Surface Protein Profiles Research Articles

P-1974. Surface Protein Profiling of Extracellular Vesicles: A Novel Biomarker Approach for Long COVID Diagnosis?

Abstract Background Extracellular vehicles (EVs) are lipid bilayer vesicles secreted by all cell types into the circulation. EVs carry an array of biomolecules indicative of their origin cells and act as cargo vesicles capable of delivering proteins, mitochondria, and viral particles. However, whether long COVID affects the profile of plasma EVs remains unknown. Methods Plasma samples were selected from the Stanford repositories of Long COVID participants—seventy-six plasma samples from patients with documented SARS-CoV-2 infection. Twenty-five participants had full recovery (10 months post-infection, Stanford Lambda Trial), 24 patients with Long COVID with CFS phenotype and 27 with ME/CFS from the Stanford Long COVID clinic. Plasma EVs were isolated using size-exclusion columns (qEV, Izon Science). The positivity for surface markers and mitochondrial membrane potentials of the plasma EVs were evaluated by flow cytometry. Samples were stained with pre-titrated volumes of the following fluorochrome-conjugated monoclonal antibodies: CD45 leukocytes, CD3/T cells, CD20/B cells, CD14/Monocytes, CD16/NK cells, CD144/endothelial cells, CD41a/Platelets, CD56/neural cell adhesion molecule (NCAM), and Mitotracker deep red/mitochondrial membrane potential. Results The total EV counts and the proportion of mitochondria-positive were decreased in Long COVID group compared to the COVID recovery group (5.02 x 106 vs. 1.92 x 107 /mL, and 42.7 vs. 56.7%, respectively). Compared with the recovery group, Monocyte and NK cell-derived EVs were significantly increased, whereas Endothelial cell-derived EVs were decreased in the Long COVID group. The EVs from the Long COVID group consistently exhibited lower positivity of mitochondrial membrane potential regardless of the parental cells. No difference was observed in plasma EVs derived from NCAM positive cells. NK cell proportion is the only difference between LC/CSF and CSF (P= 0.046). Conclusion Patients with Long COVID showed alterations in plasma EV profiles. These findings may offer insights into the pathogenesis of long COVID. Disclosures All Authors: No reported disclosures

Integrating All-rounder TiO2 Accelerated Electrochemiluminescence with Dual-Quenching PDA@COF Probes for Sensitive Quantification and Protein Profiling of Tumorous Exosomes.

Exosomes have been perceived as promising biomarkers for noninvasive cancer diagnosis and treatment monitoring. However, the sensitive and accurate quantification and phenotyping of exosomes remains challenging. Herein, a versatile electrochemiluminescence (ECL) aptasensor was proposed for the sensitive analysis of tumorous exosomes. Specifically, a ternary nanohybrid (Ru-HAuTiO2), by covalently linking ECL luminophore Ru(dcbpy)32+ with gold nanoparticles (AuNPs)-decorated hollow urchin-like TiO2 (HTiO2), was ingeniously designed as a highly luminescent and self-enhanced ECL nanoemitter. Notably, the porous HTiO2 played an "all-rounder" role, including the carrier for ECL luminophores and AuNPs, coreaction accelerator, and specific exosome capturing scaffold through Ti-phosphate coordination interaction. On the other hand, a polydopamine modified covalent organic framework (PDA@COF) was employed as a quencher to remarkably attenuate the ECL of Ru-HAuTiO2 through a dual-quenching mechanism, and further labeled with a specific aptamer (Apt) of exosomal surface protein. Based on forming a Ru-HAuTiO2/exosome/Apt-PDA@COF sandwich structure on the electrode, a "signal on-off" ECL platform for tumorous exosomes was constructed, realizing sensitive detection within the range of 3.1 × 103 particles/mL to 1 × 108 particles/mL and a low limit of detection of 1.41 × 103 particles/mL, achieving phenotypic profiling of surface proteins on different tumorous exosomes. This work provides a promising alternative method for the detection and analysis of exosomes.

