Patient-derived tumor-like cell clusters for personalized chemo- and immunotherapies in non-small cell lung cancer.

Many patient-derived tumor models have emerged recently. However, their potential to guide personalized drug selection remains unclear. Here, we report patient-derived tumor-like cell clusters (PTCs) for non-small cell lung cancer (NSCLC), capable of conducting 100-5,000 drug tests within 10 days. We have established 283 PTC models with an 81% success rate. PTCs contain primary tumor epithelium self-assembled with endogenous stromal and immune cells and show a high degree of similarity to the original tumors in phenotypic and genotypic features. Utilizing standardized culture and drug-response assessment protocols, PTC drug-testing assays reveal 89% overall consistency in prospectively predicting clinical outcomes, with 98.1% accuracy distinguishing complete/partial response from progressive disease. Notably, PTCs enable accurate prediction of clinical outcomes for patients undergoing anti-PD1 therapy by combining cell viability and IFN-γ value assessments. These findings suggest that PTCs could serve as a valuable preclinical model for personalized medicine and basic research in NSCLC.

Rational design of a 'two-in-one' immunogen DAM drives potent immune response against mpox virus.

The global outbreak of the mpox virus (MPXV) in 2022 highlights the urgent need for safer and more accessible new-generation vaccines. Here, we used a structure-guided multi-antigen fusion strategy to design a 'two-in-one' immunogen based on the single-chain dimeric MPXV extracellular enveloped virus antigen A35 bivalently fused with the intracellular mature virus antigen M1, called DAM. DAM preserved the natural epitope configuration of both components and showed stronger A35-specific and M1-specific antibody responses and in vivo protective efficacy against vaccinia virus (VACV) compared to co-immunization strategies. The MPXV-specific neutralizing antibodies elicited by DAM were 28 times higher than those induced by live VACV vaccine. Aluminum-adjuvanted DAM vaccines protected mice from a lethal VACV challenge with a safety profile, and pilot-scale production confirmed the high yield and purity of DAM. Thus, our study provides innovative insights and an immunogen candidate for the development of alternative vaccines against MPXV and other orthopoxviruses.

Computational prediction and experimental validation identify functionally conserved lncRNAs from zebrafish to human.

Functional studies of long noncoding RNAs (lncRNAs) have been hindered by the lack of methods to assess their evolution. Here we present lncRNA Homology Explorer (lncHOME), a computational pipeline that identifies a unique class of long noncoding RNAs (lncRNAs) with conserved genomic locations and patterns of RNA-binding protein (RBP) binding sites (coPARSE-lncRNAs). Remarkably, several hundred human coPARSE-lncRNAs can be evolutionarily traced to zebrafish. Using CRISPR-Cas12a knockout and rescue assays, we found that knocking out many human coPARSE-lncRNAs led to cell proliferation defects, which were subsequently rescued by predicted zebrafish homologs. Knocking down coPARSE-lncRNAs in zebrafish embryos caused severe developmental delays that were rescued by human homologs. Furthermore, we verified that human, mouse and zebrafish coPARSE-lncRNA homologs tend to bind similar RBPs with their conserved functions relying on specific RBP-binding sites. Overall, our study demonstrates a comprehensive approach for studying the functional conservation of lncRNAs and implicates numerous lncRNAs in regulating vertebrate physiology.

B7H3-targeting chimeric antigen receptor modification enhances antitumor effect of Vγ9Vδ2 T cells in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. This study investigates the therapeutic potential of human Vγ9Vδ2 T cells in GBM treatment. The sensitivity of different glioma specimens to Vγ9Vδ2 T cell-mediated cytotoxicity is assessed using a patient-derived tumor cell clusters (PTCs) model. METHODS: The study evaluates the anti-tumor effect of Vγ9Vδ2 T cells in 26 glioma cases through the PTCs model. Protein expression of BTN2A1 and BTN3A1, along with gene expression related to lipid metabolism and glioma inflammatory response pathways, is analyzed in matched tumor tissue samples. Additionally, the study explores two strategies to re-sensitize tumors in the weak anti-tumor effect (WAT) group: utilizing a BTN3A1 agonistic antibody or employing bisphosphonates to inhibit farnesyl diphosphate synthase (FPPS). Furthermore, the study investigates the efficacy of genetically engineered Vγ9Vδ2 T cells expressing Car-B7H3 in targeting diverse GBM specimens. RESULTS: The results demonstrate that Vγ9Vδ2 T cells display a stronger anti-tumor effect (SAT) in six glioma cases, while showing a weaker effect (WAT) in twenty cases. The SAT group exhibits elevated protein expression of BTN2A1 and BTN3A1, accompanied by differential gene expression related to lipid metabolism and glioma inflammatory response pathways. Importantly, the study reveals that the WAT group GBM can enhance Vγ9Vδ2 T cell-mediated killing sensitivity by incorporating either a BTN3A1 agonistic antibody or bisphosphonates. Both approaches support TCR-BTN mediated tumor recognition, which is distinct from the conventional MHC-peptide recognition by αß T cells. Furthermore, the study explores an alternative strategy by genetically engineering Vγ9Vδ2 T cells with Car-B7H3, and both non-engineered and Car-B7H3 Vγ9Vδ2 T cells demonstrate promising efficacy in vivo, underscoring the versatile potential of Vγ9Vδ2 T cells for GBM treatment. CONCLUSIONS: Vγ9Vδ2 T cells demonstrate a robust anti-tumor effect in some glioma cases, while weaker in others. Elevated BTN2A1 and BTN3A1 expression correlates with improved response. WAT group tumors can be sensitized using a BTN3A1 agonistic antibody or bisphosphonates. Genetically engineered Vγ9Vδ2 T cells, i.e.,  Car-B7H3, show promising efficacy. These results together highlight the versatility of Vγ9Vδ2 T cells for GBM treatment.

Comprehensive Detection of PD-L1 Protein and mRNA in Tumor Cells and Extracellular Vesicles through a Real-Time qPCR Assay.

A growing number of studies have shown that tumor cells secrete extracellular vesicles (EVs) containing programmed death-ligand 1 (PD-L1) protein. These vesicles can travel to lymph nodes and remotely inactivate T cells, thereby evading immune system attack. Therefore, the simultaneous detection of PD-L1 protein expression in cells and EVs is of great significance in guiding immunotherapy. Herein, we developed a method based on qPCR for the simultaneous detection of PD-L1 protein and mRNA in EVs and their parental cells (PREC-qPCR assay). Lipid probes immobilized on magnetic beads were used to capture EVs directly from samples. For RNA assay, EVs were directly broken by heating and quantified with qPCR. As to protein assay, EVs were recognized and bound with specific probes (such as aptamers), which were used as templates in subsequent qPCR analysis. This method was used to analyze EVs of patient-derived tumor clusters (PTCs) and plasma samples from patients and healthy volunteers. The results revealed that the expression of exosomal PD-L1 in PTCs was correlated with tumor types and significantly higher in plasma-derived EVs from tumor patients than that of healthy individuals. When extended to cells and PD-L1 mRNAs, the results showed that the expression of PD-L1 protein was consistent with mRNA in cancer cell lines, while PTCs demonstrated significant heterogeneity. This comprehensive detection of PD-L1 at four levels (cell, EVs, protein, and mRNA) is believed to enhance our understanding of the relationship among PD-L1, tumors, and the immune system and to provide a promising tool for predicting the benefits of immunotherapy.

High-throughput screening of spike variants uncovers the key residues that alter the affinity and antigenicity of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has elicited a worldwide pandemic since late 2019. There has been ~675 million confirmed coronavirus disease 2019 (COVID-19) cases, leading to more than 6.8 million deaths as of March 1, 2023. Five SARS-CoV-2 variants of concern (VOCs) were tracked as they emerged and were subsequently characterized. However, it is still difficult to predict the next dominant variant due to the rapid evolution of its spike (S) glycoprotein, which affects the binding activity between cellular receptor angiotensin-converting enzyme 2 (ACE2) and blocks the presenting epitope from humoral monoclonal antibody (mAb) recognition. Here, we established a robust mammalian cell-surface-display platform to study the interactions of S-ACE2 and S-mAb on a large scale. A lentivirus library of S variants was generated via in silico chip synthesis followed by site-directed saturation mutagenesis, after which the enriched candidates were acquired through single-cell fluorescence sorting and analyzed by third-generation DNA sequencing technologies. The mutational landscape provides a blueprint for understanding the key residues of the S protein binding affinity to ACE2 and mAb evasion. It was found that S205F, Y453F, Q493A, Q493M, Q498H, Q498Y, N501F, and N501T showed a 3-12-fold increase in infectivity, of which Y453F, Q493A, and Q498Y exhibited at least a 10-fold resistance to mAbs REGN10933, LY-CoV555, and REGN10987, respectively. These methods for mammalian cells may assist in the precise control of SARS-CoV-2 in the future.

Pharmacological boosting of cGAS activation sensitizes chemotherapy by enhancing antitumor immunity.

Enhancing chemosensitivity is one of the largest unmet medical needs in cancer therapy. Cyclic GMP-AMP synthase (cGAS) connects genome instability caused by platinum-based chemotherapeutics to type I interferon (IFN) response. Here, by using a high-throughput small-molecule microarray-based screening of cGAS interacting compounds, we identify brivanib, known as a dual inhibitor of vascular endothelial growth factor receptor and fibroblast growth factor receptor, as a cGAS modulator. Brivanib markedly enhances cGAS-mediated type I IFN response in tumor cells treated with platinum. Mechanistically, brivanib directly targets cGAS and enhances its DNA binding affinity. Importantly, brivanib synergizes with cisplatin in tumor control by boosting CD8+ T cell response in a tumor-intrinsic cGAS-dependent manner, which is further validated by a patient-derived tumor-like cell clusters model. Taken together, our findings identify cGAS as an unprecedented target of brivanib and provide a rationale for the combination of brivanib with platinum-based chemotherapeutics in cancer treatment.

Assessment of Drug Susceptibility for Patient-Derived Tumor Models through Lactate Biosensing and Machine Learning.

A patient-derived tumor model (PDM) is a practical tool to rapidly screen chemotherapeutics for individual patients. The evaluation method of cell viability directly determines the application of PDMs for drug susceptibility testing. As one of the metabolites of "glycosis", the lactate content was used to evaluate cell viability, but these assays were not specific for tumor cells. Based on the "Warburg effect", wherein tumor cells preferentially rely on "aerobic glycolysis" to produce lactate instead of pyruvate in "anaerobic glycolysis" of normal cells, we reported a gold lactate sensor (GLS) to estimate the cell viability of PDMs in drug susceptibility testing. It demonstrated high consistency between the GLS and commercial cell viability assay. Unlike either imaging or cell viability assay, the GLS characterizes the cell viability, enables dynamic monitoring, and distinguishes tumor cells from other cells. Moreover, machine learning (ML) was employed to perform a multi-index assessment for drug susceptibility of PDMs, which proved to be accurate and practical for clinical application. Therefore, the GLS provides an ideal drug susceptibility testing tool for individualized medicine.

High-throughput saturation mutagenesis generates a high-affinity antibody against SARS-CoV-2 variants using protein surface display assay on a human cell.

As new mutations continue to emerge, the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus to evade the human immune system and neutralizing antibodies remains a huge challenge for vaccine development and antibody research. The majority of neutralizing antibodies have reduced or lost activity against SARS-CoV-2 variants. In this study, we reported a novel protein surface display system on a mammalian cell for obtaining a higher-affinity antibody in high-throughput manner. Using a saturation mutagenesis strategy through integrating microarray-based oligonucleotide synthesis and single-cell screening assay, we generated a group of new antibodies against diverse prevalent SARS-CoV-2 variants through high-throughput screening the human antibody REGN10987 within 2 weeks. The affinity of those optimized antibodies to seven prevalent mutants was greatly improved, and the EC50 values were no higher than 5 ng/mL. These results demonstrate the robustness of our screening system in the rapid generation of an antibody with higher affinity against a new SARS-CoV-2 variant, and provides a potential application to other protein molecular interactions.

ACE2 decoy receptor generated by high-throughput saturation mutagenesis efficiently neutralizes SARS-CoV-2 and its prevalent variants.

The recent global pandemic was a spillover from the SARS-CoV-2 virus. Viral entry involves the receptor binding domain (RBD) of the viral spike protein interacting with the protease domain (PD) of the cellular receptor, ACE2. We hereby present a comprehensive mutational landscape of the effects of ACE2-PD point mutations on RBD-ACE2 binding using a saturation mutagenesis approach based on microarray-based oligo synthesis and a single-cell screening assay. We observed that changes in glycosylation sites and directly interacting sites of ACE2-PD significantly influenced ACE2-RBD binding. We further engineered an ACE2 decoy receptor with critical point mutations, D30I, L79W, T92N, N322V, and K475F, named C4-1. C4-1 shows a 200-fold increase in neutralization for the SARS-CoV-2 D614G pseudotyped virus compared to wild-type soluble ACE2 and a sevenfold increase in binding affinity to wild-type spike compared to the C-terminal Ig-Fc fused wild-type soluble ACE2. Moreover, C4-1 efficiently neutralized prevalent variants, especially the omicron variant (EC50=16 ng/mL), and rescued monoclonal antibodies, vaccine, and convalescent sera neutralization from viral immune-escaping. We hope to next investigate translating the therapeutic potential of C4-1 for the treatment of SARS-CoV-2.

Identification and characterization of long non-coding RNA Carip in modulating spatial learning and memory.

CaMKII has long been known to be a key effector for synaptic plasticity. Recent studies have shown that a variety of modulators interact with the subunits of CaMKII to regulate the long-term potentiation (LTP) of hippocampal neurons. However, whether long non-coding RNAs modulate the activity of CaMKII and affect synaptic plasticity is still elusive. Here, we identify a previously uncharacterized long non-coding RNA Carip that functions as a scaffold, specifically interacts with CaMKIIß, and regulates the phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptor subunits in the hippocampus. The absence of Carip causes dysfunction of synaptic transmission and attenuates LTP in hippocampal CA3-CA1 synapses, which further leads to impairment of spatial learning and memory. In summary, our findings demonstrate that Carip modulates long-term synaptic plasticity by changing AMPA receptor and NMDA receptor activities, thereby affecting spatial learning and memory in mice.

Synthetic liver fibrotic niche extracts achieve in vitro hepatoblasts phenotype enhancement and expansion.

It is still a challenge for synthesizing 'cellular niche-mimics' in vitro with satisfactory reproducibility and fidelity to recreate the natural niche components (e.g., extracellular matrices and soluble factors) for stem cell cultivation. Inspired by the massive amplification of hepatic progenitor cells during liver fibrosis in vivo, here we optimized the in vitro liver fibrotic niches and subsequently harvested their bioactive ingredients as niche extracts (NEs). The fibrosis-relevant NE marginally outperformed Matrigel for phenotype maintenance of human embryonic stem cell (hESC)-derived hepatoblasts (HBs) and recapitulation of the pathological angiogenesis of hESC-derived endothelial cells both in 2D culture and 3D liver organoids. Finally, defined NE components (i.e., collagen III, IV, IL-17, IL-18 and M-CSF) were resolved by the quantitative proteomics which exhibited advantage over Matrigel for multi-passaged HB expansion. The pathology-relevant and tissue-specific NEs provide innovative and generalizable strategies for the discovery of optimal cellular niche and bioactive niche compositions.

Tumors exploit FTO-mediated regulation of glycolytic metabolism to evade immune surveillance.

The ever-increasing understanding of the complexity of factors and regulatory layers that contribute to immune evasion facilitates the development of immunotherapies. However, the diversity of malignant tumors limits many known mechanisms in specific genetic and epigenetic contexts, manifesting the need to discover general driver genes. Here, we have identified the m6A demethylase FTO as an essential epitranscriptomic regulator utilized by tumors to escape immune surveillance through regulation of glycolytic metabolism. We show that FTO-mediated m6A demethylation in tumor cells elevates the transcription factors c-Jun, JunB, and C/EBPß, which allows the rewiring of glycolytic metabolism. Fto knockdown impairs the glycolytic activity of tumor cells, which restores the function of CD8+ T cells, thereby inhibiting tumor growth. Furthermore, we developed a small-molecule compound, Dac51, that can inhibit the activity of FTO, block FTO-mediated immune evasion, and synergize with checkpoint blockade for better tumor control, suggesting reprogramming RNA epitranscriptome as a potential strategy for immunotherapy.

Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair.

Homologous recombination (HR) is often used to achieve targeted gene integration because of its higher precision and operability compared with microhomology-mediated end-joining (MMEJ) or non-homologous end-joining (NHEJ). It appears to be inefficient for gene integration in animal cells and embryos due to occurring only during cell division. Here we developed genome-wide high-throughput screening and a subsequently paired crRNA library screening to search for genes suppressing homology-directed repair (HDR). We found that, in the reporter system, HDR cells with knockdown of SHROOM1 were enriched as much as 4.7-fold than those with control. Down regulating SHROOM1 significantly promoted gene integration in human and mouse cells after cleavage by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), regardless of the donor types. The knock-in efficiency of mouse embryos could also be doubled by the application of SHROOM1 siRNA during micro-injection. The increased HDR efficiency of SHROOM1 deletion in HEK293T cells could be counteracted by YU238259, an HDR inhibitor, but not by an NHEJ inhibitor. These results indicated that SHROOM1 was an HDR-suppressed gene and that the SHROOM1 knockdown strategy may be useful for a variety of applications, including gene editing to generate cell lines and animal models for studying gene function and human diseases.

Patient-derived tumor-like cell clusters for drug testing in cancer therapy.

Several patient-derived tumor models emerged recently as robust preclinical drug-testing platforms. However, their potential to guide clinical therapy remained unclear. Here, we report a model called patient-derived tumor-like cell clusters (PTCs). PTCs result from the self-assembly and proliferation of primary epithelial, fibroblast, and immune cells, which structurally and functionally recapitulate original tumors. PTCs enabled us to accomplish personalized drug testing within 2 weeks after obtaining the tumor samples. The defined culture conditions and drug concentrations in the PTC model facilitate its clinical application in precision oncology. PTC tests of 59 patients with gastric, colorectal, or breast cancers revealed an overall accuracy of 93% in predicting their clinical outcomes. We implemented PTC to guide chemotherapy selection for a patient with mucinous rectal adenocarcinoma who experienced recurrence with metastases after conventional therapy. After three cycles of a nonconventional therapy identified by the PTC, the patient showed a positive response. These findings need to be validated in larger clinical trials, but they suggest that the PTC model could be prospectively implemented in clinical decision-making for therapy selection.

CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1.

BACKGROUND: CRISPR/Cas9 systems have been repurposed as canonical genome editing tools in a variety of species, but no application for the model strain Rhodobacter sphaeroides 2.4.1 was unveiled. RESULTS: Here we showed two kinds of programmable base editing systems, cytosine base editors (CBEs) and adenine base editors (ABEs), generated by fusing endonuclease Cas9 variant to cytosine deaminase PmCDA1 or heterodimer adenine deaminase TadA-TadA*, respectively. Using CBEs, we were able to obtain C-to-T mutation of single and double targets following the first induction step, with the efficiency of up to 97% and 43%; while the second induction step was needed in the case of triple target, with the screening rate of 47%. Using ABEs, we were only able to gain A-to-G mutation of single target after the second induction step, with the screening rate of 30%. Additionally, we performed a knockout analysis to identify the genes responsible for coenzyme Q10 biosynthesis and found that ubiF, ubiA, ubiG, and ubiX to be the most crucial ones. CONCLUSIONS: Together, CBEs and ABEs serve as alternative methods for genetic manipulation in Rhodobacter sphaeroides and will shed light on the fundamental research of other bacteria that are hard to be directly edited by Cas9-sgRNA.

Engineered Maturation Approaches of Human Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes.

Heart diseases such as myocardial infarction and myocardial ischemia are paroxysmal and fatal in clinical practice. Cardiomyocytes (CMs) differentiated from human pluripotent stem cells provide a promising approach to myocardium regeneration therapy. Identifying the maturity level of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is currently the main challenge for pathophysiology and therapeutics. In this review, we describe current maturity indicators for cardiac microtissue and microdevice cultivation technologies that accelerate cardiac maturation. It may provide insights into regenerative medicine, drug cardiotoxicity testing, and preclinical safety testing.

High-Throughput Identification of miR-596 Inducing p53-Mediated Apoptosis in HeLa and HCT116 Cells Using Cell Microarray.

miRNAs play a key role in the regulation of gene networks in mammalian cells. However, little is known about their roles and functions in the apoptosis pathway. Here, we conducted a whole-genome miRNA screening for apoptosis and identified more than 100 miRNAs as apoptosis inducers. To further explain the roles of these mRNAs in apoptosis, a second round of screening was conducted between p53 +/+ and -/- cells. Among the hits, miR-596 was identified as a regulator of p53. The overexpression of miR-596 significantly increased p53 at the protein level, thereby inducing apoptosis. We also demonstrated that Smurf1 was the direct target of miR-596. Previously, Smurf1 was reported to attenuate the level of p53 through binding and stabilizing MDM2, a p53 inhibitor. Consequently, by targeting Smurf1, miR-596 indirectly increased the p53 level in mammalian cells. Moreover, our study demonstrated that miR-596 had other antitumor characteristics, such as inhibiting migration and proliferation. The data from the GEO dataset revealed that the high expression of miR-596 contributed to survival benefits among cancer patients. These results make miR-596 a potential antitumor factor for future biomedical applications.