NeoDesign: A Computational Tool for Optimal Selection of Polyvalent Neoantigen Combinations.

MOTIVATION: Tumor polyvalent neoantigen mRNA vaccines are gaining prominence in immunotherapy. The design of sequences in vaccine development is crucial for enhancing both the immunogenicity and safety of vaccines. However, a major challenge lies in selecting the optimal sequences from the large pools generated by multiple peptide combinations and synonymous codons. RESULTS: We introduce NeoDesign, a computational tool designed to tackle the challenge of sequence design. NeoDesign comprises four modules: Library Construction, Optimal Path Filtering, Linker Addition, and λ-Evaluation. It aims to identify the optimal protein sequence for tumor polyvalent neoantigen vaccines by minimizing linker usage, avoiding unexpected neoantigens and functional domains, and simplifying the structure. It also provides a preference scheme to balance mRNA stability and protein expression when designing mRNA sequences for the optimal protein sequence. This tool can potentially improve the sequence design of tumor polyvalent neoantigen mRNA vaccines, thereby significantly advancing immunotherapy strategies. AVAILABILITY: NeoDesign is freely available on https://github.com/HuangLab-Fudan/neoDesign and https://figshare.com/projects/NeoDesign/221704. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Diagnostic Utility of a 90-Gene Expression Assay (Canhelp-Origin) for Patients with Metastatic Cancer with an Unclear or Unknown Diagnosis.

BACKGROUND: Metastatic cancers with unclear or unknown origins pose significant challenges in diagnosis and management, frequently leading to suboptimal outcomes. Studies have demonstrated that a 90-gene expression assay is effective in predicting the primary origin and guiding the site-specific therapy to improve prognosis. This study aimed to evaluate the clinical effectiveness of a 90-gene expression assay in patients with unclear or unknown diagnoses. METHODS: The study encompassed patients for whom a 90-gene expression assay was requested as part of standard care. Data on patient demographics, tumor characteristics, and clinical history were collected. The assay's performance was evaluated by comparing its predicted tumor type with the final histopathological diagnosis. RESULTS: Among 303 cases analyzed, a 90-gene expression assay successfully identified a molecular-based tumor type for 295 (97.4%) patients. Comparison with histopathological diagnosis revealed an overall agreement of 88.5% (170/192). In patients with a single suspected primary site (n = 140), the assay confirmed the suspected diagnosis in 90.7% of cases. For those with a differential diagnosis (n = 52), the assay narrowed down the possibilities in 82.7% of cases. Moreover, in cases where the histopathology report indicated cancer of unknown primary (n = 103), the assay offered a molecular tumor type prediction with potential clinical significance. CONCLUSIONS: This study demonstrates the significant impact of a 90-gene expression assay on diagnosis and potential treatment selection for difficult-to-diagnose patients, highlighting its clinical value as a standardized molecular approach to streamline further diagnostic testing for patients with metastatic cancer of unclear or unknown origin. Further prospective study is required to assess whether employing molecular diagnostic classifiers enhances clinical outcomes in these patients.

MR radiomics unveils neoadjuvant chemo-responsiveness with insights into selective treatment de-intensification in HPV-positive oropharyngeal carcinoma.

