Product Citations: 8

The isolation of circulating tumor cells (CTCs) directly from blood, as a liquid biopsy, could lead to a paradigm shift in cancer clinical care by providing an earlier diagnosis, a more accurate prognosis, and personalized treatment. Nevertheless, CTC-specific challenges, including their rarity and heterogeneity, have hampered the wider use of CTCs in clinical studies. Microfluidic-based isolation technologies have emerged as promising tools to circumvent these limitations but still fail to meet the constraints of high purity and short processing time required to ensure compatibility with clinical follow-up. In this study, we developed an immunomagnetic-based microfluidic device, the MagPure chip, to achieve the negative selection of CTCs through the depletion of white blood cells (WBCs) and provide highly purified samples for subsequent analysis. We demonstrate that the MagPure chip depletes all magnetically labeled WBCs (85% of WBCs were successfully labeled) and ensures a CTC recovery rate of 81%. In addition, we show its compatibility with conventional biological studies, including 2D and 3D cell culture, as well as phenotypic and genotypic analyses. Finally, we successfully implemented a two-step separation workflow for whole blood processing by combining a size-based pre-enrichment system (ClearCell FX1®) with the MagPure chip as a subsequent purification step. The total workflow led to high throughput (7.5 mL blood in less than 4 h) and high purity (947 WBCs per mL remaining, 99.99% depletion rate), thus enabling us to quantify CTC heterogeneity in size and tumor marker expression level. This tumor-marker-free liquid biopsy workflow could be used in a clinical context to assess phenotype aggressiveness and the prognosis rate.

  • Cancer Research

NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia.

In Nature Communications on 5 September 2022 by Leung, H., Perdomo, J., et al.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of the adenoviral vector vaccines ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen) against COVID-19. The mechanisms involved in clot formation and thrombocytopenia in VITT are yet to be fully determined. Here we show neutrophils undergoing NETosis and confirm expression markers of NETs in VITT patients. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets, neutrophils, fibrin, extracellular DNA and citrullinated histone H3 in a flow microfluidics system and in vivo. Inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast, in vivo blockage of FcγRIIa abrogates both thrombosis and thrombocytopenia suggesting these are distinct processes. Our findings indicate that anti-PF4 antibodies activate blood cells via FcγRIIa and are responsible for thrombosis and thrombocytopenia in VITT. Future development of NETosis and FcγRIIa inhibitors are needed to treat VITT and similar immune thrombotic thrombocytopenia conditions more effectively, leading to better patient outcomes.
© 2022. The Author(s).

  • FC/FACS
  • Immunology and Microbiology

Pharmaco-Pheno-Multiomic Integration Reveals Biomarker Profiles and Therapeutic Response Prediction Models in Leukemia and Ovarian Cancer

Preprint on BioRxiv : the Preprint Server for Biology on 17 June 2022 by Silberberg, G., Walling, B., et al.

Despite considerable progress made in improving therapeutic strategies, the overall survival for patients diagnosed with various cancer types remains low. Further, patients often cycle through multiple therapeutic options before finding an effective regimen for the specific malignancy being treated. A focus on building enhanced computational models, which prioritize therapeutic regimens based on a tumor’s complete molecular profile, will improve the patient experience and augment initial outcomes. In this study, we present an integrative analysis of multiple omic datasets coupled with phenotypic and therapeutic response profiles of Cytarabine from a cohort of primary AML tumors, and Olaparib from a cohort of Patient-Derived Xenograft (PDX) models of ovarian cancer. These analyses, termed P harmaco- P heno- M ulti o mic (PPMO) Integration, established novel complex biomarker profiles that were used to accurately predict prospective therapeutic response profiles in cohorts of newly profiled AML and ovarian tumors. Results from the computational analyses also provide new insights into disease etiology and the mechanisms of therapeutic resistance. Collectively, this study provides proof-of-concept in the use of PPMO to establish highly accurate predictive models of therapeutic response, and the power of leveraging this method to unveil cancer disease mechanisms.

  • Cancer Research

IL-22 Plays a Dual Role in the Amniotic Cavity: Tissue Injury and Host Defense against Microbes in Preterm Labor.

In The Journal of Immunology on 1 April 2022 by Gershater, M., Romero, R., et al.

IL-22 is a multifaceted cytokine with both pro- and anti-inflammatory functions that is implicated in multiple pathologies. However, the role of IL-22 in maternal-fetal immunity in late gestation is poorly understood. In this study, we first showed that IL-22+ T cells coexpressing retinoic acid-related orphan receptor γt (ROR-γt) are enriched at the human maternal-fetal interface of women with preterm labor and birth, which was confirmed by in silico analysis of single-cell RNA sequencing data. T cell activation leading to preterm birth in mice was preceded by a surge in IL-22 in the maternal circulation and amniotic cavity; however, systemic administration of IL-22 in mice did not induce adverse perinatal outcomes. Next, using an ex vivo human system, we showed that IL-22 can cross from the choriodecidua to the intra-amniotic space, where its receptors (Il22ra1, Il10rb, and Il22ra2) are highly expressed by murine gestational and fetal tissues in late pregnancy. Importantly, amniotic fluid concentrations of IL-22 were elevated in women with sterile or microbial intra-amniotic inflammation, suggesting a dual role for this cytokine. The intra-amniotic administration of IL-22 alone shortened gestation and caused neonatal death in mice, with the latter outcome involving lung maturation and inflammation. IL-22 plays a role in host response by participating in the intra-amniotic inflammatory milieu preceding Ureaplasma parvum-induced preterm birth in mice, which was rescued by the deficiency of IL-22. Collectively, these data show that IL-22 alone is capable of causing fetal injury leading to neonatal death and can participate in host defense against microbial invasion of the amniotic cavity leading to preterm labor and birth.
Copyright © 2022 by The American Association of Immunologists, Inc.

  • Immunology and Microbiology

NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia

Preprint on Research Square on 30 September 2021 by Chong, B., Leung, H., et al.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of adenoviral vector vaccines (AstraZeneca and Johnson Johnson) against COVID-19 1 . Anti-platelet factor 4 (PF4) antibodies are present in VITT patients 2,3 . Although the current view suggests that platelet activation by anti-PF4 antibodies is the cause of thrombosis there is as yet no direct evidence that the antibodies induce clot formation and thrombocytopenia (reduction in platelet counts) in VITT and the mechanisms involved remain unknown 4 . Here we show that VITT antibodies induce thrombosis and thrombocytopenia, and that thrombus formation is mediated by neutrophil extracellular traps (NETs). We found markers of NETosis, abundance of neutrophil/platelet aggregates and presence of neutrophils undergoing NETosis in patients with active VITT. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets, neutrophils, fibrin, extracellular DNA and citrullinated histone H3 using an in vitro blood flow microfluidic system. In transgenic mice expressing human PF4 and FcγRIIa, VITT antibodies lead to thrombosis, thrombocytopenia and formation of low density granulocytes. Pharmacological and genetic inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast, in vivo blockage of FcγRIIa abrogates both thrombosis and thrombocytopenia suggesting they are distinct processes. Our findings indicate that VITT antibodies activate cells via FcγRIIa and are responsible for thrombosis and thrombocytopenia. This study identifies NETosis as a pathogenic mechanism for thrombus formation in VITT. We anticipate our findings will motivate future development of NETosis and FcγRIIa inhibitors as potential specific therapies for VITT and consequently better patient outcomes.

  • FC/FACS
  • Immunology and Microbiology
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