New insights into red blood cells in tumor precision diagnosis and treatment.

Red blood cells (RBCs), which function as material transporters in organisms, are rich in materials that are exchanged with metabolically active tumor cells. Recent studies have demonstrated that tumor cells can regulate biological changes in RBCs, including influencing differentiation, maturation, and morphology. RBCs play an important role in tumor development and immune regulation. Notably, the novel scientific finding that RBCs absorb fragments of tumor-carrying DNA overturns the conventional wisdom that RBCs do not contain nucleic acids. RBC membranes are excellent biomimetic materials with significant advantages in terms of their biocompatibility, non-immunogenicity, non-specific adsorption resistance, and biodegradability. Therefore, RBCs provide a new research perspective for the development of tumor liquid biopsies, molecular imaging, drug delivery, and other tumor precision diagnosis and treatment technologies.

Crosstalk of non-apoptotic RCD panel in hepatocellular carcinoma reveals the prognostic and therapeutic optimization.

Non-apoptotic regulated cell death (RCD) of tumor cells profoundly affects tumor progression and plays critical roles in determining response to immune checkpoint inhibitors (ICIs). Prognosis-distinctive HCC subtypes were identified by consensus cluster analysis based on the expressions of 507 non-apoptotic RCD genes obtained from databases and literature. Meanwhile, a set of bioinformatic tools was integrated to analyze the differences of the tumor immune microenvironment infiltration, genetic mutation, copy number variation, and epigenetics alternations within two subtypes. Finally, a non-apoptotic RCDRS signature was constructed and its reliability was evaluated in HCC patients' tissues. The high-RCDRS HCC subgroup showed a significantly lower overall survival and less sensitivity to ICIs compared to low-RCDRS subgroup, but higher sensitivity to cisplatin, paclitaxel, and sorafenib. Overall, we established an RCDRS panel consisting of four non-apoptotic RCD genes, which might be a promising predictor for evaluating HCC prognosis, guiding therapeutic decision-making, and ultimately improving patient outcomes.

PD-1/CD80+ small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression.

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity.

Interleukin-6 classic and trans-signaling utilize glucose metabolism reprogramming to achieve anti- or pro-inflammatory effects.

Interleukin (IL)-6 has anti- and pro-inflammatory functions, controlled by IL-6 classic and trans-signaling, respectively. Differences in the downstream signaling mechanism between IL-6 classic and trans-signaling have not been identified. Here, we report that IL-6 activates glycolysis to regulate the inflammatory response. IL-6 regulates glucose metabolism by forming a complex containing signal-transducing activators of transcription 3 (STAT3), hexokinase 2 (HK2), and voltage-dependent anion channel 1 (VDAC1). The IL-6 classic signaling directs glucose flux to oxidative phosphorylation (OxPhos), while IL-6 trans-signaling directs glucose flux to anaerobic glycolysis. Classic IL-6 signaling promotes STAT3 translocation into mitochondria to interact with pyruvate dehydrogenase kinase-1 (PDK1), leading to pyruvate dehydrogenase α (PDHA) dissociation from PDK1. As a result, PDHA is dephosphorylated, and STAT3 is phosphorylated at Ser727. By contrast, IL-6 trans-signaling promotes the interaction of sirtuin 2 (SIRT2) and lactate dehydrogenase A (LDHA), leading to the dissociation of STAT3 from SIRT2. As a result, LDHA is deacetylated, and STAT3 is acetylated and phosphorylated at Tyr705. IL-6 classic signaling promotes the differentiation of regulatory T cells via the PDK1/STAT3/PDHA axis, whereas IL-6 trans-signaling promotes the differentiation of Th17 cells via the SIRT2/STAT3/LDHA axis. Conclusion: IL-6 classic signaling generates anti-inflammatory functions by shifting energy metabolism to OxPhos, while IL-6 trans-signaling generates pro-inflammatory functions by shifting energy metabolism to anaerobic glycolysis.

Transcriptomic Signature of 3D Hierarchical Porous Chip Enriched Exosomes for Early Detection and Progression Monitoring of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly lethal malignant tumor, and the current non-invasive diagnosis method based on serum markers, such as α-fetoprotein (AFP), and des-γ-carboxy-prothrombin (DCP), has limited efficacy in detecting it. Therefore, there is a critical need to develop novel biomarkers for HCC. Recent studies have highlighted the potential of exosomes as biomarkers. To enhance exosome enrichment, a silicon dioxide (SiO2) microsphere-coated three-dimensional (3D) hierarchical porous chip, named a SiO2-chip is designed. The features of the chip, including its continuous porous 3D scaffold, large surface area, and nanopores between the SiO2 microspheres, synergistically improved the exosome capture efficiency. Exosomes from both non-HCC and HCC subjects are enriched using an SiO2-chip and performed RNA sequencing to identify HCC-related long non-coding RNAs (lncRNAs) in the exosomes. This study analysis reveales that LUCAT-1 and EGFR-AS-1 are two HCC-related lncRNAs. To further detect dual lncRNAs in exosomes, quantitative real time polymerase chain reaction (qRT-PCR) is employed. The integration of dual lncRNAs with AFP and DCP significantly improves the diagnostic accuracy. Furthermore, the integration of dual lncRNAs with DCP effectively monitors the prognosis of patients with HCC and detects disease progression. In this study, a liquid biopsy-based approach for noninvasive and reliable HCC detection is developed.

Next-Generation Preclinical Functional Testing Models in Cancer Precision Medicine: CTC-Derived Organoids.

Basic and clinical cancer research requires tumor models that consistently recapitulate the characteristics of prima tumors. As ex vivo 3D cultures of patient tumor cells, patient-derived tumor organoids possess the biological properties of primary tumors and are therefore excellent preclinical models for cancer research. Patient-derived organoids can be established using primary tumor tissues, peripheral blood, pleural fluid, ascites, and other samples containing tumor cells. Circulating tumor cells acquired by non-invasive sampling feature dynamic circulation and high heterogeneity. Circulating tumor cell-derived organoids are prospective tools for the dynamic monitoring of tumor mutation evolution profiles because they reflect the heterogeneity of the original tumors to a certain extent. This review discusses the advantages and applications of patient-derived organoids. Meanwhile, this work highlights the biological functions of circulating tumor cells, the latest advancement in research of circulating tumor cell-derived organoids, and potential application and challenges of this technology.

Tumor aerobic glycolysis confers immune evasion through modulating sensitivity to T cell-mediated bystander killing via TNF-α.

Metabolic reprogramming toward glycolysis is a hallmark of cancer malignancy. The molecular mechanisms by which the tumor glycolysis pathway promotes immune evasion remain to be elucidated. Here, by performing genome-wide CRISPR screens in murine tumor cells co-cultured with cytotoxic T cells (CTLs), we identified that deficiency of two important glycolysis enzymes, Glut1 (glucose transporter 1) and Gpi1 (glucose-6-phosphate isomerase 1), resulted in enhanced killing of tumor cells by CTLs. Mechanistically, Glut1 inactivation causes metabolic rewiring toward oxidative phosphorylation, which generates an excessive amount of reactive oxygen species (ROS). Accumulated ROS potentiate tumor cell death mediated by tumor necrosis factor alpha (TNF-α) in a caspase-8- and Fadd-dependent manner. Genetic and pharmacological inactivation of Glut1 sensitizes tumors to anti-tumor immunity and synergizes with anti-PD-1 therapy through the TNF-α pathway. The mechanistic interplay between tumor-intrinsic glycolysis and TNF-α-induced killing provides new therapeutic strategies to enhance anti-tumor immunity.

Integrated plasma and exosome long noncoding RNA profiling is promising for diagnosing non-small cell lung cancer.

OBJECTIVES: Non-small cell lung cancer (NSCLC) accounts for more than 80 % of all lung cancers, and its 5-year survival rate can be greatly improved by early diagnosis. However, early diagnosis remains elusive because of the lack of effective biomarkers. In this study, we aimed to develop an effective diagnostic model for NSCLC based on a combination of circulating biomarkers. METHODS: Tissue-deregulated long noncoding RNAs (lncRNAs) in NSCLC were identified in datasets retrieved from the Gene Expression Omnibus (GEO, n=727) and The Cancer Genome Atlas (TCGA, n=1,135) databases, and their differential expression was verified in paired local plasma and exosome samples from NSCLC patients. Subsequently, LASSO regression was used to screen for biomarkers in a large clinical population, and a logistic regression model was used to establish a multi-marker diagnostic model. The area under the receiver operating characteristic (ROC) curve (AUC), calibration plots, decision curve analysis (DCA), clinical impact curves, and integrated discrimination improvement (IDI) were used to evaluate the efficiency of the diagnostic model. RESULTS: Three lncRNAs-PGM5-AS1, SFTA1P, and CTA-384D8.35 were consistently expressed in online tissue datasets, plasma, and exosomes from local patients. LASSO regression identified nine variables (Plasma CTA-384D8.35, Plasma PGM5-AS1, Exosome CTA-384D8.35, Exosome PGM5-AS1, Exosome SFTA1P, Log10CEA, Log10CA125, SCC, and NSE) in clinical samples that were eventually included in the multi-marker diagnostic model. Logistic regression analysis revealed that Plasma CTA-384D8.35, exosome SFTA1P, Log10CEA, Exosome CTA-384D8.35, SCC, and NSE were independent risk factors for NSCLC (p<0.01), and their results were visualized using a nomogram to obtain personalized prediction outcomes. The constructed diagnostic model demonstrated good NSCLC prediction ability in both the training and validation sets (AUC=0.97). CONCLUSIONS: In summary, the constructed circulating lncRNA-based diagnostic model has good NSCLC prediction ability in clinical samples and provides a potential diagnostic tool for NSCLC.

The potential crosstalk between tumor and plasma cells and its association with clinical outcome and immunotherapy response in bladder cancer.

BACKGROUND: Although immunotherapy is effective in improving the clinical outcomes of patients with bladder cancer (BC), it is only effective in a small percentage of patients. Intercellular crosstalk in the tumor microenvironment strongly influences patient response to immunotherapy, while the crosstalk patterns of plasma cells (PCs) as endogenous antibody-producing cells remain unknown. Here, we aimed to explore the heterogeneity of PCs and their potential crosstalk patterns with BC tumor cells. METHODS: Crosstalk patterns between PCs and tumor cells were revealed by performing integrated bulk and single-cell RNA sequencing (RNA-seq) and spatial transcriptome data analysis. A risk model was constructed based on ligand/receptor to quantify crosstalk patterns by stepwise regression Cox analysis. RESULTS: Based on cell infiltration scores inferred from bulk RNA-seq data (n = 728), we found that high infiltration of PCs was associated with better overall survival (OS) and response to immunotherapy in BC. Further single-cell transcriptome analysis (n = 8; 41,894 filtered cells) identified two dominant types of PCs, IgG1 and IgA1 PCs. Signal transduction from tumor cells of specific states (stress-like and hypoxia-like tumor cells) to PCs, for example, via the LAMB3/CD44 and ANGPTL4/SDC1 ligand/receptor pairs, was validated by spatial transcriptome analysis and associated with poorer OS as well as nonresponse to immunotherapy. More importantly, a ligand/receptor pair-based risk model was constructed and showed excellent performance in predicting patient survival and immunotherapy response. CONCLUSIONS: PCs are an important component of the tumor microenvironment, and their crosstalk with tumor cells influences clinical outcomes and response to immunotherapies in BC patients.

A self-monitoring microneedle patch for light-controlled synergistic treatment of melanoma.

Melanoma is the most aggressive and malignant form of skin cancer. Current melanoma treatment methods generally suffer from frequent drug administration as well as difficulty in direct monitoring of drug release. Here, a self-monitoring microneedle (MN)-based drug delivery system, which integrates a dissolving MN patch with aggregation-induced emission (AIE)-active PATC microparticles, is designed to achieve light-controlled pulsatile chemo-photothermal synergistic therapy of melanoma. The PATC polymeric particles, termed D/I@PATC, encapsulate both of chemotherapeutic drug doxorubicin (DOX) and the photothermal agent indocyanine green (ICG). Upon light illumination, PATC gradually dissociates into smaller particles, causing the release of encapsulated DOX and subsequent fluorescence intensity change of PATC particles, thereby not only enabling direct observation of the drug release process under light stimuli, but also facilitating verification of drug release by fluorescence recovery after light trigger. Moreover, encapsulation of ICG in PATC particles displays significant improvement of its photothermal stability both in vitro and in vivo. In a tumor-bearing mouse, the application of one D/I@PATC MN patch combining with two cycles of light irradiation showed excellent controllable chemo-photothermal efficacy and exhibited ∼97% melanoma inhibition rate without inducing any evident systemic toxicity, suggesting a great potential for skin cancer treatment in clinics.

Glutamine metabolism in breast cancer and possible therapeutic targets.

Cancer is characterized by metabolic reprogramming, which is a hot topic in tumor treatment research. Cancer cells alter metabolic pathways to promote their growth, and the common purpose of these altered metabolic pathways is to adapt the metabolic state to the uncontrolled proliferation of cancer cells. Most cancer cells in a state of nonhypoxia will increase the uptake of glucose and produce lactate, called the Warburg effect. Increased glucose consumption is used as a carbon source to support cell proliferation, including nucleotide, lipid and protein synthesis. In the Warburg effect, pyruvate dehydrogenase activity decreases, thereby disrupting the TCA cycle. In addition to glucose, glutamine is also an important nutrient for the growth and proliferation of cancer cells, an important carbon bank and nitrogen bank for the growth and proliferation of cancer cells, providing ribose, nonessential amino acids, citrate, and glycerin necessary for cancer cell growth and proliferation and compensating for the reduction in oxidative phosphorylation pathways in cancer cells caused by the Warburg effect. In human plasma, glutamine is the most abundant amino acid. Normal cells produce glutamine via glutamine synthase (GLS), but the glutamine synthesized by tumor cells is insufficient to meet their high growth needs, resulting in a "glutamine-dependent phenomenon." Most cancers have an increased glutamine demand, including breast cancer. Metabolic reprogramming not only enables tumor cells to maintain the reduction-oxidation (redox) balance and commit resources to biosynthesis but also establishes heterogeneous metabolic phenotypes of tumor cells that are distinct from those of nontumor cells. Thus, targeting the metabolic differences between tumor and nontumor cells may be a promising and novel anticancer strategy. Glutamine metabolic compartments have emerged as promising candidates, especially in TNBC and drug-resistant breast cancer. In this review, the latest discoveries of breast cancer and glutamine metabolism are discussed, novel treatment methods based on amino acid transporters and glutaminase are discussed, and the relationship between glutamine metabolism and breast cancer metastasis, drug resistance, tumor immunity and ferroptosis are explained, which provides new ideas for the clinical treatment of breast cancer.

Phosphomevalonate Kinase Controls ß-Catenin Signaling via the Metabolite 5-Diphosphomevalonate.

ß-catenin signaling is abnormally activated in cancer. Here, this work screens the mevalonate metabolic pathway enzyme PMVK to stabilize ß-catenin signaling using a human genome-wide library. On the one hand, PMVK-produced MVA-5PP competitively binds to CKIα to prevent ß-catenin Ser45 phosphorylation and degradation. On the other hand, PMVK functions as a protein kinase to directly phosphorylate ß-catenin Ser184 to increase its protein nuclear localization. This synergistic effect of PMVK and MVA-5PP together promotes ß-catenin signaling. In addition, PMVK deletion impairs mouse embryonic development and causes embryonic lethal. PMVK deficiency in liver tissue alleviates DEN/CCl4 -induced hepatocarcinogenesis. Finally, the small molecule inhibitor of PMVK, PMVKi5, is developed and PMVKi5 inhibits carcinogenesis of liver and colorectal tissues. These findings reveal a non-canonical function of a key metabolic enzyme PMVK and a novel link between the mevalonate pathway and ß-catenin signaling in carcinogenesis providing a new target for clinical cancer therapy.

Dynamic gene screening enabled identification of a 10-gene panel for early detection and progression assessment of gastric cancer.

Early diagnosis and progression assessment are critical for the timely detection and treatment of gastric cancer (GC) patients. Identification of diagnostic biomarkers for early detection of GC represents an unmet clinical need, and how these markers further influence GC progression is explored rarely. We performed dynamic gene screening based on high-throughput data analysis from patients with precancerous lesions and early gastric cancer (EGC) and identified a 10-gene panel by the lasso regression model. This panel demonstrated good diagnostic performance in TCGA (AUC = 0.95, sensitivity = 86.67 %, specificity = 90.63 %) and GEO (AUC = 0.84, sensitivity = 91.67 %, specificity = 78.13 %) cohorts. Moreover, three GC subtypes were clustered based on this panel, in which cluster 2 (C2) demonstrated the highest tumor progression level with a high expression of 10 genes, showing a decreased tumor mutation burden, significantly enriched epithelial-mesenchymal transition hallmark and increased immune exclusion/exhausted features. Finally, the cell localization of these panel genes was explored in scRNA-seq data based on more than 40,000 cells. The 10-gene panel is expected to be a new clinical early detection signature for GC and may aid in progression assessment and personalized treatment of patients.

P53 deficiency affects cholesterol esterification to exacerbate hepatocarcinogenesis.

BACKGROUND AND AIMS: Cholesterol ester (CE) biosynthesis and homeostasis play critical roles in many cancers, including HCC, but their exact mechanistic contributions to HCC disease development require further study. APPROACH AND RESULTS: Here, we report on a proposed role of tumor suppressor P53 in its repressing ubiquitin-specific peptidase 19 (USP19) and sterol O-acyltransferase (SOAT) 1, which maintains CE homeostasis. USP19 enhances cholesterol esterification and contributes to hepatocarcinogenesis (HCG) by deubiquitinating and stabilizing SOAT1. Loss of either SOAT1 or USP19 dramatically attenuates cholesterol esterification and HCG in P53-deficient mice fed with either a normal chow diet or a high-cholesterol, high-fat diet (HCHFD). SOAT1 inhibitor avasimibe has more inhibitory effect on HCC progression in HCHFD-maintained P53-deficient mice when compared to the inhibitors of de novo cholesterol synthesis. Consistent with our findings in the mouse model, the P53-USP19-SOAT1 signaling axis is also dysregulated in human HCCs. CONCLUSIONS: Collectively, our findings demonstrate that SOAT1 participates in HCG by increasing cholesterol esterification, thus indicating that SOAT1 is a potential biomarker and therapeutic target in P53-deficient HCC.

De Novo Design of Reversibly pH-Switchable NIR-II Aggregation-Induced Emission Luminogens for Efficient Phototheranostics of Patient-Derived Tumor Xenografts.

Phototheranostics has received sustained attention due to its great potential in revolutionizing conventional strategies of cancer treatment. However, trapped by the complexity, poor reproducibility, insufficient phototheranostic outputs, and inevitable damage to normal tissue of most multicomponent phototheranostic systems, its clinical translation has been severely hindered. Therefore, the exploration of "one for all" smart phototheranostic agents with versatile functionalities remains an appealing yet enormously challenging task. Herein, a reversibly pH-switchable and near-infrared second photosensitizer featuring aggregation-induced emission was tactfully designed by molecular engineering for precise tumor-targeting fluorescence imaging-guided phototherapy. Thanks to the strong intramolecular charge transfer, enhanced highly efficient intersystem crossing, and sufficient intramolecular motion, the developed agent DTTVBI was endowed with boosted type-I superoxide anion radical generation and excellent photothermal performance under 808 nm laser irradiation. More importantly, DTTVBI nanoparticles with high biocompatibility exhibit remarkably enhanced type-I photodynamic/photothermal therapy in the tumor region, thus offering significant antitumor effects both in vitro and in the patient-derived tumor xenograft model of colon cancer. This work sheds new light on the development of superior versatile phototheranostics for cancer therapy.

Major vault protein regulates tumor-associated macrophage polarization through interaction with signal transducer and activator of transcription 6.

Tumor-associated macrophages (TAMs) are critical in the tumor microenvironment (TME) of hepatocellular carcinoma (HCC). Major vault protein (MVP) mediates multidrug resistance, cell growth and development, and viral immunity. However, the relationship between MVP and TAMs polarization has not been clarified in HCC. We found that MVP significantly increased M2-TAMs infiltration levels in tumor tissues of HCC patients. MVP promoted HCC proliferation, metastasis, and invasion by regulating M2 polarization in vivo and in vitro. Mechanistically, MVP associated with signal transducer and activator of transcription 6 (STAT6) and enhanced STAT6 phosphorylation. STAT6 translocated from the cytosol to the nucleus and regulated M2 macrophage-associated gene transcription. These findings suggest that MVP modulates the macrophage M2 transcriptional program, revealing its potential role in the TAMs of TME.

Posttranslational control of lipogenesis in the tumor microenvironment.

Metabolic reprogramming of cancer cells within the tumor microenvironment typically occurs in response to increased nutritional, translation and proliferative demands. Altered lipid metabolism is a marker of tumor progression that is frequently observed in aggressive tumors with poor prognosis. Underlying these abnormal metabolic behaviors are posttranslational modifications (PTMs) of lipid metabolism-related enzymes and other factors that can impact their activity and/or subcellular localization. This review focuses on the roles of these PTMs and specifically on how they permit the re-wiring of cancer lipid metabolism, particularly within the context of the tumor microenvironment.

A novel nine-microRNA-based model to improve prognosis prediction of renal cell carcinoma.

BACKGROUND: With the improved knowledge of disease biology and the introduction of immune checkpoints, there has been significant progress in treating renal cell carcinoma (RCC) patients. Individual treatment will differ according to risk stratification. As the clinical course varies in RCC, it has developed different predictive models for assessing patient's individual risk. However, among other prognostic scores, no transparent preference model was given. MicroRNA as a putative marker shown to have prognostic relevance in RCC, molecular analysis may provide an innovative benefit in the prophetic prediction and individual risk assessment. Therefore, this study aimed to establish a prognostic-related microRNA risk score model of RCC and further explore the relationship between the model and the immune microenvironment, immune infiltration, and immune checkpoints. This practical model has the potential to guide individualized surveillance protocols, patient counseling, and individualized treatment decision for RCC patients and facilitate to find more immunotherapy targets. METHODS: Downloaded data of RCC from the TCGA database for difference analysis and divided it into a training set and validation set. Then the prognostic genes were screened out by Cox and Lasso regression analysis. Multivariate Cox regression analysis was used to establish a predictive model that divided patients into high-risk and low-risk groups. The ENCORI online website and the results of the RCC difference analysis were used to search for hub genes of miRNA. Estimate package and TIMER database were used to evaluate the relationship between risk score and tumor immune microenvironment (TME) and immune infiltration. Based on Kaplan-Meier survival analysis, search for immune checkpoints related to the prognosis of RCC. RESULTS: There were nine miRNAs in the established model, with a concordance index of 0.702 and an area under the ROC curve of 0.701. Nine miRNAs were strongly correlated with the prognosis (P < 0.01), and those with high expression levels had a poor prognosis. We found a common target gene PDGFRA of hsa-miR-6718, hsa-miR-1269b and hsa-miR-374c, and five genes related to ICGs (KIR2DL3, TNFRSF4, LAG3, CD70 and TNFRSF9). The immune/stromal score, immune infiltration, and immune checkpoint genes of RCC were closely related to its prognosis and were positively associated with a risk score. CONCLUSIONS: The established nine-miRNAs prognostic model has the potential to facilitate prognostic prediction. Moreover, this model was closely related to the immune microenvironment, immune infiltration, and immune checkpoint genes of RCC.

Engineered exosomes as an in situ DC-primed vaccine to boost antitumor immunity in breast cancer.

