Biology subjects

El-Shennawy, L.

Publications and source records attributed to El-Shennawy, L..

7 recordsLinked to original sources

bioRxiv2025

Rare Subset of T Cells Form Heterotypic Clusters with Circulating Tumor Cells to Foster Cancer Metastasis

The immune ecosystem is central to maintaining effective defensive responses. However, how immune cells in the periphery blood interact with circulating tumor cells (CTCs) - seeds of metastasis - remains largely understudied. Here, our analysis of the blood specimens (N=1,529) from patients with advanced breast cancer revealed that over 75% of the CTC-positive blood specimens contained heterotypic CTC clusters with CD45+ white blood cells (WBCs). Detection of CTC-WBC clusters correlates with breast cancer subtypes (triple negative and luminal B), racial groups (Black), and decreased survival rates. Flow cytometry and ImageStream analyses revealed diverse WBC composition of heterotypic CTC-WBC clusters, including overrepresented T cells and underrepresented neutrophils. Most strikingly, a rare subset of CD4 and CD8 double positive T (DPT) cells showed an up to 140-fold enrichment in the CTC clusters versus its frequency in WBCs. DPT cells shared part of the profiles with CD4+ T cells and others with CD8+ T cells but exhibited unique features of T cell exhaustion and immune suppression with higher expression of TIM-3 and PD-1. Single-cell RNA sequencing and genetic perturbation studies further pinpointed the integrin VLA4 (4{beta}1) in DPT cells and its ligand VCAM1 in tumor cells as essential mediators of heterotypic WBC-CTC clusters. Neoadjuvant administration of anti-4 (VLA4) neutralizing antibodies markedly blocked CTC-DPT cell clustering and inhibited metastasis for extended survival in preclinical mouse models in vivo. These findings uncover a pivotal role of rare DPT cells with immune suppressive features in fostering cancer dissemination through direct interactive clustering with CTCs. It lays a foundation for developing innovative biomarkers and therapeutic strategies to prevent and target cancer metastasis, ultimately benefiting cancer care. Brief summaryOur findings uncover a fostering role of immune-suppressive T cells in contact with circulating tumor cells and identify therapeutic approaches to eliminate devastating cancer metastasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/646421v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@13b7862org.highwire.dtl.DTLVardef@15c97b2org.highwire.dtl.DTLVardef@1eb4fd2org.highwire.dtl.DTLVardef@1c588e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Physics-driven structural docking and protein language models accelerate antibody screening and design for broad-spectrum antiviral therapy

Therapeutic antibodies have become one of the most influential therapeutics in modern medicine to fight against infectious pathogens, cancer, and many other diseases. However, experimental screening for highly efficacious targeting antibodies is labor-intensive and of high cost, which is exacerbated by evolving antigen targets under selective pressure such as fast-mutating viral variants. As a proof-of-concept, we developed a machine learning-assisted antibody generation pipeline AbGen that greatly accelerates the screening and re-design of immunoglobulins G (IgGs) against a broad spectrum of SARS-CoV-2 coronavirus variant strains. Our AbGen centers around a novel antibody language model (AbLM) that is pretrained on 12 million generic protein domain sequences and fine-tuned on 4,000+ paired VH-VL sequences, with IgG-specific CDR-masking and VH-VL cross-attention. AbLM provides a latent space of IgG sequence embeddings for AbGen, including (a) landscapes of IgGs activities in neutralizing the wild-type virus are analyzed through structure prediction for IgG and IgG-antigen (viral protein spikes receptor binding domain, RBD) interactions; and (b) landscapes of IgGs susceptibility in neutralizing variant viruses are predicted through Gaussian process regression, despite that as few as 14 clinical antibodies responses to variants of concern are available. The AbGen pipeline was applied to over 1300 IgG sequences we collected from RBD-binding B cells of convalescent patients. With experimental validations, AbGen efficiently prioritized IgG candidates against a broad spectrum of viral variants (wildtype, Delta, and Omicron), preventing the infection of host cells in vitro and hACE2 transgenic mice in vivo. Compared to other existing protein language models that require 10-100 times more model parameters, AbLM improved the precision from around 50% to 75% to predict IgGs with low variant susceptibility. Furthermore, AbGen enables structure-based computational protein redesign for selected IgG clones with single amino acid substitutions at the RBD-binding interface that doubled the IgG blockade efficacy for one of the severe, therapy-resistant strains - Delta (B.1.617). Our work expedites applications of artificial intelligence in antibody screen and re- design combining data-driven protein language models and Kriging for antibody sequence analysis and activity prediction, in synergy with physics-driven protein docking and design for antibody-antigen interface analyses and functional optimization.

