High PD-1/PD-L1 Checkpoint Interaction Infers Tumor Selection and Therapeutic Sensitivity to Anti-PD-1/PD-L1 Treatment

Many cancers are termed immunoevasive due to expression of immunomodulatory ligands. Programmed death ligand-1 (PD-L1) and cluster of differentiation 80/86 (CD80/86) interact with their receptors, programmed death receptor-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4), respectively, on tumor-infiltrating leukocytes eliciting immunosuppression. Immunotherapies aimed at blocking these interactions are revolutionizing cancer treatments, albeit in an inadequately described patient subset. To address the issue of patient stratification for immune checkpoint intervention, we quantitatively imaged PD-1/PD-L1 interactions in tumor samples from patients, employing an assay that readily detects these intercellular protein-protein interactions in the less than or equal to 10 nm range. These analyses across multiple patient cohorts demonstrated the intercancer, interpatient, and intratumoral heterogeneity of interacting immune checkpoints. The PD-1/PD-L1 interaction was not correlated with clinical PD-L1 expression scores in malignant melanoma. Crucially, among anti-PD-1-treated patients with metastatic non-small cell lung cancer, those with lower PD-1/PD-L1 interaction had significantly worsened survival. It is surmised that within tumors selecting for an elevated level of PD-1/PD-L1 interaction, there is a greater dependence on this pathway for immune evasion and hence, they exhibit more impressive patient response to intervention. SIGNIFICANCE: Quantitation of immune checkpoint interaction by direct imaging demonstrates that immunotherapy-treated patients with metastatic NSCLC with a low extent of PD-1/PD-L1 interaction show significantly worse outcome.This work was supported, in part, by Department of Education, Basque Government- IT1270-19, Elkartek grant (BG18), and the Spanish Ministry grant (MINECO) PROJECTS of EXCELLENCE (BFU2015-65625-P). P.J. Parker was supported by a core grant to the Francis Crick Institute, from Cancer Research UK (FC001130), the UK Medical Research Council (FC001130), and the Wellcome Trust (FC001130).Peer reviewe

Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation

Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments, ranging from pathogens in the human body to phytoplankton in the oceans, imposes strong selection for improved metal acquisition systems. In this study, we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source, but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B12, a cobalt-containing cofactor, to sustain two vitamin B12–dependent enzymatic reactions essential to methanol, but not succinate, metabolism. Interestingly, this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth, indicating growth-rate–dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism, suggest that certain molecular features may promote the emergence of parallel mutations, and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences

Dynamics of PLCγ and Src family kinase 1 interactions during nuclear envelope formation revealed by FRET-FLIM.

The nuclear envelope (NE) breaks down and reforms during each mitotic cycle. A similar process happens to the sperm NE following fertilisation. The formation of the NE in both these circumstances involves endoplasmic reticulum membranes enveloping the chromatin, but PLCγ-dependent membrane fusion events are also essential. Here we demonstrate the activation of PLCγ by a Src family kinase (SFK1) during NE assembly. We show by time-resolved FRET for the first time the direct in vivo interaction and temporal regulation of PLCγ and SFK1 in sea urchins. As a prerequisite for protein activation, there is a rapid phosphorylation of PLCγ on its Y783 residue in response to GTP in vitro. This phosphorylation is dependent upon SFK activity; thus Y783 phosphorylation and NE assembly are susceptible to SFK inhibition. Y783 phosphorylation is also observed on the surface of the male pronucleus (MPN) in vivo during NE formation. Together the corroborative in vivo and in vitro data demonstrate the phosphorylation and activation of PLCγ by SFK1 during NE assembly. We discuss the potential generality of such a mechanism

Spatial Functional Mapping of Hypoxia Inducible Factor Heterodimerisation and Immune checkpoint regulators in clear cell Renal Cell Carcinoma

Background: Clear cell renal cell carcinoma (ccRCC) is a highly malignant subtype of kidney cancer. Ninety percent of ccRCC have inactivating mutations of VHL that stabilise transcription factors, HIF1α and HIF2α, only stabilised in hypoxia. The varied response to HIF2 inhibition, in the preclinical and clinical settings, suggests that assessment of HIF2α activation state, not just expression levels is required as a biomarker of sensitivity to enable optimal clinical use. Methods: Two-site amplified time-resolved Förster Resonance Energy Transfer (aiFRET), with FRET-Efficiency, Ef, as its read out, provides functional proteomics quantification, a precise step forward from protein expression as a tool for patient stratification. To enhance the clinical accessibility of Ef, we have devised a new computational approach, Functional Oncology map (FuncOmap). Results: FuncOmap directly maps functional states of oncoproteins and allows functional states quantification at an enhanced spatial resolution. The innovative contributions in FuncOmap are the means to co-analyse and map expressional and functional state images and the enhancement of spatial resolution to facilitate clinical application. We show the spatial interactive states HIF2α and HIF1β in ccRCC patient samples. Conclusion: FuncOmap can be used to quantify heterogeneity in patient response and improve accurate patient stratification, thus enhancing the power of precision

Schematic model and data summary of molecular events leading to NE formation.

