Region-Specific CD16+ Neutrophils Promote Colorectal Cancer Progression by Inhibiting Natural Killer Cells.

The colon is the largest compartment of the immune system, with innate immune cells exposed to antigens in the environment. However, the mechanisms by which the innate immune system is instigated are poorly defined in colorectal cancer (CRC). Here, a population of CD16+ neutrophils that specifically accumulate in CRC tumor tissues by imaging mass cytometry (IMC), immune fluorescence, and flow cytometry, which demonstrated pro-tumor activity by disturbing natural killer (NK) cells are identified. It is found that these CD16+ neutrophils possess abnormal cholesterol accumulation due to activation of the CD16/TAK1/NF-κB axis, which upregulates scavenger receptors for cholesterol intake including CD36 and LRP1. Consequently, these region-specific CD16+ neutrophils not only competitively inhibit cholesterol intake of NK cells, which interrupts NK lipid raft formation and blocks their antitumor signaling but also release neutrophil extracellular traps (NETs) to induce the death of NK cells. Furthermore, CD16-knockout reverses the pro-tumor activity of neutrophils and restored NK cell cytotoxicity. Collectively, the findings suggest that CRC region-specific CD16+ neutrophils can be a diagnostic marker and potential therapeutic target for CRC.

Heterogeneous Analysis of Extracellular Vesicles for Osteosarcoma Diagnosis.

Osteosarcoma (OS) is the most prevalent primary tumor of bones, often diagnosed late with a poor prognosis. Currently, few effective biomarkers or diagnostic methods have been developed for early OS detection with high confidence, especially for metastatic OS. Tumor-derived extracellular vesicles (EVs) are emerging as promising biomarkers for early cancer diagnosis through liquid biopsy. Here, we report a plasmonic imaging-based biosensing technique, termed subpopulation protein analysis by single EV counting (SPASEC), for size-dependent EV subpopulation analysis. In our SPASEC platform, EVs are accurately sized and counted on plasmonic sensor chips coated with OS-specific antibodies. Subsequently, EVs are categorized into distinct subpopulations based on their sizes, and the membrane proteins of each size-dependent subpopulation are profiled. We measured the heterogeneous expression levels of the EV markers (CD63, BMP2, GD2, and N-cadherin) in each of the EV subsets from both OS cell lines and clinical plasma samples. Using the linear discriminant analysis (LDA) model, the combination of four markers is applied to classify the healthy donors (n = 37), nonmetastatic OS patients (n = 13), and metastatic patients (n = 12) with an area under the curve of 0.95, 0.92, and 0.99, respectively. SPASEC provides accurate EV sensing technology for early OS diagnosis.

Ultrasensitive Proteomics of Trace Cardiac Tissues with Anchor-Nanoparticles.

Pathological cardiac hypertrophy is a complex process that often leads to heart failure. Label-free proteomics has emerged as an important platform to reveal protein variations and to elucidate the mechanisms of cardiac hypertrophy. Endomyocardial biopsy is a minimally invasive technique for sampling cardiac tissue, but it yields only limited amounts of an ethically permissible specimen. After regular pathological examination, the remaining trace samples pose significant challenges for effective protein extraction and mass spectrometry analysis. Herein, we developed trace cardiac tissue proteomics based on the anchor-nanoparticles (TCPA) method. We identified an average of 6666 protein groups using ∼50 µg of myocardial interventricular septum samples by TCPA. We then applied TCPA to acquire proteomics from patients' cardiac samples both diagnosed as hypertrophic hearts and myocarditis controls and identified significant alterations in pathways such as regulation of actin cytoskeleton, oxidative phosphorylation, and cGMP-PKG signaling pathway. Moreover, we found multiple lipid metabolic pathways to be dysregulated in transthyretin cardiac amyloidosis compared to other types of cardiac hypertrophy. TCPA offers a new technique for studying pathological cardiac hypertrophy and can serve as a platform toolbox for proteomic research in other cardiac diseases.

Boosting humoral and cellular immunity with enhanced STING activation by hierarchical mesoporous metal-organic framework adjuvants.

