scholarly journals Applying Single-Cell Technology in Uveal Melanomas: Current Trends and Perspectives for Improving Uveal Melanoma Metastasis Surveillance and Tumor Profiling

2021 ◽  
Vol 7 ◽  
Author(s):  
Mona Meng Wang ◽  
Chuanfei Chen ◽  
Myoe Naing Lynn ◽  
Carlos R. Figueiredo ◽  
Wei Jian Tan ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Uveal Melanoma ◽  
Vision Loss ◽  
Single Cells ◽  
Chromosomal Rearrangements ◽  
Distant Metastases ◽  
Current Approach ◽  
Melanoma Metastasis ◽  
Molecular Features

Uveal melanoma (UM) is the most common primary adult intraocular malignancy. This rare but devastating cancer causes vision loss and confers a poor survival rate due to distant metastases. Identifying clinical and molecular features that portend a metastatic risk is an important part of UM workup and prognostication. Current UM prognostication tools are based on determining the tumor size, gene expression profile, and chromosomal rearrangements. Although we can predict the risk of metastasis fairly accurately, we cannot obtain preclinical evidence of metastasis or identify biomarkers that might form the basis of targeted therapy. These gaps in UM research might be addressed by single-cell research. Indeed, single-cell technologies are being increasingly used to identify circulating tumor cells and profile transcriptomic signatures in single, drug-resistant tumor cells. Such advances have led to the identification of suitable biomarkers for targeted treatment. Here, we review the approaches used in cutaneous melanomas and other cancers to isolate single cells and profile them at the transcriptomic and/or genomic level. We discuss how these approaches might enhance our current approach to UM management and review the emerging data from single-cell analyses in UM.

scholarly journals Haplotype-aware single-cell multiomics uncovers functional effects of somatic structural variation

2021 ◽  
Author(s):  
Hyobin Jeong ◽  
Karen Grimes ◽  
Peter-Martin Bruch ◽  
Tobias Rausch ◽  
Patrick Hasenfeld ◽  
...  
Keyword(s):  
Single Cell ◽  
Nucleosome Occupancy ◽  
Single Cells ◽  
Chromosomal Rearrangements ◽  
Regulatory Elements ◽  
Computational Method ◽  
Tumour Heterogeneity ◽  
Cancer Genomes ◽  
Functional Consequences ◽  
Oncogenic Transcription Factor

Somatic structural variants (SVs) are widespread in cancer genomes, however, their impact on tumorigenesis and intra-tumour heterogeneity is incompletely understood, since methods to functionally characterize the broad spectrum of SVs arising in cancerous single-cells are lacking. We present a computational method, scNOVA, that couples SV discovery with nucleosome occupancy analysis by haplotype-resolved single-cell sequencing, to systematically uncover SV effects on cis-regulatory elements and gene activity. Application to leukemias and cell lines uncovered SV outcomes at several loci, including dysregulated cancer-related pathways and mono-allelic oncogene expression near SV breakpoints. At the intra-patient level, we identified different yet overlapping subclonal SVs that converge on aberrant Wnt signaling. We also deconvoluted the effects of catastrophic chromosomal rearrangements resulting in oncogenic transcription factor dysregulation. scNOVA directly links SVs to their functional consequences, opening the door for single-cell multiomics of SVs in heterogeneous cell populations.

Capillary Force Driven Single-Cell Spiking Apparatus for Studying Circulating Tumor Cells

2018 ◽  
Author(s):  
Jacob Amontree ◽  
Kangfu Chen ◽  
Jose Varillas ◽  
Z. Hugh Fan
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Single Cells ◽  
Lymphoblastic Leukemia ◽  
Surface Membrane ◽  
Critical Element ◽  
Cell Capture ◽  
Biomedical Sciences ◽  
Low Concentrations