BACKGROUND: Accurate prediction of neoadjuvant chemotherapy (NAC) response allows for NAC-guided personalized treatment de-intensification in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC). In this study, we aimed to apply baseline MR radiomic features to predict NAC response to help select NAC-guided de-intensification candidates, and to explore biological underpinnings of response-oriented radiomics. METHODS: Pre-treatment MR images and clinical data of 131 patients with HPV-positive OPSCC were retrieved from Fudan University Shanghai Cancer Center. Patients were divided into training cohort (n = 47), validation cohort 1 (n = 49) from NAC response-adapted de-intensification trial (IChoice-01, NCT04012502) and real-world validation cohort 2 (n = 35). NAC prediction model using linear support vector machine (SVM) was built and validated. Subsequent nomograms combined radiomics and clinical characteristics were established to predict survival outcomes. RNA-seq and proteomic data were compared to interpret the molecular features underlying radiomic signatures with differential NAC response. FINDINGS: For NAC response prediction, the fusion model with both oropharyngeal and nodal signatures achieved encouraging performance to predict good responders in the training cohort (AUC 0·89, 95% CI, 0·79-0·95) and validation cohort 1 (AUC 0·71, 95% CI, 0·59-0·83). For prognosis prediction, radiomics-based nomograms exhibited satisfactory discriminative ability between low-risk and high-risk patients (PFS, C-index 0·85, 0·76 and 0·83; OS, C-index 0·79, 0·76 and 0·87, respectively) in three cohorts. Expression analysis unveiled NAC poor responders had predominantly enhanced keratinization while good responders were featured by upregulated immune response and oxidative stress. INTERPRETATION: The MR-based radiomic models and prognostic models efficiently discriminate among patients with different NAC response and survival risk, which help candidate selection in HPV-positive OPSCC with regard to personalized treatment de-intensification.

Oncofetal TRIM71 drives liver cancer carcinogenesis through remodeling CEBPA-mediated serine/glycine metabolism.

Rationale: Tumor cells remodel transcriptome to construct an ecosystem with stemness features, which maintains tumor growth and highly malignant characteristics. However, the core regulatory factors involved in this process still need to be further discovered. Methods: Single cell RNA-sequncing (scRNA-seq) and bulk RNA-sequencing profiles derived from fetal liver, normal liver, liver tumors, and their adjacent samples were collected to analyze the ecosystem of liver cancer. Mouse models were established to identify molecular functions of oncofetal-related oncogenes using hydrodynamic tail vein injection. Results: We found that liver cancer rebuilt oncofetal ecosystem to maintain malignant features. Interestingly, we identified a group of RNA-binding proteins (RBPs) that were highly overexpressed with oncofetal features. Among them, TRIM71 was specifically expressed in liver cancers and was associated with poor outcomes. TRIM71 drove the carcinogenesis of hepatocellular carcinoma (HCC), and knockdown of TRIM71 significantly abolished liver cancer cell proliferation. Mechanistically, TRIM71 formed a protein complex with IGF2BP1, bound to and stabilized the mRNA of CEBPA in an m6A-dependent manner, enhance the serine/glycine metabolic pathway, and ultimately promoted liver cancer progression. Furthermore, we identified that all-trans-retinoic acid (ATRA) combined with e1A binding protein p300 (EP300) inhibitor A-485 repressed TRIM71, attenuated glycine/serine metabolism, and inhibited liver cancer cell proliferation with high TRIM71 levels. Conclusions: We demonstrated the oncofetal status in liver cancer and highlighted the crucial role of TRIM71 and provided potential therapeutic strategies and liver cancer-specific biomarker for liver cancer patients.

Elevated choline drives KLF5-dominated transcriptional reprogramming to facilitate liver cancer progression.

An increase in the total choline-containing compound content is a common characteristic of cancer cells, and aberrant choline metabolism in cancer is closely associated with malignant progression. However, the potential role of choline-induced global transcriptional changes in cancer cells remains unclear. In this study, we reveal that an elevated choline content facilitates hepatocellular carcinoma (HCC) cell proliferation by reprogramming Krüppel-like factor 5 (KLF5)-dominated core transcriptional regulatory circuitry (CRC). Mechanistically, choline administration leads to elevated S-adenosylmethionine (SAM) levels, inducing the formation of H3K4me1 within the super-enhancer (SE) region of KLF5 and activating its transcription. KLF5, as a key transcription factor (TF) of CRC established by choline, further transactivates downstream genes to facilitate HCC cell cycle progression. Additionally, KLF5 can increase the expression of choline kinase-α (CHKA) and CTP:phosphocholine cytidylyltransferase (CCT) resulting in a positive feedback loop to promote HCC cell proliferation. Notably, the histone deacetylase inhibitor (HDACi) vorinostat (SAHA) significantly suppressed KLF5 expression and liver tumor growth in mice, leading to a prolonged lifespan. In conclusion, these findings highlight the epigenetic regulatory mechanism of the SE-driven key regulatory factor KLF5 conducted by choline metabolism in HCC and suggest a potential therapeutic strategy for HCC patients with high choline content.