immunology↗

bioRxiv2023

Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

Metastasis is the cause of over 90% of all deaths associated with breast cancer, yet the strategies to predict cancer spreading based on primary tumor profiles and therefore prevent metastasis are egregiously limited. As rare precursor cells to metastasis, circulating tumor cells (CTCs) in multicellular clusters in the blood are 20-50 times more likely to produce viable metastasis than single CTCs. However, the molecular mechanisms underlying various CTC clusters, such as homotypic tumor cell clusters and heterotypic tumor-immune cell clusters, are yet to be fully elucidated. Combining machine learning-assisted computational ranking with experimental demonstration to assess cell adhesion candidates, we identified a transmembrane protein Plexin- B2 (PB2) as a new therapeutic target that drives the formation of both homotypic and heterotypic CTC clusters. High PB2 expression in human primary tumors predicts an unfavorable distant metastasis-free survival and is enriched in CTC clusters compared to single CTCs in advanced breast cancers. Loss of PB2 reduces formation of homotypic tumor cell clusters as well as heterotypic tumor-myeloid cell clusters in triple-negative breast cancer. Interactions between PB2 and its ligand Sema4C on tumor cells promote homotypic cluster formation, and PB2 binding with Sema4A on myeloid cells (monocytes) drives heterotypic CTC cluster formation, suggesting that metastasizing tumor cells hijack the PB2/Sema family axis to promote lung metastasis in breast cancer. Additionally, using a global proteomic analysis, we identified novel downstream effectors of the PB2 pathway associated with cancer stemness, cell cycling, and tumor cell clustering in breast cancer. Thus, PB2 is a novel therapeutic target for preventing new metastasis.

cancer biology↗

bioRxiv2022

Unique molecular signatures sustained in circulating monocytes and regulatory T cells in Convalescent COVID-19 patients

Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized multi-omic single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including covenlesent COVID-19 and sero-negative controls. The reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells are significantly associated with the patients recovered from severe COVID-19. Consistently, sc-RNA seq analysis reveals seven heterogeneous clusters of monocytes (M0-M6) and ten Treg clusters (T0-T9) featuring distinct molecular signatures and associated with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocyte and Treg expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters with S100 family genes: S100A8 & A9 with high HLA-I whereas S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, and a unique TGF-{beta} high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-ived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (>= 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.

immunology↗

bioRxiv2022

CD81 partners with CD44 in promoting exosome biogenesis, tumor cluster formation, and lung metastasis in triple negative breast cancer

Tumor-initiating cells with reprogramming plasticity are thought to be essential for cancer development and metastatic regeneration in many cancers; however, the molecular mechanisms are not fully understood. This study reports that CD81, a tetraspanin protein marker of small extracellular vesicles (exosomes), functions as a binding partner of CD44 and facilitates self-renewal of tumor initiating cells. Using machine learning-assisted protein structure modeling, co-immunoprecipitation, and mutagenesis approaches, we further demonstrate that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. In-depth global and phosphoproteomic analyses of clustering tumor cells unveils endocytosis-related signature pathways of proteins and phosphorylation patterns regulated by CD81 and CD44 specifically or shared between two. Notably, CRISPR Cas9-mediated depletion of either CD44 or CD81 results in loss of both proteins in cancer cell-secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells for mammosphere formation. CD81 is expressed in >80% of human circulating tumor cells (CTCs) and specifically enriched in clustered CTCs along with CD44 isolated from breast cancer patients. Mimicking the phenotypes of CD44 deficiency, loss of CD81 also inhibits tumor cluster aggregation, tumorigenesis, and lung metastasis of triple negative breast cancer (TNBC), supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights the novel role of CD81 and its partnership with CD44 in cancer exosomes, self-renewal, CTC clustering, and metastasis initiation of TNBC.

cancer biology↗

bioRxiv2020

Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Virus Infection as an Innate Antiviral Mechanism

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50-150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

immunology↗

bioRxiv2019

The Arabian camel, Camelus dromedarius interferon epsilon: functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines MDA-MB-231 and MCF-7

The current study highlights for the first time cloning, overexpression, purification, and assessing the cytotxcity of the novel interferon epsilon (IFN{varepsilon}), from the Arabian camel Camelus dromedarius, against two human breast cancer cell lines MDA-MB-231 and MCF-7. Full-length cDNA encoding interferon epsilon (IFN{varepsilon}) was isolated and cloned from the liver of the Arabian camel, C. dromedarius using reverse transcription-polymerase chain reaction. The sequence analysis of the camel IFN{varepsilon} cDNA showed a 582-bp open reading frame encoding a protein of 193 amino acids with an estimated molecular weight of 22.953 kDa. A BLAST search analysis revealed that the C. dromedarius IFN{varepsilon} shared high sequence identity with the IFN genes of other species, such as Camelus ferus, Vicugna pacos, and Homo sapiens. Expression of the camel IFN{varepsilon} cDNA in Escherichia coli gave a fusion protein band of 22.73 kDa after induction with either isopropyl {beta}-D-1-thiogalactopyranoside or lactose for 5 h. Recombinant IFN{varepsilon} protein was overexpressed in the form of inclusion bodies that were easily solubilized and refolded using SDS and KCl. The solubilized inclusion bodies were purified to apparent homogeneity using nickel affinity chromatography. We examined the effect of IFN{varepsilon} on two breast cancer cell lines MDA-MB-231 and MCF-7. In both cell lines, IFN{varepsilon} inhibited cell survival in a dose dependent manner as observed by MTT assay, morphological changes and apoptosis assay. Caspase-3 expression level was found to be increased in MDA-MB-231 treated cells as compared to untreated cells.

molecular biology↗