Cancer cell states: Lessons from ten years of single-cell RNA-sequencing of human tumors.

Human tumors are intricate ecosystems composed of diverse genetic clones and malignant cell states that evolve in a complex tumor micro-environment. Single-cell RNA-sequencing (scRNA-seq) provides a compelling strategy to dissect this intricate biology and has enabled a revolution in our ability to understand tumor biology over the last ten years. Here we reflect on this first decade of scRNA-seq in human tumors and highlight some of the powerful insights gleaned from these studies. We first focus on computational approaches for robustly defining cancer cell states and their diversity and highlight some of the most common patterns of gene expression intra-tumor heterogeneity (eITH) observed across cancer types. We then discuss ambiguities in the field in defining and naming such eITH programs. Finally, we highlight critical developments that will facilitate future research and the broader implementation of these technologies in clinical settings.

A spatial expression atlas of the adult human proximal small intestine.

The mouse small intestine shows profound variability in gene expression along the crypt-villus axis1,2. Whether similar spatial heterogeneity exists in the adult human gut remains unclear. Here we use spatial transcriptomics, spatial proteomics and single-molecule fluorescence in situ hybridization to reconstruct a comprehensive spatial expression atlas of the adult human proximal small intestine. We describe zonated expression and cell type representation for epithelial, mesenchymal and immune cell types. We find that migrating enterocytes switch from lipid droplet assembly and iron uptake at the villus bottom to chylomicron biosynthesis and iron release at the tip. Villus tip cells are pro-immunogenic, recruiting γδ T cells and macrophages to the tip, in contrast to their immunosuppressive roles in mouse. We also show that the human small intestine contains abundant serrated and branched villi that are enriched at the tops of circular folds. Our study presents a detailed resource for understanding the biology of the adult human small intestine.

Single-cell atlas of the human brain vasculature across development, adulthood and disease.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Oncogenic Calreticulin Induces Immune Escape by Stimulating TGFß Expression and Regulatory T-cell Expansion in the Bone Marrow Microenvironment.

Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA sequencing revealed that CALRdel52 led to the expansion of TGFß1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGFß antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, whereas T-cell depletion abrogated the protective effects conferred by neutralizing TGFß. TGFß1 reduced perforin and TNFα production by T cells in vitro. TGFß1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGFß1 expression. In four independent patient cohorts, TGFß1 expression was increased in the BM of patients with MPN compared with healthy individuals, and the BM of patients with MPN contained a higher frequency of Treg compared with healthy individuals. Together, this study identified an ERK/Sp1/TGFß1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity. Significance: Targeting the mutant calreticulin/TGFß1 axis increases T-cell activity and glycolytic capacity, providing the rationale for conducting clinical trials on TGFß antagonists as an immunotherapeutic strategy in patients with myeloproliferative neoplasms.

A hybrid epithelial/mesenchymal state in head and neck cancer: A biomarker for survival with differential prognosis by self-reported race.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the 6th leading cause of cancer-related mortality, with racial disparities amplifying the challenges in treatment. Although the relationship between hybrid epithelial/mesenchymal (E/M) states and tumor progression is of interest, no studies have characterized the clinical relevance of hybrid E/M states in head and neck cancer outcomes among self-reported racial cohorts. METHODS: Given the overlap in gene expression between hybrid E/M malignant cells and cancer-associated fibroblasts, we utilized deconvolution of bulk RNA sequencing data from oral cavity and laryngeal squamous cell carcinoma tumors from The Cancer Genome Atlas. We utilized our previously collected single-cell profiles to generate inferred malignant profiles and then scored these for hybrid E/M. We then conducted a survival analysis on overall and disease-free survival among self-reported Black and White Americans. FINDINGS: The hybrid E/M state was differentially associated with head and neck cancer survival by self-reported race and ethnicity, with a stronger association in non-Hispanic Black patients. Black patients with a high hybrid E/M score had a higher risk of death or recurrence (hazard ratio [HR]: 4.18 [95% confidence interval (CI): 2.06, 8.49]) than White patients with a high hybrid E/M score (HR: 1.58 [95% CI: 1.11, 2.26]). CONCLUSION: Our results suggest a complex interplay of social structure, racism, and genetic diversity. We implore researchers to consider the social and biological context contributing to disparities. FUNDING: A.L.M. received support from the National Institute of Minority Health and Health Disparities (K01MD013897 [principal investigator (PI), A.L.M.]). S.V.P. received support from the National Institute of Dental and Craniofacial Research (R01DE032865 [PI, S.V.P.] and R01DE032371 [PI, S.V.P.]).

