Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas

Single-cell RNA sequencing (scRNA-seq) enables the systematic identification of cell populations in a tissue, but characterizing their spatial organization remains challenging. We combine a microarray-based spatial transcriptomics method that reveals spatial patterns of gene expression using an array of spots, each capturing the transcriptomes of multiple adjacent cells, with scRNA-Seq generated from the same sample. To annotate the precise cellular composition of distinct tissue regions, we introduce a method for multimodal intersection analysis. Applying multimodal intersection analysis to primary pancreatic tumors, we find that subpopulations of ductal cells, macrophages, dendritic cells and cancer cells have spatially restricted enrichments, as well as distinct coenrichments with other cell types. Furthermore, we identify colocalization of inflammatory fibroblasts and cancer cells expressing a stress-response gene module. Our approach for mapping the architecture of scRNA-seq-defined subpopulations can be applied to reveal the interactions inherent to complex tissues. Combining single-cell RNA-seq data and microarray-based spatial transcriptomics maps the location of different cell types and cell states in pancreatic tumors.

[1]  L. Cai,et al.  In Situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus , 2016, Neuron.

[2]  I. Hellmann,et al.  Comparative Analysis of Single-Cell RNA Sequencing Methods , 2016, bioRxiv.

[3]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[4]  Renaud Gaujoux,et al.  A flexible R package for nonnegative matrix factorization , 2010, BMC Bioinformatics.

[5]  R. White,et al.  Cancer archetypes co-opt and adapt the transcriptional programs of existing cellular states , 2018, bioRxiv.

[6]  Tracy T Batchelor,et al.  Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq , 2018, Science.

[7]  Andrey Alexeyenko,et al.  Spatially resolved transcriptome profiling in model plant species , 2017, Nature Plants.

[8]  J. Pastorek,et al.  Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating machinery in tumors , 2013, Front. Physiol..

[9]  Chih-Yang Wang,et al.  Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells , 2015, Nature.

[10]  Robert H Singer,et al.  Quantitative mRNA imaging throughout the entire Drosophila brain , 2016, Nature Methods.

[11]  R. Satija,et al.  Single-cell RNA sequencing to explore immune cell heterogeneity , 2017, Nature Reviews Immunology.

[12]  Steven D Chang,et al.  Single-Cell RNAseq analysis of infiltrating neoplastic cells at the migrating front of human glioblastoma , 2017, bioRxiv.

[13]  Charles H. Yoon,et al.  Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq , 2016, Science.

[14]  G. Macchiarelli,et al.  Histology of the exocrine pancreas , 1997, Microscopy research and technique.

[15]  Shawn M. Gillespie,et al.  Single-Cell Transcriptomic Analysis of Primary and Metastatic Tumor Ecosystems in Head and Neck Cancer , 2017, Cell.

[16]  Pradeep S Rajendran,et al.  Single-cell dissection of transcriptional heterogeneity in human colon tumors , 2011, Nature Biotechnology.

[17]  Aleksandra A. Kolodziejczyk,et al.  The technology and biology of single-cell RNA sequencing. , 2015, Molecular cell.

[18]  T. Rőszer,et al.  Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms , 2015, Mediators of inflammation.

[19]  Mariella G. Filbin,et al.  Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma , 2016, Nature.

[20]  R. Sandberg,et al.  Laser capture microscopy coupled with Smart-seq2 for precise spatial transcriptomic profiling , 2016, Nature Communications.

[21]  A. Jaiswal,et al.  Nrf2 and Nrf1 in association with Jun proteins regulate antioxidant response element-mediated expression and coordinated induction of genes encoding detoxifying enzymes , 1998, Oncogene.

[22]  Shawn M. Gillespie,et al.  Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma , 2014, Science.

[23]  A. Regev,et al.  Spatial reconstruction of single-cell gene expression , 2015, Nature Biotechnology.

[24]  Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer , 2019, Cell.

[25]  K. Flaherty,et al.  Toward Minimal Residual Disease-Directed Therapy in Melanoma , 2018, Cell.

[26]  S. Akira,et al.  A nuclear factor for IL‐6 expression (NF‐IL6) is a member of a C/EBP family. , 1990, The EMBO journal.

[27]  P. Robson,et al.  Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts. , 2019, Cancer discovery.

[28]  Shuqiang Li,et al.  CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq , 2016, Genome Biology.

[29]  Jeong Eon Lee,et al.  Single-cell RNA-seq enables comprehensive tumour and immune cell profiling in primary breast cancer , 2017, Nature Communications.

[30]  C. Slingluff,et al.  The heterogeneity of tumor-infiltrating CD8+ T cells in metastatic melanoma distorts their quantification: how to manage heterogeneity? , 2017, Melanoma research.

[31]  J. Lundeberg,et al.  Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics , 2018, Scientific Reports.