<p>Once the sperm has entered the egg in vivo, or upon the sperm nucleus mixing with egg cytoplasmic extract and ATP-GS in vitro, the sperm nucleus decondenses (not shown), with concomitant membrane vesicle binding to the nucleus surface (grey). <b>Left</b>: MV1 vesicles enriched in SFK1, PLCγ and PtdIns(4,5)P₂ bind in a polar fashion near the two sperm NERs. SFK1 and PLCγ directly interact. The premature phosphorylation of PLCγ by SFK1 is prevented by Csk inhibition of SFK1. <b>Middle</b>: Upon addition of GTP, Csk inhibition of SFK1 is removed, and SFK1 phosphorylates PLCγ on its Y783 residue, a pre-requisite for full PLCγ activation. Once active, PLCγ hydrolyses PtdIns(4,5)P₂ to the fusogenic lipid DAG, which initially promotes membrane fusion events between MV1 vesicles and possibly NERs. <b>Right</b>: Fusion events subsequently spread over the surface of the sperm nucleus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040669#pone.0040669-Dumas1" target="_blank">[19]</a> until it is enclosed by a continuous double bilayer perforated with nuclear pore complexes (not shown). After fusion is initiated, PLCγ and SFK1 dissociate but remain on the NE. L and C denote the luminal and cytoplasmic faces of vesicles respectively. ER denotes endoplasmic reticulum derived membranes.</p

PLCγ is transiently phosphorylated on Y783 at the NE <i>in vitro</i>.

<p>Demembranated sperm nuclei were fixed alone (top row) or decondensed in fertilised egg cytoplasmic extract and ATP-GS (lower panels). Nuclei were additionally treated with 1 mM GTP for the times indicated (minutes) and were fixed and stained with Hoechst (blue), DiOC₆ (green) and anti-pY783 of PLCγ (red). Nuclei were imaged by confocal microscopy. Note the staining of the NERs with anti-pY783 is retained in nuclei decondensed in ATP-GS (2^nd row). Data are representative of those obtained in three independent experiments. Scale bar is 1 µm.</p

PLCγ and SFK1 interact during the early stages of male pronuclear envelope formation <i>in vivo</i>.

<p>(<b>A</b>) Unfertilised <i>L. pictus</i> eggs (T0) or five minutes post-fertilisation (T+5) were fixed and labelled with anti-PLCγ-OG488 alone or together with anti-SFK1-Cy5. Samples were subjected to two-photon time domain FLIM and the lifetime of the donor chromophore (OG488) determined in the absence and presence of acceptor (Cy5). Donor (PLCγ-OG488) two-photon images, donor lifetime ‘heat maps’ and Hg-lamp epifluoresence acceptor (SFK-Cy5) images are shown. At T+5 the donor lifetimes in the whole egg (middle panel) and in the vicinity of the MPN only (bottom panel) were determined. In the donor two-photon image the MPN region of interest is indicated (dashed circle). The donor lifetime heat map and corresponding two-photon image are at the same scale. The images shown are from a donor alone labelled egg. The same process was repeated with eggs labelled with donor and acceptor. Data are representative of at least three independent experiments performed. (<b>B</b>) The FRET efficiency for each condition was calculated. Solid green boxes are the donor alone condition, green/red stripes for donor and acceptor conditions. Data are from at least three independent experiments, with a total of 6–18 eggs analysed per condition. Boxes display the median, upper and lower quartiles and whiskers the maximum and minimum values.</p

Summary of FRET experiments <i>in vivo.</i>

<p>Experiments were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040669#s2" target="_blank">methods</a> section and results text. Data show the mean FRET efficiency ± SEM, calculated for each region of interest (ROI), the egg or male pronucleus (MPN).</p

The unprecedented membrane deformation of the human nuclear envelope, in a magnetic field, indicates formation of nuclear membrane invaginations

International audienceHuman nuclear membrane (hNM) invaginations are thought to be crucial in fusion, fission and remodeling of cells and present in many human diseases. There is however little knowledge, if any, about their lipid composition and dynamics. We therefore isolated nuclear envelope lipids from human kidney cells, analyzed their composition and determined the membrane dynamics after resuspension in buffer. The hNM lipid extract was composed of a complex mixture of phospholipids, with high amounts of phosphatidylcholines, phosphatidylinositols (PI) and cholesterol. hNM dynamics was determined by solid-state NMR and revealed that the lamellar gel-to-fluid phase transition occurs below 0 °C, reflecting the presence of elevated amounts of unsaturated fatty acid chains. Fluidity was higher than the plasma membrane, illustrating the dual action of Cholesterol (ordering) and PI lipids (disordering). The most striking result was the large magnetic field-induced membrane deformation allowing to determine the membrane bending elasticity, a property related to hydrodynamics of cells and organelles. Human Nuclear Lipid Membranes were at least two orders of magnitude more elastic than the classical plasma membrane suggesting a physical explanation for the formation of nuclear membrane invaginations

NE formation <i>in vitro</i> is dependent upon SFK kinase activity.

<p>(<b>A</b>) Experiments were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040669#pone-0040669-g006" target="_blank">Figure 6</a> with incubation of 1 mM GTP for 2 hours. After this time nuclear membranes were stained with DiOC₆, and the nuclei scored as having bound vesicles (punctate signal) or a complete NE (continuous rim). Epifluoresence images and quantification (mean+s.e.m) are for three independent experiments. (<b>B</b>) Experiment performed as in A with GTP replaced with either normal IgY or anti-SFK1 serum (both at 1 µg/ml). Nuclei were scored as above. Scale bar is 5 µm.</p