Single-cell analysis of patient-derived PDAC organoids reveals cell state heterogeneity and a conserved developmental hierarchy

Pancreatic tumors are frequently divided into basal and classical subtypes. Here, the authors use single cell sequencing to investigate organoids derived from pancreatic cancer tissue and find a hierarchy of distinct cell states, and classical and basal cells existing within the same tumor. Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer mortality by 2030. Bulk transcriptomic analyses have distinguished 'classical' from 'basal-like' tumors with more aggressive clinical behavior. We derive PDAC organoids from 18 primary tumors and two matched liver metastases, and show that 'classical' and 'basal-like' cells coexist in individual organoids. By single-cell transcriptome analysis of PDAC organoids and primary PDAC, we identify distinct tumor cell states shared across patients, including a cycling progenitor cell state and a differentiated secretory state. Cell states are connected by a differentiation hierarchy, with 'classical' cells concentrated at the endpoint. In an imaging-based drug screen, expression of 'classical' subtype genes correlates with better drug response. Our results thus uncover a functional hierarchy of PDAC cell states linked to transcriptional tumor subtypes, and support the use of PDAC organoids as a clinically relevant model for in vitro studies of tumor heterogeneity

Blm10 binds to pre-activated proteasome core particles with open gate conformation

Blm10, a crucial protease of eukaryotic cells, is bound to yeast proteasome core particles (CPs). Two gates, at both ends of the CP, control the access of protein substrates to the catalytic cavity of the CP. Normally, substrate access is auto-inhibited by a closed gate conformation unless regulatory complexes are bound to the CP and translocate protein substrates in an ATP-dependent manner. Here, we provide evidence that Blm10 recognizes pre-activated open gate CPs, which are assumed to exist in an equilibrium with inactive closed gate CP. Consequently, single-capped Blm10-CP shows peptide hydrolysis activity. Under conditions of disturbed CP assembly, as well as in open gate mutants, pre-activated CP or constitutively active CP, respectively, prevail. Then, Blm10 sequesters disordered and open gate CP by forming double-capped Blm102-CP in which peptide hydrolysis activity is repressed. We conclude that Blm10 distinguishes between gate conformations and regulates the activation of CP

Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish

EGFP-tagged PrP constructs used in this study.

<p>The structural domains of zebrafish (zf) PrP-1, PrP-2 and mouse (m) PrP are represented as follows: leader peptide containing the polybasic motif (L) in violet, repetitive domain (Rep) in blue, hydrophobic region (Hyd) in red, globular domain (Glob) in light blue and GPI-anchored signal (GPI) in yellow. Amino acid (aa) positions of mouse and fish PrP domains are indicated. The EGFP fluorescence tags are depicted as green triangles. Deletion constructs lacking Rep (ΔRep), Hyd (ΔHyd), Glob (ΔGlob), Rep+Hyd+Glob (ΔCore), GPI (GPI⁻) and N-glycosylation sites (Glyc⁻) are shown for mouse PrP only. PrP domains were defined by evolutionary criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070327#pone.0070327-RiveraMilla1" target="_blank">[13]</a>.</p

Accumulation of mouse and zebrafish PrP constructs at established MCF-7 cell cell contacts.

<p>Wild type (WT) and mutant EGFP-tagged constructs of mouse PrP (A) and zebrafish PrP-1 (B) localize differently at E-cadherin-positive cell contact sites (in red). Marked areas on the overlays are enlarged (right) to show detailed views of the contact sites. Cell nuclei are stained with DAPI (blue). Scale bars = 10 µm.</p

Rescue of PrP-1 knockdown embryos by mutant PrP-1 constructs.

<p>PrP-1 morphant embryos were microinjected with mRNAs encoding selected EGFP-tagged PrP-1 constructs, and their rescue activity was evaluated morphologically and molecularly. A) Quantitative differences in rescue activity between untreated (control) or morphant embryos (PrP-1 MO), and embryos expressing WT, ΔRep, ΔGlob and Glyc⁻ PrP-1 constructs. Data are given as the proportion of embryos showing normal-to-mild gastrulation phenotypes at 6 hpf. Three independent experiments were analyzed (average n = 30 embryos). Triple asterisks [***] indicate statistically significant rescues at <i>p</i><0.001; one-way ANOVA test; error bars represent SEM. B) Confocal images of deep cells from embryos immunostained against E-cadherin. Rescue is indicated by the recovery of E-cadherin cell-surface localization. Scale bar = 10 µm.</p

Overexpression (OE) of mouse and zebrafish PrP constructs in early zebrafish embryos.

<p>Embryos were microinjected with mRNAs encoding all mouse and zebrafish EGFP-tagged PrP constructs. A) OE of mouse or zebrafish WT PrPs in early embryos produces a gain-of-function phenotype characterized by asymmetric gastrulation at 6 hpf. B–D) The activities of mouse and zebrafish constructs were evaluated morphologically by quantifying the proportion of 6 hpf embryos exhibiting the OE phenotype (asymmetric gastrulation). Three independent experiments were analyzed (average n = 30 embryos). Triple asterisks [***] indicate statistically significant reduction in activity at <i>p</i><0.001; one-way ANOVA test; error bars represent SEM.</p

Light Pollution, Circadian Photoreception, and Melatonin in Vertebrates

Artificial light at night (ALAN) is increasing exponentially worldwide, accelerated by the transition to new efficient lighting technologies. However, ALAN and resulting light pollution can cause unintended physiological consequences. In vertebrates, production of melatonin—the “hormone of darkness” and a key player in circadian regulation—can be suppressed by ALAN. In this paper, we provide an overview of research on melatonin and ALAN in vertebrates. We discuss how ALAN disrupts natural photic environments, its effect on melatonin and circadian rhythms, and different photoreceptor systems across vertebrate taxa. We then present the results of a systematic review in which we identified studies on melatonin under typical light-polluted conditions in fishes, amphibians, reptiles, birds, and mammals, including humans. Melatonin is suppressed by extremely low light intensities in many vertebrates, ranging from 0.01–0.03 lx for fishes and rodents to 6 lx for sensitive humans. Even lower, wavelength-dependent intensities are implied by some studies and require rigorous testing in ecological contexts. In many studies, melatonin suppression occurs at the minimum light levels tested, and, in better-studied groups, melatonin suppression is reported to occur at lower light levels. We identify major research gaps and conclude that, for most groups, crucial information is lacking. No studies were identified for amphibians and reptiles and long-term impacts of low-level ALAN exposure are unknown. Given the high sensitivity of vertebrate melatonin production to ALAN and the paucity of available information, it is crucial to research impacts of ALAN further in order to inform effective mitigation strategies for human health and the wellbeing and fitness of vertebrates in natural ecosystems.Peer reviewe

Functional States in Tumor-Initiating Cell Differentiation in Human Colorectal Cancer

Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC