Analysis of the Single-Cell Heterogeneity of Adenocarcinoma Cell Lines and the Investigation of Intratumor Heterogeneity Reveals the Expression of Transmembrane Protein 45A (TMEM45A) in Lung Adenocarcinoma Cancer Patients

Neuperger, Patrícia; Balog, J.; Tiszlavicz, László; Furák, József; Gémes, Nikolett; Kotogány, Edit; Szalontai, Klára; Puskás, László G.; Szebeni, Gábor J.

doi:10.3390/cancers14010144

Cited by 11 publications

(8 citation statements)

References 80 publications

(93 reference statements)

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“…Due to this precise performance of ZeptoCTC, we argue that the observed signal variabilities observed between single cells – also of the same cell line – are pointing toward well-known cell-to-cell heterogeneity, a phenomenon that has been previously noted in other single cell experiments (43–45). For instance, EpCAM expression levels in two MCF7 cells were highly different, matching observations we have made previously suggesting a continuum of EpCAM-positivity in MCF-7 cells (46).…”

Section: Discussionsupporting

confidence: 54%

ZeptoCTC - Sensitive Protein Analysis of True Single Cell Lysates using Reverse Phase Protein Arrays (RPPA)

Rivandi,

Franken,

Yang

et al. 2023

Preprint

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Circulating Tumor Cells (CTCs) are commonly analyzed through genomic profiling, which does not capture posttranslational and functional alterations of encoded proteins. To address this limitation, we developed ZeptoCTC, a single-cell protein analysis workflow that combines established technologies for single-cell isolation and sensitive Reverse Phase Protein Array (RPPA) analysis to assess multiple protein expression and activation in individual CTCs. The workflow involves single cell labeling, isolation, lysis, and printing of the true single cell lysates onto a ZeptoChip using a modified micromanipulator CellCelector™. Subsequently, the printed lysates undergo fluorescence immunoassay RPPA protein detection using a ZeptoReader followed by signal quantification with Image J software. ZeptoCTC was successfully optimized, beginning with the measurement of EpCAM protein expression, a standard marker for CTC detection. As expected, mean fluorescence signals for EpCAM levels were significantly higher in single MCF-7 cells compared to MDA-MB-231 cells. Next, Capivasertib-treated MCF-7 cells exhibited an approximately 2-fold increase in the pAkt/Akt ratio compared to non-treated control cells. This finding was consistent with a co-performed western blot analysis of pooled MCF-7 cells. Application of ZeptoCTC to the analysis of single CTCs derived from a metastasized breast cancer (MBC) patient indicated a significantly higher level of pAkt, accompanied by a corresponding increase in pErk level when compared to patient-matched WBC. Finally, the current workflow successfully indicated the detectable pAkt and Akt signal difference in CTCs from two MBC patients: one with an Akt1 wild-type genotype, and the other harboring approximately 80% Akt1(E17K) mutated CTCs. The mutated CTCs revealed clearly elevated pAkt levels (1.8-fold), along with an even more strongly elevated total Akt (3.4-fold) when compared to the respective signals measured in wild-type CTCs. In conclusion, ZeptoCTC is a highly sensitive method for measuring the expression and phosphorylation of treatment-relevant proteins in key cancer-driving signaling pathways from true single cell samples.

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Section: Discussionsupporting

confidence: 54%

ZeptoCTC - Sensitive Protein Analysis of True Single Cell Lysates using Reverse Phase Protein Arrays (RPPA)

Rivandi,

Franken,

Yang

et al. 2023

Preprint

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“…Mass cytometry was performed as previously described by our group with minor modifications ( 38 , 39 ). Briefly, cells were resuspended in 50 µl of MCSB supplemented with 1:20 v/v Human TruStain FcX Fc Receptor Blocking Solution (BioLegend, San Diego, California, USA) and incubated at RT for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Comparative single-cell multiplex immunophenotyping of therapy-naive patients with rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus shed light on disease-specific composition of the peripheral immune system

Balog,

Zvara,

Bukovinszki

et al. 2024

Front. Immunol.

