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Phenotypic Mapping of Pathologic Cross-Talk Between Glioblastoma and Innate Immune Cells by Synthetic Genetic Tracing

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Phenotypic Mapping of Pathologic Cross-Talk Between Glioblastoma and Innate Immune Cells by Synthetic Genetic Tracing Published OnlineFirst December 23, 2020; DOI: 10.1158/2159-8290.CD-20-0219 RESEARCH ARTICLE Phenotypic Mapping of Pathologic Cross-Talk between Glioblastoma and Innate Immune Cells by Synthetic Genetic Tracing Matthias Jürgen Schmitt1, Carlos Company1, Yuliia Dramaretska1, Iros Barozzi2, Andreas Göhrig1, Sonia Kertalli1, Melanie Großmann1, Heike Naumann1, Maria Pilar Sanchez-Bailon1, Danielle Hulsman3, Rainer Glass4, Massimo Squatrito5, Michela Serresi1, and Gaetano Gargiulo1 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst December 23, 2020; DOI: 10.1158/2159-8290.CD-20-0219 ABSTRACT Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cel- lular and molecular levels, in vitro and in vivo. Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural- to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing meth- odology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment. SIGNIFICANCE: Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways’ activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses. INTRODUCTION plasticity of genotype and phenotype, in which a dominant mutation and expression profile changes after treatment The cellular and molecular heterogeneity of cancer is (2, 10). This makes it difficult to discern whether GBM sub- thought to contribute to resistance to targeted and immune types represent hardwired entities or transient states imposed therapies. Glioblastoma (GBM) is the most common, het- by differences in signaling or tumor regions. Yet, several erogeneous, and resistant primary adult brain tumor (1). studies established correlations between subtype-specific Compared with most cancers, GBM is highly genomically gene-expression signatures, differential response to therapy, and epigenomically characterized (2–5). Lineage tracing has and overall patient survival; the latter is particularly poor provided important insights into GBM biology in the mouse, in highly mesenchymal tumors that exhibit infiltration of including the cellular origins of individual subtypes (6), and innate immune cells at recurrence (5). A better understanding how aberrant homeostatic regulation can affect responses to of GBM subtype identities and fate changes might be crucial treatments in vivo (7). to develop effective therapies. Transcriptome analyses of human GBM biopsies have Technological advances in single-cell biology confirmed repeatedly yielded a general classification into three subtypes, and extended our understanding of the complexity of brain classic (CL), mesenchymal (MES), and proneural (PN), across tumor homeostasis (11, 12). Yet, as single-cell RNA sequenc- cohorts (2–5). However, a single GBM tumor may exhibit the ing (scRNA-seq) has increasingly expanded the catalogs of coexistence of a predominant subtype along with tumor cells cell populations within tumors, the biological interpretation of other subtypes (8, 9). In addition, recurrent tumors exhibit of novel cell types and disease states has remained difficult, due to a lack of experimental approaches for validation (13). Advanced experimental approaches will be required. 1Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany. Genetic tracing is extremely informative in developmental 2Department of Surgery and Cancer, Imperial College London, London, settings and diseases involving alterations in tissue home- United Kingdom. 3Division of Molecular Genetics, The Netherlands Cancer ostasis including metabolic, immunologic, neurologic, or Institute, Amsterdam, the Netherlands. 4Neurosurgical Research, Depart- psychiatric disorders, as well as inflammation and cancer ment of Neurosurgery, University Hospital, Munich, Germany. 5Seve Ballesteros Foundation Brain Tumor Group, Molecular Oncology Pro- (14–17). However, despite the expansion of sophisticated gramme, Spanish National Cancer Research Center, Madrid, Spain. perturbation tools, such as CRISPR/Cas9 and optogenetics Note: Supplementary data for this article are available at Cancer Discovery (14), genetic tracing has yet to be fully exploited in the dissec- Online (http://cancerdiscovery.aacrjournals.org/). tion of complex disease traits. The pressing need for a novel M.J. Schmitt, C. Company, and Y. Dramaretska are the co–first authors of strategy to characterize brain tumor heterogeneity prompted this article. us to design synthetic reporters based on gene-expression Corresponding Author: Gaetano Gargiulo, Cancer Research, Max-Delbrück signatures. These reporters are designed to integrate multiple Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin 13125, Germany. pathways into a single genetic cassette, thereby mimicking Phone: 49-30-9406-3861; E-mail: [email protected] endogenous regulatory elements. As an example, the β-globin Cancer Discov 2021;11:1–24 locus control region shows cell type– and developmental doi: 10.1158/2159-8290.CD-20-0219 stage–specific expression and engages transcription factors ©2020 American Association for Cancer Research. independently of its genomic position (15, 16). March 2021 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst December 23, 2020; DOI: 10.1158/2159-8290.CD-20-0219 RESEARCH ARTICLE Schmitt et al. Our method permits us to genetically label individual cell The GBM subtype–specific reporters we generated can populations that share a similar state or undergo similar inform on the transcriptional identity of subsets of patients’ fate transitions within a heterogeneous tissue in vitro and in GBM single cells. Consistently, single-sample gene set enrich- vivo, and to discover mechanisms regulating proneural-to- ment analysis (ssGSEA) showed a high correspondence mesenchymal transition. Whereas a hierarchical and direc- between the potential expression pattern of each individual tional organization of the subtypes is continuously revised reporter, the known cell states of freshly purified single GBM (2, 5, 12, 17), through synthetic genetic tracing in vitro and cells (12), and their corresponding TCGA subtype (Fig. 1C). in vivo, we observed that interconversion between proneural Each sLCR encodes the subtype-specific expression of a fluo- and mesenchymal states is bidirectional. Our results are rescent reporter (mVenus or mCherry) and a second cassette clinically relevant because they expose a causal connection expressing the nuclear H2B–CFP fusion via a ubiquitous between radiotherapy or innate immune cell infiltration and viral promoter enabling reporter-independent selection (Fig. mesenchymal transdifferentiation, which was previously 1D). Reporter expression was validated in live transiently hypothesized on the basis of correlative analyses. Notably, we transfected cells, in stably transduced and cryosectioned link innate immunity and mesenchymal commitment to the tumorspheres, and in fixed tumor cells (Supplementary Fig. acquisition of selective resistance to therapies, which holds S1B–S1D). In the latter, dual RNA FISH and immunofluores- translational implications. cence demonstrated colocalization between nascent MGT#1 RNA and MED1, a master regulator organized in coactivator RESULTS puncta and regulating endogenous cell-identity LCRs (also known as superenhancers; ref. 18). GBM Subtype Genetic Tracing by Synthetic Locus To test the relative expression of synthetic reporters, which Control Regions are representative of two opposite GBM subtypes, we next To trace complex GBM expression subtype identities, we transduced proneural and mesenchymal sLCRs into a near- developed a method that generates synthetic reporters. Our isogenic pair of human glioma-initiating cells (hGIC). These method relies on robust evidence that transcription factors cells were engineered by genetically manipulating sponta- govern cell identity or states through binding to cis-regulatory neously immortalized human neural progenitor cells (19) elements, which in turn control downstream target genes. From and have a proneural-like expression signature, possibly The Cancer Genome Atlas (TCGA) data sets (3), we annotated inherited from the cell of origin (Supplementary Fig. S1E). the genes specific to mesenchymal, classic, and proneural sub- In addition to preexisting and shared aberrations, the near- types. Cell-intrinsic signature genes [i.e., differentially regulated isogenic
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