BACKGROUND: Dendritic cells (DCs) are central for the initiation and regulation of innate and adaptive immunity in the tumor microenvironment. As such, many kinds of DC-targeted vaccines have been developed to improve cancer immunotherapy in numerous clinical trials. Targeted delivery of antigens and adjuvants to DCs in vivo represents an important approach for the development of DC vaccines. However, nonspecific activation of systemic DCs and the preparation of optimal immunodominant tumor antigens still represent major challenges. METHODS: We loaded the immunogenic cell death (ICD) inducers human neutrophil elastase (ELANE) and Hiltonol (TLR3 agonist) into α-lactalbumin (α-LA)-engineered breast cancer-derived exosomes to form an in situ DC vaccine (HELA-Exos). HELA-Exos were identified by transmission electron microscopy, nanoscale flow cytometry, and Western blot analysis. The targeting, killing, and immune activation effects of HELA-Exos were evaluated in vitro. The tumor suppressor and immune-activating effects of HELA-Exos were explored in immunocompetent mice and patient-derived organoids. RESULTS: HELA-Exos possessed a profound ability to specifically induce ICD in breast cancer cells. Adequate exposure to tumor antigens and Hiltonol following HELA-Exo-induced ICD of cancer cells activated type one conventional DCs (cDC1s) in situ and cross-primed tumor-reactive CD8+ T cell responses, leading to potent tumor inhibition in a poorly immunogenic triple negative breast cancer (TNBC) mouse xenograft model and patient-derived tumor organoids. CONCLUSIONS: HELA-Exos exhibit potent antitumor activity in both a mouse model and human breast cancer organoids by promoting the activation of cDC1s in situ and thus improving the subsequent tumor-reactive CD8+ T cell responses. The strategy proposed here is promising for generating an in situ DC-primed vaccine and can be extended to various types of cancers. Scheme 1. Schematic illustration of HELA-Exos as an in situ DC-primed vaccine for breast cancer. (A) Allogenic breast cancer-derived exosomes isolated from MDA-MB-231 cells were genetically engineered to overexpress α-LA and simultaneously loaded with the ICD inducers ELANE and Hiltonol (TLR3 agonist) to generate HELA-Exos. (B) Mechanism by which HELA-Exos activate DCs in situ in a mouse xenograft model ofTNBC. HELA-Exos specifically homed to the TME and induced ICD in cancer cells, which resulted in the increased release of tumor antigens, Hiltonol, and DAMPs, as well as the uptake of dying tumor cells by cDC1s. The activated cDC1s then cross-primed tumor-reactive CD8+ T cell responses. (C) HELA-Exos activated DCs in situ in the breast cancer patient PBMC-autologous tumor organoid coculture system. ABBREVIATIONS: DCs: dendritic cells; α-LA: α-lactalbumin; HELA-Exos: Hiltonol-ELANE-α-LA-engineered exosomes; ICD: immunogenic cell death; ELANE: human neutrophil elastase; TLR3: Toll-like receptor 3; TNBC: triple-negative breast cancer; TME: tumor microenvironment; DAMPs: damage-associated molecular patterns; cDC1s: type 1 conventional dendritic cells; PBMCs: peripheral blood mononuclear cells.

Single-cell sequencing and its applications in bladder cancer.

Bladder cancer is the most common malignant tumour of the urinary system that is characterised by significant intra-tumoural heterogeneity. While large-scale sequencing projects have provided a preliminary understanding of tumour heterogeneity, these findings are based on the average signals obtained from the pooled populations of diverse cells. Recent advances in single-cell sequencing (SCS) technologies have been critical in this regard, opening up new ways of understanding the nuanced tumour biology by identifying distinct cellular subpopulations, dissecting the tumour microenvironment, and characterizing cellular genomic mutations. By integrating these novel insights, SCS technologies are expected to make powerful and meaningful changes to the current diagnosis and treatment of bladder cancer through the identification and usage of novel biomarkers as well as targeted therapeutics. SCS can discriminate complex heterogeneity in a large population of tumour cells and determine the key molecular properties that influence clinical outcomes. Here, we review the advances in single-cell technologies and discuss their applications in cancer research and clinical practice, with a specific focus on bladder cancer.