Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, although their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity. Here, following a negative selection strategy combined with single-cell RNA sequencing of 768 transcriptomes of mesenchymal cells from a genetically engineered mouse model of breast cancer, we define three distinct subpopulations of CAFs. Validation at the transcriptional and protein level in several experimental models of cancer and human tumors reveal spatial separation of the CAF subclasses attributable to different origins, including the peri-vascular niche, the mammary fat pad and the transformed epithelium. Gene profiles for each CAF subtype correlate to distinctive functional programs and hold independent prognostic capability in clinical cohorts by association to metastatic disease. In conclusion, the improved resolution of the widely defined CAF population opens the possibility for biomarker-driven development of drugs for precision targeting of CAFs.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of β-cells. Pancreatic β-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature β-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger β-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for β-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional β-cell heterogeneity and induce β-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional β-cell mass in diabetic patients.

We performed fine-mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in/near KCNQ1. “Credible sets” of variants most likely to drive each distinct signal mapped predominantly to non-coding sequence, implying that T2D association is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine-mapping implicated rs10830963 as driving T2D association. We confirmed that this T2D-risk allele increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D-risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease.

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, key tissues and cell-types required for functional inference are absent from large-scale resources. Here we explore the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using data from 420 donors. We find: (a) 7741 cis-eQTLs in islets with a replication rate across 44 GTEx tissues between 40% and 73%; (b) marked overlap between islet cis-eQTL signals and active regulatory sequences in islets, with reduced eQTL effect size observed in the stretch enhancers most strongly implicated in GWAS signal location; (c) enrichment of islet cis-eQTL signals with T2D risk variants identified in genome-wide association studies; and (d) colocalization between 47 islet cis-eQTLs and variants influencing T2D or glycemic traits, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in disease relevant tissues.