Vaccination is essential for preventing and controlling infectious diseases, along with reducing mortality. Developing safe and versatile adjuvants to enhance humoral and cellular immune responses to vaccines remains a key challenge in vaccine development. Here, we designed hierarchical mesoporous MOF-801 (HM801) using a Cocamidopropyl betaine (CAPB) and a Pluronics F127 in an aqueous phase system. Meanwhile, we synthesized a novel SARS-CoV-2 nanovaccine (R@M@HM801) with a high loading capacity for both the STING agonist (MSA-2) and the Delta receptor binding domain (Delta-RBD) antigen. R@M@HM801 enhanced MSA-2 and RBD utilization and effectively co-delivered MSA-2 and RBD antigens to antigen-presenting cells in the draining lymph nodes, thereby promoting the activation of both T and B cells. Lymphocyte single-cell analysis showed that R@M@HM801 stimulated robust CD11b+CD4+ T cells, CXCR5+CD4+ T follicular helper (Tfh), and durable CD4+CD44+CD62L-, CD8+CD44+CD62L- effector memory T cell (TEM) immune responses, and promoted the proliferative activation of CD26+ B cells in vivo. Meanwhile, R@M@HM801 induced stronger specific antibodies and neutralization of pseudovirus against Delta compared to the RBD + MAS-2 and RBD + MAS-2 + Alum vaccines. Our study demonstrated the efficacy of a hierarchical mesoporous HM801 and its potential immune activation mechanism in enhancing adaptive immune responses against viruses and other diseases.

Bio-inspired hierarchical wearable patch for fast sweat collection.

Sweat contains abundant physiological and metabolic data to evaluate an individual's physical health. Since the non-exercise sweat secretion rate is low, with an average value of 1-10 µl h-1 cm-2, sweat is generally collected during exercise for existing wearable sweat sensors. To expand their applications to include daily scenarios, these sensors developed for sports and fitness are challenged by the difficulty of collecting trace amounts of sweat. This study proposes a wearable patch inspired by the hierarchical structure of Sarracenia trichomes, allowing for the spontaneous and fast collection of a small amount of secreted sweat. The patch contains microfluidic channels featuring a 20 µm-wide rib structure, fully utilizing the capillary force, thereby eliminating the issue of sweat hysteresis. Furthermore, with only 0.5 µl of the sweat secreted at the collection site, it can converge on the detection medium located within the center reservoir. Volunteer verification demonstrated a twofold increase in sweat collection efficiency compared to traditional wearable patches. This patch serves as an efficient sweat-collection configuration, promising potential for diverse in situ sweat colorimetric analyses.

Codetection of Proteins and RNAs on Extracellular Vesicles for Pancreatic Cancer Early Diagnosis.

Tumor-derived extracellular vesicles (EVs) carry tumor-specific proteins and RNAs, thus becoming prevalent targets for early cancer diagnosis. However, low expression of EV cargos and insufficient diagnostic power of individual biomarkers hindered EVs application in clinical practice. Herein, we propose a multiplex Codetection platform of proteins and RNAs (Co-PAR) for EVs. Co-PAR adopted a pair of antibody-DNA probes to recognize the same target protein, which in turn formed a double-stranded DNA. Thus, the target protein could be quantified by detecting the double-stranded DNA via qPCR. Meanwhile, qRT-PCR simultaneously quantified the target RNAs. Thus, with a regular qPCR instrument, Co-PAR enabled the codetection of multiplex proteins and RNAs, with the sensitivity of 102 EVs/µL (targeting CD63) and 1 EV/µL (targeting snRNA U6). We analyzed the coexpressions of three protein markers (CD63, GPC-1, HER2) and three RNA markers (snRNA U6, GPC-1 mRNA, miR-10b) on EVs from three pancreatic cell lines and 30 human plasma samples using Co-PAR. The diagnostic accuracy of the 6-biomarker combination reached 92.9%, which was at least 6.2% higher than that of 3-biomarker combinations and at least 13.5% higher than that of 6 single biomarkers. Co-PAR, as a multiparameter detection platform for EVs, has great potential in early disease diagnosis.

SpiDe-Sr: blind super-resolution network for precise cell segmentation and clustering in spatial proteomics imaging.

Spatial proteomics elucidates cellular biochemical changes with unprecedented topological level. Imaging mass cytometry (IMC) is a high-dimensional single-cell resolution platform for targeted spatial proteomics. However, the precision of subsequent clinical analysis is constrained by imaging noise and resolution. Here, we propose SpiDe-Sr, a super-resolution network embedded with a denoising module for IMC spatial resolution enhancement. SpiDe-Sr effectively resists noise and improves resolution by 4 times. We demonstrate SpiDe-Sr respectively with cells, mouse and human tissues, resulting 18.95%/27.27%/21.16% increase in peak signal-to-noise ratio and 15.95%/31.63%/15.52% increase in cell extraction accuracy. We further apply SpiDe-Sr to study the tumor microenvironment of a 20-patient clinical breast cancer cohort with 269,556 single cells, and discover the invasion of Gram-negative bacteria is positively correlated with carcinogenesis markers and negatively correlated with immunological markers. Additionally, SpiDe-Sr is also compatible with fluorescence microscopy imaging, suggesting SpiDe-Sr an alternative tool for microscopy image super-resolution.

Photosensitive Hydrogel with Temperature-Controlled Reversible Nano-Apertures for Single-Cell Protein Analysis.

Single cell western blot (scWB) is one of the most important methods for cellular heterogeneity profiling. However, current scWB based on conventional photoactive polyacrylamide hydrogel material suffers from the tradeoff between in-gel probing and separation resolution. Here, a highly sensitive temperature-controlled single-cell western blotting (tc-scWB) method is introduced, which is based on a thermo/photo-dualistic-sensitive polyacrylamide hydrogel, namely acrylic acid-functionalized graphene oxide (AFGO) assisted, N-isopropylacrylamide modified polyacrylamide (ANP) hydrogel. The ANP hydrogel is contracted at high-temperature to constrain protein band diffusion during microchip electrophoretic separation, while the gel aperture is expanded under low-temperature for better antibody penetration into the hydrogel. The tc-scWB method enables the separation and profiling of small-molecule-weight proteins with highly crosslinked gel (12% T) in SDS-PAGE. The tc-scWB is demonstrated on three metabolic and ER stress-specific proteins (CHOP, MDH2 and FH) in four pancreatic cell subtypes, revealing the expression of key enzymes in the Krebs cycle is upregulated with enhanced ER stress. It is found that ER stress can regulate crucial enzyme (MDH2 and FH) activities of metabolic cascade in cancer cells, boosting aerobic respiration to attenuate the Warburg effect and promote cell apoptosis. The tc-scWB is a general toolbox for the analysis of low-abundance small-molecular functional proteins at the single-cell level.

Protective effects of Pt-N-C single-atom nanozymes against myocardial ischemia-reperfusion injury.

Effective therapeutic strategies for myocardial ischemia/reperfusion (I/R) injury remain elusive. Targeting reactive oxygen species (ROS) provides a practical approach to mitigate myocardial damage following reperfusion. In this study, we synthesize an antioxidant nanozyme, equipped with a single-Platinum (Pt)-atom (PtsaN-C), for protecting against I/R injury. PtsaN-C exhibits multiple enzyme-mimicking activities for ROS scavenging with high efficiency and stability. Mechanistic studies demonstrate that the excellent ROS-elimination performance of the single Pt atom center precedes that of the Pt cluster center, owing to its better synergistic effect and metallic electronic property. Systematic in vitro and in vivo studies confirm that PtsaN-C efficiently counteracts ROS, restores cellular homeostasis and prevents apoptotic progression after I/R injury. PtsaN-C also demonstrates good biocompatibility, making it a promising candidate for clinical applications. Our study expands the scope of single-atom nanozyme in combating ROS-induced damage and offers a promising therapeutic avenue for the treatment of I/R injury.

Engineered CRISPRa System for Precise and Multiplex Gene Regulation Using a Hybrid dCas12a Variant and Hairpin-Spacer crRNAs.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a nucleases have emerged as a promising alternative to CRISPR-Cas9 in gene editing and expression regulation. However, the adoption of Cas12a has been hindered due to general off-target activities and limited efficiency. Here, we utilized a hybrid engineered Cas12a variant and hairpin-spacer crRNAs (h-CAP) to enhance the specificity and efficiency of the CRISPR-Cas12a system. Leveraging the h-CAP strategy, we demonstrate both single-base-specific and multiplex gene expression regulation in human cells.

Single-Cell Study Unveils Lead Lifespan in Blood Cell Populations Follows a Universal Lognormal Distribution with Individual Skewness.

Lead is a widespread environmental hazard that can adversely affect multiple biological functions. Blood cells are the initial targets that face lead exposure. However, a systematic assessment of lead dynamics in blood cells at single-cell resolution is still absent. Herein, C57BL/6 mice were fed with lead-contaminated food. Peripheral blood was harvested at different days. Extracted red blood cells and leukocytes were stained with 19 metal-conjugated antibodies and analyzed by mass cytometry. We quantified the time-lapse lead levels in 12 major blood cell subpopulations and established the distribution of lead heterogeneity. Our results show that the lead levels in all major blood cell subtypes follow lognormal distributions but with distinctively individual skewness. The lognormal distribution suggests a multiplicative accumulation of lead with stochastic turnover of cells, which allows us to estimate the lead lifespan of different blood cell populations by calculating the distribution skewness. These findings suggest that lead accumulation by single blood cells follows a stochastic multiplicative process.

Oxali-palladium nanoparticle synthesis, characterization, protein binding, and apoptosis induction in colorectal cancer cells.

This paper focuses on the synthesis of nano-oxali-palladium coated with turmeric extract (PdNPs) using a green chemistry technique based on the reduction in the Pd (II) complex by phytochemicals inherent in turmeric extract. PdNPs were examined and characterized using Field Emission Scanning Electron Microscopy (FESEM), Dynamic Light Scattering (DLS), Fourier Transform Infrared (FTIR), and Atomic Force Microscopy (AFM). Using different spectroscopic and molecular dynamics simulations, a protein-binding analysis of the produced nanoparticle was conducted by observing its interaction with human serum albumin (HSA). Lastly, the cytotoxic effects and apoptotic processes of PdNPs were studied against the HCT116 human colorectal cell line using the MTT assay and flow cytometry tests. According to the findings, PdNPs with spherical and homogenous morphology and a size smaller than 100 nm were generated. In addition, they can induce apoptosis in colorectal cancer cells in a dose-dependent manner with a lower Cc50 (78 µL) than cisplatin and free oxali-palladium against HCT116 cells. The thermodynamic characteristics of protein binding of nanoparticles with HSA demonstrated that PdNPs had a great capacity for quenching and interacting with HSA through hydrophobic forces. In addition, molecular dynamics simulations revealed that free oxali-palladium and PdNP attach to the same area of HSA via non-covalent interactions. It is conceivable to indicate that the synthesized PdNPs are a potential candidate for the construction of novel, nature-based anticancer treatments with fewer side effects and a high level of eco-friendliness.

Proteome signatures of joint toxicity to arsenic (As) and lead (Pb) in human brain organoids with optic vesicles.

Arsenic (As) and lead (Pb) are toxins found in the natural surroundings, and the harmful health outcomes caused by the co-exposure of such toxins have become a considerable problem. However, the joint neurotoxicity of As and Pb to neurodevelopment and the underlying mechanisms remain unclear. Pluripotent stem cell-derived human brain organoids are emerging animal model alternatives for understanding neurological-related diseases. Therefore, we utilized brain organoids with optic vesicles (OVB-organoids) to systematically analyze the neurotoxicity of As and Pb. After 24 h of As and/or Pb exposure, hematoxylin-eosin staining revealed that As and Pb exposure could cause disorders in the structure of the ventricular zone and general cell disarrangement in OVB-organoids. Immunostaining displayed that OVB-organoids are more susceptible to As and Pb co-exposure than independent exposure in apoptosis, proliferation, and cell differentiation. Meanwhile, even though As and Pb could both hinder cell proliferation, contrary to Pb, As could induce an increasing proportion of mitotic (G2/M) cells. The proteome landscape of OVB-organoids illustrated that Pb synergized with As in G2/M arrest and the common role of As and Pb in carcinogenesis. Besides, proteomics analyses suggested the consequential role of autophagy and Wnt pathway in the neurotoxicity of As and Pb co-exposure. Overall, our findings provide penetrating insights into the cell cycle, carcinogenesis, autophagy, and Wnt pathway underlying the As and Pb binary exposure scenarios, which could enhance our understanding of the mixture neurotoxicity mechanisms.

A self-assembled graphene oxide adjuvant induces both enhanced humoral and cellular immune responses in influenza vaccine.

Antiviral vaccine is essential for preventing and controlling virus spreading, along with declining morbidity and mortality. A major challenge in effective vaccination lies in the ability to enhance both the humoral and cellular immune responses by adjuvants. Herein, self-assembled nanoparticles based on graphene oxide quantum dots with components of carnosine, resiquimod and Zn2+ ions, namely ZnGC-R, are designed as a new adjuvant for influenza vaccine. With its high capability for antigen-loading, ZnGC-R enhances antigen utilization, improves DC recruitment, and activates antigen-presenting cells. Single cell analysis of lymphocytes after intramuscular vaccination revealed that ZnGC-R generated multifaceted immune responses. ZnGC-R stimulated robust CD4+CCR7loPD-1hi Tfh and durable CD8+CD44hiCD62L- TEM immune responses, and simultaneously promoted the proliferation of CD26+ germinal center B cells. Besides, ZnGC-R elicited 2.53-fold higher hemagglutination-inhibiting antibody than commercial-licensed aluminum salt adjuvant. ZnGC-R based vaccine induced 342% stronger IgG antibody responses compared with vaccines with inactivated virus alone, leading to 100% in vivo protection efficacy against the H1N1 influenza virus challenge.

Correlation between membrane proteins and sizes of extracellular vesicles and particles: A potential signature for cancer diagnosis.

Extracellular vesicles and particles (EVPs) are recognized as ideal liquid biopsy tools for cancer detection, and membrane proteins are commonly used EVP biomarkers. However, bulk analysis of EVP membrane protein biomarkers typically fails to meet the clinical requirement for diagnostic accuracy. We investigated the correlation between the membrane protein expression level, the binding kinetics to aptamers and the sizes of EVPs with interferometric plasmonic microscopy (iPM), and demonstrated the implementation of the correlative signature to determine cancer types. Using EVPs collected from both cell model and clinical plasma samples with liver, lung, breast, or prostate cancer, we found that the selective set of membrane protein expression levels of five protein markers and their binding kinetics were highly heterogeneous across various sizes of EVPs, resulting in the low overall accuracy (<50%) in cancer classification with bulk analysis of all populations. By grouping the EVPs into three subpopulations according to their sizes, the overall accuracy could be increased to about 70%. We further grouped the EVPs into subpopulations with a 10 nm interval in sizes and analysed the correlation between the membrane proteins and sizes with a machine learning algorithm. The results show that the overall accuracy to discriminate cancer types could be improved to 85%. Therefore, this work highlights the significance of size-dependent subtyping of EVPs and suggests that the correlation between the selective set of membrane proteins and sizes of EVP can serve as a signature for clinical cancer diagnosis.

SETD2 deficiency accelerates sphingomyelin accumulation and promotes the development of renal cancer.

Patients with polycystic kidney disease (PKD) encounter a high risk of clear cell renal cell carcinoma (ccRCC), a malignant tumor with dysregulated lipid metabolism. SET domain-containing 2 (SETD2) has been identified as an important tumor suppressor and an immunosuppressor in ccRCC. However, the role of SETD2 in ccRCC generation in PKD remains largely unexplored. Herein, we perform metabolomics, lipidomics, transcriptomics and proteomics within SETD2 loss induced PKD-ccRCC transition mouse model. Our analyses show that SETD2 loss causes extensive metabolic reprogramming events that eventually results in enhanced sphingomyelin biosynthesis and tumorigenesis. Clinical ccRCC patient specimens further confirm the abnormal metabolic reprogramming and sphingomyelin accumulation. Tumor symptom caused by Setd2 knockout is relieved by myriocin, a selective inhibitor of serine-palmitoyl-transferase and sphingomyelin biosynthesis. Our results reveal that SETD2 deficiency promotes large-scale metabolic reprogramming and sphingomyelin biosynthesis during PKD-ccRCC transition. This study introduces high-quality multi-omics resources and uncovers a regulatory mechanism of SETD2 on lipid metabolism during tumorigenesis.

