Abstract 1483: Ex vivo single cell RNA-sequencing of tumor derived organotypic spheroids identifies a unique mesenchymal resistance program to PD-1 blockade

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

Download Full-text

The novel BET inhibitor UM-002 reduces glioblastoma cell proliferation and invasion

Scientific Reports ◽

10.1038/s41598-021-02584-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anna M. Jermakowicz ◽

Matthew J. Rybin ◽

Robert K. Suter ◽

Jann N. Sarkaria ◽

Zane Zeier ◽

...

Keyword(s):

Cell Proliferation ◽

Single Cell ◽

Rna Sequencing ◽

Ex Vivo ◽

Adult Brain ◽

Bet Inhibitor ◽

Bet Inhibitors ◽

Single Cell Rna Sequencing

AbstractBromodomain and extraterminal domain (BET) proteins have emerged as therapeutic targets in multiple cancers, including the most common primary adult brain tumor glioblastoma (GBM). Although several BET inhibitors have entered clinical trials, few are brain penetrant. We have generated UM-002, a novel brain penetrant BET inhibitor that reduces GBM cell proliferation in vitro and in a human cerebral brain organoid model. Since UM-002 is more potent than other BET inhibitors, it could potentially be developed for GBM treatment. Furthermore, UM-002 treatment reduces the expression of cell-cycle related genes in vivo and reduces the expression of invasion related genes within the non-proliferative cells present in tumors as measured by single cell RNA-sequencing. These studies suggest that BET inhibition alters the transcriptional landscape of GBM tumors, which has implications for designing combination therapies. Importantly, they also provide an integrated dataset that combines in vitro and ex vivo studies with in vivo single-cell RNA-sequencing to characterize a novel BET inhibitor in GBM.

Download Full-text

Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling

Life Science Alliance ◽

10.26508/lsa.201900561 ◽

2019 ◽

Vol 2 (6) ◽

pp. e201900561 ◽

Cited By ~ 9

Author(s):

Dong Seong Cho ◽

Bolim Lee ◽

Jason D Doles

Keyword(s):

Gene Expression ◽

Adipose Tissue ◽

Single Cell ◽

Rna Sequencing ◽

Visceral Adipose Tissue ◽

Ex Vivo ◽

Cellular Heterogeneity ◽

Health Concern ◽

Single Cell Rna Sequencing ◽

Visceral Adipose

Obesity is a serious health concern and is associated with a reduced quality of life and a number of chronic diseases, including diabetes, heart disease, stroke, and cancer. With obesity rates on the rise worldwide, adipose tissue biology has become a top biomedical research priority. Despite steady growth in obesity-related research, more investigation into the basic biology of adipose tissue is needed to drive innovative solutions aiming to curtail the obesity epidemic. Adipose progenitor cells (APCs) play a central role in adipose tissue homeostasis and coordinate adipose tissue expansion and remodeling. Although APCs are well studied, defining and characterizing APC subsets remains ambiguous because of ill-defined cellular heterogeneity within this cellular compartment. In this study, we used single-cell RNA sequencing to create a cellular atlas of APC heterogeneity in mouse visceral adipose tissue. Our analysis identified two distinct populations of adipose tissue–derived stem cells (ASCs) and three distinct populations of preadipocytes (PAs). We identified novel cell surface markers that, when used in combination with traditional ASC and preadipocyte markers, could discriminate between these APC subpopulations by flow cytometry. Prospective isolation and molecular characterization of these APC subpopulations confirmed single-cell RNA sequencing gene expression signatures, and ex vivo culture revealed differential expansion/differentiation capabilities. Obese visceral adipose tissue featured relative expansion of less mature ASC and PA subpopulations, and expression analyses revealed major obesity-associated signaling alterations within each APC subpopulation. Taken together, our study highlights cellular and transcriptional heterogeneity within the APC pool, provides new tools to prospectively isolate and study these novel subpopulations, and underscores the importance of considering APC diversity when studying the etiology of obesity.

Download Full-text

Abstract 1483: Ex vivo single cell RNA-sequencing of tumor derived organotypic spheroids identifies a unique mesenchymal resistance program to PD-1 blockade

10.1158/1538-7445.am2019-1483 ◽

2019 ◽

Author(s):

Andrew J. Portell ◽

Jonathan Greene ◽

Luke J. Taus ◽

Patrick Lizotte ◽

Elena Ivanova ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Ex Vivo ◽

Single Cell Rna Sequencing

Download Full-text

Phenotypic and Epigenetic Adaptations of Cord Blood CD4+ T Cells to Maternal Obesity

Frontiers in Immunology ◽

10.3389/fimmu.2021.617592 ◽

2021 ◽

Vol 12 ◽

Author(s):

Suhas Sureshchandra ◽

Norma Mendoza ◽

Allen Jankeel ◽

Randall M. Wilson ◽

Nicole E. Marshall ◽

...

Keyword(s):

T Cells ◽

Cord Blood ◽

Single Cell ◽

Rna Sequencing ◽

Cd4 T Cells ◽

Maternal Obesity ◽

Ex Vivo ◽

Microbial Infection ◽

Single Cell Rna Sequencing ◽

The Impact

Pregravid obesity has been shown to disrupt the development of the offspring’s immune system and increase susceptibility to infection. While the mechanisms underlying the impact of maternal obesity on fetal myeloid cells are emerging, the consequences for T cells remain poorly defined. In this study, we collected umbilical cord blood samples from infants born to lean mothers and mothers with obesity and profiled CD4 T cells using flow cytometry and single cell RNA sequencing at resting and following ex vivo polyclonal stimulation. We report that maternal obesity is associated with higher frequencies of memory CD4 T cells suggestive of in vivo activation. Moreover, single cell RNA sequencing revealed expansion of an activated subset of memory T cells with maternal obesity. However, ex vivo stimulation of purified CD4 T cells resulted in poor cytokine responses, suggesting functional defects. These phenotypic and functional aberrations correlated with methylation and chromatin accessibility changes in loci associated with lymphocyte activation and T cell receptor signaling, suggesting a possible link between maternal obesogenic environment and fetal immune reprogramming. These observations offer a potential explanation for the increased susceptibility to microbial infection in babies born to mothers with obesity.

Download Full-text

A novel anti-influenza combined therapy assessed by single cell RNA-sequencing

10.1101/2021.07.27.453967 ◽

2021 ◽

Author(s):

Chiara Medaglia ◽

Ilya Kolpakov ◽

Yong Zhu ◽

Samuel Constant ◽

Song Huang ◽

...

Keyword(s):

Viral Replication ◽

Single Cell ◽

Rna Sequencing ◽

Ex Vivo ◽

Combined Therapy ◽

Combined Treatment ◽

Cell Types ◽

Host Cells ◽

Sequencing Analysis ◽

Single Cell Rna Sequencing

Influenza makes millions of people ill every year, placing a large burden on the healthcare system and the economy. Combination therapies with two or more compounds hindering different mechanisms of viral replication represent a suitable approach to not only enhance the effectiveness of the individual drugs, but also to reduce the likelihood of resistance emergence. To develop a novel treatment against influenza, we combine virucidal sialylated cyclodextrins with interferon lambda and demonstrate, in ex vivo human airway epithelia, that the two compounds inhibit the replication of a clinical H1N1 strain more efficiently when administered together rather than alone. We investigate the mechanism of action of the combined treatment and its effects on the host cells in absence of infection, by performing single cell RNA sequencing analysis. We find that both the single and combined treatments impair viral replication to different extents across distinct epithelial cell types. We also show that each cell type comprises multiple sub-types, whose proportions are altered by H1N1 infection, and assess the ability of the treatments to restore them. To the best of our knowledge this is the first study investigating the effectiveness of an antiviral therapy by transcriptomic studies at the single cell level.

Download Full-text

Elucidation of tumor-stromal heterogeneity and the ligand-receptor interactome by single cell transcriptomics in real-world pancreatic cancer biopsies

10.1101/2020.07.28.225813 ◽

2020 ◽

Author(s):

Jaewon J. Lee ◽

Vincent Bernard ◽

Alexander Semaan ◽

Maria E. Monberg ◽

Jonathan Huang ◽

...

Keyword(s):

Pancreatic Cancer ◽

Single Cell ◽

Rna Sequencing ◽

Clinical Decision Making ◽

De Novo ◽

Ex Vivo ◽

Ductal Adenocarcinoma ◽

Precision Oncology ◽

Single Cell Rna Sequencing

AbstractPrecision medicine approaches in pancreatic ductal adenocarcinoma (PDAC) are imperative for improving disease outcomes. However, the long-term fidelity of recently deployed ex vivo preclinical platforms, such as patient-derived organoids (PDOs) remains unknown. Through single-cell RNA sequencing (scRNA-seq), we identify substantial transcriptomic evolution of PDOs propagated from the parental tumor, which may alter predicted drug sensitivity. In contrast, scRNA-seq is readily applicable to limited biopsies from human primary and metastatic PDAC and identifies most cancers as being an admixture of previously described epithelial transcriptomic subtypes. Integrative analyses of our data provide an in-depth characterization of the heterogeneity within the tumor microenvironment, including cancer-associated fibroblast (CAF) subclasses, and predicts for a multitude of ligand-receptor interactions, revealing potential targets for immunotherapy approaches. While PDOs continue to enable prospective therapeutic prediction, our analysis also demonstrates the complementarity of using orthogonal de novo biopsies from PDAC patients paired with scRNA-seq to inform clinical decision-making.Statement of SignificanceThe application of single-cell RNA sequencing to diagnostic pancreatic cancer biopsies provides in-depth transcriptomic characterization of the tumor epithelium and microenvironment, while minimizing potential artifacts introduced by an intervening ex vivo passaging step. Thus, this approach can complement the use of patient-derived organoids in implementing precision oncology.

Download Full-text