P1.04-06 Tumor Microenvironment Landscape in Lung Adenocarcinoma by 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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M6a Regulator-Associated Methylation Modification Patterns Shape Tumor Microenvironment Characteristics in Hepatocellular Carcinoma

10.21203/rs.3.rs-778749/v1 ◽

2021 ◽

Author(s):

Bobin Ning ◽

Yonggan Xue ◽

Hongyi Liu ◽

Hongyu Sun ◽

Baoqing Jia

Keyword(s):

Hepatocellular Carcinoma ◽

Tumor Microenvironment ◽

Single Cell ◽

Signaling Pathways ◽

Principal Component ◽

Unsupervised Clustering ◽

Survival Prediction ◽

Multiple Perspectives ◽

Single Cell Sequencing ◽

Survival Expectations

Abstract Although substantial achievements in the tumor microenvironment (TME) of hepatocellular carcinoma (HCC) have led to fundamental improvements both in the basic research and clinical management, the potential mechanisms and regulatory relationships between m6A regulators and the TME are still unknown. We first conducted unsupervised clustering on the samples according to the core m6A expression, and then compared the signaling pathways, differential genes (DEGs), and TME between the m6A phenotypes, and re-validated the relationship between m6A regulators and TME by single cell sequencing. Then, the geneCluster was obtained by another unsupervised clustering of the DEGs, and the clinical as well as TME traits were evaluated among the geneClusters. Finally, the m6A scores of individual patients were calculated by principal component analysis (PCA) to verify the correlation from multiple perspectives, including survivals, clinical characters, mutations, TME, immunotherapy, and chemotherapy. Through a comprehensive analysis of 729 samples, we classified HCC patients into three m6A clusters and three geneClusters. Each group exhibited remarkable variations in terms of signaling pathways, clinical traits, and survival expectations. Notably, the m6A phenotypes corresponded to three different types of TME, namely immune-inflamed, immune-excluded, and immune-desert, respectively. In addition, the m6A regulator can accurately reflect the individualized microenvironment in HCC, and present supreme expression levels in the stromal microenvironment. However, the m6A score system is able to make accurate predictions not only in terms of clinical traits, survival prediction, and TME mentioned above, but also in the sensitivity of HCC patients to immunotherapy and chemotherapy. This study revealed the uniqueness and pluripotency of m6A regulators in the TME of HCC by combining single-cell sequencing and bulk sequencing. The quantified m6A modification indices were able to accurately predict patient survival expectations, clinical traits, TME, and sensitivity to immunotherapy and chemotherapy.

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Applications of Single-Cell Omics to Dissect Tumor Microenvironment

Frontiers in Genetics ◽

10.3389/fgene.2020.548719 ◽

2020 ◽

Vol 11 ◽

Author(s):

Tingting Guo ◽

Weimin Li ◽

Xuyu Cai

Keyword(s):

Tumor Microenvironment ◽

Single Cell ◽

Immune Cells ◽

Immune Cell ◽

Cell Types ◽

Immune Checkpoint Blockade ◽

Lineage Tracing ◽

Great Success ◽

Novel Technologies ◽

Single Cell Sequencing

The recent technical and computational advances in single-cell sequencing technologies have significantly broaden our toolkit to study tumor microenvironment (TME) directly from human specimens. The TME is the complex and dynamic ecosystem composed of multiple cell types, including tumor cells, immune cells, stromal cells, endothelial cells, and other non-cellular components such as the extracellular matrix and secreted signaling molecules. The great success on immune checkpoint blockade therapy has highlighted the importance of TME on anti-tumor immunity and has made it a prime target for further immunotherapy strategies. Applications of single-cell transcriptomics on studying TME has yielded unprecedented resolution of the cellular and molecular complexity of the TME, accelerating our understanding of the heterogeneity, plasticity, and complex cross-interaction between different cell types within the TME. In this review, we discuss the recent advances by single-cell sequencing on understanding the diversity of TME and its functional impact on tumor progression and immunotherapy response driven by single-cell sequencing. We primarily focus on the major immune cell types infiltrated in the human TME, including T cells, dendritic cells, and macrophages. We further discuss the limitations of the existing methodologies and the prospects on future studies utilizing single-cell multi-omics technologies. Since immune cells undergo continuous activation and differentiation within the TME in response to various environmental cues, we highlight the importance of integrating multimodal datasets to enable retrospective lineage tracing and epigenetic profiling of the tumor infiltrating immune cells. These novel technologies enable better characterization of the developmental lineages and differentiation states that are critical for the understanding of the underlying mechanisms driving the functional diversity of immune cells within the TME. We envision that with the continued accumulation of single-cell omics datasets, single-cell sequencing will become an indispensable aspect of the immune-oncology experimental toolkit. It will continue to drive the scientific innovations in precision immunotherapy and will be ultimately adopted by routine clinical practice in the foreseeable future.