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IntroductionSystemic autoimmune diseases (SADs) are a significant burden on the healthcare system. Understanding the complexity of the peripheral immunophenotype in SADs may facilitate the differential diagnosis and identification of potential therapeutic targets.MethodsSingle-cell mass cytometric immunophenotyping was performed on peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and therapy-naive patients with rheumatoid arthritis (RA), progressive systemic sclerosis (SSc), and systemic lupus erythematosus (SLE). Immunophenotyping was performed on 15,387,165 CD45+ live single cells from 52 participants (13 cases/group), using an antibody panel to detect 34 markers.ResultsUsing the t-SNE (t-distributed stochastic neighbor embedding) algorithm, the following 17 main immune cell types were determined: CD4+/CD57– T cells, CD4+/CD57+ T cells, CD8+/CD161– T cells, CD8+/CD161+/CD28+ T cells, CD8dim T cells, CD3+/CD4–/CD8– T cells, TCRγ/δ T cells, CD4+ NKT cells, CD8+ NKT cells, classic NK cells, CD56dim/CD98dim cells, B cells, plasmablasts, monocytes, CD11cdim/CD172dim cells, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs). Seven of the 17 main cell types exhibited statistically significant frequencies in the investigated groups. The expression levels of the 34 markers in the main populations were compared between HCs and SADs. In summary, 59 scatter plots showed significant differences in the expression intensities between at least two groups. Next, each immune cell population was divided into subpopulations (metaclusters) using the FlowSOM (self-organizing map) algorithm. Finally, 121 metaclusters (MCs) of the 10 main immune cell populations were found to have significant differences to classify diseases. The single-cell T-cell heterogeneity represented 64MCs based on the expression of 34 markers, and the frequency of 23 MCs differed significantly between at least twoconditions. The CD3– non-T-cell compartment contained 57 MCs with 17 MCs differentiating at least two investigated groups. In summary, we are the first to demonstrate the complexity of the immunophenotype of 34 markers over 15 million single cells in HCs vs. therapy-naive patients with RA, SSc, and SLE. Disease specific population frequencies or expression patterns of peripheral immune cells provide a single-cell data resource to the scientific community.

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“…High-throughput analysis for single cells with high-dimensional information, including viability, cell morphologies, proteins, and even mRNA transcripts [ 83 ], makes CyTOF a powerful analytical technique to address biological questions in many applications, such as broad-scale immune profiling [ 84 , 85 , 86 ], T and NK cell subtyping [ 87 , 88 , 89 ], therapeutic responses [ 75 , 90 ], antiviral T cell response [ 91 , 92 , 93 ], biomarker discovery for diseases [ 94 , 95 , 96 , 97 ], and patient profiling involving COVID-19 [ 98 , 99 ]. CyTOF can also help to analyze the cellular heterogeneity related to clinical trials [ 100 , 101 , 102 ], autoimmune disorders [ 103 , 104 , 105 ], and cancers [ 106 , 107 , 108 ]. We will further discuss the current applications of CyTOF in the section on the application of single-cell proteomics.…”

Section: Cytof For Single-cell Proteomicsmentioning

confidence: 99%

Advances in Mass Spectrometry-Based Single Cell Analysis

Lee

et al. 2023

Biology

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Technological developments and improvements in single-cell isolation and analytical platforms allow for advanced molecular profiling at the single-cell level, which reveals cell-to-cell variation within the admixture cells in complex biological or clinical systems. This helps to understand the cellular heterogeneity of normal or diseased tissues and organs. However, most studies focused on the analysis of nucleic acids (e.g., DNA and RNA) and mass spectrometry (MS)-based analysis for proteins and metabolites of a single cell lagged until recently. Undoubtedly, MS-based single-cell analysis will provide a deeper insight into cellular mechanisms related to health and disease. This review summarizes recent advances in MS-based single-cell analysis methods and their applications in biology and medicine.

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Analysis of the Single-Cell Heterogeneity of Adenocarcinoma Cell Lines and the Investigation of Intratumor Heterogeneity Reveals the Expression of Transmembrane Protein 45A (TMEM45A) in Lung Adenocarcinoma Cancer Patients

Cited by 11 publications

References 80 publications

ZeptoCTC - Sensitive Protein Analysis of True Single Cell Lysates using Reverse Phase Protein Arrays (RPPA)

ZeptoCTC - Sensitive Protein Analysis of True Single Cell Lysates using Reverse Phase Protein Arrays (RPPA)

Comparative single-cell multiplex immunophenotyping of therapy-naive patients with rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus shed light on disease-specific composition of the peripheral immune system

Advances in Mass Spectrometry-Based Single Cell Analysis

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