Mutant IDH inhibitors induce lineage differentiation in IDH-mutant oligodendroglioma.

A subset of patients with IDH-mutant glioma respond to inhibitors of mutant IDH (IDHi), yet the molecular underpinnings of such responses are not understood. Here, we profiled by single-cell or single-nucleus RNA-sequencing three IDH-mutant oligodendrogliomas from patients who derived clinical benefit from IDHi. Importantly, the tissues were sampled on-drug, four weeks from treatment initiation. We further integrate our findings with analysis of single-cell and bulk transcriptomes from independent cohorts and experimental models. We find that IDHi treatment induces a robust differentiation toward the astrocytic lineage, accompanied by a depletion of stem-like cells and a reduction of cell proliferation. Furthermore, mutations in NOTCH1 are associated with decreased astrocytic differentiation and may limit the response to IDHi. Our study highlights the differentiating potential of IDHi on the cellular hierarchies that drive oligodendrogliomas and suggests a genetic modifier that may improve patient stratification.

Integrative spatial analysis reveals a multi-layered organization of glioblastoma.

Glioma contains malignant cells in diverse states. Here, we combine spatial transcriptomics, spatial proteomics, and computational approaches to define glioma cellular states and uncover their organization. We find three prominent modes of organization. First, gliomas are composed of small local environments, each typically enriched with one major cellular state. Second, specific pairs of states preferentially reside in proximity across multiple scales. This pairing of states is consistent across tumors. Third, these pairwise interactions collectively define a global architecture composed of five layers. Hypoxia appears to drive the layers, as it is associated with a long-range organization that includes all cancer cell states. Accordingly, tumor regions distant from any hypoxic/necrotic foci and tumors that lack hypoxia such as low-grade IDH-mutant glioma are less organized. In summary, we provide a conceptual framework for the organization of cellular states in glioma, highlighting hypoxia as a long-range tissue organizer.

Glioma synapses recruit mechanisms of adaptive plasticity.

The role of the nervous system in the regulation of cancer is increasingly appreciated. In gliomas, neuronal activity drives tumour progression through paracrine signalling factors such as neuroligin-3 and brain-derived neurotrophic factor1-3 (BDNF), and also through electrophysiologically functional neuron-to-glioma synapses mediated by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors4,5. The consequent glioma cell membrane depolarization drives tumour proliferation4,6. In the healthy brain, activity-regulated secretion of BDNF promotes adaptive plasticity of synaptic connectivity7,8 and strength9-15. Here we show that malignant synapses exhibit similar plasticity regulated by BDNF. Signalling through the receptor tropomyosin-related kinase B16 (TrkB) to CAMKII, BDNF promotes AMPA receptor trafficking to the glioma cell membrane, resulting in increased amplitude of glutamate-evoked currents in the malignant cells. Linking plasticity of glioma synaptic strength to tumour growth, graded optogenetic control of glioma membrane potential demonstrates that greater depolarizing current amplitude promotes increased glioma proliferation. This potentiation of malignant synaptic strength shares mechanistic features with synaptic plasticity17-22 that contributes to memory and learning in the healthy brain23-26. BDNF-TrkB signalling also regulates the number of neuron-to-glioma synapses. Abrogation of activity-regulated BDNF secretion from the brain microenvironment or loss of glioma TrkB expression robustly inhibits tumour progression. Blocking TrkB genetically or pharmacologically abrogates these effects of BDNF on glioma synapses and substantially prolongs survival in xenograft models of paediatric glioblastoma and diffuse intrinsic pontine glioma. Together, these findings indicate that BDNF-TrkB signalling promotes malignant synaptic plasticity and augments tumour progression.

Spectrum of Response to Platinum and PARP Inhibitors in Germline BRCA-Associated Pancreatic Cancer in the Clinical and Preclinical Setting.

Germline BRCA-associated pancreatic ductal adenocarcinoma (glBRCA PDAC) tumors are susceptible to platinum and PARP inhibition. The clinical outcomes of 125 patients with glBRCA PDAC were stratified based on the spectrum of response to platinum/PARP inhibition: (i) refractory [overall survival (OS) <6 months], (ii) durable response followed by acquired resistance (OS <36 months), and (iii) long-term responders (OS >36 months). Patient-derived xenografts (PDX) were generated from 25 patients with glBRCA PDAC at different clinical time points. Response to platinum/PARP inhibition in vivo and ex vivo culture (EVOC) correlated with clinical response. We deciphered the mechanisms of resistance in glBRCA PDAC and identified homologous recombination (HR) proficiency and secondary mutations restoring partial functionality as the most dominant resistant mechanism. Yet, a subset of HR-deficient (HRD) patients demonstrated clinical resistance. Their tumors displayed basal-like molecular subtype and were more aneuploid. Tumor mutational burden was high in HRD PDAC and significantly higher in tumors with secondary mutations. Anti-PD-1 attenuated tumor growth in a novel humanized glBRCA PDAC PDX model. This work demonstrates the utility of preclinical models, including EVOC, to predict the response of glBRCA PDAC to treatment, which has the potential to inform time-sensitive medical decisions. SIGNIFICANCE: glBRCA PDAC has a favorable response to platinum/PARP inhibition. However, most patients develop resistance. Additional treatment options for this unique subpopulation are needed. We generated model systems in PDXs and an ex vivo system (EVOC) that faithfully recapitulate these specific clinical scenarios as a platform to investigate the mechanisms of resistance for further drug development. This article is highlighted in the In This Issue feature, p. 1749.

Hallmarks of transcriptional intratumour heterogeneity across a thousand tumours.

Each tumour contains diverse cellular states that underlie intratumour heterogeneity (ITH), a central challenge of cancer therapeutics1. Dozens of recent studies have begun to describe ITH by single-cell RNA sequencing, but each study typically profiled only a small number of tumours and provided a narrow view of transcriptional ITH2. Here we curate, annotate and integrate the data from 77 different studies to reveal the patterns of transcriptional ITH across 1,163 tumour samples covering 24 tumour types. Among the malignant cells, we identify 41 consensus meta-programs, each consisting of dozens of genes that are coordinately upregulated in subpopulations of cells within many tumours. The meta-programs cover diverse cellular processes including both generic (for example, cell cycle and stress) and lineage-specific patterns that we map into 11 hallmarks of transcriptional ITH. Most meta-programs of carcinoma cells are similar to those identified in non-malignant epithelial cells, suggesting that a large fraction of malignant ITH programs are variable even before oncogenesis, reflecting the biology of their cell of origin. We further extended the meta-program analysis to six common non-malignant cell types and utilize these to map cell-cell interactions within the tumour microenvironment. In summary, we have assembled a comprehensive pan-cancer single-cell RNA-sequencing dataset, which is available through the Curated Cancer Cell Atlas website, and leveraged this dataset to carry out a systematic characterization of transcriptional ITH.

Cellular states are coupled to genomic and viral heterogeneity in HPV-related oropharyngeal carcinoma.

Head and neck squamous cell carcinoma (HNSCC) includes a subset of cancers driven by human papillomavirus (HPV). Here we use single-cell RNA-seq to profile both HPV-positive and HPV-negative oropharyngeal tumors, uncovering a high level of cellular diversity within and between tumors. First, we detect diverse chromosomal aberrations within individual tumors, suggesting genomic instability and enabling the identification of malignant cells even at pathologically negative margins. Second, we uncover diversity with respect to HNSCC subtypes and other cellular states such as the cell cycle, senescence and epithelial-mesenchymal transitions. Third, we find heterogeneity in viral gene expression within HPV-positive tumors. HPV expression is lost or repressed in a subset of cells, which are associated with a decrease in HPV-associated cell cycle phenotypes, decreased response to treatment, increased invasion and poor prognosis. These findings suggest that HPV expression diversity must be considered during diagnosis and treatment of HPV-positive tumors, with important prognostic ramifications.

Elucidating the diversity of malignant mesenchymal states in glioblastoma by integrative analysis.

BACKGROUND: Multiple glioblastoma studies have described a mesenchymal (MES) state, with each study defining the MES program by distinct sets of genes and highlighting distinct functional associations, including both immune activation and suppression. These variable descriptions complicate our understanding of the MES state and its implications. Here, we hypothesize that there is a range of glioma MES states, possibly reflecting distinct prior states in which a MES program can be induced, and/or distinct mechanisms that induce the MES states in those cells. METHODS: We integrated multiple published single-cell and bulk RNA sequencing datasets and MES signatures to define a core MES program that recurs across studies, as well as multiple function-specific MES signatures that vary across MES cells. We then examined the co-occurrence of these signatures and their associations with genetic and microenvironmental features. RESULTS: Based on co-occurrence of MES signatures, we found three main variants of MES states: hypoxia-related (MES-Hyp), astrocyte-related (MES-Ast), and an intermediate state. Notably, the MES states are differentially associated with genetic and microenvironmental features. MES-Hyp is preferentially associated with NF1 deletion, overall macrophage abundance, a high macrophage/microglia ratio, and M2-related macrophages, consistent with previous studies that associated MES with immune suppression. In contrast, MES-Ast is associated with T cell abundance and cytotoxicity, consistent with immune activation through expression of MHC-I/II. CONCLUSIONS: Diverse MES states occur in glioblastoma. These states share a subset of core genes but differ primarily in their association with hypoxia vs. astrocytic expression programs, and with immune suppression vs. activation, respectively.

An activation to memory differentiation trajectory of tumor-infiltrating lymphocytes informs metastatic melanoma outcomes.

There is a need for better classification and understanding of tumor-infiltrating lymphocytes (TILs). Here, we applied advanced functional genomics to interrogate 9,000 human tumors and multiple single-cell sequencing sets using benchmarked T cell states, comprehensive T cell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared with other T cell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8+ TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early T cell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity.

Genome-wide CRISPR screen identifies PRC2 and KMT2D-COMPASS as regulators of distinct EMT trajectories that contribute differentially to metastasis.

Epithelial-mesenchymal transition (EMT) programs operate within carcinoma cells, where they generate phenotypes associated with malignant progression. In their various manifestations, EMT programs enable epithelial cells to enter into a series of intermediate states arrayed along the E-M phenotypic spectrum. At present, we lack a coherent understanding of how carcinoma cells control their entrance into and continued residence in these various states, and which of these states favour the process of metastasis. Here we characterize a layer of EMT-regulating machinery that governs E-M plasticity (EMP). This machinery consists of two chromatin-modifying complexes, PRC2 and KMT2D-COMPASS, which operate as critical regulators to maintain a stable epithelial state. Interestingly, loss of these two complexes unlocks two distinct EMT trajectories. Dysfunction of PRC2, but not KMT2D-COMPASS, yields a quasi-mesenchymal state that is associated with highly metastatic capabilities and poor survival of patients with breast cancer, suggesting that great caution should be applied when PRC2 inhibitors are evaluated clinically in certain patient cohorts. These observations identify epigenetic factors that regulate EMP, determine specific intermediate EMT states and, as a direct consequence, govern the metastatic ability of carcinoma cells.

Interactions between cancer cells and immune cells drive transitions to mesenchymal-like states in glioblastoma.