[32]  Victor X. Jin,et al.  Single-Cell RNA-seq Reveals a Subpopulation of Prostate Cancer Cells with Enhanced Cell-Cycle-Related Transcription and Attenuated Androgen Response. , 2018, Cancer research.

[33]  F. Azuaje,et al.  Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment , 2019, Nature Communications.

[34]  Patrik L. Ståhl,et al.  Spatial detection of fetal marker genes expressed at low level in adult human heart tissue , 2017, Scientific Reports.

[35]  M. Kornek,et al.  The Contribution of Non-Professional Antigen-Presenting Cells to Immunity and Tolerance in the Liver , 2018, Front. Immunol..

[36]  Florian Wagner,et al.  K-nearest neighbor smoothing for high-throughput single-cell RNA-Seq data , 2017, bioRxiv.

[37]  Hans Clevers,et al.  Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer , 2017, The Journal of experimental medicine.

[38]  J. Maaskola,et al.  Spatially Resolved Transcriptomics Enables Dissection of Genetic Heterogeneity in Stage III Cutaneous Malignant Melanoma. , 2018, Cancer research.

[39]  X. Zhuang,et al.  Spatially resolved, highly multiplexed RNA profiling in single cells , 2015, Science.

[40]  Samuel L. Wolock,et al.  A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure. , 2016, Cell systems.

[41]  Evan Z. Macosko,et al.  Single-Cell Multi-omic Integration Compares and Contrasts Features of Brain Cell Identity , 2019, Cell.

[42]  Francisco Tirado,et al.  Modulating the Expression of Disease Genes with RNA-Based Therapy , 2006, BMC Bioinformatics.

[43]  A. Oudenaarden,et al.  Genome-wide RNA Tomography in the Zebrafish Embryo , 2014, Cell.

[44]  Catherine E. Braine,et al.  Spatiotemporal dynamics of molecular pathology in amyotrophic lateral sclerosis , 2018, Science.

[45]  L. Pilarski,et al.  MS4A4A: a novel cell surface marker for M2 macrophages and plasma cells , 2017, Immunology and cell biology.

[46]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[47]  Geoffrey E. Hinton,et al.  Visualizing Data using t-SNE , 2008 .

[48]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[49]  Paul J. Hoffman,et al.  Comprehensive Integration of Single-Cell Data , 2018, Cell.

[50]  George M. Church,et al.  Highly Multiplexed Subcellular RNA Sequencing in Situ , 2014, Science.

[51]  Hideaki Takahashi,et al.  JunB promotes cell invasion, migration and distant metastasis of head and neck squamous cell carcinoma , 2016, Journal of experimental & clinical cancer research : CR.

[52]  Jun Chen,et al.  Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq , 2017, Nature Protocols.

[53]  J. Marioni,et al.  High-throughput spatial mapping of single-cell RNA-seq data to tissue of origin , 2015, Nature Biotechnology.

[54]  Takaya Nagasaki,et al.  Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth , 2015, Cancers.

[55]  Decade in review—genomics: A decade of discovery in cancer genomics , 2014, Nature Reviews Clinical Oncology.

[56]  E. Feinstein,et al.  Ero1-Lα plays a key role in a HIF-1-mediated pathway to improve disulfide bond formation and VEGF secretion under hypoxia: implication for cancer , 2005, Oncogene.

[57]  J. Wosen,et al.  Epithelial MHC Class II Expression and Its Role in Antigen Presentation in the Gastrointestinal and Respiratory Tracts , 2018, Front. Immunol..

[58]  Patrik L. Ståhl,et al.  Visualization and analysis of gene expression in tissue sections by spatial transcriptomics , 2016, Science.

[59]  Mariella G. Filbin,et al.  Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq , 2017, Science.

[60]  Patrik L. Ståhl,et al.  Spatial maps of prostate cancer transcriptomes reveal an unexplored landscape of heterogeneity , 2018, Nature Communications.

[61]  Kevin Petrecca,et al.  A Targetable EGFR-Dependent Tumor-Initiating Program in Breast Cancer. , 2017, Cell reports.

[62]  E. Vellenga,et al.  c-Jun and c-Fos cooperate with STAT3 in IL-6-induced transactivation of the IL-6 respone element (IRE). , 2001, Cytokine.

[63]  Hui Sun Leong,et al.  Longitudinal single-cell RNA sequencing of patient-derived primary cells reveals drug-induced infidelity in stem cell hierarchy , 2018, Nature Communications.

[64]  S. Bohlander,et al.  Consistent chromosome abnormalities in adenocarcinoma of the pancreas. , 1995, Cancer research.

[65]  Z. Ronai,et al.  Emerging roles of ATF2 and the dynamic AP1 network in cancer , 2010, Nature Reviews Cancer.

[66]  D. Larsimont,et al.  Transcriptional output, cell-type densities, and normalization in spatial transcriptomics , 2018, bioRxiv.