A comprehensive single-cell map of T cell exhaustion-associated immune environments in human breast cancer

Immune checkpoint therapy in breast cancer remains restricted to triple negative patients, and long-term clinical benefit is rare. The primary aim of immune checkpoint blockade is to prevent or reverse exhausted T cell states, but T cell exhaustion in breast tumors is not well understood. Here, we use single-cell transcriptomics combined with imaging mass cytometry to systematically study immune environments of human breast tumors that either do or do not contain exhausted T cells, with a focus on luminal subtypes. We find that the presence of a PD-1high exhaustion-like T cell phenotype is associated with an inflammatory immune environment with a characteristic cytotoxic profile, increased myeloid cell activation, evidence for elevated immunomodulatory, chemotactic, and cytokine signaling, and accumulation of natural killer T cells. Tumors harboring exhausted-like T cells show increased expression of MHC-I on tumor cells and of CXCL13 on T cells, as well as altered spatial organization with more immature rather than mature tertiary lymphoid structures. Our data reveal fundamental differences between immune environments with and without exhausted T cells within luminal breast cancer, and show that expression of PD-1 and CXCL13 on T cells, and MHC-I - but not PD-L1 - on tumor cells are strong distinguishing features between these environments

Tripartite multidrug efflux pumps in the human pathogen Salmonella typhimurium

Der intrazelluläre Krankheitserreger Salmonella infiziert sowohl menschliche als auch tierische Wirte und ist eine häufige Ursache von Lebensmittelinfektionen. Abhängig von der Subspezies kann er entweder eine Magen-Darm-Infektion oder eine systemische Erkrankung verursachen. Während in leichten Fällen meist keine besondere Behandlung notwendig ist, werden schwerere Formen der Krankheit mit Antibiotika behandelt. Dabei ist die Evolution von multiresistenten Stämmen ein wiederkehrendes klinisches Problem bei Salmonella wie auch bei anderen Gram-negativen Bakterien, etwa E. coli oder Pseudomonas aeruginosa. Die Multiresistenz betrifft nicht nur Antibiotika, sondern auch andere toxische Substanzen wie Detergentien und Desinfektionslösungen und ist oft verbunden mit einer erhöhten Fähigkeit der Bakterien, diese Substanzen mit Hilfe von aktiven Membrantransportern zu exportieren. Diese sogenannten „multidrug efflux pumps“ reichen in Gram-negativen Bakterien oft durch beide Membranen und bestehen aus drei Komponenten in unterschiedlicher Kopienzahl: i) einem Transporter in der inneren Membran, der die Energie für den Substrattransport liefert, ii) einem Kanal in der äußeren Membran, und iii) einem periplasmatischen Adaptorprotein, welches als verbindendes Element zwischen den beiden dient. Basierend auf ihrer Funktionsweise werden diese Effluxpumpen eingeteilt in primäre Pumpen, die ATP als primäre Energiequelle verwenden, und sekundäre Pumpen, die als Ionen/Substrat Antiporter wirken und die elektrochemische Potentialdifferenz an der inneren Membran ausnutzen. Die vorliegende Studie untersucht je ein Mitglied beider Klassen: Ausgewählt wurden MacAB-TolC als der wichtigste Repräsentant der tripartiten Pumpen vom ABC-Typ sowie der Proton/Substrat Antiporter AcrAB-TolC, der die klinisch relevanteste und am besten untersuchte tripartite Effluxpumpe ist. Die detaillierte Struktur und der Assemblierungsmodus des MacAB-TolC Komplexes ist großteils unbekannt, während zur AcrAB-TolC Pumpe bereits einige strukturelle Studien durchgeführt wurden – allerdings keine davon mit dem nativen Proteinkomplex. Während meiner experimentellen Arbeit konnte ich beide Effluxpumpen direkt im Ursprungsorganismus (S. enterica serovar Typhimurium) überexprimieren, wodurch die Assemblierung unter nativen Bedingungen erfolgen kann. Ein etabliertes Protokoll ermöglichte die Isolierung aus Membranfraktionen und die Visualisierung von beinahe-nativen Effluxpumpen in „negative-stain“ Elektronenmikroskopie sowie im Falle von AcrAB-TolC auch in der Kryo-Elektronenmikroskopie. Für die individuelle Expression der zwei Pumpen wurde ein Deletionsstamm generiert, in dem keine anderen wichtigen tripartiten Effluxpumpen stark exprimiert werden, die die verlässliche Identifikation und Zuordnung der sichtbaren Partikel stören könnten. Weiters wurde die periplasmatische Komponente von MacAB-TolC aufgereinigt und für die Produktion polyklonaler Antikörper verwendet, und die Funktionalität der Plasmid-exprimierten Pumpen wurde bestätigt. Wir hoffen, dass unsere Bemühungen zu einem besseren strukturellen und mechanistischem Verständnis der tripartiten Effluxpumpen führen und zur Entwicklung neuer Strategien zur Bekämpfung multiresistenter Keime beitragen könnenThe intracellular pathogen Salmonella infects both human and animal hosts and is a common cause of foodborne diseases, causing gastrointestinal illness or systemic infection depending on the serovar. While for light cases treatment is usually not necessary, patients with more severe forms of disease depend on antibiotic treatment. However, evolution of multidrug resistant strains is a recurring clinical problem for Salmonella as well as for other Gram-negative bacteria such as E. coli and Pseudomonas aeruginosa. This multidrug resistance includes not only antibiotics but also other toxic substances such as dyes, detergents and disinfectants and is often associated with increased ability of the bacterial cell to expel these substances via active membrane transporters. These multidrug efflux pumps in Gram-negative bacteria often span both membranes and are built up of three components in different copy numbers: i) an inner membrane transporter which provides the energy for substrate transport, ii) an outer membrane channel and iii) a periplasmic adaptor protein that acts as a connecting element between the two. Based on their mode of action, these tripartite efflux pumps are classified into primary-type pumps that use ATP as their primary energy source and secondary-type pumps which work as ion/drug antiporters and exploit the electrochemical potential gradient at the inner membrane. In this study, I investigated one member of each group: MacAB-TolC was chosen for its role as the major representative of the ABC-type tripartite pumps, while the proton/drug antiporter AcrAB-TolC was chosen as the most clinically relevant and best characterized of the tripartite efflux pumps. The detailed structure and assembly mode of the MacAB-TolC complex is largely unknown, while for AcrAB-TolC, some structural studies have been made, though none of them of the native protein. During my experimental work I managed to overexpress both efflux pumps directly in the organism they stem from (S. enterica serovar Typhimurium), therefore allowing assembly in native conditions. An established protocol allowed the isolation from membrane fractions and visualization of the near-native efflux pumps in negative-stain electron microscopy and for AcrAB-TolC also in cryo-EM. For individual expression of the two pumps, a mutant strain was constructed where presumably no other major tripartite efflux pumps are highly expressed, ensuring a more reliable identification and assignment of visualized pump-shaped particles. Furthermore, the periplasmic component of MacAB-TolC was purified and used for production of polyclonal antibodies, and the functionality of plasmid-expressed pump operons was assessed. Ultimately, we hope that our efforts will lead to a better structural and mechanistic understanding of these tripartite efflux pumps and contribute to the development of new strategies to fight multidrug resistance

Cancer-associated fibroblast classification in single-cell and spatial proteomics data

Abstract Cancer-associated fibroblasts (CAFs) are a diverse cell population within the tumour microenvironment, where they have critical effects on tumour evolution and patient prognosis. To define CAF phenotypes, we analyse a single-cell RNA sequencing (scRNA-seq) dataset of over 16,000 stromal cells from tumours of 14 breast cancer patients, based on which we define and functionally annotate nine CAF phenotypes and one class of pericytes. We validate this classification system in four additional cancer types and use highly multiplexed imaging mass cytometry on matched breast cancer samples to confirm our defined CAF phenotypes at the protein level and to analyse their spatial distribution within tumours. This general CAF classification scheme will allow comparison of CAF phenotypes across studies, facilitate analysis of their functional roles, and potentially guide development of new treatment strategies in the future

A comprehensive single-cell map of T cell exhaustion-associated immune environments in human breast cancer

Immune checkpoint therapy in breast cancer remains restricted to triple negative patients, and long-term clinical benefit is rare. The primary aim of immune checkpoint blockade is to prevent or reverse exhausted T cell states, but T cell exhaustion in breast tumors is not well understood. Here, we use single-cell transcriptomics combined with imaging mass cytometry to systematically study immune environments of human breast tumors that either do or do not contain exhausted T cells, with a focus on luminal subtypes. We find that the presence of a PD-1high exhaustion-like T cell phenotype is associated with an inflammatory immune environment with a characteristic cytotoxic profile, increased myeloid cell activation, evidence for elevated immunomodulatory, chemotactic, and cytokine signaling, and accumulation of natural killer T cells. Tumors harboring exhausted-like T cells show increased expression of MHC-I on tumor cells and of CXCL13 on T cells, as well as altered spatial organization with more immature rather than mature tertiary lymphoid structures. Our data reveal fundamental differences between immune environments with and without exhausted T cells within luminal breast cancer, and show that expression of PD-1 and CXCL13 on T cells, and MHC-I – but not PD-L1 – on tumor cells are strong distinguishing features between these environments.ISSN:2041-172

Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology

Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them. Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors. Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts

Clonal fitness inferred from time-series modelling of single-cell cancer genomes

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours