Drug screening on digital microfluidics for cancer precision medicine.

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Cellulose Nanofibrils of High Immunoaffinity for Efficient Enrichment of Small Extracellular Vesicles.

Extracellular vesicles (EVs), crucial in facilitating the transport of diverse molecular cargoes for intercellular communication, have shown great potential in diagnostics, therapeutics, and drug delivery. The challenge of developing effective preparation methods for EVs is heightened by their intrinsic heterogeneity and complexity. Here, a novel strategy for high EV enrichment is developed by utilizing EV-affinitive-modified cellulose nanofibrils. Specifically, modified cellulose with rich carboxyl groups has outstanding dispersing properties, able to be dispersed into cellulose nanofibrils in solution. These cellulose nanofibrils are utilized as scaffolds for the immobilization of EV-affinitive antibody of CD63 by chemical conjugation. The CD63-modified nanofibrils demonstrate a superior EV capture efficiency of 86.4% compared with other reported methods. The high performance of this system is further validated by the efficient capture of EVs from biological blood plasma, allowing the detection of bioactive markers from EV-derived miRNAs and proteins. The authors envision that these modified cellulose nanofibrils of enhanced capability on EV enrichment will open new avenues in various biomedical applications.

Automatic high-throughput and non-invasive selection of sperm at the biochemical level.

BACKGROUND: Sperm selection, a key step in assisted reproductive technology (ART), has long been restrained at the preliminary physical level (morphology or motility); however, subsequent fertilization and embryogenesis are complicated biochemical processes. Such an enormous "gap" poses tough problems for couples dealing with infertility, especially patients with severe/total asthenozoospermia . METHODS: We developed a biochemical-level, automatic-screening/separation, smart droplet-TO-hydrogel chip (BLASTO-chip) for sperm selection. The droplet can sense the pH change caused by sperm's respiration products and then transforms into a hydrogel to be selected out. FINDINGS: The BLASTO-chip system can select biochemically active sperm with an accuracy of over 90%, and its selection efficiency can be flexibly tuned by nearly 10-fold. All the substances in the system were proven to be biosafe via evaluating mice fertilization and offspring health. Live sperm down to 1% could be enriched by over 76-fold to 76%. For clinical application to patients with severe/total asthenozoospermia, the BLASTO-chip could select live sperm from human semen samples containing 10% live but 100% immotile sperm. The rates of fertilization, cleavage, early embryos, and blastocysts were drastically elevated from 15% to 70.83%, 10% to 62.5%, 5% to 37.5%, and 0% to 16.67%, respectively. CONCLUSIONS: The BLASTO-chip represents a real biochemical-level technology for sperm selection that is completely independent of sperm's motility. It can be a powerful tool in ART, especially for patients with severe/total asthenozoospermia. FUNDING: This work was funded by the Ministry of Science and Technology of China, the Ministry of Education of China, and the Shenzhen-Hong Kong Hetao Cooperation Zone.

An integrated and multi-functional droplet-based microfluidic platform for digital DNA amplification.

Digital DNA amplification is a powerful method for detecting and quantifying rare nucleic acids. In this study, we developed a multi-functional droplet-based platform that integrates the traditional digital DNA amplification workflow into a one-step device. This platform enables efficient droplet generation, transition, and signal detection within a 5-min timeframe, distributing the sample into a uniform array of 4 × 104 droplets (variation <2%) within a chamber. Subsequent in-situ DNA amplification, fluorescence detection, and signal analysis were carried out. To assess the platform's performance, we quantitatively detected the human epidermal growth factor receptor (EGFR) mutation and human papillomavirus (HPV) mutation using digital polymerase chain reaction (dPCR) and digital loop-mediated isothermal amplification (dLAMP), respectively. The fluorescence results exhibited a positive, linear, and statistically significant correlation with target DNA concentrations ranging from 101 to 105 copies/µL, demonstrating the capability and feasibility of the integrated device for dPCR and dLAMP. This platform offers high-throughput droplet generation, eliminates droplet fusion and transition, is user-friendly, reduces costs compared to current methods, and holds potential for thermocycling and isothermal nucleic acid quantification with high sensitivity and accuracy.

Magnetic Particle Imaging: From Tracer Design to Biomedical Applications in Vasculature Abnormality.

Magnetic particle imaging (MPI) is an emerging non-invasive tomographic technique based on the response of magnetic nanoparticles (MNPs) to oscillating drive fields at the center of a static magnetic gradient. In contrast to magnetic resonance imaging (MRI), which is driven by uniform magnetic fields and projects the anatomic information of the subjects, MPI directly tracks and quantifies MNPs in vivo without background signals. Moreover, it does not require radioactive tracers and has no limitations on imaging depth. This article first introduces the basic principles of MPI and important features of MNPs for imaging sensitivity, spatial resolution, and targeted biodistribution. The latest research aiming to optimize the performance of MPI tracers is reviewed based on their material composition, physical properties, and surface modifications. While the unique advantages of MPI have led to a series of promising biomedical applications, recent development of MPI in investigating vascular abnormalities in cardiovascular and cerebrovascular systems, and cancer are also discussed. Finally, recent progress and challenges in the clinical translation of MPI are discussed to provide possible directions for future research and development.

Unraveling the whole genome DNA methylation profile of zebrafish kidney marrow by Oxford Nanopore sequencing.

Zebrafish is a widely used model organism for investigating human diseases, including hematopoietic disorders. However, a comprehensive methylation baseline for zebrafish primary hematopoietic organ, the kidney marrow (KM), is still lacking. We employed Oxford Nanopore Technologies (ONT) sequencing to profile DNA methylation in zebrafish KM by generating four KM datasets, with two groups based on the presence or absence of red blood cells. Our findings revealed that blood contamination in the KM samples reduced read quality and altered methylation patterns. Compared with whole-genome bisulfite sequencing (WGBS), the ONT-based methylation profiling can cover more CpG sites (92.4% vs 70%-80%), and exhibit less GC bias with more even genomic coverage. And the ONT methylation calling results showed a high correlation with WGBS results when using shared sites. This study establishes a comprehensive methylation profile for zebrafish KM, paving the way for further investigations into epigenetic regulation and the development of targeted therapies for hematopoietic disorders.

Wnt3a signaling with serum supply induces replication stress in cultured cells.

Wnt signaling plays a central role in tissue development and homeostasis, and its deregulation is implicated in many human diseases, including cancer. As an essential posttranslational modification, protein phosphorylation is critical in Wnt signaling and has been a focus of investigation using systematic approaches, including proteomics. Typically, studies were conducted by applying purified Wnt ligands to cells in a "starvation" condition to minimize the background noise. Despite leading to many important discoveries, such an approach may omit pivotal integrative effects of Wnt signaling in a complex physiological environment. In this study, we investigated the temporal dynamics of the phosphoproteome following treatments of Wnt3a conditioned medium (CM) with serum supply. This revealed three clusters of phosphoproteome changes with distinct temporal profiles with implications in gene expressions and chromatin organizations. Among these, we observed enhanced phosphorylation at the Thr543 residue of 53BP1, which is a key event in the cellular response to DNA damage. Functionally, it triggered the replication stress response pathway mediated by γH2AX accumulation and Chk1 activation, leading to a significant reduction of cells in the S phase of the cell cycle. Intriguingly, Wnt3a treatment in the serum-free condition did not activate 53BP1-Chk1 and replication stress response. Our study indicates the importance of noting the presence or absence of serum supply when studying the signaling pathways.

Near-infrared-activated anticancer platinum(IV) complexes directly photooxidize biomolecules in an oxygen-independent manner.

Conventional light-driven cancer therapeutics require oxygen and visible light to indirectly damage biomolecules, limiting their efficacy in deep, hypoxic tumours. Here we report the use of near-infrared-activated small-molecule Pt(IV) photooxidants to directly oxidize intracellular biomolecules in an oxygen-independent manner, achieving controllable and effective elimination of cancer stem cells. These Pt(IV) complexes accumulate in the endoplasmic reticulum and show low toxicity in the dark. Upon irradiation, the resultant metal-enhanced photooxidation effect causes them to robustly photooxidize survival-related biomolecules, induce intense oxidative stress, disrupt intracellular pH (pHi) homeostasis and initiate nonclassical necrosis. In vivo experiments confirm that the lead photooxidant can effectively inhibit tumour growth, suppress metastasis and activate the immune system. Our study validates the concept of metal-enhanced photooxidation and the subsequent chemotherapeutic applications, supporting the development of such localized photooxidants to directly damage intracellular biomolecules and decrease pHi as a strategy for effective metal-based drugs.

Benchmarking of Nanopore R10.4 and R9.4.1 flow cells in single-cell whole-genome amplification and whole-genome shotgun sequencing.

Third-generation sequencing can be used in human cancer genomics and epigenomic research. Oxford Nanopore Technologies (ONT) recently released R10.4 flow cell, which claimed an improved read accuracy compared to R9.4.1 flow cell. To evaluate the benefits and defects of R10.4 flow cell for cancer cell profiling on MinION devices, we used the human non-small-cell lung-carcinoma cell line HCC78 to construct libraries for both single-cell whole-genome amplification (scWGA) and whole-genome shotgun sequencing. The R10.4 and R9.4.1 reads were benchmarked in terms of read accuracy, variant detection, modification calling, genome recovery rate and compared with the next generation sequencing (NGS) reads. The results highlighted that the R10.4 outperforms R9.4.1 reads, achieving a higher modal read accuracy of over 99.1%, superior variation detection, lower false-discovery rate (FDR) in methylation calling, and comparable genome recovery rate. To achieve high yields scWGA sequencing in the ONT platform as NGS, we recommended multiple displacement amplification with a modified T7 endonuclease Ⅰ cutting procedure as a promising method. In addition, we provided a possible solution to filter the likely false positive sites among the whole genome region with R10.4 by using scWGA sequencing result as a negative control. Our study is the first benchmark of whole genome single-cell sequencing using ONT R10.4 and R9.4.1 MinION flow cells by clarifying the capacity of genomic and epigenomic profiling within a single flow cell. A promising method for scWGA sequencing together with the methylation calling results can benefit researchers who work on cancer cell genomic and epigenomic profiling using third-generation sequencing.

3D Biomimetic Models to Reconstitute Tumor Microenvironment In Vitro: Spheroids, Organoids, and Tumor-on-a-Chip.

Decades of efforts in engineering in vitro cancer models have advanced drug discovery and the insight into cancer biology. However, the establishment of preclinical models that enable fully recapitulating the tumor microenvironment remains challenging owing to its intrinsic complexity. Recent progress in engineering techniques has allowed the development of a new generation of in vitro preclinical models that can recreate complex in vivo tumor microenvironments and accurately predict drug responses, including spheroids, organoids, and tumor-on-a-chip. These biomimetic 3D tumor models are of particular interest as they pave the way for better understanding of cancer biology and accelerating the development of new anticancer therapeutics with reducing animal use. Here, the recent advances in developing these in vitro platforms for cancer modeling and preclinical drug screening, focusing on incorporating hydrogels are reviewed to reconstitute physiologically relevant microenvironments. The combination of spheroids/organoids with microfluidic technologies is also highlighted to better mimic in vivo tumors and discuss the challenges and future directions in the clinical translation of such models for drug screening and personalized medicine.

The versatile roles of testrapanins in cancer from intracellular signaling to cell-cell communication: cell membrane proteins without ligands.

The tetraspanins (TSPANs) are a family of four-transmembrane proteins with 33 members in mammals. They are variably expressed on the cell surface, various intracellular organelles and vesicles in nearly all cell types. Different from the majority of cell membrane proteins, TSPANs do not have natural ligands. TSPANs typically organize laterally with other membrane proteins to form tetraspanin-enriched microdomains (TEMs) to influence cell adhesion, migration, invasion, survival and induce downstream signaling. Emerging evidence shows that TSPANs can regulate not only cancer cell growth, metastasis, stemness, drug resistance, but also biogenesis of extracellular vesicles (exosomes and migrasomes), and immunomicroenvironment. This review summarizes recent studies that have shown the versatile function of TSPANs in cancer development and progression, or the molecular mechanism of TSPANs. These findings support the potential of TSPANs as novel therapeutic targets against cancer.

Lysyl hydroxylase LH1 promotes confined migration and metastasis of cancer cells by stabilizing Septin2 to enhance actin network.

BACKGROUND: Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors such as hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC). These conditions create confined spaces for tumor cell migration and metastasis. The regulatory mechanism of confined migration remains unclear. METHODS: LC-MS was applied to determine the differentially expressed proteins between HCC tissues and corresponding adjacent tissue. Collective migration and single cell migration microfluidic devices with 6 µm-high confined channels were designed and fabricated to mimic the in vivo confined space. 3D invasion assay was created by Matrigel and Collagen I mixture treat to adherent cells. 3D spheroid formation under various stiffness environment was developed by different substitution percentage GelMA. Immunoprecipitation was performed to pull down the LH1-binding proteins, which were identified by LC-MS. Immunofluorescent staining, FRET, RT-PCR, Western blotting, FRAP, CCK-8, transwell cell migration, wound healing, orthotopic liver injection mouse model and in vivo imaging were used to evaluate the target expression and cellular phenotype. RESULTS: Lysyl hydroxylase 1 (LH1) promoted the confined migration of cancer cells at both collective and single cell levels. In addition, LH1 enhanced cell invasion in a 3D biomimetic model and spheroid formation in stiffer environments. High LH1 expression correlated with poor prognosis of both HCC and PDAC patients, while it also promoted in vivo metastasis. Mechanistically, LH1 bound and stabilized Septin2 (SEPT2) to enhance actin polymerization, depending on the hydroxylase domain. Finally, the subpopulation with high expression of both LH1 and SEPT2 had the poorest prognosis. CONCLUSIONS: LH1 promotes the confined migration and metastasis of cancer cells by stabilizing SEPT2 and thus facilitating actin polymerization.

High Throughput Confined Migration Microfluidic Device for Drug Screening.

Cancer metastasis is the major cause of cancer-related death. Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors, creating confined spaces for tumor cell migration and metastasis. Confined migration is involved in all metastasis steps. However, confined and unconfined migration inhibitors are different and drugs available to inhibit confined migration are rare. The main challenges are the modeling of confined migration, the suffering of low throughput, and others. Microfluidic device has the advantage to reduce reagent consumption and enhance throughput. Here, a microfluidic chip that can achieve multi-function drug screening against the collective migration of cancer cells under confined environment is designed. This device is applied to screen out effective drugs on confined migration among a novel mechanoreceptors compound library (166 compounds) in hepatocellular carcinoma, non-small lung cancer, breast cancer, and pancreatic ductal adenocarcinoma cells. Three compounds that can significantly inhibit confined migration in pan-cancer: mitochonic acid 5 (MA-5), SB-705498, and diphenyleneiodonium chloride are found. Finally, it is elucidated that these drugs targeted mitochondria, actin polymerization, and cell viability, respectively. In sum, a high-throughput microfluidic platform for screening drugs targeting confined migration is established and three novel inhibitors of confined migration in multiple cancer types are identified.

Dually stimulative single-chain polymeric nano lock with dynamic ligands for sensitive detection of circulating tumor cells.

Circulating tumor cells (CTCs) are important markers for cancer diagnosis and monitoring. However, CTCs detection remains challenging due to their scarcity, where most of the detection methods are compromised by the loss of CTCs in pre-enrichment, and by the lack of universal antibodies for capturing different kinds of cancer cells. Herein, we report a single-chain based nano lock (SCNL) polymer incorporating dually stimulative dynamic ligands that can bind with a broad spectrum of cancer cells and CTCs overexpressing sialic acid (SA) with high sensitivity and selectivity. The high sensitivity is realized by the polymeric single chain structure and the multi-valent functional moieties, which improve the accessibility and binding stability between the target cells and the SCNL. The highly selective targeting of cancer cells is achieved by the dynamic and dually stimulative nano lock structures, which can be unlocked and functionalized upon simultaneous exposure to overexpressed SA and acidic microenvironment. We applied the SCNL to detecting cancer cells and CTCs in clinical samples, where the detection threshold of SCNL reached 4 cells/mL. Besides CTCs enumeration, the SCNL approach could also be extended to metastasis assessment through monitoring the expressing level of surface SA on cancer cells.

Molecular Basis of Mink ACE2 Binding to SARS-CoV-2 and Its Mink-Derived Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted between humans and minks, and some mutations in the spike (S) protein, especially in the receptor-binding domain (RBD), have been identified in mink-derived viruses. Here, we examined binding of the mink angiotensin-converting enzyme 2 (ACE2) receptor to mink-derived and important human-originating variants, and we demonstrated that most of the RBD variants increased the binding affinities to mink ACE2 (mkACE2). Cryo-electron microscopy structures of the mkACE2-RBD Y453F (with a Y-to-F change at position 453) and mkACE2-RBD F486L complexes helped identify the key residues that facilitate changes in mkACE2 binding affinity. Additionally, the data indicated that the Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and human vaccinated sera efficiently prevented infection of human cells by pseudoviruses expressing Y453F, F486L, or N501T RBD. Our findings provide an important molecular mechanism for the rapid adaptation of SARS-CoV-2 in minks and highlight the potential influence of the main mink-originating variants for humans. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a broad range of hosts. Mink-derived SARS-CoV-2 can transmit back to humans. There is an urgent need to understand the binding mechanism of mink-derived SARS-CoV-2 variants to mink receptor. In this study, we identified all mutations in the receptor-binding domain (RBD) of spike (S) protein from mink-derived SARS-CoV-2, and we demonstrated the enhanced binding affinity of mink angiotensin-converting enzyme 2 (ACE2) to most of the mink-derived RBD variants as well as important human-originating RBD variants. Cryo-electron microscopy structures revealed that the Y453F and F486L mutations enhanced the binding forces in the interaction interface. In addition, Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and the SARS-CoV-2 pseudoviruses with Y453F, F486L, or N501T mutations were neutralized by human vaccinated sera. Therefore, our results provide valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Single cell analysis of mechanical properties and EMT-related gene expression profiles in cancer fingers.

Collective cell migration is associated with cancer metastasis. Cancer fingers are formed when groups of migrating cancer cells follow the leader cells in the front. Epithelial to mesenchymal transition (EMT) is a critical process of cancer metastasis. However, the role of EMT in cancer finger formation remains unclear. In this work, we investigated the EMT-associated mechanical properties and gene expression at single-cell levels in non-small lung cancer fingers. We found that leader cells were more elastic and less sticky than follower cells. Spatial EMT-related gene expression profiling in cancer fingers revealed cellular heterogeneity. Particularly, SNAIL and VIM were found to be two key genes that positively correlated with leader cell phenotypes and controlled cancer finger formation. Silencing either SNAIL or VIM, decreased cancer cell elasticity, cancer finger formation and migration, and increased adhesiveness. These findings indicated that SNAIL and VIM are two driver genes for cancer finger formation.

Molecular basis of differential receptor usage for naturally occurring CD55-binding and -nonbinding coxsackievirus B3 strains.

Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.

Single-cell RNA-seq recognized the initiator of epithelial ovarian cancer recurrence.

Epithelial ovarian cancers (EOCs) are sensitive to chemotherapy but will ultimately relapse and develop drug resistance. The origin of EOC recurrence has been elusive due to intra-tumor heterogeneity. Here we performed single-cell RNA sequencing (scRNA-seq) in 13,369 cells from primary, untreated peritoneal metastasis, and relapse tumors. We used time-resolved analysis to chart the developmental sequence of cells from the metastatic tumors, then traced the earliest replanting cells back to the primary tumors. We discovered seven distinct subpopulations in primary tumors where the CYR61+ "stress" subpopulation was identified as the relapse-initiators. Furthermore, a subpopulation of RGS5+ cancer-associated fibroblasts (CAFs) was found to strongly support tumor metastasis. The combined CYR61/RGS5 expression scores significantly correlated with the relapse-free-survival of EOC patients and can be used as predictors of EOC recurrence. Our study provides insights into the mechanism of EOC recurrence and presents CYR61+ relapse-initiating cells as potential therapeutic targets to prevent EOC relapse.

Deep RNA Sequencing Revealed Fusion Junctional Heterogeneity May Predict Crizotinib Treatment Efficacy in ALK-Rearranged NSCLC.

INTRODUCTION: Gene fusion variants in ALK-rearranged NSCLC may predict patient outcomes, but previous results have been inconclusive. Fusion isoforms coexisting in the same tumor may affect the efficacy of targeted therapy, but they have not been investigated. METHODS: Patients with ALK-rearranged NSCLC who received crizotinib treatments were recruited. Precrizotinib tumor tissues were analyzed by the anchored multiplex polymerase chain reaction for targeted RNA sequencing. Kaplan-Meier and Cox regression were used to compare overall and progression-free survivals. RESULTS: Of the 51 studied subjects, EML4-ALK variant types v1, v2, v3, and others were detected in 23 (45.1%), five (9.8%), 19 (37.3%), and four patients (7.8%), respectively. Multiple EML4-ALK RNA isoforms were detected in 24 tumors (47.1%), and single isoform in 27 (52.9%). Most of the v3 tumors (16 of 19) harbored both v3a and v3b RNA isoforms. Multiple isoforms were also detected in eight non-v3 tumors (33.3% of all 24 multiple isoforms; five v1, two v5', and one v2). Compared with patients with single isoform, those with multiple isoforms had worse progression-free (hazard ratio and 95% confidence interval: 2.45 [1.06-5.69]) and overall (hazard ratio [95% confidence interval]: 3.74 [1.26-11.13]) survivals after adjusting for potential confounders including variant type. Using the patient-derived H2228 cells known to express v3a and v3b, our single-cell polymerase chain reaction detected either v3a or v3b in most single cells. Treatment of H2228 cells by three ALK inhibitors revealed increased ratios of v3a-to-v3b expression over time. CONCLUSIONS: Intratumoral EML4-ALK isoforms may predict the efficacy of targeted therapy in ALK-rearranged NSCLC. Temporal changes of intratumoral fusion isoforms may result from differential selection pressures that a drug might have on one isoform over another. Larger studies on fusion heterogeneity using RNA sequencing are warranted.

Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants.

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.