Single-cell sequencing | EPFL Graph Search

Single-cell sequencing examines the nucleic acid sequence information from individual cells with optimized next-generation sequencing technologies, providing a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment. For example, in cancer, sequencing the DNA of individual cells can give information about mutations carried by small populations of cells. In development, sequencing the RNAs expressed by individual cells can give insight into the existence and behavior of different cell types. In microbial systems, a population of the same species can appear genetically clonal. Still, single-cell sequencing of RNA or epigenetic modifications can reveal cell-to-cell variability that may help populations rapidly adapt to survive in changing environments. Background A typical human cell consists of about 2 x 3.3 billion base pairs of DNA and 600 million mRNA bases. Usually, a mix of millions

Real-time monitoring of single-cell secretion with a high-throughput nanoplasmonic microarray

Hatice Altug, Saeid Ansaryan, Xiaokang Li, Yen-Cheng Liu, Eduardo Romero Arvelo

Proteins secreted by cells play significant roles in mediating many physiological, developmental, and pathological processes due to their functions in intra/intercellular communication and signaling. Conventional end-point methods are insufficient for understanding the temporal response in cell secretion process, which is often highly dynamic. Furthermore, cellular heterogeneity makes it essential to analyze secretory proteins from single cells. To uncover individual cellular activities and the underlying kinetics, new technologies are needed for real-time analysis of the secretomes of many cells at single-cell resolution. This study reports a high-throughput biosensing microarray platform, which is capable of label-free and real-time secretome monitoring from a large number of living single cells using a biochip integrating ultrasensitive nanoplasmonic substrate and microwell compartments having volumes of ∼0.4 nL. Precise synchronization of image acquisition and microscope stage movement of the developed optical platform enables spectroscopic analysis with high temporal and spectral resolution. In addition, our system allows simultaneous optical imaging of cells to track morphology changes for a comprehensive understanding of cellular behavior. We demonstrated the platform performance by detecting interleukin-2 secretion from hundreds of single lymphoma cells in real-time over many hours. Significantly, the analysis of the secretion kinetics allows us to study cellular response to the stimulations in a statistical way. The new platform is a promising tool for the characterization of single-cell functionalities given its versatility, throughput and label-free configuration.

2022

Immunosuppressive roles of Activin-A in melanoma and characterization of its precursor cleavage

Katarina Pinjusic

Activin-A, a transforming growth factor êµ family member, is a pleiotropic cytokine with diverse functions in development, fertility, adult tissue homeostasis, and aging. Accordingly, deregulation of Activin-A signaling has been associated with many pathological conditions, including cancer and cancer-associated muscle wasting (cachexia). Activin-A was shown to signal in endocrine, but also local autocrine and paracrine manner. Its secretion in many cancer types is associated with a poor prognosis. Previous studies suggest that Activin-A expression in melanoma promotes tumor growth and metastasis indirectly by inhibiting adaptive anti-tumor immunity, leading to a decrease in the intratumoral frequency of cytotoxic CD8+ T cells (CTLs). However, the underlying mechanisms and the relevant direct target cells of Activin-A and their significance for cancer therapies remained unknown. Similar to other TGFêµ-related ligands, proActivin-A is cleaved by proprotein convertases (PCs) to release mature ligands. However, unlike other family members whose signaling is tightly regulated, Activin-A apparently does not have latency.In this thesis, I characterize proActivin-A cleavage and show that one prodomain can be cleaved independently of known PCs. The resulting hemi-cleaved Activin-A can bind activin type II receptors and induce SMAD3 signaling in reporter cells. A protein interactome screen and analysis of cleavage intermediates in plasma revealed that the hemi-cleaved Activin-A likely interacts with extracellular matrix proteins via the remaining prodomain where it promotes local signaling and regulates trafficking. Luciferase reporter assay showed that hemi-cleaved Activin-A can avoid, at least partially, inhibition by endogenous inhibitors FSTL3 and FST, and enable parallel activation of BMP and Activin signaling pathways. Finally, analysis of B16-F1 melanoma models revealed that only fully cleaved Activin-A in source cells increased tumor growth by acting locally in the tumor microenvironment suggesting that the context of Activin-A precursor processing regulates its tumor growth-promoting effect.The second part of this thesis addresses the mechanisms of Activin-induced immunosuppression in melanoma. We found that Activin-A impairs CTL function indirectly and independently of CD4+ T cell, CSF1R+ macrophages, and IL4 signaling. At least in part, anti-tumor CTL responses are impaired by the inhibitory effects of Activin-A on the secretion of critical CTL-recruiting chemokines CXCL9 and CXCL10 by macrophages and dendritic cells (DCs). Furthermore, Activin-A conferred resistance to immune checkpoint blockade therapy. Characterization of Activin-induced changes in the gene expression of tumor-infiltrating cells by Single cell RNA sequencing revealed that INHBA expression induced the most changes in DCs and least in T cells. Interestingly, the most prominently induced hallmark in Activin-secreting tumors across cell types was the IFN signature. In vitro analyses showed that Activin-A increases IFNÎ³-induced activation of STAT1 and confers resistance to cytostatic IFN signaling in cancer cells.Altogether, the findings of this thesis expand our knowledge on the regulation of Activin-A signaling by precursor cleavage and offer insights into mechanisms of Activin-induced immunosuppression in melanoma. Inhibition of Activin-A maturation thus emerges as a potential new strategy to improve responses to immunotherapies in melanoma and likely other cancers