Single Vesicle Surface Protein Profiling and Machine Learning-Based Dual Image Analysis for Breast Cancer Detection.

Single-vesicle molecular profiling of cancer-associated extracellular vesicles (EVs) is increasingly being recognized as a powerful tool for cancer detection and monitoring. Mask and target dual imaging is a facile method to quantify the fraction of the molecularly targeted population of EVs in biofluids at the single-vesicle level. However, accurate and efficient dual imaging vesicle analysis has been challenging due to the interference of false signals on the mask images and the need to analyze a large number of images in clinical samples. In this work, we report a fully automatic dual imaging analysis method based on machine learning and use it with dual imaging single-vesicle technology (DISVT) to detect breast cancer at different stages. The convolutional neural network Resnet34 was used along with transfer learning to produce a suitable machine learning model that could accurately identify areas of interest in experimental data. A combination of experimental and synthetic data were used to train the model. Using DISVT and our machine learning-assisted image analysis platform, we determined the fractions of EpCAM-positive EVs and CD24-positive EVs over captured plasma EVs with CD81 marker in the blood plasma of pilot HER2-positive breast cancer patients and compared to those from healthy donors. The amount of both EpCAM-positive and CD24-positive EVs was found negligible for both healthy donors and Stage I patients. The amount of EpCAM-positive EVs (also CD81-positive) increased from 18% to 29% as the cancer progressed from Stage II to III. No significant increase was found with further progression to Stage IV. A similar trend was found for the CD24-positive EVs. Statistical analysis showed that both EpCAM and CD24 markers can detect HER2-positive breast cancer at Stages II, III, or IV. They can also differentiate individual cancer stages except those between Stage III and Stage IV. Due to the simplicity, high sensitivity, and high efficiency, the DISVT with the AI-assisted dual imaging analysis can be widely used for both basic research and clinical applications to quantitatively characterize molecularly targeted EV subtypes in biofluids.

Snorkel-tag based affinity chromatography for recombinant extracellular vesicle purification.

Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell-cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine is still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue-specific EV cargo information. Here, we present the Snorkel-tag, for which we have genetically fused the EV surface marker protein CD81, to a series of tags with an additional transmembrane domain to be displayed on the EV surface, resembling a snorkel. This system enables the affinity purification of EVs from complex matrices in a non-destructive form while maintaining EV characteristics in terms of surface protein profiles, associated miRNA patterns and uptake into a model cell line. Therefore, we consider the Snorkel-tag to be a widely applicable tool in EV research, allowing for efficient preparation of EV standards and reference materials, or dissecting EVs with different surface markers when fusing to other tetraspanins in vitro or in vivo.

Storage of plasma-derived exosomes: evaluation of anticoagulant use and preserving temperatures

Exosomes carry large cargo of proteins, lipids, and nucleic acids, serving as versatile biomarkers for disease diagnosis and vehicles for drug delivery. However, up to date, no well recognized standard procedures for exosome storage were available for clinical application. This study aimed to determine the optimal storage conditions and the anticoagulants for plasma-derived exosome isolation. Fresh whole blood samples were collected from healthy participants and preserved in four different anticoagulants including sodium citrate (SC1/4), sodium citrate (SC1/9), lithium heparin (LH), or Ethylenediamine tetraacetic acid (EDTA), respectively. Exosomes were extracted from the plasma by differential ultracentrifugation and stored at three different temperatures, 4°C, −20°C or − 80°C for a duration ranging from one week to six months. All plasma samples for storage conditions comparison were pretreated with LH anticoagulant. Exosome features including morphological characteristics, pariticles size diameter, and surface protein profiles (TSG101, CD63, CD81, CD9, CALNEXIN) were assessed by transmission electron microscopy, Nanoparticle Tracking Analysis, and Western Blotting, respectively. Exosomes preserved in LH and SC1/4 group tended to remain intact microstructure with highly abundant protein biomarkers. Exosomes stored at 4°C for short time were prone to be more stable compared to thos at −80°C. Exosomes stored in plasma were superior in terms of ultrastructure, size diameter and surface protein expression to those stored in PBS. In conclusion, plasma-dervied exosome characteristics strictly depend on the anticoagulants and storage temperature and duration.

Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

Surface protein profiling and subtyping of extracellular vesicles in body fluids reveals non-CSF biomarkers of Alzheimer's disease.

Noninvasive and effortless diagnosis of Alzheimer's disease (AD) remains challenging. Here we report the multiplexed profiling of extracellular vesicle (EV) surface proteins at the single EV level in five types of easily accessible body fluids using a proximity barcoding assay (PBA). A total of 183 surface proteins were detected on the EVs from body fluids collected from APP/PS1 transgenic mice and patients with AD. The AD-associated differentially expressed EV proteins could discriminate between the control and AD/AD model samples with high accuracy. Based on machine learning predictive models, urinary EV proteins exhibited the highest diagnostic potential compared to those on other biofluid EVs, both in mice and humans. Single EV analysis further revealed AD-associated EV subpopulations in the tested body fluids, and a urinary EV subpopulation with the signature proteins PLAU, ITGAX and ANXA1 could diagnose patients with AD in blinded datasets with 88% accuracy. Our results suggest that EVs and their subpopulations from noninvasive body fluids, particularly urine, are potential diagnostic biomarkers for AD.

THE DIFFERENTIAL EXTRACTION METHOD OF NEISSERIA MENINGITIDIS AND NON-PATHOGENIC SPECIES FOR PROTEIN PROFILING BY SDS-PAGE

Invasive meningococcal disease (IMD) is an acute, severe, and potentially fatal infection caused by Neisseria meningitidis and is a global public health burden. Given the high fatality rate linked to acute bacterial meningitis, it is imperative to initiate treatment and diagnosis concurrently in most cases. While a culture demonstrating the growth of N. meningitidis from blood or cerebrospinal fluid (CSF) remains the gold standard, however, its sensitivity diminishes following antibiotic administration. Therefore, in this study, differentially extracted whole cell protein (WCP), surface depleted whole cell protein (sdWCP), and cell surface protein (CSP) profiles were analysed by using SDS-PAGE to characterise and classify pathogenic N. meningitidis and non-pathogenic species. This study provides clear evidence that SDS-PAGE yields distinct protein patterns across Men A, B, C, and Y, clinical isolates 1 and 2, N. sicca, N. cinerea, and M. catarrhalis. It effectively demonstrates that SDS-PAGE protein profiling can serve as a reliable method for characterising and separating bacterial proteins at both the species and strain levels. Despite the availability of more advanced technologies, SDS-PAGE remains a valuable tool, particularly in settings where advanced equipment and knowledgeable personnel are lacking. Our approach employs various extraction techniques, has successfully defined and characterised N. meningitidis, non-pathogenic Neisseria species, and M. catarrhalis.

Abstract 3984: Full-length TCR and BCR repertoire analysis, integrated with mRNA and surface proteins characterization, in a tumor mouse model

Abstract Full-length TCR and BCR analysis with single-cell multiomic assays provides an understanding of immune responses, offering insights into the complete receptor structure and functionality that is crucial for accurately assessing the diversity and specificity of immune repertoires. Studying mouse immune cell heterogeneity can be achieved by simultaneous mRNA and surface protein profiling. However, when coupled with full-length TCR and BCR analysis, this integrated approach provides a more comprehensive understanding of immune responses and cellular function in healthy and diseased conditions. In this study, we used the MC38 colon carcinoma model to study T and B cell heterogeneity and clonotype diversity. We stained single-cell suspensions prepared from bone marrow, thymus, spleen and tumor tissues with 20 AbSeq (CITE-Seq) antibody oligos and sample-multiplexing tags. We then loaded 50,000 isolated T and B cells into two lanes of an 8-lane microwell cartridge for single-cell capture by a single-cell analysis system. To aid in the simultaneous analysis of clonality and gene expression, we carried out library preparation using a full-length TCR/BCR multiomic assay and a mouse immune response panel. The combination of mRNA and protein expression confirmed the elevated expression of exhaustion and activation markers in tumor-infiltrating lymphocytes (TILs). We identified high-frequency clonotypes within TILs and tracked their frequency across other tissues. Tumor-burdened mice splenic T cells showed shared TCR combinations with TILs, which were absent in healthy tissue. The results show the ability to successfully profile T and B cell receptors using a full-length TCR/BCR multiomic assay in a mouse tumor model and the use of transcriptome, protein and TCR/BCR clonal information to investigate immune cell characteristics across multiple samples. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo and BD Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. NPM-2536 (v1.0) 1023 Citation Format: Ali Rezvan, Xiaoshan Shi, Moen Sen, Stephanie Widmann, Aaron Tyznik. Full-length TCR and BCR repertoire analysis, integrated with mRNA and surface proteins characterization, in a tumor mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3984.

Abstract 4682: Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma

Abstract The B-cell Receptor (BCR), comprising membrane Ig (mIg) and CD79a/b, controls normal signaling in B-cells, but dysregulation of this signaling is the dominant driver of leukemia/lymphoma. The class-specific mIg subunit of the BCR is a rational drug target for B-cell malignancies. It plays a critical role in the functioning of these cells, and allows for restricted targeting of only B-cells expressing an Ig class, offering a more tumor-specific approach than current pan-B-cell treatments. We developed a first-in-class antibody, WBMP-4, to the mIgM-BCR, which inhibits cell growth and induces apoptosis via widespread down-regulation of tyrosine kinase activity. Given the potent inhibition of intracellular signaling induced upon WBMP-4 binding to the mIgM-BCR, we expect that translation of membrane proteins will be impacted. Here we investigate the expression and phosphorylation profile of cell surface proteins following treatment of malignant B-cells with WBMP-4. Using western blot and flow cytometry, we measure levels of CD79a/b, CD19, CD20, CD21, CD22, CD32a, CD81, and CXCR4 at 6, 24, and 48 hours after low (5ug/mL) and high (20ug/mL) dose WBMP-4 treatment, as compared to control, untreated Burkitt lymphoma (CA46) cells. We analyze phosphorylation levels of those surface proteins that serve as kinases in this system (CD79a/b, CD19, CXCR4, etc.). Using these same methods, we also examine the expression and phosphorylation of key B-cell transcription factors (e.g. NF-kB, c-MYC, STAT5, PAX5, EBF1, FOXO, and BCL-2). Preliminary analyses show a complete absence of CD79a protein (western blot), and decreased phospho-kinase activity of CD79b (Pamgene phosphokinase array) following treatment of Burkitt lymphoma cells with WBMP-4, as compared to untreated cells. While the effect of an antibody binding to mIg may be most pronounced for CD79a/b relative to other proteins, as mIg and CD79a/b form a complex, we expect that the absence of CD79a/b is a result of WBMP-4-induced mIgM inhibition modulating downstream signaling and transcription factor activity. By examining cell surface markers, we may gain insight into how WBMP-4 impacts a cell’s interaction with the tumor microenvironment, and this information is valuable for diagnostic and patient monitoring purposes, and to determine drug combination strategies. This work will be considered in the context of our ongoing research on WBMP-4’s inhibition of intracellular signaling pathways, and the resulting biologic effects, and will contribute to the continued development of WBMP-4 by providing an understanding of the effects achieved (cell growth inhibition, cell differentiation, apoptosis) at different dosages and if these effects are maintained over time. As a novel, efficacious, and low toxicity treatment strategy for B-cell leukemia and lymphoma, WBMP-4 has significant clinical potential. Citation Format: Rachel S. Welt, David Kostyal, Virginia Raymond, Jose M. Lobo, Sydney Welt. Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4682.

An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

Directing the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells.

Vγ9Vδ2 T cells represent a promising cancer therapy platform because the implementation of allogenic, off-the-shelf product candidates is possible. However, intravenous administration of human Vγ9Vδ2 T cells manufactured under good manufacturing practice (GMP)-compliant, serum-free conditions are not tested easily in most mouse models, mainly because they lack the ability to migrate from the blood to tissues or tumors. We demonstrate that these T cells do not migrate from the circulation to the mouse bone marrow (BM), the site of many malignancies. Thus, there is a need to better characterize human γδ T-cell migration in vivo and develop strategies to direct these cells to in vivo sites of therapeutic interest. To better understand the migration of these cells and possibly influence their migration, NSG mice were conditioned with agents to clear BM cellular compartments, i.e., busulfan or total body irradiation (TBI), or promote T-cell migration to inflamed BM, i.e., incomplete Freund's adjuvant (IFA), prior to administering γδ T cells. Conditioning with TBI, unlike busulfan or IFA, increases the percentage and number of γδ T cells accumulating in the mouse BM, and cells in the peripheral blood (PB) and BM display identical surface protein profiles. To better understand the mechanism by which cells migrate to the BM, mice were conditioned with TBI and administered γδ T cells or tracker-stained red blood cells. The mechanism by which γδ T cells enter the BM after radiation is passive migration from the circulation, not homing. We tested if these ex vivo-expanded cells can migrate based on chemokine expression patterns and showed that it is possible to initiate homing by utilizing highly expressed chemokine receptors on the expanded γδ T cells. γδ T cells highly express CCR2, which provides chemokine attraction to C-C motif chemokine ligand 2 (CCL2)-expressing cells. IFNγ-primed mesenchymal stromal cells (MSCs) (γMSCs) express CCL2, and we developed in vitro and in vivo models to test γδ T-cell homing to CCL2-expressing cells. Using an established neuroblastoma NSG mouse model, we show that intratumorally-injected γMSCs increase the homing of γδ T cells to this tumor. These studies provide insight into the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells in NSG mice, which is critical to understanding the fundamental properties of these cells.

Aptamer-Based Multiparameter Analysis for Molecular Profiling of Hematological Malignancies.

The subtypes of hematological malignancies (HM) with minimal molecular profile differences display an extremely heterogeneous clinical course and a discrepant response to certain treatment regimens. Profiling the surface protein markers offers a potent solution for precision diagnosis of HM by differentiating among the subtypes of cancer cells. Herein, we report the use of Cell-SELEX technology to generate a panel of high-affinity aptamer probes that are able to discriminate subtle differences among surface protein profiles between different HM cells. Experimental results show that these aptamers with apparent dissociation constants (Kd) below 10 nM display a unique recognition pattern on different HM subtypes. By combining a machine learning model on the basis of partial least-squares discriminant analysis, 100% accuracy was achieved for the classification of different HM cells. Furthermore, we preliminarily validated the effectiveness of the aptamer-based multiparameter analysis strategy from a clinical perspective by accurately classifying complex clinical samples, thus providing a promising molecular tool for precise HM phenotyping.

Miniaturized Laser Probe for Exosome-Based Cancer Liquid Biopsy.

Exosomes have been established as a valuable tool for clinical applications for the purpose of liquid biopsy and therapy. However, the clinical practice of exosomes as cancer biopsy markers is still to a very low extent. Active mode optical microcavity with microlaser emission has aroused as a versatile approach for chemical and biological sensing due to its benefits of larger photon population, increased effective Q-factor, decreased line width, and improved sensitivity. Herein, we report a label-free and precise quantification of exosome vesicles and surface protein profiling of breast cancer exosomes using functionalized active whispering gallery mode (WGM) microlaser probes. A detection limit of 40 exosomes per microresonator was achieved. The proposed system enabled a pilot assay of quantitative exosome analysis in cancer patients' blood with only a few microliters of sample consumption, holding good potential for large-scale cancer liquid biopsy. Multiplexed functionalization of the optical microresonator allowed us to profile cancer exosomal surface markers and distinct subclasses of breast cancer-associated exosomes and monitor drug treatment outcomes. Our findings speak volumes about the advantages of the WGM microlaser sensor, including very small sample consumption, low detection limit, high specificity, and ease of operation, offering a promising means for precious clinical sample analysis.

Multiplexed Surface Protein Detection and Cancer Classification Using Gap-Enhanced Magnetic-Plasmonic Core-Shell Raman Nanotags and Machine Learning Algorithm.

Cancer is the second leading cause of death attributed to disease worldwide. Current standard detection methods often rely on a single cancer marker, which can lead to inaccurate results, including false negatives, and an inability to detect multiple cancers simultaneously. Here, we developed a multiplex method that can effectively detect and classify surface proteins associated with three distinct types of breast cancer by utilizing gap-enhanced Raman scattering nanotags and machine learning algorithm. We synthesized anisotropic magnetic core-gold shell gap-enhanced Raman nanotags incorporating three different Raman reporters. These multicolor Raman nanotags were employed to distinguish specific surface protein markers in breast cancer cells. The acquired signals were deconvoluted and analyzed using classical least-squares regression to generate a surface protein profile and characterize the breast cancer cells. Furthermore, computational data obtained via finite-difference time-domain and discrete dipole approximation showed the amplification of the electric fields within the gap region due to plasmonic coupling between the two gold layers. Finally, a random forest classifier achieved an impressive classification and profiling accuracy of 93.9%, enabling effective distinguishing between the three different types of breast cancer cell lines in a mixed solution. With the combination of immunomagnetic multiplex target specificity and separation, gap-enhancement Raman nanotags, and machine learning, our method provides an accurate and integrated platform to profile and classify different cancer cells, giving implications for identification of the origin of circulating tumor cells in the blood system.

Iron-poly(tannic acid) biomimetic nanoparticles for targeted magnetic resonance imaging and drug delivery

The artificial nanomaterials with surface modification are promising platform in targeted therapy, but the nanomaterials are easily tracked by the reticuloendothelial system (RES) before reaching the target sites. Here we report a biomimetic camouflage system, macrophage membrane (MM) coated iron-poly(tannic acid) nanoparticels (Fe-PTA NPs) for targeted drug delivery and magnetic resonance imaging (MRI). The MM inherits the surface protein profiles from macrophage (RAW 264.7 cells), which would escape from RES and target the homotypic cells. The anticancer drug doxorubicin (DOX) was loaded into MM-Fe-PTA NPs, and demonstrated pH-responsive release ability. In addition, the MM-Fe-PTA NPs can be used as T1-weighted MRI contrast agents for targeted MRI. This cell membrane camouflage strategy has great potential and challenge for clinic targeted therapy.

Precise Identification and Profiling of Surface Proteins of Ultra Rare Tumor Specific Extracellular Vesicle with Dynamic Quantitative Plasmonic Imaging.

Specific detection of tumor-derived EVs (tEVs) in plasma is complicated by nontumor EVs and non-EV particles. To accurately identify tEVs and profile their surface protein expression at single tEV resolution directly with clinical plasma is still an unmet need. Here, we present a Dynamic Immunoassay for Single tEV surface protein Profiling (DISEP), a kinetic assay based on surface plasmon resonance microscopy (SPRM) for specific single tEV profiling. DISEP adopts a pair of low-affinity oligonucleotide probes to respectively label EV surface proteins and functionalize an SPRM biosensor interface. tEVs labeled with the oligonucleotide probes possess distinctive binding kinetics from nonspecific particles in plasma, which permits accurate digital plasmonic counting of single EVs. We demonstrate DISEP for recognizing target EVs among 350-fold background plasma particles with high sensitivity (4677 EVs per μL). Clinical plasma samples were analyzed to discriminate between pancreatic cancer patients (n = 40) and healthy donors (n = 45). With a panel of biomarker signatures (EpCAM, HER2, and GPC1), DISEP only requires 10 μL primary sample from each donor to classify tumor patients with an area under the curve of 0.98. DISEP provides a highly specific EV detection and surface protein profiling strategy for early cancer diagnosis.

Profiling of single-vesicle surface proteins via droplet digital immuno-PCR for multi-subpopulation extracellular vesicles counting towards cancer diagnostics

Quantification of protein-specific extracellular vesicle (EV) subpopulations at the single-vesicle level is of great significance for cancer diagnosis. Although individual vesicle analysis has been implemented by novel emerging technologies, developing an easy-to-operate and cost-effective single EV analysis approach to promote clinical applications is still challenging. Herein, we constructed a versatile droplet digital immuno-PCR (ddiPCR) assay which integrates the high specificity of immuno-PCR and superior sensitivity of droplet digital PCR to profile the surface proteins of single EVs for multi-subpopulation EVs counting. The clinical application of the ddiPCR assay was validated by simultaneous profiling the EV proteins of CD9/CD63/CD81, HER2, EpCAM in a breast cancer cohort, and CD9/CD63/CD81, GPC-3, EpCAM in a hepatocellular carcinoma cohort (HCC). The results demonstrated that the counting of multi-subpopulation EVs could significantly distinguish patients with breast tumor or HCC from healthy controls. Furthermore, with the assistance of machine learning algorithm and under the best combination of sEV subpopulations, our method exhibited great performance in differentiating breast cancer from healthy individuals. Therefore, this study provides a promising strategy to count multi-subpopulation EVs at the single-vesicle level for cancer diagnosis.

Surface protein profiling of milk and serum extracellular vesicles unveils body fluid-specific signatures

Cell-derived extracellular vesicles (EVs) are currently in the limelight as potential disease biomarkers. The promise of EV-based liquid biopsy resides in the identification of specific disease-associated EV signatures. Knowing the reference EV profile of a body fluid can facilitate the identification of such disease-associated EV-biomarkers. With this aim, we purified EVs from paired human milk and serum samples and used the MACSPlex bead-based flow-cytometry assay to capture EVs on bead-bound antibodies specific for a certain surface protein, followed by EV detection by the tetraspanins CD9, CD63, and CD81. Using this approach we identified body fluid-specific EV signatures, e.g. breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, as well as body fluid-specific markers associated to immune cells and stem cells. Interestingly, comparison of pan-tetraspanin detection (simultaneous CD9, CD63 and CD81 detection) and single tetraspanin detection (detection by CD9, CD63 or CD81) also unveiled body fluid-specific tetraspanin distributions on EVs. Moreover, certain EV surface proteins were associated with a specific tetraspanin distribution, which could be indicative of the biogenesis route of this EV subset. Altogether, the identified body fluid-specific EV profiles can contribute to study EV profile deviations in these fluids during disease processes.

The BD Rhapsody&[trade] HT Xpress System supports dynamic and high-throughput single-cell capture enabling a seamless workflow for analysis of cellular immune responses

Abstract The BD Rhapsody™ Single-Cell Analysis System is an innovative platform that aids high-throughput sequencing of mRNA and proteins in thousands of single cells. Here we present a new eight-lane cartridge design concept, the BD Rhapsody™ HT Xpress System. This novel device architecture can reduce both hands-on and assay time by enabling the users to simultaneously load multiple cell samples while preserving the flexibility to use one lane at a time. To demonstrate the increased throughput capabilities and workflow flexibility of the system, we measured in vitro T cell activation over time. To do this, peripheral blood mononuclear cells from four different subjects were prepared for cell culture in IL-2 or IL-2/anti-CD3/anti-CD28. After one day in culture, cell samples from each culture condition were barcoded using the BD® Single-Cell Multiplexing Kit. Cells from the same subject were pooled and stained with the BD® AbSeq Immune Discovery Panel. Pooled cells from each subject were loaded into the cartridge (one subject per lane, four lanes total). Remaining cells were cultured for another day and similarly barcoded, stained and loaded into the other four lanes of the cartridge. Eight different next generation sequencing libraries were generated and analyzed with the BD Rhapsody™ Analysis Pipeline and SeqGeq™ Software. This strategy enabled us to assess dynamic changes in both T cell surface protein profiles and transcriptome during cell activation while using a streamlined workflow for concomitant analysis of multiple samples. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, BD Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved.