scholarly journals Deciphering cell lineage specification of human lung adenocarcinoma with single-cell RNA sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhoufeng Wang ◽  
Zhe Li ◽  
Kun Zhou ◽  
Chengdi Wang ◽  
Lili Jiang ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Precancerous Lesions ◽  
Cellular Heterogeneity ◽  
Alveolar Type ◽  
Adenocarcinoma In Situ ◽  
Atypical Adenomatous Hyperplasia ◽  
Adenomatous Hyperplasia ◽  
Invasive Adenocarcinoma ◽  
Single Cell Rna Sequencing

AbstractLung adenocarcinomas (LUAD) arise from precancerous lesions such as atypical adenomatous hyperplasia, which progress into adenocarcinoma in situ and minimally invasive adenocarcinoma, then finally into invasive adenocarcinoma. The cellular heterogeneity and molecular events underlying this stepwise progression remain unclear. In this study, we perform single-cell RNA sequencing of 268,471 cells collected from 25 patients in four histologic stages of LUAD and compare them to normal cell types. We detect a group of cells closely resembling alveolar type 2 cells (AT2) that emerged during atypical adenomatous hyperplasia and whose transcriptional profile began to diverge from that of AT2 cells as LUAD progressed, taking on feature characteristic of stem-like cells. We identify genes related to energy metabolism and ribosome synthesis that are upregulated in early stages of LUAD and may promote progression. MDK and TIMP1 could be potential biomarkers for understanding LUAD pathogenesis. Our work shed light on the underlying transcriptional signatures of distinct histologic stages of LUAD progression and our findings may facilitate early diagnosis.

scholarly journals Deciphering cell lineage specification of human lung adenocarcinoma with single-cell RNA sequencing

2021 ◽  
Author(s):  
Zhoufeng Wang ◽  
Zhe Li ◽  
Kun Zhou ◽  
Li Zhang ◽  
Ying Yang ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Early Stage ◽  
Precancerous Lesions ◽  
Cell Types ◽  
Marker Genes ◽  
Adenocarcinoma In Situ ◽  
Invasive Adenocarcinoma ◽  
Single Cell Rna Sequencing ◽  
Clinical Stages

Abstract Lung adenocarcinomas (LUAD) start as precancerous lesions such as atypical adenomatous hyperplasia (AAH), develop stepwise into adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA), then eventually progress toward invasive adenocarcinoma (IA). To date the cellular heterogeneity across these distinct clinical stages and the underlying molecular events driving tumor progression remain largely unclear. In this study, we performed single-cell RNA sequencing on 52 specimens from 25 patients spanning the four clinical stages. By assessing the expression pattern of marker genes among 268,471 cells, we identified 16 major cell types. We demonstrated that AT2 feature cell types (AT2-like cells) were associated with malignant composition. AT2-like subcluster emerged first in AAH and partially lost AT2 cell transcriptional identity, accompanied with a gain of stemness during cell transition. In addition, genes related to energy metabolism, ribosome synthesis were upregulated in the early stage of LUAD, leading us to identify new markers including miRNA10 and β-hydroxybutyric acid to diagnose early-stage LUAD noninvasively in the blood. We also identified MDK and TIMP1 as potential biomarkers to facilitate our understanding of LUAD pathogenesis. Taken together, our data identified a new mechanism in LUAD evolution, and provided a robust basis for diagnosis and treatment of LUAD.

scholarly journals Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sunny Z. Wu ◽  
Daniel L. Roden ◽  
Ghamdan Al-Eryani ◽  
Nenad Bartonicek ◽  
Kate Harvey ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Tumour Heterogeneity ◽  
Fresh Tissue ◽  
Human Cancers ◽  
Cryopreserved Cell ◽  
Single Cell Rna Sequencing

Abstract Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

scholarly journals MBRS-46. CHARTING NEOPLASTIC AND IMMUNE CELL HETEROGENEITY IN HUMAN AND GEM MODELS OF MEDULLOBLASTOMA USING scRNAseq

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii406
Author(s):  
Andrew Donson ◽  
Kent Riemondy ◽  
Sujatha Venkataraman ◽  
Ahmed Gilani ◽  
Bridget Sanford ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Genetically Engineered ◽  
Cellular Heterogeneity ◽  
Cell Heterogeneity ◽  
Transcriptomic Data ◽  
Single Cell Rna Sequencing ◽  
Transcript Profiles

Abstract We explored cellular heterogeneity in medulloblastoma using single-cell RNA sequencing (scRNAseq), immunohistochemistry and deconvolution of bulk transcriptomic data. Over 45,000 cells from 31 patients from all main subgroups of medulloblastoma (2 WNT, 10 SHH, 9 GP3, 11 GP4 and 1 GP3/4) were clustered using Harmony alignment to identify conserved subpopulations. Each subgroup contained subpopulations exhibiting mitotic, undifferentiated and neuronal differentiated transcript profiles, corroborating other recent medulloblastoma scRNAseq studies. The magnitude of our present study builds on the findings of existing studies, providing further characterization of conserved neoplastic subpopulations, including identification of a photoreceptor-differentiated subpopulation that was predominantly, but not exclusively, found in GP3 medulloblastoma. Deconvolution of MAGIC transcriptomic cohort data showed that neoplastic subpopulations are associated with major and minor subgroup subdivisions, for example, photoreceptor subpopulation cells are more abundant in GP3-alpha. In both GP3 and GP4, higher proportions of undifferentiated subpopulations is associated with shorter survival and conversely, differentiated subpopulation is associated with longer survival. This scRNAseq dataset also afforded unique insights into the immune landscape of medulloblastoma, and revealed an M2-polarized myeloid subpopulation that was restricted to SHH medulloblastoma. Additionally, we performed scRNAseq on 16,000 cells from genetically engineered mouse (GEM) models of GP3 and SHH medulloblastoma. These models showed a level of fidelity with corresponding human subgroup-specific neoplastic and immune subpopulations. Collectively, our findings advance our understanding of the neoplastic and immune landscape of the main medulloblastoma subgroups in both humans and GEM models.

scholarly journals Single-cell RNA sequencing of cultured human endometrial CD140b+CD146+ perivascular cells highlights the importance of in vivo microenvironment

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dandan Cao ◽  
Rachel W. S. Chan ◽  
Ernest H. Y. Ng ◽  
Kristina Gemzell-Danielsson ◽  
William S. B. Yeung
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Marker Gene ◽  
Cellular Heterogeneity ◽  
Stromal Fibroblast ◽  
Perivascular Cells ◽  
Endometrial Cells ◽  
Single Cell Rna Sequencing

Abstract Background Endometrial mesenchymal-like stromal/stem cells (eMSCs) have been proposed as adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b&CD146) has been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity, relationship to primary counterpart, remains largely unclear. Methods In this study, we applied 10X genomics single-cell RNA sequencing (scRNA-seq) to cultured human CD140b+CD146+ endometrial perivascular cells (ePCs) from menstrual and secretory endometrium. We also analyzed publicly available scRNA-seq data of primary endometrium and performed transcriptome comparison between cultured ePCs and primary ePCs at single-cell level. Results Transcriptomic expression-based clustering revealed limited heterogeneity within cultured menstrual and secretory ePCs. A main subpopulation and a small stress-induced subpopulation were identified in secretory and menstrual ePCs. Cell identity analysis demonstrated the similar cellular composition in secretory and menstrual ePCs. Marker gene expression analysis showed that the main subpopulations identified from cultured secretory and menstrual ePCs simultaneously expressed genes marking mesenchymal stem cell (MSC), perivascular cell, smooth muscle cell, and stromal fibroblast. GO enrichment analysis revealed that genes upregulated in the main subpopulation enriched in actin filament organization, cellular division, etc., while genes upregulated in the small subpopulation enriched in extracellular matrix disassembly, stress response, etc. By comparing subpopulations of cultured ePCs to the publicly available primary endometrial cells, it was found that the main subpopulation identified from cultured ePCs was culture-unique which was unlike primary ePCs or primary endometrial stromal fibroblast cells. Conclusion In summary, these data for the first time provides a single-cell atlas of the cultured human CD140b+CD146+ ePCs. The identification of culture-unique relatively homogenous cell population of CD140b+CD146+ ePCs underscores the importance of in vivo microenvironment in maintaining cellular identity.

scholarly journals Single-Cell RNA-Sequencing Identifies Infrapatellar Fat Pad Macrophage Polarization in Acute Synovitis/Fat Pad Fibrosis and Cell Therapy

2021 ◽  
Vol 8 (11) ◽  
pp. 166
Author(s):  
Dimitrios Kouroupis ◽  
Thomas M. Best ◽  
Lee D. Kaplan ◽  
Diego Correa ◽  
Anthony J. Griswold
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Transcriptional Profiling ◽  
Macrophage Polarization ◽  
Myeloid Cells ◽  
Cellular Heterogeneity ◽  
Joint Disease ◽  
Infrapatellar Fat Pad ◽  
Fat Pad ◽  
Single Cell Rna Sequencing

The pathogenesis and progression of knee inflammatory pathologies is modulated partly by residing macrophages in the infrapatellar fat pad (IFP), thus, macrophage polarization towards pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes is important in joint disease pathologies. Alteration of M1/M2 balance contributes to the initiation and progression of joint inflammation and can be potentially altered with mesenchymal stem cell (MSC) therapy. In an acute synovial/IFP inflammation rat model a single intra-articular injection of IFP-MSC was performed, having as controls (1) diseased rats not receiving IFP-MSC and (2) non-diseased rats. After 4 days, cell specific transcriptional profiling via single-cell RNA-sequencing was performed on isolated IFP tissue from each group. Eight transcriptomically distinct cell populations were identified within the IFP across all three treatment groups with a noted difference in the proportion of myeloid cells across the groups. Largely myeloid cells consisted of macrophages (>90%); one M1 sub-cluster highly expressing pro-inflammatory markers and two M2 sub-clusters with one of them expressing higher levels of canonical M2 markers. Notably, the diseased samples (11.9%) had the lowest proportion of cells expressing M2 markers relative to healthy (14.8%) and MSC treated (19.4%) samples. These results suggest a phenotypic polarization of IFP macrophages towards the pro-inflammatory M1 phenotype in an acute model of inflammation, which are alleviated by IFP-MSC therapy inducing a switch towards an alternate M2 status. Understanding the IFP cellular heterogeneity and associated transcriptional programs may offer insights into novel therapeutic strategies for disabling joint disease pathologies.

scholarly journals Biological and Medical Importance of Cellular Heterogeneity Deciphered by Single-Cell RNA Sequencing

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1751 ◽  
Author(s):  
Rishikesh Kumar Gupta ◽  
Jacek Kuznicki
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Future Directions ◽  
Strong Basis ◽  
Bodily Fluids ◽  
Single Cell Rna Sequencing

The present review discusses recent progress in single-cell RNA sequencing (scRNA-seq), which can describe cellular heterogeneity in various organs, bodily fluids, and pathologies (e.g., cancer and Alzheimer’s disease). We outline scRNA-seq techniques that are suitable for investigating cellular heterogeneity that is present in cell populations with very high resolution of the transcriptomic landscape. We summarize scRNA-seq findings and applications of this technology to identify cell types, activity, and other features that are important for the function of different bodily organs. We discuss future directions for scRNA-seq techniques that can link gene expression, protein expression, cellular function, and their roles in pathology. We speculate on how the field could develop beyond its present limitations (e.g., performing scRNA-seq in situ and in vivo). Finally, we discuss the integration of machine learning and artificial intelligence with cutting-edge scRNA-seq technology, which could provide a strong basis for designing precision medicine and targeted therapy in the future.