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Deciphering the Prognostic Implications of the Components and Signatures in the Immune Microenvironment of Pancreatic Ductal Adenocarcinoma

Frontiers in Immunology ◽

10.3389/fimmu.2021.648917 ◽

2021 ◽

Vol 12 ◽

Author(s):

Rong Tang ◽

Xiaomeng Liu ◽

Chen Liang ◽

Jie Hua ◽

Jin Xu ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Single Cell ◽

Pancreatic Ductal Adenocarcinoma ◽

Cd8 T Cells ◽

Nkt Cells ◽

Ductal Adenocarcinoma ◽

Hematopoietic Stem ◽

Single Cell Sequencing ◽

Prognostic Implications

Background: The treatment modalities for pancreatic ductal adenocarcinoma (PDAC) are limited and unsatisfactory. Although many novel drugs targeting the tumor microenvironment, such as immune checkpoint inhibitors, have shown promising efficacy for some tumors, few of them significantly prolong the survival of patients with PDAC due to insufficient knowledge on the tumor microenvironment.Methods: A single-cell RNA sequencing (scRNA-seq) dataset and seven PDAC cohorts with complete clinical and bulk sequencing data were collected for bioinformatics analysis. The relative proportions of each cell type were estimated using the gene set variation analysis (GSVA) algorithm based on the signatures identified by scRNA-seq or previous literature.Results: A meta-analysis of 883 PDAC patients showed that neutrophils are associated with worse overall survival (OS) for PDAC, while CD8+ T cells, CD4+ T cells, and B cells are related to prolonged OS for PDAC, with marginal statistical significance. Seventeen cell categories were identified by clustering analysis based on single-cell sequencing. Among them, CD8+ T cells and NKT cells were universally exhausted by expressing exhaustion-associated molecular markers. Interestingly, signatures of CD8+ T cells and NKT cells predicted prolonged OS for PDAC only in the presence of “targets” for pyroptosis and ferroptosis induction. Moreover, a specific state of T cells with overexpression of ribosome-related proteins was associated with a good prognosis. In addition, the hematopoietic stem cell (HSC)-like signature predicted prolonged OS in PDAC. Weighted gene co-expression network analysis identified 5 hub genes whose downregulation may mediate the observed survival benefits of the HSC-like signature. Moreover, trajectory analysis revealed that myeloid cells evolutionarily consisted of 7 states, and antigen-presenting molecules and complement-associated genes were lost along the pseudotime flow. Consensus clustering based on the differentially expressed genes between two states harboring the longest pseudotime span identified two PDAC groups with prognostic differences, and more infiltrated immune cells and activated immune signatures may account for the survival benefits.Conclusion: This study systematically investigated the prognostic implications of the components of the PDAC tumor microenvironment by integrating single-cell sequencing and bulk sequencing, and future studies are expected to develop novel targeted agents for PDAC treatment.

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A Systematic Review on the Therapeutic Potentiality of PD-L1-Inhibiting MicroRNAs for Triple-Negative Breast Cancer: Toward Single-Cell Sequencing-Guided Biomimetic Delivery

Genes ◽

10.3390/genes12081206 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1206

Author(s):

Mahdi Abdoli Shadbad ◽

Sahar Safaei ◽

Oronzo Brunetti ◽

Afshin Derakhshani ◽

Parisa Lotfinejad ◽

...

Keyword(s):

Tumor Microenvironment ◽

Single Cell ◽

Immune Checkpoint ◽

Triple Negative ◽

De Novo ◽

Checkpoint Inhibitors ◽

Oncogenic Signaling ◽

Single Cell Sequencing ◽

Oncogenic Signaling Pathways ◽

Tumoral Cells

The programmed death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) is a well-established inhibitory immune checkpoint axis in triple-negative breast cancer (TNBC). Growing evidence indicates that tumoral PD-L1 can lead to TNBC development. Although conventional immune checkpoint inhibitors have improved TNBC patients’ prognosis, their effect is mainly focused on improving anti-tumoral immune responses without substantially regulating oncogenic signaling pathways in tumoral cells. Moreover, the conventional immune checkpoint inhibitors cannot impede the de novo expression of oncoproteins, like PD-L1, in tumoral cells. Accumulating evidence has indicated that the restoration of specific microRNAs (miRs) can downregulate tumoral PD-L1 and inhibit TNBC development. Since miRs can target multiple mRNAs, miR-based gene therapy can be an appealing approach to inhibit the de novo expression of oncoproteins, like PD-L1, restore anti-tumoral immune responses, and regulate various intracellular singling pathways in TNBC. Therefore, we conducted the current systematic review based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) to provide a comprehensive and unbiased synthesis of currently available evidence regarding the effect of PD-L1-inhibiting miRs restoration on TNBC development and tumor microenvironment. For this purpose, we systematically searched the Cochrane Library, Embase, Scopus, PubMed, ProQuest, Web of Science, Ovid, and IranDoc databases to obtain the relevant peer-reviewed studies published before 25 May 2021. Based on the current evidence, the restoration of miR-424-5p, miR-138-5p, miR-570-3p, miR-200c-3p, miR-383-5p, miR-34a-5p, miR-3609, miR-195-5p, and miR-497-5p can inhibit tumoral PD-L1 expression, transform immunosuppressive tumor microenvironment into the pro-inflammatory tumor microenvironment, inhibit tumor proliferation, suppress tumor migration, enhance chemosensitivity of tumoral cells, stimulate tumor apoptosis, arrest cell cycle, repress the clonogenicity of tumoral cells, and regulate various oncogenic signaling pathways in TNBC cells. Concerning the biocompatibility of biomimetic carriers and the valuable insights provided by the single-cell sequencing technologies, single-cell sequencing-guided biomimetic delivery of these PD-L1-inhibiting miRs can decrease the toxicity of traditional approaches, increase the specificity of miR-delivery, enhance the efficacy of miR delivery, and provide the affected patients with personalized cancer therapy.

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Single-cell RNA sequencing reveals distinct tumor microenvironmental patterns in lung adenocarcinoma

Oncogene ◽

10.1038/s41388-021-02054-3 ◽

2021 ◽

Author(s):

Philip Bischoff ◽

Alexandra Trinks ◽

Benedikt Obermayer ◽

Jan Patrick Pett ◽

Jennifer Wiederspahn ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Tumor Microenvironment ◽

Lung Adenocarcinoma ◽

Single Cell ◽

Rna Sequencing ◽

Histological Grade ◽

Cell Populations ◽

Marker Genes ◽

Single Cell Rna Sequencing

AbstractRecent developments in immuno-oncology demonstrate that not only cancer cells, but also the tumor microenvironment can guide precision medicine. A comprehensive and in-depth characterization of the tumor microenvironment is challenging since its cell populations are diverse and can be important even if scarce. To identify clinically relevant microenvironmental and cancer features, we applied single-cell RNA sequencing to ten human lung adenocarcinomas and ten normal control tissues. Our analyses revealed heterogeneous carcinoma cell transcriptomes reflecting histological grade and oncogenic pathway activities, and two distinct microenvironmental patterns. The immune-activated CP²E microenvironment was composed of cancer-associated myofibroblasts, proinflammatory monocyte-derived macrophages, plasmacytoid dendritic cells and exhausted CD8+ T cells, and was prognostically unfavorable. In contrast, the inert N³MC microenvironment was characterized by normal-like myofibroblasts, non-inflammatory monocyte-derived macrophages, NK cells, myeloid dendritic cells and conventional T cells, and was associated with a favorable prognosis. Microenvironmental marker genes and signatures identified in single-cell profiles had progonostic value in bulk tumor profiles. In summary, single-cell RNA profiling of lung adenocarcinoma provides additional prognostic information based on the microenvironment, and may help to predict therapy response and to reveal possible target cell populations for future therapeutic approaches.

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IMMU-01. SINGLE CELL SEQUENCING OF MELANOMA BRAIN METASTASES UNVEILS HETEROGENEITY OF THE TUMOR MICROENVIRONMENT IN RESPONSE TO IMMUNE CHECKPOINT BLOCKADE

Neuro-Oncology ◽

10.1093/neuonc/noaa215.432 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii104-ii104

Author(s):

Christopher Alvarez-Breckenridge ◽

Samuel Markson ◽

Jackson Stocking ◽

Matt Lastrapes ◽

Naema Nayyar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Brain Metastases ◽

Single Cell ◽

Immune Checkpoint ◽

Heterogeneous Distribution ◽

Single Cell Sequencing ◽

Melanoma Brain Metastases ◽

The Brain

Abstract Immune checkpoint inhibitors (ICI) have revolutionized oncologic treatment for metastatic melanoma. With improved systemic control, there has been increasing prevalence of patients with brain metastases. Recent evidence has demonstrated intracranial responses in a subset of these patients treated with ICI. We hypothesize that the response to ICI in melanoma brain metastases (MBM) is reflective of unique features within the tumor microenvironment of the brain. A cohort of 27 patients, encompassing 8 pre- and 19 post-immunotherapy MBM underwent single cell RNA sequencing (Smart-Seq2). The cohort includes patients with longitudinal cranial resections and simultaneously resected, spatially distinct tumors. Each tumor underwent unsupervised transcriptomic analysis, differential gene expression, inferred copy number variation, and T-cell receptor (TCR) clonotyping. Published extracranial melanoma single cell datasets were used to compare the tumor microenvironment of the brain and periphery in response to ICI. A total of 14,027 cells (6,189 malignant, 7,838 non-malignant) were sequenced. Brain metastases demonstrated a heterogeneous distribution of macrophage states. Intracranial macrophages were found to be more tumor-supportive than their extracranial counterparts. MBM also included a distribution of reactive neutrophils and astrocytes. Analysis across pre- and post-treatment MBM demonstrated an increase in clonally expanded T cells in patients responding to ICI. Across longitudinal brain metastases collected from the same patients, there was evidence of identical T cell clones across timepoints and locations. Single cell sequencing of MBM provides insights into the cellular composition of the tumor and microenvironment. Our data suggest the cellular heterogeneity within MBM is unique when compared to extracranial disease. Additionally, T cell clonal expansion is found following ICI and T cells of the same clonotype infiltrate spatially and temporally separated brain metastases. These findings raise potential therapeutic implications as we learn to target the differential features of the innate and adaptive immune system within brain metastases and their extracranial counterparts.

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Resolving the spatial and cellular architecture of lung adenocarcinoma by multi-region single-cell sequencing

10.1101/2020.09.04.283739 ◽

2020 ◽

Author(s):

Ansam Sinjab ◽

Guangchun Han ◽

Kieko Hara ◽

Warapen Treekitkarnmongkol ◽

Patrick Brennan ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Single Cell ◽

Early Stage ◽

Normal Lung ◽

Cell Populations ◽

T Regulatory Cell ◽

Single Cell Sequencing ◽

Normal Tissues ◽

Inflammatory Dendritic Cells ◽

Antigen Presenting

ABSTRACTLittle is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and fourteen multi-region normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to the LUADs. LUADs exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs driven by KRAS mutations. T regulatory cell phenotypes are increased in normal tissues with closer proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages and inflammatory dendritic cells. Further, the LUAD ecosystem harbors gain of ligand-receptor based interactions involving increased expression of CD24 antigen on epithelial cells and SIGLEC10 on myeloid subsets. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for treatment.Statement of significanceThe geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multi-region single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecological evolution LUAD from the lung that comprise high-potential targets for early interception.

