Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern

BACKGROUND. The role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome. METHODS. Using SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients’ plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution. RESULTS. We identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811–825, S-881–895, and N-156–170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes. CONCLUSION. Epitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats. FUNDING. Ontario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/ Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between fulllength Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit a-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein- protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions

Implication of Microenvironment on the Onset of Metastasis & Initiation of Residual Disease in Breast Adenocarcinoma

Le cancer du sein est le cancer le plus fréquemment diagnostiqué et la deuxième cause de décès par cancer dans les pays développé. Récemment le rôle du microenvironnement a été mis en évidence dans l’oncogenèse et la progression tumorale. Plusieurs études ont montré que le microenvironnement tumoral est un élément dynamique en constant dialogue avec les cellules tumorales. Parmi les éléments du microenvironnement les cellules endothéliales jouent un rôle particulier. Effectivement ces cellules constituent la paroi des vaisseaux et permettent l’acheminement des nutriments et de l’oxygène vers la tumeur. Ainsi ces dernières années des thérapies visant a détruire les vaisseaux sanguins ont vu le jour mais n’ont pas permis d’atteindre les progrès thérapeutiques escomptés. Notre groupe ainsi que d’autres ont mis en évidence des rôles des cellules endothéliales indépendant de la perfusion tumorale. Dans ce travail de thèse nous avons caractérisé l’interaction entre cellules endothéliales et tumorales et mis en évidence le rôle pro-tumoral de la niche vasculaire. Nous avons tout d’abord pu montrer en utilisant une stratégie de souris transgénique où nous annulons l’expression de Jag1 des cellules endothéliales la réduction drastique de l’occurrence des métastases. Nous avons pu montrer que cela n’est pas dépendant de la perfusion tumorale mais dépend de la modification de nombreux gènes pro-métastatiques dont Id1 dans les cellules tumorales par les cellules endothéliales. Parallèlement nous avons montré que le dialogue entre cellules tumorales et endothéliales induit une transition mésenchymateuse des cellules endothéliales avec pour conséquence une augmentation de leur survie ainsi que de leur migration et de leur capacité angiocrine.Ainsi nous avons montré comment le dialogue entre cellules tumorales et endothéliales induit une modification du phénotype tumoral et endothéliale et le rôle de la voie Notch dans ce dialogue. Notre travail suggère la possibilité de moduler l’agressivité tumorale en interrompant le dialogue entre cellules endothéliales et tumorales.Breast cancer is a heterogeneous disease, which is characterized by distinct morphological features and clinical behaviors and is the most commonly diagnosed cancer among women in the United States and worldwide and the second cause of cancer-related mortality in women. Several years of investigation have demonstrated that tumor initiation, progression and metastasis are closely regulated by the adjacent non-neoplastic tissues that are collectively referred to as tumor microenvironment (stroma). The components of tumor stroma such as mesenchymal stem cells have been shown to enhance cancer stem cell population in breast tumor. Also, the endothelial cells (ECs) conventional role in tumor angiogenesis is crucial in determining the tumor fate as microscopic and asymptomatic versus aggressive. This outstanding characteristic of ECs has set them as promising targets in cancer therapy. However, failure of anti-angiogenic therapies despite vessel disruption suggests the blood flow-independent ability of ECs to facilitate tumor growth. In this study, we show that ECs promoted breast cancer cell self-renewal, stemness, migratory characteristic and lung metastasis through Jagged1/Notch dependent Id1 modulation. ECs with Jag1 knock down (ECsJag1-) failed to sustain breast cancer cell proliferation and stemness in vitro and during xenografted tumor growth. Furthermore, we established a breast tumor mouse model with EC specific Jag1 mutation, by crossing the MMTV-PyMT mice with Cdh5-Cre+/-Jag1loxP/loxP mice. It demonstrated significant decrease in primary tumor growth and dramatic reduction in lung metastasis in Cdh5-Cre+/-Jag1loxP/loxPPyMT+ mice. Transcriptome sequencing analysis of the sorted primary tumor cells identified Notch downstream targets, specifically, Id1, which was reported to be essential for lung metastasis of breast tumors. Additionally, we were interested in determining the mechanisms that derive the activation of ECs toward supporting tumor growth and expansion. Previous studies have shown that ECs show tremendous degree of plasticity when placed under different conditions. Here, we showed that ECs show EndMT phenotypes upon having contact with tumor cells. Interestingly, the EndMT transforms the ECs into activated entities with increased proliferation, migration and angiogenesis properties. Our results demonstrated that the EndMT was reversible and dependent on EC-tumor cell contacts. Moreover, we were able to show that the tumor-induced EndMT in ECs is synergistically regulated by TGFβ and notch signaling pathways. Overall, our findings implicate the significance of endothelial-tumor cells perfusion-independent interaction in cancer progression, stemness, and metastasis. Besides, this study might have determined novel targets in combating cancer in a more effective way

Implication of Microenvironment on the Onset of Metastasis & Initiation of Residual Disease in Breast Adenocarcinoma

Le cancer du sein est le cancer le plus fréquemment diagnostiqué et la deuxième cause de décès par cancer dans les pays développé. Récemment le rôle du microenvironnement a été mis en évidence dans l oncogenèse et la progression tumorale. Plusieurs études ont montré que le microenvironnement tumoral est un élément dynamique en constant dialogue avec les cellules tumorales. Parmi les éléments du microenvironnement les cellules endothéliales jouent un rôle particulier. Effectivement ces cellules constituent la paroi des vaisseaux et permettent l acheminement des nutriments et de l oxygène vers la tumeur. Ainsi ces dernières années des thérapies visant a détruire les vaisseaux sanguins ont vu le jour mais n ont pas permis d atteindre les progrès thérapeutiques escomptés. Notre groupe ainsi que d autres ont mis en évidence des rôles des cellules endothéliales indépendant de la perfusion tumorale. Dans ce travail de thèse nous avons caractérisé l interaction entre cellules endothéliales et tumorales et mis en évidence le rôle pro-tumoral de la niche vasculaire. Nous avons tout d abord pu montrer en utilisant une stratégie de souris transgénique où nous annulons l expression de Jag1 des cellules endothéliales la réduction drastique de l occurrence des métastases. Nous avons pu montrer que cela n est pas dépendant de la perfusion tumorale mais dépend de la modification de nombreux gènes pro-métastatiques dont Id1 dans les cellules tumorales par les cellules endothéliales. Parallèlement nous avons montré que le dialogue entre cellules tumorales et endothéliales induit une transition mésenchymateuse des cellules endothéliales avec pour conséquence une augmentation de leur survie ainsi que de leur migration et de leur capacité angiocrine.Ainsi nous avons montré comment le dialogue entre cellules tumorales et endothéliales induit une modification du phénotype tumoral et endothéliale et le rôle de la voie Notch dans ce dialogue. Notre travail suggère la possibilité de moduler l agressivité tumorale en interrompant le dialogue entre cellules endothéliales et tumorales.Breast cancer is a heterogeneous disease, which is characterized by distinct morphological features and clinical behaviors and is the most commonly diagnosed cancer among women in the United States and worldwide and the second cause of cancer-related mortality in women. Several years of investigation have demonstrated that tumor initiation, progression and metastasis are closely regulated by the adjacent non-neoplastic tissues that are collectively referred to as tumor microenvironment (stroma). The components of tumor stroma such as mesenchymal stem cells have been shown to enhance cancer stem cell population in breast tumor. Also, the endothelial cells (ECs) conventional role in tumor angiogenesis is crucial in determining the tumor fate as microscopic and asymptomatic versus aggressive. This outstanding characteristic of ECs has set them as promising targets in cancer therapy. However, failure of anti-angiogenic therapies despite vessel disruption suggests the blood flow-independent ability of ECs to facilitate tumor growth. In this study, we show that ECs promoted breast cancer cell self-renewal, stemness, migratory characteristic and lung metastasis through Jagged1/Notch dependent Id1 modulation. ECs with Jag1 knock down (ECsJag1-) failed to sustain breast cancer cell proliferation and stemness in vitro and during xenografted tumor growth. Furthermore, we established a breast tumor mouse model with EC specific Jag1 mutation, by crossing the MMTV-PyMT mice with Cdh5-Cre+/-Jag1loxP/loxP mice. It demonstrated significant decrease in primary tumor growth and dramatic reduction in lung metastasis in Cdh5-Cre+/-Jag1loxP/loxPPyMT+ mice. Transcriptome sequencing analysis of the sorted primary tumor cells identified Notch downstream targets, specifically, Id1, which was reported to be essential for lung metastasis of breast tumors. Additionally, we were interested in determining the mechanisms that derive the activation of ECs toward supporting tumor growth and expansion. Previous studies have shown that ECs show tremendous degree of plasticity when placed under different conditions. Here, we showed that ECs show EndMT phenotypes upon having contact with tumor cells. Interestingly, the EndMT transforms the ECs into activated entities with increased proliferation, migration and angiogenesis properties. Our results demonstrated that the EndMT was reversible and dependent on EC-tumor cell contacts. Moreover, we were able to show that the tumor-induced EndMT in ECs is synergistically regulated by TGFb and notch signaling pathways. Overall, our findings implicate the significance of endothelial-tumor cells perfusion-independent interaction in cancer progression, stemness, and metastasis. Besides, this study might have determined novel targets in combating cancer in a more effective way.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Discovery of Nedd4 auto-ubiquitination inhibitors