Study on membrane fouling mechanisms and mitigation strategies in a pilot-scale anaerobic membrane bioreactor (P-AnMBR) treating digestate.

Anaerobic Membrane Bioreactor (AnMBR) are employed for solid-liquid separation in wastewater treatment, enhancing process efficiency of digestion systems treating digestate. However, membrane fouling remains a primary challenge. This study operated a pilot-scale AnMBR (P-AnMBR) to treat high-concentration organic digestate, investigating system performance and fouling mechanisms. P-AnMBR operation reduced acid-producing bacteria and increased methane-producing bacteria on the membrane, preventing acid accumulation and ensuring stable operation. The P-AnMBR effectively removed COD and VFA, achieving removal rates of 82.3 % and 92.0 %, respectively. Higher retention of organic nitrogen and lower retention of ammonia nitrogen were observed. The membrane fouling consisted of organic substances (20.3 %), predominantly polysaccharides, and inorganic substances (79.7 %), primarily Mg ions (10.1 %) and Ca ions (4.5 %). To reduce the increased transmembrane pressure (TMP) caused by fouling (a 10.6-fold increase in filtration resistance), backwash frequency experiment was conducted. It revealed a 30-min backwash frequency minimized membrane flux decline, facilitating recovery to higher flux levels. The water produced amounted to 70.3 m³ over 52 days. The research provided theoretical guidance and practical support for engineering applications, offering practical insights for scaling up P-AnMBR.

Fibrillarin reprograms glucose metabolism by driving the enhancer-mediated transcription of PFKFB4 in liver cancer.

DNA- and RNA-binding proteins (DRBPs) are versatile proteins capable of binding to both DNA and RNA molecules. In this study, we identified fibrillarin (FBL) as a key DRBP that is upregulated in liver cancer tissues vs. normal tissues and is correlated with patient prognosis. FBL promotes the proliferation of liver cancer cells both in vitro and in vivo. Mechanistically, FBL interacts with the transcription factor KHSRP, thereby regulating the expression of genes involved in glucose metabolism and leading to the reprogramming of glucose metabolism. Specifically, FBL and KHSRP work together to transcriptionally activate the glycolytic enzyme PFKFB4 by co-occupying enhancer and promoter elements, thereby further promoting liver cancer growth. Collectively, these findings provide compelling evidence highlighting the role of FBL as a transcriptional regulator in liver cancer cells, working in conjunction with KHSRP. The FBL/KHSRP-PFKFB4 regulatory axis holds potential as both a prognostic indicator and a therapeutic target for liver cancer. SIGNIFICANCE: A novel role of FBL in the transcriptional activation of PFKFB4, leading to glucose metabolism reprogramming in liver cancer.

Identifying an immunosenescence-associated gene signature in gastric cancer by integrating bulk and single-cell sequencing data.

It has been believed that immunosenescence plays a crucial role in tumorigenesis and cancer therapy. Nevertheless, there is still a lack of understanding regarding its role in determining clinical outcomes and therapy selection for gastric cancer patients, due to the lack of a feasible immunosenescence signature. Therefore, this research aims to develop a gene signature based on immunosenescence, which is used for stratification of gastric cancer. By integrative analysis of bulk transcriptome and single-cell data, we uncovered immunosenescence features in gastric cancer. Random forest algorithm was used to select hub genes and multivariate Cox algorithm was applied to construct a scoring system to evaluate the prognosis and the response to immunotherapy and chemotherapy. The Cancer Genome Atlas of Stomach Adenocarcinoma (TCGA-STAD) cohort was implemented as the training cohort and two independent cohorts from the Gene Expression Omnibus (GEO) database were used for validation. The model was further tested by our Fudan cohort. In this study, immunosenescence was identified as a hallmark of gastric cancer that is linked with transcriptomic features, genomic variations, and distinctive tumor microenvironment (TME). Four immunosenescence genes, including APOD, ADIPOR2, BRAF, and C3, were screened out to construct a gene signature for risk stratification. Higher risk scores indicated strong predictive power for poorer overall survival. Notably, the risk score signature could reliably predict response to chemotherapy and immunotherapy, with patients with high scores benefiting from immunotherapy and patients with low scores responding to chemotherapy. We report immunosenescence as a hitherto unheralded hallmark of gastric cancer that affects prognosis and treatment efficiency.

The neoantigens derived from transposable elements - A hidden treasure for cancer immunotherapy.

Neoantigen-based therapy is a promising approach that selectively activates the immune system of the host to recognize and eradicate cancer cells. Preliminary clinical trials have validated the feasibility, safety, and immunogenicity of personalized neoantigen-directed vaccines, enhancing their effectiveness and broad applicability in immunotherapy. While many ongoing oncological trials concentrate on neoantigens derived from mutations, these targets do not consistently provoke an immune response in all patients harboring the mutations. Additionally, tumors like ovarian cancer, which have a low tumor mutational burden (TMB), may be less amenable to mutation-based neoantigen therapies. Recent advancements in next-generation sequencing and bioinformatics have uncovered a rich source of neoantigens from non-canonical RNAs associated with transposable elements (TEs). Considering the substantial presence of TEs in the human genome and the proven immunogenicity of TE-derived neoantigens in various tumor types, this review investigates the latest findings on TE-derived neoantigens, examining their clinical implications, challenges, and unique advantages in enhancing tumor immunotherapy.

Identification of stable reference genes for relative quantification of long RNA expression in urinary extracellular vesicles.

Urinary extracellular vesicles (uEVs) are rich in valuable biomolecule information which are increasingly recognized as potential biomarkers for various diseases. uEV long RNAs are among the critical cargos capable of providing unique transcriptome information of the source cells. However, consensus regarding ideal reference genes for relative long RNAs quantification in uEVs is not available as of date. Here we explored stable reference genes through profiling the long RNA expression by RNA-seq following unsupervised analysis and validation studies. Candidate reference genes were identified using four algorithms: NormFinder, GeNorm, BestKeeper and the Delta Ct method, followed by validation. RNA profile showed uEVs contained abundant long RNAs information and the core transcriptome was related to cellular structures, especially ribosome which functions mainly as translation, protein and RNA binding molecules. Analysis of RNA-seq data identified RPL18A, RPL11, RPL27, RACK1, RPSA, RPL41, H1-2, RPL4, GAPDH, RPS27A as candidate reference genes. RT-qPCR validation revealed that RPL41, RPSA and RPL18A were reliable reference genes for long RNA quantification in uEVs from patients with diabetes mellitus (DM), diabetic nephropathy (DN), IgA nephropathy (IgAN) and prostate cancer (PCA). Interestingly, RPL41 also outperformed traditional reference genes in renal tissues of DN and IgAN, as well as in plasma EVs of several types of cancers. The stable reference genes identified in this study may facilitate development of uEVs as novel biomarkers and increase the accuracy and comparability of biomarker studies.

LTR retrotransposon-derived LncRNA LINC01446 promotes hepatocellular carcinoma progression and angiogenesis by regulating the SRPK2/SRSF1/VEGF axis.

The causal link between long terminal repeat (LTR) retrotransposon-derived lncRNAs and hepatocellular carcinoma (HCC) remains elusive and whether these cancer-exclusive lncRNAs contribute to the effectiveness of current HCC therapies is yet to explore. Here, we investigated the activation of LTR retrotransposon-derived lncRNAs in a broad range of liver diseases. We found that LTR retrotransposon-derived lncRNAs are mainly activated in HCC and is correlated with the proliferation status of HCC. Furthermore, we discovered that an LTR retrotransposon-derived lncRNA, LINC01446, exhibits specific expression in HCC. HCC patients with higher LINC01446 expression had shorter overall survival times. In vitro and in vivo assays showed that LINC01446 promoted HCC growth and angiogenesis. Mechanistically, LINC01446 bound to serine/arginine protein kinase 2 (SRPK2) and activated its downstream target, serine/arginine splicing factor 1 (SRSF1). Furthermore, activation of the SRPK2-SRSF1 axis increased the splicing and expression of VEGF isoform A165 (VEGFA165). Notably, inhibiting LINC01446 expression dramatically impaired tumor growth in vivo and resulted in better therapeutic outcomes when combined with antiangiogenic agents. In addition, we found that the transcription factor MESI2 bound to the cryptic MLT2B3 LTR promoter and drove LINC01446 transcription in HCC cells. Taken together, our findings demonstrate that LTR retrotransposon-derived LINC01446 promotes the progression of HCC by activating the SRPK2/SRSF1/VEGFA165 axis and highlight targeting LINC01446 as a potential therapeutic strategy for HCC patients.

Single cell and bulk RNA sequencing identifies tumor microenvironment subtypes and chemoresistance-related IGF1+ cancer-associated fibroblast in gastric cancer.

BACKGROUND: The tumor microenvironment (TME) significantly influences prognosis and drug resistance in various tumors, yet its heterogeneity and the mechanisms affecting therapeutic response remain unclear in gastric cancer (GC). METHODS: The heterogenous TME were explored with single-cell RNA-sequencing (scRNA-seq) data of 50 primary GC samples. We then identified four GC TME subtypes with nonnegative matrix factorization (NMF) and constructed a pearson nearest-centroid classifier based on subtype-specific upregulated genes. Genomic features and clinical significance of four subtypes were comprehensively evaluated. We reclustered fibroblasts to identify cancer-associated fibroblast (CAF) subtype associated with poor clinical outcomes. RT-qPCR and double immunofluorescence staining were applied to validate the findings. Cellchat analysis elucidated potential molecular mechanisms of the CAF subtype in GC disease progression and chemotherapy resistance. FINDINGS: The GC TME exhibited high heterogeneity, influencing chemo-sensitivity. Four TME-based subtypes predicting response to immunotherapy and chemotherapy were identified and validated in 1406 GC patients. Among which, ISG1 subtype displayed higher fibroblasts infiltration and heightened oncogenic pathways, and inferior response to chemotherapy with unfavorable prognosis. Microsatellite instability-high (MSI-H) GCs within four TME subtypes showed immunological heterogeneity. We then reported an IGF1-overexpressing CAF was associated with chemo-resistance and GC recurrence. Cell communication analysis revealed IGF1+ CAF may induce drug-resistant phenotypes in tumor cells through IGF1-α6ß4 integrin ligand-receptor binding and activation of EMT biological process. INTERPRETATION: We identified four TME-based subtypes with different clinical outcomes and IGF1+ CAFs contributing to poor clinical outcomes in GC, which might provide guidance for individualized treatment and facilitate the development of novel therapeutic targets.

Exploring Circular RNA Profile and Expression in Extracellular Vesicles.

Extracellular vesicles (EVs) are small vesicles secreted by various cell types and are enriched in multiple body fluids. EVs containing RNA have the potential to modulate biological processes and are being investigated for their diagnostic and therapeutic applications. Circular RNAs (circRNAs), generated through back-splicing of exons, are enriched in EVs. Given their unique characteristics and diverse functions, EV-circRNAs are important players in disease pathology. This chapter describes a workflow for investigating the expression profile of EV-circRNAs, which includes EVs separation, library preparation, and bioinformatics analysis. This workflow can aid the investigation of EV-circRNAs and their potential role in disease pathology.

Induction Toripalimab and Chemotherapy for Organ Preservation in Locally Advanced Laryngeal and Hypopharyngeal Cancer: A Single-Arm Phase II Clinical Trial.

PURPOSE: The aim of this study was to assess the efficacy, toxicities, and potential role of larynx preservation of induction chemotherapy combined with programmed cell death protein 1 (PD-1) inhibitor in locally advanced laryngeal and hypopharyngeal cancer. PATIENTS AND METHODS: This is a single-arm phase II study. Patients with histopathologically confirmed, resectable locally advanced laryngeal/hypopharyngeal squamous cell carcinoma and Eastern Cooperative Oncology Group Performance Status 0-1 were eligible. Three cycles of induction chemotherapy (paclitaxel 175 mg/m2 d1, cisplatin 25 mg/m2 d1-3) combined with PD-1 inhibitor (toripalimab 240 mg d0) were administered. Response assessment was performed after induction chemoimmunotherapy using RECIST 1.1 criteria. Patients with a complete/partial response of the primary tumor received concurrent chemoradiation, followed by maintenance therapy of toripalimab. Otherwise, patients were referred to surgery, followed by adjuvant (chemo) radiation and maintenance therapy of toripalimab. The primary endpoint is a larynx preservation rate at 3 months postradiation. RESULTS: Twenty-seven patients were enrolled. Most cases exhibited stage IV disease (81.5%), with T4 representing 37.0%. Five patients underwent pretreatment tracheostomy because of impaired larynx function. Overall response rate of induction chemoimmunotherapy was 85.2%. At 3 months postradiation, the larynx preservation rate was 88.9%. With a median follow-up of 18.7 months, the 1-year overall survival rate, progression-free survival rate, and larynx preservation rate were 84.7%, 77.6%, and 88.7%, respectively. When excluding those with pretreatment tracheostomy, the 1-year larynx preservation rate was 95.5%. Exploratory analysis revealed that relapse correlated with enrichment of RNA signature of hypoxia and M2 macrophage-associated genes. CONCLUSIONS: Induction toripalimab combined with chemotherapy provided encouraging activity, promising larynx preservation rate and acceptable toxicity in this cohort of extensively locally advanced laryngeal and hypopharyngeal cancer.

A liquid biopsy signature of circulating extracellular vesicles-derived RNAs predicts response to first line chemotherapy in patients with metastatic colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most threatening tumors in the world, and chemotherapy remains dominant in the treatment of metastatic CRC (mCRC) patients. The purpose of this study was to develop a biomarker panel to predict the response of the first line chemotherapy in mCRC patients. METHODS: Totally 190 mCRC patients treated with FOLFOX or XEOLX chemotherapy in 3 different institutions were included. We extracted the plasma extracellular vesicle (EV) RNA, performed RNA sequencing, constructed a model and generated a signature through shrinking the number of variables by the random forest algorithm and the least absolute shrinkage and selection operator (LASSO) algorithm in the training cohort (n = 80). We validated it in an internal validation cohort (n = 62) and a prospective external validation cohort (n = 48). RESULTS: We established a signature consisted of 22 EV RNAs which could identify responders, and the area under the receiver operating characteristic curve (AUC) values was 0.986, 0.821, and 0.816 in the training, internal validation, and external validation cohort respectively. The signature could also identify the progression-free survival (PFS) and overall survival (OS). Besides, we constructed a 7-gene signature which could predict tumor response to first-line oxaliplatin-containing chemotherapy and simultaneously resistance to second-line irinotecan-containing chemotherapy. CONCLUSIONS: The study was first to develop a signature of EV-derived RNAs to predict the response of the first line chemotherapy in mCRC with high accuracy using a non-invasive approach, indicating that the signature could help to select the optimal regimen for mCRC patients.

A pyroptosis-related signature in colorectal cancer: exploring its prognostic value and immunological characteristics.

Background: The heterogeneity of colorectal cancer (CRC) is the main cause of the disparity of drug sensitivity and the variability of prognosis. Pyroptosis is closely associated with the development and prognosis of various tumors, including CRC. Dividing CRC into distinct subgroups based on pyroptosis is a worthwhile topic for improving the precision treatment and prognosis prediction of CRC. Methods: We classified patients into two clusters using the consensus clustering based on the pyroptosis-related genes (PRGs). Next, the prognostic signature was developed with LASSO regression analysis using the screened genes from differentially expressed genes (DEGs) by univariate and multivariate Cox analyses. According to the pyroptosis-related score (PR score) calculated with the signature, patients belonged to two groups with distinct prognosis. Moreover, we assessed the immune profile to explore the relationship between the signature and immunological characteristics. Two single cell sequencing databases were adopted for further exploration of tumor immune microenvironment (TME). In addition, we applied our own cohort and Drugbank to explore the correlation of the signature and clinical therapies. We also studied the expression of key genes by immunohistochemistry. Results: The signature performed well in predicting the prognosis of CRC as the high area under curve (AUC) value demonstrated. Patients with a higher PR score had poorer prognosis and higher expression of immune checkpoints but more abundant infiltration of immune cells. Combining with the indicator of therapeutic analysis, they might benefit more from immune checkpoint blockade (ICB) and neo-adjuvant chemoradiotherapy (nCRT). Conclusion: In conclusion, our study is based on genomics and transcriptomics to investigate the role of PRGs in CRC. We have established a prognostic signature and integrated single-cell data to study the relationship between the signature with the TME in CRC. Its clinical application in reliable prediction of prognosis and personalized treatment was validated by public and own sequencing cohort. It provided a new insight for the personalized treatment of CRC.

Extracellular vesicles long RNA profiling identifies abundant mRNA, circRNA and lncRNA in human bile as potential biomarkers for cancer diagnosis.

Extracellular vesicles (EVs) are bilayered membrane vesicles produced by living cells and secreted into the extracellular matrix. Bile is a special body fluid that is secreted by the liver cells, and extracellular vesicles long RNAs (exLRs) have not been explored in bile. In this study, exLR sequencing (exLR-seq) was performed on 19 bile samples from patients with malignant cancer or patients with biliary stones. A total of 8649 mRNAs, 13 823 circRNAs and 1105 lncRNAs were detected. The KEGG pathway analysis revealed that differentially expressed exLRs were enriched in mTOR and AMPK signaling pathway. We identified five mRNAs (EID2, LLPH, ATP6V0A2, RRP9 and MTRNR2L10), three lncRNAs (AC015922.2, AL135905.1 and LINC00921) and six circRNAs (circASH1L, circATP9A, circCLIP1, circRNF138, circTIMMDC1 and circANKRD12) were enriched in bile EV samples with cancer, and these exLRs may be potential markers used to distinguish malignant cancers from benign biliary diseases. Moreover, the tissue/cellular source components of EVs were analyzed using the EV-origin algorithm. The absolute abundance of CD4_naive and Th1 cell source in bile EVs from cancer patients were significantly increased. In summary, our study presented abundant exLRs in human bile EVs and provides some basis for the selection of tumor diagnostic markers.

KLF5 Promotes Tumor Progression and Parp Inhibitor Resistance in Ovarian Cancer.

