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Rapid non-uniform adaptation to conformation-specific KRAS(G12C) inhibition

KRAS GTPases are activated in one-third of cancers, and KRAS(G12C) is one of the most common activating alterations in lung adenocarcinoma. KRAS(G12C) inhibitors are in phase-I clinical trials and early data show partial responses in nearly half of patients with lung cancer. How cancer cells bypass inhibition to prevent maximal response to therapy is not understood. Because KRAS(G12C) cycles between an active and inactive conformation4-6, and the inhibitors bind only to the latter, we tested whether isogenic cell populations respond in a non-uniform manner by studying the effect of treatment at a single-cell resolution. Here we report that, shortly after treatment, some cancer cells are sequestered in a quiescent state with low KRAS activity, whereas others bypass this effect to resume proliferation. This rapid divergent response occurs because some quiescent cells produce new KRAS(G12C) in response to suppressed mitogen-activated protein kinase output. New KRAS(G12C) is maintained in its active, drug-insensitive state by epidermal growth factor receptor and aurora kinase signalling. Cells without these adaptive changes-or cells in which these changes are pharmacologically inhibited-remain sensitive to drug treatment, because new KRAS(G12C) is either not available or exists in its inactive, drug-sensitive state. The direct targeting of KRAS oncoproteins has been a longstanding objective in precision oncology. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome if we are to achieve complete and durable responses in the clinic

In vivo developmental trajectories of human podocyte inform in vitro differentiation of pluripotent stem cell-derived podocytes

The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions.

Decoding the development of the human hippocampus

The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and the consolidation of information from short-term memory to long-term memory1,2. Here we use single-cell RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC–seq) analysis to illustrate the cell types, cell linage, molecular features and transcriptional regulation of the developing human hippocampus. Using the transcriptomes of 30,416 cells from the human hippocampus at gestational weeks 16–27, we identify 47 cell subtypes and their developmental trajectories. We also identify the migrating paths and cell lineages of PAX6+ and HOPX+ hippocampal progenitors, and regional markers of CA1, CA3 and dentate gyrus neurons. Multiomic data have uncovered transcriptional regulatory networks of the dentate gyrus marker PROX1. We also illustrate spatially specific gene expression in the developing human prefrontal cortex and hippocampus. The molecular features of the human hippocampus at gestational weeks 16–20 are similar to those of the mouse at postnatal days 0–5 and reveal gene expression differences between the two species. Transient expression of the primate-specific gene NBPF1 leads to a marked increase in PROX1+ cells in the mouse hippocampus. These data provides a blueprint for understanding human hippocampal development and a tool for investigating related diseases.

Single-cell RNA sequencing reveals stromal evolution into LRRC15+ myofibroblasts as a determinant of patient response to cancer immunotherapy

With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAFs) programmed by transforming growth factor beta and expressing the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15+ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15+ CAFs in human patients was confirmed in >80,000 single-cells from 22 PDAC patients as well as immunohistochemistry on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising over 600 patients across 6 cancer types revealed elevated levels of the LRRC15+ CAF signature correlated with poor response to anti-PD-L1 therapy. This work has important implications for targeting non-immune elements of the tumor microenvironment to boost responses of cancer patients to immune checkpoint blockade therapy.

Landscape and dynamics of single immune cells in hepatocellular carcinoma (Smart-seq2)

The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45 + immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3 + dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3 + DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45 + cell types revealed by this study add new dimensions to the immune landscape of HCC.

Landscape and dynamics of single immune cells in hepatocellular carcinoma (10X)

The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45 + immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3 + dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3 + DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45 + cell types revealed by this study add new dimensions to the immune landscape of HCC.

The Dynamic Transcriptional Cell Atlas of Testis Development during Human Puberty

The human testis undergoes dramatic developmental and structural changes during puberty, including proliferation and maturation of somatic niche cells, and the onset of spermatogenesis. To characterize this understudied process, we profiled and analyzed single-cell transcriptomes of ∼10,000 testicular cells from four boys spanning puberty and compared them to those of infants and adults. During puberty, undifferentiated spermatogonia sequentially expand and differentiate prior to the initiation of gametogenesis. Notably, we identify a common pre-pubertal progenitor for Leydig and myoid cells and delineate candidate factors controlling pubertal differentiation. Furthermore, pre-pubertal Sertoli cells exhibit two distinct transcriptional states differing in metabolic profiles before converging to an alternative single mature population during puberty. Roles for testosterone in Sertoli cell maturation, antimicrobial peptide secretion, and spermatogonial differentiation are further highlighted through single-cell analysis of testosterone-suppressed transfemale testes. Taken together, our transcriptional atlas of the developing human testis provides multiple insights into developmental changes and key factors accompanying male puberty.

(Fibroblast-enriched) Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts

Cancer-associated fibroblasts (CAF) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we use single-cell RNA sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human and mouse PDAC tumors. We corroborate the presence of myofibroblastic CAFs and inflammatory CAFs and define their unique gene signatures in vivo. Moreover, we describe a new population of CAFs that express MHC class II and CD74, but do not express classic costimulatory molecules. We term this cell population “antigen-presenting CAFs” and find that they activate CD4+ T cells in an antigen-specific fashion in a model system, confirming their putative immune-modulatory capacity. Our cross-species analysis paves the way for investigating distinct functions of CAF subtypes in PDAC immunity and progression. SIGNIFICANCE: Appreciating the full spectrum of fibroblast heterogeneity in pancreatic ductal adenocarcinoma is crucial to developing therapies that specifically target tumor-promoting CAFs. This work identifies MHC class II–expressing CAFs with a capacity to present antigens to CD4+ T cells, and potentially to modulate the immune response in pancreatic tumors.

(Viable cell) Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts

Cancer-associated fibroblasts (CAF) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we use single-cell RNA sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human and mouse PDAC tumors. We corroborate the presence of myofibroblastic CAFs and inflammatory CAFs and define their unique gene signatures in vivo. Moreover, we describe a new population of CAFs that express MHC class II and CD74, but do not express classic costimulatory molecules. We term this cell population “antigen-presenting CAFs” and find that they activate CD4+ T cells in an antigen-specific fashion in a model system, confirming their putative immune-modulatory capacity. Our cross-species analysis paves the way for investigating distinct functions of CAF subtypes in PDAC immunity and progression. SIGNIFICANCE: Appreciating the full spectrum of fibroblast heterogeneity in pancreatic ductal adenocarcinoma is crucial to developing therapies that specifically target tumor-promoting CAFs. This work identifies MHC class II–expressing CAFs with a capacity to present antigens to CD4+ T cells, and potentially to modulate the immune response in pancreatic tumors.

Integrated single cell analysis of blood and cerebrospinal fluid leukocytes in multiple sclerosis

Cerebrospinal fluid (CSF) protects the central nervous system (CNS) and analyzing CSF aids the diagnosis of CNS diseases, but our understanding of CSF leukocytes remains superficial. Here, using single cell transcriptomics, we identify a specific location-associated composition and transcriptome of CSF leukocytes. Multiple sclerosis (MS) – an autoimmune disease of the CNS – increases transcriptional diversity in blood, but increases cell type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells. An analytical approach, named cell set enrichment analysis (CSEA) identifies a cluster-independent increase of follicular (TFH) cells potentially driving the known expansion of B lineage cells in the CSF in MS. In mice, TFH cells accordingly promote B cell infiltration into the CNS and the severity of MS animal models. Immune mechanisms in MS are thus highly compartmentalized and indicate ongoing local T/B cell interaction.

Mucosal Profiling of Pediatric-Onset Colitis and IBD Reveals Common Pathogenics and Therapeutic Pathways

Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn’s disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.

The immune cell landscape in kidneys of patients with lupus nephritis

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing

Cancer immunotherapies have shown sustained clinical responses in treating non-small-cell lung cancer1,2,3, but efficacy varies and depends in part on the amount and properties of tumor infiltrating lymphocytes4,5,6. To depict the baseline landscape of the composition, lineage and functional states of tumor infiltrating lymphocytes, here we performed deep single-cell RNA sequencing for 12,346 T cells from 14 treatment-naïve non-small-cell lung cancer patients. Combined expression and T cell antigen receptor based lineage tracking revealed a significant proportion of inter-tissue effector T cells with a highly migratory nature. As well as tumor-infiltrating CD8+ T cells undergoing exhaustion, we observed two clusters of cells exhibiting states preceding exhaustion, and a high ratio of “pre-exhausted” to exhausted T cells was associated with better prognosis of lung adenocarcinoma. Additionally, we observed further heterogeneity within the tumor regulatory T cells (Tregs), characterized by the bimodal distribution of TNFRSF9, an activation marker for antigen-specific Tregs. The gene signature of those activated tumor Tregs, which included IL1R2, correlated with poor prognosis in lung adenocarcinoma. Our study provides a new approach for patient stratification and will help further understand the functional states and dynamics of T cells in lung cancer.

Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution

The human bronchial epithelium is composed of multiple, distinct cell types that cooperate to perform functions, such as mucociliary clearance, that defend against environmental insults. While studies have shown that smoking alters bronchial epithelial function and morphology, the precise effects of this exposure on specific cell types are not well-understood. We used single-cell RNA sequencing to profile bronchial epithelial cells from six never- and six current smokers. Unsupervised analyses identified thirteen cell clusters defined by unique combinations of nineteen distinct gene sets. Expression of a set of toxin metabolism genes localized to ciliated cells from smokers. Smoking-induced airway remodeling was characterized by a loss of club cells and extensive goblet cell hyperplasia. Finally, we identified a novel peri-goblet epithelial subpopulation in smokers that expressed a marker of bronchial premalignant lesions. Our data demonstrates that smoke exposure drives a complex landscape of cellular and molecular alterations in the human bronchial epithelium that may contribute to the onset of smoking-associated lung diseases.

Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity of Aortic Macrophages in Murine Atherosclerosis

Rationale: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they have been defined by the expression of a restricted number of markers. Objective: We have applied single-cell RNA sequencing as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis. Method and Results: We performed single-cell RNA sequencing of total aortic CD45+ cells extracted from the nondiseased (chow fed) and atherosclerotic (11 weeks of high-fat diet) aorta of low-density lipoprotein receptor–deficient (Ldlr−/−) mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortas, whereas monocytes, monocyte-derived dendritic cells, and 2 populations of macrophages were almost exclusively detectable in atherosclerotic aortas, comprising inflammatory macrophages showing enrichment in Il1b and previously undescribed TREM2hi (triggered receptor expressed on myeloid cells 2) macrophages showing enrichment in Trem2. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these 3 macrophage subsets and monocyte-derived dendritic cells and uncovered putative functions of each cell type. Notably, TREM2hi macrophages seemed to be endowed with specialized functions in lipid metabolism and catabolism and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in Apoe−/− aortas, indicating relevance of our findings in different stages of atherosclerosis and mouse models.