GeoMx Assays are for RESEARCH ONLY, not for diagnostics.

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Single-Cell Expression Landscape of SARS-CoV-2 Receptor ACE2 and Host Proteases in Normal and Malignant Lung Tissues from Pulmonary Adenocarcinoma Patients

Cancers ◽

10.3390/cancers13061250 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1250

Author(s):

Guangchun Han ◽

Ansam Sinjab ◽

Kieko Hara ◽

Warapen Treekitkarnmongkol ◽

Patrick Brennan ◽

...

Keyword(s):

Lung Cancer ◽

Single Cell ◽

Cancer Patients ◽

Expression Profiles ◽

Lung Cells ◽

Alveolar Type ◽

Specific Expression ◽

Lung Cancer Patients ◽

Normal Tissues ◽

Cell Expression

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

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