scholarly journals Cellular heterogeneity of circulating CD4+CD8+ double-positive T cells characterized by single-cell RNA sequencing

2021 ◽  
Author(s):  
Sung Min Choi ◽  
Hi Jung Park ◽  
Eun A Choi ◽  
Kyeong Cheon Jung ◽  
Jae Il Lee
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cellular Heterogeneity ◽  
Flow Cytometry Analysis ◽  
Double Positive ◽  
Cynomolgus Monkeys ◽  
Disease States ◽  
Single Cell Rna Sequencing ◽  
Regulatory Functions

Abstract Circulating CD4+CD8+ double-positive (DP) T cells are associated with a variety of disease states. However, unlike conventional T cells, the composition of this population is poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq) to analyze the composition and characteristics of the DP T cell population circulating in the peripheral blood of cynomolgus monkeys. We found that circulating DP T cells not only contain a large number of naïve cells, but also comprise a heterogeneous population (Th1-, Th2-, Th17-, Tfh-, Treg-, Eomes+ Tr1-, CD4 CTL-, CD8 CTL-, and innate-like cells) with multiple potential functions. Flow cytometry analysis revealed that a substantial number of the naïve DP T cells expressed CD8αβ, as well as CD8αα, along with high expression of CD31. Moreover, the CD4hiCD8lo and CD4hiCD8hi populations, which express high levels of the CD4 coreceptor, comprised subsets characterized by helper and regulatory functions, some of which also exhibited cytotoxic functions. By contrast, the CD4loCD8hi population with high CD8 coreceptor expression comprised a subset characterized by CD8 CTL- and innate-like properties. Taken together, the data show that scRNA-seq analysis identified a more diverse subset of the circulating DP cells than is currently known, despite this population being very small.

scholarly journals RNA-seq library preparation from single pancreatic acinar cells

2016 ◽  
Author(s):  
Damian Wollny ◽  
Sheng Zhao ◽  
Ana Martin-Villalba
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Acinar Cells ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Pancreatic Acinar Cells ◽  
Library Preparation ◽  
Cell Type ◽  
Promising Tool ◽  
Single Cell Rna Sequencing

Single cell RNA sequencing technology has emerged as a promising tool to uncover previously neglected cellular heterogeneity. Multiple methods and protocols have been developed to apply single cell sequencing to different cell types from various organs. However, library preparation for RNA sequencing remains challenging for cell types with high RNAse content due to rapid degradation of endogenous RNA molecules upon cell lysis. To this end, we developed a protocol based on the SMART-seq2 technology for single cell RNA sequencing of pancreatic acinar cells, the cell type with one of the highest ribonuclease concentration measured to date. This protocol reliably produces high quality libraries from single acinar cells reaching a total of 5x106 reads / cell and ∼ 80% transcript mapping rate with no detectable 3´end bias. Thus, our protocol makes single cell transcriptomics accessible to cell type with very high RNAse content.

scholarly journals STARTRAC analyses of scRNAseq data from tumor models reveal T cell dynamics and therapeutic targets

2021 ◽  
Vol 218 (6) ◽  
Author(s):  
Dev Bhatt ◽  
Boxi Kang ◽  
Deepali Sawant ◽  
Liangtao Zheng ◽  
Kristy Perez ◽  
...  
Keyword(s):  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Target Genes ◽  
Cellular Heterogeneity ◽  
T Cell Subsets ◽  
Mouse Tumor ◽  
Tumor Models ◽  
Antibody Treatment ◽  
Single Cell Rna Sequencing

Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8+ cells, CD4+ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/β sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti–PD-1 treatment in MC38 and B16F10 tumor models.

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