Identification and Characterization of CD8+ CD27+ CXCR3- T Cell Dysregulation and Progression-Associated Biomarkers in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) etiopathogenesis highlights the contributions of overproduction of CD4+ T cells and loss of immune tolerance. However, the involvement of CD8+ T cells in SLE pathology and disease progression remains unclear. Here, the comprehensive immune cell dysregulation in total 263 clinical peripheral blood samples composed of active SLE (aSLE), remission SLE (rSLE) and healthy controls (HCs) is investigated via mass cytometry, flow cytometry and single-cell RNA sequencing. This is observed that CD8+ CD27+ CXCR3- T cells are increased in rSLE compare to aSLE. Meanwhile, the effector function of CD8+ CD27+ CXCR3- T cells are overactive in aSLE compare to HCs and rSLE, and are positively associated with clinical SLE activity. In addition, the response of peripheral blood mononuclear cells (PBMCs) is monitored to interleukin-2 stimulation in aSLE and rSLE to construct dynamic network biomarker (DNB) model. It is demonstrated that DNB score-related parameters can faithfully predict the remission of aSLE and the flares of rSLE. The abundance and functional dysregulation of CD8+ CD27+ CXCR3- T cells can be potential biomarkers for SLE prognosis and concomitant diagnosis. The DNB score with accurate prediction to SLE disease progression can provide clinical treatment suggestions especially for drug dosage determination.

Profiling extracellular vesicle surface proteins with 10 µL peripheral plasma within 4 h.

Extracellular vesicle (EV) surface proteins, expressed by primary tumours, are important biomarkers for early cancer diagnosis. However, the detection of these EV proteins is complicated by their low abundance and interference from non-EV components in clinical samples. Herein, we present a MEmbrane-Specific Separation and two-step Cascade AmpLificatioN (MESS2CAN) strategy for direct detection of EV surface proteins within 4 h. MESS2CAN utilises novel lipid probes (long chains linked by PEG2K with biotin at one end, and DSPE at the other end) and streptavidin-coated magnetic beads, permitting a 49.6% EV recovery rate within 1 h. A dual amplification strategy with a primer exchange reaction (PER) cascaded by the Cas12a system then allows sensitive detection of the target protein at 10 EV particles per microliter. Using 4 cell lines and 90 clinical test samples, we demonstrate MESS2CAN for analysing HER2, EpCAM and EGFR expression on EVs derived from cells and patient plasma. MESS2CAN reports the desired specificity and sensitivity of EGFR (AUC = 0.98) and of HER2 (AUC = 1) for discriminating between HER2-positive breast cancer, triple-negative breast cancer and healthy donors. MESS2CAN is a pioneering method for highly sensitive in vitro EV diagnostics, applicable to clinical samples with trace amounts of EVs.

Pairwise synthetic cytotoxicity between Paxlovid and 100 frequently prescribed FDA-approved small molecule drugs on liver cells.

Paxlovid is a recent FDA approved specific drug for COVID-19. Extensive prescription of Paxlovid could induce potential synthetic cytotoxicity with drugs. Herein, we aimed to examine pairwise synthetic cytotoxicity between Paxlovid and 100 frequently FDA approved small molecule drugs. Liver cell line HL-7702 or L02 was adopted to evaluate synthetic cytotoxicity between Paxlovid and the 100 small molecule drugs. Inhibitory concentration IC-10 and IC-50 doses for all the 100 small molecule drugs and Paxlovid were experimentally acquired. Then, pairwise synthetic cytotoxicity was examined with the fixed dose IC-10 for each drug. The most 4 significant interactive pairs (2 positively interactive and 2 negatively interactive) were further subjected to molecular docking simulation to reveal the structural modulation with Caspase-8, a key mediator for cell apoptosis.

Platinum-Chimeric Carrier Cells for Ultratrace Cell Analysis in Mass Cytometry.

Mass cytometry by time-of-flight (CyTOF), a high-dimensional single-cell analysis platform, detects up to 50 biomarkers at single-cell resolution. However, CyTOF analysis of biological samples with a minimal number of available cells or rare cell subsets remains a major technical challenge due to the extensive loss of cells during cell recovery, staining, and acquisition. Here, we introduce a platinum-chimeric carrier cell strategy for mass cytometry profiling of ultratrace cell samples. Cisplatin can rapidly enter broken plasma membranes of dead cells and form a chimeric interaction with cellular proteins, peptides, and amino acids. Thus, 198Pt-cisplatin is adopted to tag carrier cells in the pretreatment stage. We investigated 8 cell lines that are commonly accessible in laboratories for their potential as carrier cells to preserve rare target cells for CyTOF analysis. We designed a panel of 35 protein biomarkers to evaluate the comprehensive single-cell subtype classification capability with or without the carrier cell strategy. We further demonstrated the detection and analysis of as few as 1 × 104 immune cells using our method. The proposed method thus allows CyTOF analysis on precious clinical samples with less abundant cells.