The characterization of single cells within heterogeneous populations has great impact on both biomedical sciences and cancer research. By investigating cellular compositions on a broad scale, pertinent outliers may be lost in the sample set. Alternatively, an investigation focused on the behavior of specific cells, such as circulating tumor cells (CTCs), will reveal genetic biomarkers or phenotypic characteristics associated with cancer and metastasis. On average, CTC concentration in peripheral blood is extremely low, as few as one to two per billion of healthy blood cells. Consequently, the critical element lacking in many methods of CTC detection is accurate cell capture efficiency at low concentrations. To simulate CTC isolation, researchers usually spike small amounts of tumor cells to healthy blood for separation. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. We report our study on an innovative apparatus and method designed for low-cost, precise, and replicable single-cell spiking (SCS). Our SCS method operates solely from capillary aspiration without the reliance on external laboratory equipment. To ensure that our method does not affect the viability of each cell, we investigated the effects of surface membrane tensions induced by aspiration. Finally, we performed affinity-based CTC isolation using human acute lymphoblastic leukemia cells (CCRF-CEM) spiked into healthy whole blood with the SCS technique. The results of the isolation experiments demonstrate the reliability of our method in generating low-concentration cell samples.

scholarly journals Combined Molecular Genetic and Cytogenetic Analysis from Single Cells after Isothermal Whole-Genome Amplification

2011 ◽  
Vol 57 (7) ◽  
pp. 1032-1041 ◽  
Author(s):  
Thomas Kroneis ◽  
Jochen B Geigl ◽  
Amin El-Heliebi ◽  
Martina Auer ◽  
Peter Ulz ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Molecular Genetic ◽  
Target Cells ◽  
Whole Genome ◽  
Single Cell Level ◽  
Cell Level ◽  
Genome Amplification

BACKGROUND Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells. RESULTS DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

scholarly journals Uveal Melanoma Identified as Ocular Mass on Point-of-care Ultrasound

2021 ◽  
Vol 5 (3) ◽  
pp. 367-368
Author(s):  
Hannah Spungen ◽  
Daniel Weingrow
Keyword(s):  
Emergency Department ◽  
Retinal Detachment ◽  
Uveal Melanoma ◽  
Vision Loss ◽  
Point Of Care ◽  
Black Spot ◽  
Distant Metastases ◽  
Posterior Chamber ◽  
Point Of Care Ultrasound ◽  
Point Of Care Ultrasonography

Case Presentation: A 41-year-old man presented to the emergency department with five months of progressive monocular vision loss in his right eye, which he described as a gradually descending and enlarging black spot. He had no light perception in his right eye with elevated intraocular pressure and an afferent pupillary defect, while his left eye visual acuity and pupillary exam was normal. Point-of-care ultrasound demonstrated a hyperechoic, pedunculated mass in the posterior chamber of his right eye, consistent with a diagnosis of ocular melanoma. Ophthalmology scheduled the patient for an elective, right eye enucleation the following week, after which a diagnosis of uveal melanoma (UM) was confirmed on histopathology. Discussion: Uveal melanoma is an uncommon diagnosis that requires prompt intervention and surveillance due to the possibility of distant metastases arising in up to 50% of patients. Emergency department diagnosis of UM may be confounded by features of other intraocular pathology, such as increased ocular pressure or the finding of retinal detachment on fundoscopy. When emergency providers encounter glaucoma or retinal detachment on physical exam, point-of-care ultrasonography represents a key adjunct in the timely diagnosis and referral of this potentially vision- and life-threatening malignancy.

Approaches to the Treatment of Children with Esthesioneuroblastoma: Literature Review

2019 ◽  
Vol 6 (2) ◽  
pp. 78-86
Author(s):  
Tatiana V. Gorbunova ◽  
Anastasia D. Rodina ◽  
Ruslan V. Shishkov ◽  
Natalia V. Ivanova ◽  
Igor V. Glekov ◽  
...  
Keyword(s):  
Radiation Therapy ◽  
Surgical Treatment ◽  
Tumor Cells ◽  
Tumor Resection ◽  
Neuron Specific Enolase ◽  
Distant Metastases ◽  
Intravenous Contrast ◽  
Adult Age ◽  
Variable Expression ◽  
Molecular Features

The incidence of esthesioneuroblastoma in children under 15 years of age is 0.1 per 100.000 children. Distinctive histological features of this tumor are diffuse accumulation of neuron-specific enolase, synaptophysin, chromogranin, and variable expression of cytokeratins. Diagnosis of the tumor includes endoscopic examination of the nasal cavity and nasopharynx, magnetic resonance imaging (MRI) and computed tomography (CT) of the skull base, paranasal sinuses with intravenous contrast. PET-CT is advisable to use for the detection of regional and distant metastases, as well as for suspected relapse. In patients of adult age, a negative effect on the outcome of the disease was detected, the detection of metastases in the lymph nodes of the neck, the presence of tumor cells at the edges of tumor resection and a high degree of malignancy of the tumor according to the Hyams system. Therapeutic approaches depend on the stage of esthesioneuroblastoma by Kadish. In the A-stage, surgical treatment is advisable. In the presence of tumor cells at the edges of the resection or residual tumor, radiation therapy is performed. In case of B-stage, surgical treatment is combined with the mandatory irradiation of the primary tumor area. In patients with the C-stage, neoadjuvant chemotherapy or radiation is performed, followed by a surgical treatment, adjuvant chemotherapy and/or radiation therapy. Patients with D-stage chemoradiation therapy is indicated. There is no consensus on an effective drug regimen. Overall 5-year survival varies significantly depending on the design of the study — 55% to 98%. Further study of the features of the clinical picture, morphological and molecular features and the course of the disease will help to improve our understanding of the nature of the tumor.

scholarly journals Application of single-cell sequencing technologies in pancreatic cancer

2021 ◽  
Author(s):  
Mastan Mannarapu ◽  
Begum Dariya ◽  
Obul Reddy Bandapalli
Keyword(s):  
Pancreatic Cancer ◽  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Single Cells ◽  
Evolutionary Relationship ◽  
Intratumor Heterogeneity ◽  
Sequencing Technology ◽  
Single Cell Sequencing ◽  
Sequencing Technologies

AbstractPancreatic cancer (PC) is the third lethal disease for cancer-related mortalities globally. This is mainly because of the aggressive nature and heterogeneity of the disease that is diagnosed only in their advanced stages. Thus, it is challenging for researchers and clinicians to study the molecular mechanism involved in the development of this aggressive disease. The single-cell sequencing technology enables researchers to study each and every individual cell in a single tumor. It can be used to detect genome, transcriptome, and multi-omics of single cells. The current single-cell sequencing technology is now becoming an important tool for the biological analysis of cells, to find evolutionary relationship between multiple cells and unmask the heterogeneity present in the tumor cells. Moreover, its sensitivity nature is found progressive enabling to detect rare cancer cells, circulating tumor cells, metastatic cells, and analyze the intratumor heterogeneity. Furthermore, these single-cell sequencing technologies also promoted personalized treatment strategies and next-generation sequencing to predict the disease. In this review, we have focused on the applications of single-cell sequencing technology in identifying cancer-associated cells like cancer-associated fibroblast via detecting circulating tumor cells. We also included advanced technologies involved in single-cell sequencing and their advantages. The future research indeed brings the single-cell sequencing into the clinical arena and thus could be beneficial for diagnosis and therapy of PC patients.

STEM-27. A PERIVASCULAR STEM CELL UNDERLIES VERTEBRATE MENINGEAL TUMORIGENESIS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi26-vi27
Author(s):  
Abrar Choudhury ◽  
Martha Cady ◽  
Calixto Lucas ◽  
Brisa Palikuqi ◽  
Ophir Klein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Single Cells ◽  
Cell Types ◽  
Human Meningioma ◽  
Single Cell Rna Sequencing ◽  
Human Meningiomas

Abstract BACKGROUND Meningiomas are the most common primary intracranial tumors in humans and dogs, but biologic drivers and cell types underlying meningeal tumorigenesis are incompletely understood. Here we integrate meningioma single-cell RNA sequencing with stem cell approaches to define a perivascular stem cell underlying vertebrate meningeal tumorigenesis. METHODS Single-cell RNA sequencing was performed on 57,114 cells from 8 human meningiomas, 54,607 cells from 3 dog meningiomas, and human meningioma xenografts in mice. Results were validated using immunofluorescence (IF), immunohistochemistry (IHC), and deconvolution of bulk RNA sequencing of 200 human meningiomas. Mechanistic and functional studies were performed using clonogenic and limiting dilution assays, xenografts, and genetically engineered mouse models. RESULTS Copy number variant identification from human meningioma single cells distinguished tumor cells with loss of chr22q from non-tumor cells with intact chr22q. A single cluster distinguished by expression of Notch3 and other cancer stem cell genes had an intermediate level of loss of chr22q, suggesting this cluster may represent meningioma stem cells. In support of this hypothesis, pseudotime trajectory analysis demonstrated transcriptomic progression starting from Notch3+ cells and encompassing all other meningioma cell types. Notch3+ meningioma cells had transcriptomic concordance to mural pericytes, and IF/IHC of prenatal and adult human meninges, as well as lineage tracing using a Notch3-CreERT2 allele in mice, confirmed Notch3+ cells were restricted to the perivascular stem cell niche in mammalian meningeal development and homeostasis. Integrating human and dog meningioma single cells revealed Notch3+ cells in tumor and non-tumor clusters in dog meningiomas. Notch3 IF/IHC and cell-type deconvolution of bulk RNA sequencing showed Notch3+ cells were enriched in high-grade human meningiomas. Notch3 overexpression in human meningioma cells increased clonogenic growth in vitro, and increased tumorigenesis and tumor growth in vivo, decreasing overall survival. CONCLUSIONS Notch3+ stem cells in the perivascular niche underlie vertebrate meningeal tumorigenesis.

Concordance of PD-L1 expression detection in non-small cell lung cancer (NSCLC) tissue biopsies between OncoTect iO single cell PD-L1 quantification assay and immunohistochemistry (IHC).

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23172-e23172
Author(s):  
Amanda Chargin ◽  
Rian Janine Morgan ◽  
Bruce Kendrick Patterson
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Single Cells ◽  
Current Method ◽  
Cell Suspensions ◽  
Normal Lung ◽  
Small Cell Lung ◽  
Nsclc Tissue ◽  
Aneuploid Tumor ◽  
Tumor Types

e23172 Background: PD-1/PD-L1 therapy has been shown to be effective in patients with NSCLC, specifically those with 50% PD-L1 expression or greater by IHC. Response rates for those with lower PD-L1 expression do not consistently correlate with PD-L1 amount. The OncoTect iO Lung PD-L1 Assay was designed to provide non-subjective quantification of PD-L1 expression on tumor cells and immune cells from a multitude of tumor types. The objective of this study was to compare OncoTect iO and the current method of PD-L1 testing by immunohistochemistry (IHC) in NSCLC biopsies. Methods: Eleven NSCLC tissues were obtained from two IRB approved sites. Fresh tissues were processed into single cell suspensions using the IVD IncellPREP Kit (IncellDx, Inc). Normal lung tissue adjacent to tumor sites was obtained for 9 of the samples. Cell suspensions were tested with the OncoTect iO Lung Assay which contains antibodies directed against PD-L1 (28-8), CD45, CD3, and CD8. Suspensions are fixed and permeabilized, labeled with the antibodies, and then stained with DAPI to identify intact, single cells, and to analyze cell cycle including aneuploidy.Matched FFPE sections were taken from the tissue biopsies and were tested at BioReference Laboratories with the Dako PD-L1 IHC 28-8 PharmDx Kit. Results: A cut-off of 4% was used for the OncoTect iO Assay and a positive result for the Dako assay is ≥ 5% of the tumor cells (equivalent to 5 cells out of 100). PD-L1 expression ranged from 0% to 75% in the tumor samples which is consistent with reported ranges. Seven samples were positive by OncoTect iO Lung and 6 were positive by IHC. Twelve samples were negative by OncoTect iO Lung and 13 were negative by IHC demonstrating a concordance of 95%. Of note, one tumor that was negative by OncoTect iO and IHC demonstrated 5% of aneuploidy tumor cells that were positive and most likely very rare to be detected in the assays. Conclusions: In this study, the concordance between Oncotect iO and IHC was 95%, however, the Oncotect iO Single Cell PD-L1 Quantification Assay was fast ( < 3 hours), non-subjective, and provided addition expression information for aneuploid tumor cells.