EPFL2022

Evaluation of oxidation-sensitive polymersomes as vaccine nanocarriers to enhance humoral responses against arenavirus envelope glycoproteins

Clara Galan Navarro

Vaccines currently represent one of the most effective means to control and prevent infectious diseases affecting humans and animals and have dramatically improved public health, quality of life, and life expectancy. Over the last years several conceptual and technological advances in the field of bioengineering and immunology have allowed the development of new vaccines designs. Expanding novel domains such as structural biology, human monoclonal antibody isolation, high throughput sequencing, and design of new nanocarrier delivery platforms have changed the landscape of vaccinology. These new vaccine formulations allow the design of tailor-made antigens and particulate vehicles which allow better and fine-tuned protective immune responses against known and newly emerging pathogens. Over the past decades emerging human pathogens with the potential to cause severe epidemics have become a major public health problem. The last Ebola and Zika virus epidemics demonstrated in a dramatic manner that there is an urgent need to develop prophylactic and therapeutic approaches against pathogenic emerging viruses. Most successful vaccines rely on attenuated live microorganisms to induce protective immunity. However, changed demographics and associated safety concerns makes the development of a safer alternative a priority. Subunit vaccines composed of microbial components and proteins have emerged as a suitable alternative, however they are generally less immunogenic. To address this problematic our laboratory works on the development of immunization platforms which allow 1) the development of safe recombinant subunit vaccines and 2) the rapid generation of specific monoclonal antibodies using biosynthetic immunogens without the need for prior isolation and culture of the agent. The approach evaluated in the present studies is based on a novel polymersome (PS) platform serving as nanocarrier for efficient antigen delivery and the induction of humoral immunity. The potential of PS was assessed in the context of two important human emerging pathogens form the arenavirus family that cause severe hemorrhagic fevers and of which there are neither FDA-approved vaccines not prophylactic treatments: Lassa virus (LASV), endemic in Western Africa, and Machupo virus (MACV), the causative agent of Bolivian hemorrhagic fever. In a first part, we evaluated the PS platformâs capacity to enhance humoral immunity against the envelope glycoprotein 1 of LASV, (LASV GP1) that is notorious for its weak immunogenicity and poor antibody response. Immunization of mice with adjuvanted PS (LASV GP1) enhanced the quality of the humoral response to LASV GP1, eliciting antibodies with higher binding affinity to virion GP1, increased levels of polyfunctional anti-viral CD4 T cells, and the frequency of IgG-secreting B cells. PS (LASV GP1) elicited a more diverse epitope repertoire of anti-viral IgG. In a second part, we employed the PS platform in combination with single cell B cell sorting and cloning of recombinant IgG to generate a first set of species-specific mAbs against MACV. These new mAbs show exquisite specificity and negligible cross-reactivity to closely related arenaviruses in relevant techniques making them a unique and powerful tool for research and diagnostics purposes.

EPFL2017