P16.09 Regulation of chromatin accessibility in the hypoxic tumor microenvironment of glioblastoma

Abstract BACKGROUND Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most aggressive type of primary brain tumor. GBM has the poorest prognosis with no efficient cure to date due to diffusive growth into the brain, resistance to treatments and the immunosuppressive tumor microenvironment (TME). The growth and invasiveness of GBM is supported by the heterogeneous TME including local microglia and bone-marrow-derived macrophages (collectively known as glioma-associated microglia and macrophages, GAMs). In addition, tumor hypoxia is a key factor in the progression of GBM, as it can globally and rapidly alter gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can influence the pro-tumorigenic function of GAMs by inducing the expression of cytokines and cell surface receptors. However, little is known on the hypoxia-imposed chromatin changes of GAMs and GBM cells, which can in turn impact the interaction between these cell populations. Here we analyze these changes using a single-cell method, which preserves in situ hypoxia within the TME of GBM. MATERIAL AND METHODS Single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing) method in a GL261 murine glioma model was used to simultaneously assess genome-wide chromatin accessibility and expression of intracellular protein markers in single cells, enabling accurate selection of hypoxic and non-hypoxic tumor cells and GAMs. Pi-ATAC-seq is used on paraformaldehyde-perfused tumors and therefore allows capturing unaltered hypoxia-dependent cellular states, that often become distorted during dissociation and preparation of fresh material in most common single-cell methods. RESULTS We optimized Pi-ATAC method in a GL261 GBM mouse model, with specific sorting of GAMs using CD11b+ immunosorting followed by separation of microglia and macrophages, based on intensity of CD45 staining. HIF-1α induction and binding of pimonidazole were used to mark hypoxic populations. Currently, we are investigating the chromatin accessibility profiles of cancer cells and GAMs within the hypoxic tumor microenvironment of GBM. Exploring open chromatin profiles in GAMs and glioma-microglia co-cultures will allow to unravel the mechanisms of chromatin accessibility modulation in the oxygen-dependent manner. CONCLUSION In summary, we optimized the Pi-ATAC method in a mouse GBM model to characterize the chromatin openness changes in GAMs and cancer cells in response to hypoxic stress. Further validation of these results will provide the potential to identify novel markers for GAMs/glioma interactions in hypoxic GBMs and develop novel therapeutic targets.

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OTME-2. Regulation of chromatin accessibility in the hypoxic tumor microenvironment of glioblastoma

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab070.053 ◽

2021 ◽

Vol 3 (Supplement_2) ◽

pp. ii13-ii13

Author(s):

Monika Dzwigonska ◽

Jakub Mieczkowski ◽

Paulina Pilanc ◽

Salwador Cyranowski ◽

Agata Kominek ◽

...

Keyword(s):

Tumor Microenvironment ◽

Single Cells ◽

Glioma Cells ◽

Therapy Resistance ◽

Chromatin Accessibility ◽

Protein Markers ◽

Science Center ◽

Regulation Of Expression ◽

Genome Wide

Abstract Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most common primary brain tumor in adults. GBM has the poorest prognosis and no efficient cure to date due to diffusive growth into the surrounding brain, preventing complete surgical resection and leading to inevitable tumor relapse. Tumor microenvironment (TME) of GBM contains brain-residing microglia and bone-marrow derived macrophages (collectively known as glioma-associated microglia/macrophages, GAMs) that constitute up to 30% of the tumor mass and promote tumor invasion. Hypoxia (a shortage of oxygen) is a key factor in tumor progression of GBM as it can globally and rapidly alter the gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can enhance the pro-tumorigenic function of GAMs, e.g. by inducing expression of cytokines and cell surface receptors both in GAMs and glioma cells, but little is known about chromatin alterations of GBM under hypoxia. Since regulation of expression of such molecules could depend on the epigenetic alterations, we hypothesize that hypoxia may potently alter the chromatin accessibility and functions of GAMs and glioma cells. We determine the genome-wide changes in chromatin accessibility in GAMs and glioma cells in response to hypoxic stress using single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing), which allows simultaneous genome-wide assessment of chromatin accessibility and expression of intracellular protein markers in single cells, allowing faithful selection of hypoxic and non-hypoxic cells. Secondly, we are employing an oxygen-dependent co-culture model in vitro to study the mechanisms of chromatin alterations in GAMs and glioma cells under controlled hypoxic conditions and test how these changes depend on the glioma - GAMs cross-communication. In summary, we characterize the interactions between innate immune cells and glioma cells by looking at their chromatin alterations under hypoxia. Supported by the National Science Center grant (Poland) 2019/33/B/NZ1/01556 (KBL).