Single cell dissociation from mouse tumor samples and analysis on BD FACSLyric Flow Cytometer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15245-e15245
Author(s):  
Wei Huang ◽  
Nate Pereira ◽  
Patrick Thomas Neuhoefer ◽  
Aaron Middlebrook ◽  
Smita Ghanekar
Keyword(s):  
Sample Preparation ◽  
Single Cell ◽  
Tumor Cells ◽  
Solid Tumor ◽  
Single Cells ◽  
Flow Cytometer ◽  
Sample Processing ◽  
Dissociation Process ◽  
Cell Dissociation ◽  
Tumor Sample

e15245 Background: Single cell multiomics analysis of tumor cells and tumor infiltrating immune cells using high parameter flow cytometry and single cell genomics technologies is a powerful approach in the investigation of tumor biology and in the discovery of new drug targets and biomarkers. The preparation of single cells from solid tumor samples often involves a physical dissociation step in combination with an enzymatic digestion step. Methods: The effect of the tumor cell dissociation process on the phenotypic properties of single cells isolated from solid tumor samples were analyzed using a 12-color reagent panel on the BD FACSLyric flow cytometer. Specifically, spontaneous pancreatic tumor samples from genetically modified mice were processed using a manual dissociation process with a scalpel blade as well as an integrated tumor sample dissociation process with the prototype Singulator instrument with various protocol settings. The BD Horizon Dri Tumor & Tissue Dissociation Reagent (TTDR) reagent was used for the enzymatic step in all sample preparations. The yield, viability, and frequency of tumor cells and different subsets of leukocytes were measured for each tumor-derived single cell sample preparation. On the Singulator system, cell yield reached a plateau with highest viability at the 30-min process time and a higher percentage of tumor cells was obtained compared to the manual sample processing method. Results: The relative frequencies of T cells, B cells, myeloid cells and macrophages also changed in different sample preparation methods. However, the spatial heterogeneity in the tumor section and cell composition may contribute to these differences and would require more in-depth analysis. In summary, these results suggest that there are many variables in the tumor sample single cell dissociation process that could impact the downstream study results, particularly for multiomics studies. Conclusions: This study highlights the need for optimization and standardization of the sample processing methodologies for downstream analysis. Disclaimers: For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD, the BD Logo, FACSLyric, and Horizon are trademarks of Becton, Dickinson and Company. © 2020 BD and its subsidiaries. All rights reserved.

scholarly journals Effect of Blood Collection Tube Type and Time to Processing on the Enumeration and High-Content Characterization of Circulating Tumor Cells Using the High-Definition Single-Cell Assay

2017 ◽  
Vol 142 (2) ◽  
pp. 198-207 ◽  
Author(s):  
Mariam Rodríguez-Lee ◽  
Anand Kolatkar ◽  
Madelyn McCormick ◽  
Angel D. Dago ◽  
Jude Kendall ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Single Cells ◽  
Blood Collection ◽  
High Definition ◽  
Cell Assay ◽  
Collection Tube ◽  
Blood Collection Tube

Context.— As circulating tumor cell (CTC) assays gain clinical relevance, it is essential to address preanalytic variability and to develop standard operating procedures for sample handling in order to successfully implement genomically informed, precision health care. Objective.— To evaluate the effects of blood collection tube (BCT) type and time-to-assay (TTA) on the enumeration and high-content characterization of CTCs by using the high-definition single-cell assay (HD-SCA). Design.— Blood samples of patients with early- and advanced-stage breast cancer were collected into cell-free DNA (CfDNA), EDTA, acid-citrate-dextrose solution, and heparin BCTs. Time-to-assay was evaluated at 24 and 72 hours, representing the fastest possible and more routine domestic shipping intervals, respectively. Results.— We detected the highest CTC levels and the lowest levels of negative events in CfDNA BCT at 24 hours. At 72 hours in this BCT, all CTC subpopulations were decreased with the larger effect observed in high-definition CTCs and cytokeratin-positive cells smaller than white blood cells. Overall cell retention was also optimal in CfDNA BCT at 24 hours. Whole-genome copy number variation profiles were generated from single cells isolated from all BCT types and TTAs. Cells from CfDNA BCT at 24-hour TTA exhibited the least noise. Conclusions.— Circulating tumor cells can be identified and characterized under a variety of collection, handling, and processing conditions, but the highest quality can be achieved with optimized conditions. We quantified performance differences of the HD-SCA for specific preanalytic variables that may be used as a guide to develop best practices for implementation into patient care and/or research biorepository processes.

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