Abstract E3 ubiquitin ligases are critical to the protein degradation pathway by catalyzing the final step in protein ubiquitination by mediating ubiquitin transfer from E2 enzymes to target proteins. Nedd4 is a HECT domain-containing E3 ubiquitin ligase with a wide range of protein targets, the dysregulation of which has been implicated in myriad pathologies, including cancer and Parkinson's disease. Towards the discovery of compounds disrupting the auto-ubiquitination activity of Nedd4, we developed and optimized a TR-FRET assay for high-throughput screening. Through selective screening of a library of potentially covalent compounds, compounds 25 and 81 demonstrated apparent IC50 values of 52 µM and 31 µM, respectively. Tandem mass spectrometry (MS/MS) analysis confirmed that 25 and 81 were covalently bound to Nedd4 cysteine residues (Cys182 and Cys867). In addition, 81 also adducted to Cys627. Auto-ubiquitination assays of Nedd4 mutants featuring alanine substitutions for each of these cysteines suggested that the mode of inhibition of these compounds occurs through blocking the catalytic Cys867. The discovery of these inhibitors could enable the development of therapeutics for various diseases caused by Nedd4 E3 ligase dysregulation

Breast cancer cells promote a notch-dependent mesenchymal phenotype in endothelial cells participating to a pro-tumoral niche

International audienceBackground: Endothelial cells (ECs) are responsible for creating a tumor vascular niche as well as producing angiocrine factors. ECs demonstrate functional and phenotypic heterogeneity when located under different microenvironments. Here, we describe a tumor-stimulated mesenchymal phenotype in ECs and investigate its impact on tumor growth, stemness, and invasiveness.Methods: Xenograft tumor assay in NOD/SCID mice and confocal imaging were conducted to show the acquisition of mesenchymal phenotype in tumor-associated ECs in vivo. Immunocytochemistry, qPCR and flow cytometry techniques showed the appearance of mesenchymal traits in ECs after contact with breast tumor cell lines MDA-MB231 or MCF-7. Cell proliferation, cell migration, and sphere formation assays were applied to display the functional advantages of mesenchymal ECs in tumor growth, invasiveness, and enrichment of tumor initiating cells. qPCR and western blotting were used to investigate the mechanisms underlying EC mesenchymal transition.Results: Our results showed that co-injection of ECs and tumor cells in NOD/SCID mice significantly enhanced tumor growth in vivo with tumor-associated ECs expressing mesenchymal markers while maintaining their intrinsic endothelial trait. We also showed that a mesenchymal phenotype is possibly detectable in human neoplastic breast biopsies as well as ECs pre-exposed to tumor cells (ECs(Mes)) in vitro. The ECs(Mes) acquired prolonged survival, increased migratory behavior and enhanced angiogenic properties. In return, ECs(Mes) were capable of enhancing tumor survival and invasiveness. The mesenchymal phenotypes in ECs(Mes) were the result of a contact-dependent transient phenomenon and reversed upon removal of the neoplastic contexture. We showed a synergistic role for TGFβ and notch pathways in this phenotypic change, as simultaneous inhibition of notch and TGFβ down-regulated Smad1/5 phosphorylation and Jag1(KD) tumor cells were unable to initiate the process.Conclusions: Overall, our data proposed a crosstalk mechanism between tumor and microenvironment where tumor-stimulated mesenchymal modulation of ECs enhanced the constitution of a transient mesenchymal/endothelial niche leading to significant increase in tumor proliferation, stemness, and invasiveness. The possible involvement of notch and TGFβ pathways in the initiation of mesenchymal phenotype may propose new stromal targets

Endothelial Cells Provide a Notch-Dependent Pro-Tumoral Niche for Enhancing Breast Cancer Survival, Stemness and Pro-Metastatic Properties

<div><p>Treating metastasis has been challenging due to tumors complexity and heterogeneity. This complexity is partly related to the crosstalk between tumor and its microenvironment. Endothelial cells -the building blocks of tumor vasculature- have been shown to have additional roles in cancer progression than angiogenesis and supplying oxygen and nutrients. Here, we show an alternative role for endothelial cells in supporting breast cancer growth and spreading independent of their vascular functions. Using endothelial cells and breast cancer cell lines MDA-MB231 and MCF-7, we developed co-culture systems to study the influence of tumor endothelium on breast tumor development by both <i>in vitro</i> and <i>in vivo</i> approaches. Our results demonstrated that endothelial cells conferred survival advantage to tumor cells under complete starvation and enriched the CD44^HighCD24^Low/- stem cell population in tumor cells. Moreover, endothelial cells enhanced the pro-metastatic potential of breast cancer cells. The <i>in vitro</i> and <i>in vivo</i> results concordantly confirmed a role for endothelial Jagged1 to promote breast tumor through notch activation. Here, we propose a role for endothelial cells in enhancing breast cancer progression, stemness, and pro-metastatic traits through a perfusion-independent manner. Our findings may be beneficial in developing novel therapeutic approaches.</p></div

E4-ECs promote BCCs self-renewal and survival in a contact-dependent manner.

<p><b>A</b>) Schematic representation of the co-culture system developed for assessing tumor cell proliferation in direct contact with GFP⁺E4-ECs. BCCs and GFP⁺E4-ECs were co-cultivated at 1∶5 ratio without serum and cytokine supplementation and proliferation and survival of BCCs was evaluated 2, 4, and 7 days post co-culture by counting the GFP^-BCCs using a fluorescent microscope. <b>B</b>) Phase contrast and fluorescent microscopy images showing higher BCC proliferation (dark gray cells) in co-culture with GFP⁺E4-ECs as compared with BCCs grown without ECs. <b>C</b>) Quantitative analysis of breast cancer cells MDA-231 and MCF-7 proliferation cultured with or without E4-ECs (***<i>p</i><0.001, mean ± SEM). <b>D</b>) Schematic representation of a transwell system used for co-culturing BCCs and E4-EC without any direct contact. BCCs and E4-ECs were separately grown as monolayers in multi-well culture plates and transwell inserts respectively. Then, inserts were positioned in the multi-well plates and both cell types were continued to grow in a serum- and cytokine-free medium (conditioned medium) and the proliferation of BBCs was evaluated at 2, 4, and 7day intervals by manual counting. <b>E</b>) Phase contrast and fluorescent microscopy images demonstrating BCCs grown under starvation either alone or in E4-EC conditioned media (CM). <b>F</b>) Quantitative analysis of proliferation of breast cancer cells MDA-231 and MCF-7 with or without direct physical contact with E4-ECs demonstrating the importance of contact (***<i>p</i><0.001, mean ± SEM).</p

Inhibition of Jagged1 ligand on E4-ECs attenuated their influence on BCCs.

<p><b>A</b>) qPCR analysis showing down-regulation of Jagged1 in E4-ECs after transfection with shRNA against Jagged1. <b>B</b>) qPCR analysis confirming reduced activation of notch pathway by showing decreased expression of Hes1 and Hey1 notch downstream effector molecules in MDA-231 cells sorted from E4-ECs^Jag1KD as compared with MDA-231 cells sorted from E4-ECs^Scr (*<i>p</i><0.05, mean ± SEM). <b>C</b>) Cell proliferation assay evaluating the outcome of endothelial Jag1 silencing on BCC self-renewal and survival. Plot represents quantitative analysis of MDA-231 cells grown with either E4-ECs^Scr or E4-ECs^Jag1KD (upper panel). Fluorescent imaging (bottom panels) illustrates GFP⁺MDA-231 cells in co-culture with mCherry⁺E4-ECs^Scr or mCherry⁺E4-ECs^Jag1KD (***<i>p</i><0.001, mean ± SEM). <b>D</b>) Schematic representation of subcutaneous injection of MDA-231 with or without E4-ECs^Scr or E4-ECs^Jag1KD cells (1∶10 ratio) into NSG mice (upper panel). Quantification of tumor weight from NSG mice injected subcutaneously with GFP⁺MDA-231 cells solely or mixed with mCherry⁺E4-ECs or mCherry⁺ECs^Jag1KD 7 weeks post injection (bottom panel). <b>E</b>) Bar graph representing primary mammo-angiospheres once BCCs were co-cultured with either E4-EC^Scr or E4-EC^Jag1KD (upper panel). Phase contrast microscopy images (bottom panels) showing mammosphere formation with E4-EC^Scr or E4-EC^Jag1KD (bottom panel) (***<i>p</i><0.001, mean ± SEM). <b>F</b>) Schematic representation of mammosphere injection into NSG mice (upper panel). Quantification of tumor weight 7 weeks post subcutaneous injection of 1×10⁵ sorted MDA-231 spheroids from primary mammosphere or mammo-angiospheres (bottom panel).</p

Notch activation mediates E4-ECs induced phenotypic advantages of BCCs.

<p><b>A</b>) qPCR analysis showed significant over-expression of notch ligand Jagged1 in E4-ECs after sorting from BCCs MDA-231 and MCF-7 (**<i>p</i><0.01, mean ± SEM). <b>B</b>) Cell proliferation assay performed on MDA-231/E4-ECs co-cultures under complete starvation with or without notch inhibitor, GSI. The results showed dramatic reduction in the ability of E4-ECs to maintain growth and survival of MDA-231 cells when GSI was added (***<i>p</i><0.001, mean ± SEM). <b>C</b>) Mammo-angiosphere forming assay done with or without daily doses of GSI illustrates the effect of notch inhibition on mammosphere enrichment. <b>D</b>) Quantitative evaluation of mammosphere formation under GSI treatment. Plot shows that addition of GSI dramatically attenuates E4-ECs role in enhancing mammosphere formation (***<i>p</i><0.001, mean ± SEM). <b>E</b>) Flow cytometry analysis shows significant reduction in CD44^HighCD24^Low/- population when mammo-angiospheres were grown under GSI treatment.</p