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scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells

10.1101/138685 ◽

2017 ◽

Cited By ~ 5

Author(s):

Stephen J. Clark ◽

Ricard Argelaguet ◽

Chantriolnt-Andreas Kapourani ◽

Thomas M. Stubbs ◽

Heather J. Lee ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Single Cells ◽

Embryonic Stem ◽

Transcriptome Profiling ◽

Chromatin Accessibility ◽

Open Chromatin ◽

Dynamic Coupling ◽

Single Cell Sequencing ◽

Molecular Layers

AbstractParallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a novel single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

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High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer

Nature Communications ◽

10.1038/s41467-021-23213-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sarah E. Pierce ◽

Jeffrey M. Granja ◽

William J. Greenleaf

Keyword(s):

Transcription Factor ◽

Single Cell ◽

Cancer Cells ◽

High Throughput ◽

Regulatory Networks ◽

Temporal Dynamics ◽

Chromatin Accessibility ◽

Regulatory Regions ◽

Factor Binding ◽

Genome Wide

AbstractChromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.

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Single cell transcriptome atlas of mouse mammary epithelial cells across development

Breast Cancer Research ◽

10.1186/s13058-021-01445-4 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Bhupinder Pal ◽

Yunshun Chen ◽

Michael J. G. Milevskiy ◽

François Vaillant ◽

Lexie Prokopuk ◽

...

Keyword(s):

Epithelial Cells ◽

Single Cell ◽

Developmental Stages ◽

Mammary Epithelial Cells ◽

Expression Profiles ◽

Single Cells ◽

Chromatin Accessibility ◽

Mammary Epithelial ◽

Cell Transcriptome ◽

Single Cell Transcriptome

Abstract Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

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Targeting E-selectin to Tackle Cancer Using Uproleselan

Cancers ◽

10.3390/cancers13020335 ◽

2021 ◽

Vol 13 (2) ◽

pp. 335

Author(s):

Barbara Muz ◽

Anas Abdelghafer ◽

Matea Markovic ◽

Jessica Yavner ◽

Anupama Melam ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Microenvironment ◽

Cancer Cells ◽

Metastatic Tumor ◽

Therapy Resistance ◽

Surface Proteins ◽

Cell Trafficking ◽

Primary Role ◽

Novel Therapy ◽

Tumor Dissemination

E-selectin is a vascular adhesion molecule expressed mainly on endothelium, and its primary role is to facilitate leukocyte cell trafficking by recognizing ligand surface proteins. E-selectin gained a new role since it was demonstrated to be involved in cancer cell trafficking, stem-like properties and therapy resistance. Therefore, being expressed in the tumor microenvironment, E-selectin can potentially be used to eradicate cancer. Uproleselan (also known as GMI-1271), a specific E-selectin antagonist, has been tested on leukemia, myeloma, pancreatic, colon and breast cancer cells, most of which involve the bone marrow as a primary or as a metastatic tumor site. This novel therapy disrupts the tumor microenvironment by affecting the two main steps of metastasis—extravasation and adhesion—thus blocking E-selectin reduces tumor dissemination. Additionally, uproleselan mobilized cancer cells from the protective vascular niche into the circulation, making them more susceptible to chemotherapy. Several preclinical and clinical studies summarized herein demonstrate that uproleselan has favorable safety and pharmacokinetics and is a tumor microenvironment-disrupting agent that improves the efficacy of chemotherapy, reduces side effects such as neutropenia, intestinal mucositis and infections, and extends overall survival. This review highlights the critical contribution of E-selectin and its specific antagonist, uproleselan, in the regulation of cancer growth, dissemination, and drug resistance in the context of the bone marrow microenvironment.

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EPCO-31. EPIGENOMIC INTRATUMORAL HETEROGENEITY OF GLIOBLASTOMA IN THREE-DIMENSIONAL SPACE

Neuro-Oncology ◽

10.1093/neuonc/noaa215.310 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii76-ii76

Author(s):

Radhika Mathur ◽

Sriranga Iyyanki ◽

Stephanie Hilz ◽

Chibo Hong ◽

Joanna Phillips ◽

...

Keyword(s):

Spatial Organization ◽

Dimensional Space ◽

Three Dimensional ◽

Spatial Location ◽

Therapy Resistance ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Intratumoral Heterogeneity ◽

Tumor Evolution ◽

Open Chromatin

Abstract Treatment failure in glioblastoma is often attributed to intratumoral heterogeneity (ITH), which fosters tumor evolution and generation of therapy-resistant clones. While ITH in glioblastoma has been well-characterized at the genomic and transcriptomic levels, the extent of ITH at the epigenomic level and its biological and clinical significance are not well understood. In collaboration with neurosurgeons, neuropathologists, and biomedical imaging experts, we have established a novel topographical approach towards characterizing epigenomic ITH in three-dimensional (3-D) space. We utilize pre-operative MRI scans to define tumor volume and then utilize 3-D surgical neuro-navigation to intra-operatively acquire 10+ samples representing maximal anatomical diversity. The precise spatial location of each sample is mapped by 3-D coordinates, enabling tumors to be visualized in 360-degrees and providing unprecedented insight into their spatial organization and patterning. For each sample, we conduct assay for transposase-accessible chromatin using sequencing (ATAC-Seq), which provides information on the genomic locations of open chromatin, DNA-binding proteins, and individual nucleosomes at nucleotide resolution. We additionally conduct whole-exome sequencing and RNA sequencing for each spatially mapped sample. Integrative analysis of these datasets reveals distinct patterns of chromatin accessibility within glioblastoma tumors, as well as their associations with genetically defined clonal expansions. Our analysis further reveals how differences in chromatin accessibility within tumors reflect underlying transcription factor activity at gene regulatory elements, including both promoters and enhancers, and drive expression of particular gene expression sets, including neuronal and immune programs. Collectively, this work provides the most comprehensive characterization of epigenomic ITH to date, establishing its importance for driving tumor evolution and therapy resistance in glioblastoma. As a resource for further investigation, we have provided our datasets on an interactive data sharing platform – The 3D Glioma Atlas – that enables 360-degree visualization of both genomic and epigenomic ITH.

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RA3 is a reference-guided approach for epigenetic characterization of single cells

Nature Communications ◽

10.1038/s41467-021-22495-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shengquan Chen ◽

Guanao Yan ◽

Wenyu Zhang ◽

Jinzhao Li ◽

Rui Jiang ◽

...

Keyword(s):

Single Cell ◽

Computational Analysis ◽

Reference Data ◽

Single Cells ◽

Chromatin Accessibility ◽

Biological Variation ◽

Superior Performance ◽

High Dimensionality ◽

High Degree

AbstractThe recent advancements in single-cell technologies, including single-cell chromatin accessibility sequencing (scCAS), have enabled profiling the epigenetic landscapes for thousands of individual cells. However, the characteristics of scCAS data, including high dimensionality, high degree of sparsity and high technical variation, make the computational analysis challenging. Reference-guided approaches, which utilize the information in existing datasets, may facilitate the analysis of scCAS data. Here, we present RA3 (Reference-guided Approach for the Analysis of single-cell chromatin Accessibility data), which utilizes the information in massive existing bulk chromatin accessibility and annotated scCAS data. RA3 simultaneously models (1) the shared biological variation among scCAS data and the reference data, and (2) the unique biological variation in scCAS data that identifies distinct subpopulations. We show that RA3 achieves superior performance when used on several scCAS datasets, and on references constructed using various approaches. Altogether, these analyses demonstrate the wide applicability of RA3 in analyzing scCAS data.

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P02.10 FocuSCOPE: a single cell, multi-omics solution to simultaneously analyze tumor variants and microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.22 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A12.1-A12

Author(s):

Y Arjmand Abbassi ◽

N Fang ◽

W Zhu ◽

Y Zhou ◽

Y Chen ◽

...

Keyword(s):

Gene Expression ◽

Tumor Microenvironment ◽

Single Cell ◽

High Throughput ◽

Immune Cells ◽

Genetic Variants ◽

Expression Profiles ◽

Single Cells ◽

Gene Expression Profiles ◽

Single Cell Sequencing

Recent advances of high-throughput single cell sequencing technologies have greatly improved our understanding of the complex biological systems. Heterogeneous samples such as tumor tissues commonly harbor cancer cell-specific genetic variants and gene expression profiles, both of which have been shown to be related to the mechanisms of disease development, progression, and responses to treatment. Furthermore, stromal and immune cells within tumor microenvironment interact with cancer cells to play important roles in tumor responses to systematic therapy such as immunotherapy or cell therapy. However, most current high-throughput single cell sequencing methods detect only gene expression levels or epigenetics events such as chromatin conformation. The information on important genetic variants including mutation or fusion is not captured. To better understand the mechanisms of tumor responses to systematic therapy, it is essential to decipher the connection between genotype and gene expression patterns of both tumor cells and cells in the tumor microenvironment. We developed FocuSCOPE, a high-throughput multi-omics sequencing solution that can detect both genetic variants and transcriptome from same single cells. FocuSCOPE has been used to successfully perform single cell analysis of both gene expression profiles and point mutations, fusion genes, or intracellular viral sequences from thousands of cells simultaneously, delivering comprehensive insights of tumor and immune cells in tumor microenvironment at single cell resolution.Disclosure InformationY. Arjmand Abbassi: None. N. Fang: None. W. Zhu: None. Y. Zhou: None. Y. Chen: None. U. Deutsch: None.

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Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer

10.21203/rs.3.rs-257705/v2 ◽

2021 ◽

Author(s):

Inga-Maria Launonen ◽

Nuppu Lyytikäinen ◽

Julia Casado ◽

Ella Anttila ◽

Angéla Szabó ◽

...

Keyword(s):

Tumor Microenvironment ◽

Single Cell ◽

Single Cells ◽

High Grade ◽

Prognostic Role ◽

Immune Microenvironment ◽

Proteomic Data ◽

Tumor Immune Microenvironment ◽

Cellular Phenotypes

Abstract The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis we generated spatial proteomic data for 21 markers in 124,623 single cells from 112 tumor cores originating from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and from 13 tumors without alterations in HR genes (HRwt). We identified a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we found an opposing prognostic role of a proliferative tumor-cell phenotypic subpopulation in the HR-genotypes, which associated with enhanced spatial tumor-immune interactions by the CD8+ and CD4+T-cells in BRCA1/2mut tumors. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the premise to improve immunotherapeutic strategies and patient stratification in HGSC.

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Characterizing Intercellular Communication of Pan-Cancer Reveals SPP1+ Tumor-Associated Macrophage Expanded in Hypoxia and Promoting Cancer Malignancy Through Single-Cell RNA-Seq Data

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.749210 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jinfen Wei ◽

Zixi Chen ◽

Meiling Hu ◽

Ziqing He ◽

Dawei Jiang ◽

...

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Cancer Progression ◽

Epithelial Mesenchymal Transition ◽

Single Cells ◽

Matrix Remodeling ◽

Rna Seq ◽

Mesenchymal Transition ◽

Tumor Associated Macrophage ◽

Cancer Types

Hypoxia is a characteristic of tumor microenvironment (TME) and is a major contributor to tumor progression. Yet, subtype identification of tumor-associated non-malignant cells at single-cell resolution and how they influence cancer progression under hypoxia TME remain largely unexplored. Here, we used RNA-seq data of 424,194 single cells from 108 patients to identify the subtypes of cancer cells, stromal cells, and immune cells; to evaluate their hypoxia score; and also to uncover potential interaction signals between these cells in vivo across six cancer types. We identified SPP1+ tumor-associated macrophage (TAM) subpopulation potentially enhanced epithelial–mesenchymal transition (EMT) by interaction with cancer cells through paracrine pattern. We prioritized SPP1 as a TAM-secreted factor to act on cancer cells and found a significant enhanced migration phenotype and invasion ability in A549 lung cancer cells induced by recombinant protein SPP1. Besides, prognostic analysis indicated that a higher expression of SPP1 was found to be related to worse clinical outcome in six cancer types. SPP1 expression was higher in hypoxia-high macrophages based on single-cell data, which was further validated by an in vitro experiment that SPP1 was upregulated in macrophages under hypoxia-cultured compared with normoxic conditions. Additionally, a differential analysis demonstrated that hypoxia potentially influences extracellular matrix remodeling, glycolysis, and interleukin-10 signal activation in various cancer types. Our work illuminates the clearer underlying mechanism in the intricate interaction between different cell subtypes within hypoxia TME and proposes the guidelines for the development of therapeutic targets specifically for patients with high proportion of SPP1+ TAMs in hypoxic lesions.

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