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PNOC Expressed by B Cells in Cholangiocarcinoma Was Survival Related and LAIR2 Could Be a T Cell Exhaustion Biomarker in Tumor Microenvironment: Characterization of Immune Microenvironment Combining Single-Cell and Bulk Sequencing Technology

Frontiers in Immunology ◽

10.3389/fimmu.2021.647209 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zheng Chen ◽

Mincheng Yu ◽

Jiuliang Yan ◽

Lei Guo ◽

Bo Zhang ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Tumor Microenvironment ◽

Single Cell ◽

Malignant Cell ◽

Cell Populations ◽

Sequencing Data ◽

Sequencing Technology ◽

Immune Infiltration ◽

Single Cell Sequencing

BackgroundCholangiocarcinoma was a highly malignant liver cancer with poor prognosis, and immune infiltration status was considered an important factor in response to immunotherapy. In this investigation, we tried to locate immune infiltration related genes of cholangiocarcinoma through combination of bulk-sequencing and single-cell sequencing technology.MethodsSingle sample gene set enrichment analysis was used to annotate immune infiltration status in datasets of TCGA CHOL, GSE32225, and GSE26566. Differentially expressed genes between high- and low-infiltrated groups in TCGA dataset were yielded and further compressed in other two datasets through backward stepwise regression in R environment. Single-cell sequencing data of GSE138709 was loaded by Seurat software and was used to examined the expression of infiltration-related gene set. Pathway changes in malignant cell populations were analyzed through scTPA web tool.ResultsThere were 43 genes differentially expressed between high- and low-immune infiltrated patients, and after further compression, PNOC and LAIR2 were significantly correlated with high immune infiltration status in cholangiocarcinoma. Through analysis of single-cell sequencing data, PNOC was mainly expressed by infiltrated B cells in tumor microenvironment, while LAIR2 was expressed by Treg cells and partial GZMB+ CD8 T cells, which were survival related and increased in tumor tissues. High B cell infiltration levels were related to better overall survival. Also, malignant cell populations demonstrated functionally different roles in tumor progression.ConclusionPNOC and LAIR2 were biomarkers for immune infiltration evaluation in cholangiocarcinoma. PNOC, expressed by B cells, could predict better survival of patients, while LAIR2 was a potential marker for exhaustive T cell populations, correlating with worse survival of patients.

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Dive into Single, Seek Out Multiple: Probing Cancer Metastases via Single-Cell Sequencing and Imaging Techniques

Cancers ◽

10.3390/cancers13051067 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1067

Author(s):

Shang Su ◽

Xiaohong Li

Keyword(s):

Tumor Microenvironment ◽

Single Cell ◽

Cell Imaging ◽

Imaging Techniques ◽

Metastatic Tumor ◽

Single Cell Sequencing ◽

Complex Interactions ◽

Single Cell Imaging ◽

Cancer Metastases ◽

Transcriptomic Changes

Metastasis is the cause of most cancer deaths and continues to be the biggest challenge in clinical practice and laboratory investigation. The challenge is largely due to the intrinsic heterogeneity of primary and metastatic tumor populations and the complex interactions among cancer cells and cells in the tumor microenvironment. Therefore, it is important to determine the genotype and phenotype of individual cells so that the metastasis-driving events can be precisely identified, understood, and targeted in future therapies. Single-cell sequencing techniques have allowed the direct comparison of the genomic and transcriptomic changes among different stages of metastatic samples. Single-cell imaging approaches have enabled the live visualization of the heterogeneous behaviors of malignant and non-malignant cells in the tumor microenvironment. By applying these technologies, we are achieving a spatiotemporal precision understanding of cancer metastases and clinical therapeutic translations.

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