The mesenchymal subtype of glioblastoma is thought to be determined by both cancer cell-intrinsic alterations and extrinsic cellular interactions, but remains poorly understood. Here, we dissect glioblastoma-to-microenvironment interactions by single-cell RNA sequencing analysis of human tumors and model systems, combined with functional experiments. We demonstrate that macrophages induce a transition of glioblastoma cells into mesenchymal-like (MES-like) states. This effect is mediated, both in vitro and in vivo, by macrophage-derived oncostatin M (OSM) that interacts with its receptors (OSMR or LIFR) in complex with GP130 on glioblastoma cells and activates STAT3. We show that MES-like glioblastoma states are also associated with increased expression of a mesenchymal program in macrophages and with increased cytotoxicity of T cells, highlighting extensive alterations of the immune microenvironment with potential therapeutic implications.

Decoupling epithelial-mesenchymal transitions from stromal profiles by integrative expression analysis.

Epithelial-to-mesenchymal transition (EMT) is the most commonly cited mechanism for cancer metastasis, but it is difficult to distinguish from profiles of normal stromal cells in the tumour microenvironment. In this study we use published single cell RNA-seq data to directly compare mesenchymal signatures from cancer and stromal cells. Informed by these comparisons, we developed a computational framework to decouple these two sources of mesenchymal expression profiles using bulk RNA-seq datasets. This deconvolution offers the opportunity to characterise EMT across hundreds of tumours and examine its association with metastasis and other clinical features. With this approach, we find three distinct patterns of EMT, associated with squamous, gynaecological and gastrointestinal cancer types. Surprisingly, in most cancer types, EMT patterns are not associated with increased chance of metastasis, suggesting that other steps in the metastatic cascade may represent the main bottleneck. This work provides a comprehensive evaluation of EMT profiles and their functional significance across hundreds of tumours while circumventing the confounding effect of stromal cells.

Decoding Cancer Biology One Cell at a Time.

Human tumors are composed of diverse malignant and nonmalignant cells, generating a complex ecosystem that governs tumor biology and response to treatments. Recent technological advances have enabled the characterization of tumors at single-cell resolution, providing a compelling strategy to dissect their intricate biology. Here we describe recent developments in single-cell expression profiling and the studies applying them in clinical settings. We highlight some of the powerful insights gleaned from these studies for tumor classification, stem cell programs, tumor microenvironment, metastasis, and response to targeted and immune therapies. SIGNIFICANCE: Intratumor heterogeneity (ITH) has been a major barrier to our understanding of cancer. Single-cell genomics is leading a revolution in our ability to systematically dissect ITH. In this review, we focus on single-cell expression profiling and lessons learned in key aspects of human tumor biology.

Single-Cell Deconvolution of Head and Neck Squamous Cell Carcinoma.

Complexities in cell-type composition have rightfully led to skepticism and caution in the interpretation of bulk transcriptomic analyses. Recent studies have shown that deconvolution algorithms can be utilized to computationally estimate cell-type proportions from the gene expression data of bulk blood samples, but their performance when applied to tumor tissues, including those from head and neck, remains poorly characterized. Here, we use single-cell data (~6000 single cells) collected from 21 head and neck squamous cell carcinoma (HNSCC) samples to generate cell-type-specific gene expression signatures. We leverage bulk RNA-seq data from >500 HNSCC samples profiled by The Cancer Genome Atlas (TCGA), and using single-cell data as a reference, apply two newly developed deconvolution algorithms (CIBERSORTx and MuSiC) to the bulk transcriptome data to quantitatively estimate cell-type proportions for each tumor in TCGA. We show that these two algorithms produce similar estimates of constituent/major cell-type proportions and that a high T-cell fraction correlates with improved survival. By further characterizing T-cell subpopulations, we identify that regulatory T-cells (Tregs) were the major contributor to this improved survival. Lastly, we assessed gene expression, specifically in the Treg population, and found that TNFRSF4 (Tumor Necrosis Factor Receptor Superfamily Member 4) was differentially expressed in the core Treg subpopulation. Moreover, higher TNFRSF4 expression was associated with greater survival, suggesting that TNFRSF4 could play a key role in mechanisms underlying the contribution of Treg in HNSCC outcomes.