One major characteristic of tumor cells is the aberrant activation of epigenetic regulatory elements, which remodel the tumor transcriptome and ultimately promote cancer progression and drug resistance. However, the oncogenic functions and mechanisms of ovarian cancer (OC) remain elusive. Here, super-enhancer (SE) regulatory elements that are aberrantly activated in OC are identified and it is found that SEs drive the relative specific expression of the transcription factor KLF5 in OC patients and poly(ADP-ribose) polymerase inhibitor (PARPi)-resistant patients. KLF5 expression is associated with poor outcomes in OC patients and can drive tumor progression in vitro and in vivo. Mechanistically, KLF5 forms a transcriptional complex with EHF and ELF3 and binds to the promoter region of RAD51 to enhance its transcription, strengthening the homologous recombination repair (HRR) pathway. Notably, the combination of suberoylanilide hydroxamic acid (SAHA) and olaparib significantly inhibits tumor growth and metastasis of PARPi-resistant OC cells with high KLF5. In conclusion, it is discovered that SEs-driven KLF5 is a key regulatory factor in OC progression and PARPi resistance; and potential therapeutic strategies for OC patients with PARPi resistance and high KLF5 are identified.

Genomic and transcriptional characterization of early esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a highly heterogeneous cancer that lacks comprehensive understanding and effective treatment. Although multi-omics study has revealed features and underlying drivers of advanced ESCC, research on molecular characteristics of the early stage ESCC is quite limited. MATERIALS AND METHODS: We presented characteristics of genomics and transcriptomics in 10 matched pairs of tumor and normal tissues of early ESCC patients in the China region. RESULTS: We identified the specific patterns of cancer gene mutations and copy number variations. We also found a dramatic change in the transcriptome, with more than 4,000 genes upregulated in cancer. Among them, more than one-third of HOX family genes were specifically and highly expressed in early ESCC samples of China and validated by RT-qPCR. Gene regulation network analysis indicated that alteration of Hox family genes promoted the proliferation and metabolism remodeling of early ESCC. CONCLUSIONS: We characterized the genomic and transcriptomic landscape of 10 paired normal adjacent and early ESCC tissues in the China region, and provided a new perspective to understand the development of ESCC and insight into potential prevention and diagnostic targets for the management of early ESCC in China.

Low level of ARID1A contributes to adaptive immune resistance and sensitizes triple-negative breast cancer to immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) shed new light on triple-negative breast cancer (TNBC), but only a minority of patients demonstrate response. Therefore, adaptive immune resistance (AIR) needs to be further defined to guide the development of ICI regimens. METHODS: Databases, including The Cancer Genome Atlas, Gene Ontology Resource, University of California Santa Cruz Genome Browser, and Pubmed, were used to screen epigenetic modulators, regulators for CD8+ T cells, and transcriptional regulators of programmed cell death-ligand 1 (PD-L1). Human peripheral blood mononuclear cell (Hu-PBMC) reconstruction mice were adopted for xenograft transplantation. Tumor specimens from a TNBC cohort and the clinical trial CTR20191353 were retrospectively analyzed. RNA-sequencing, Western blotting, qPCR and immunohistochemistry were used to assess gene expression. Coculture assays were performed to evaluate the regulation of TNBC cells on T cells. Chromatin immunoprecipitation and transposase-accessible chromatin sequencing were used to determine chromatin-binding and accessibility. RESULTS: The epigenetic modulator AT-rich interaction domain 1A (ARID1A) gene demonstrated the highest expression association with AIR relative to other epigenetic modulators in TNBC patients. Low ARID1A expression in TNBC, causing an immunosuppressive microenvironment, promoted AIR and inhibited CD8+ T cell infiltration and activity through upregulating PD-L1. However, ARID1A did not directly regulate PD-L1 expression. We found that ARID1A directly bound the promoter of nucleophosmin 1 (NPM1) and that low ARID1A expression increased NPM1 chromatin accessibility as well as gene expression, further activating PD-L1 transcription. In Hu-PBMC mice, atezolizumab demonstrated the potential to reverse ARID1A deficiency-induced AIR in TNBC by reducing tumor malignancy and activating anti-tumor immunity. In CTR20191353, ARID1A-low patients derived more benefit from pucotenlimab compared to ARID1A-high patients. CONCLUSIONS: In AIR epigenetics, low ARID1A expression in TNBC contributed to AIR via the ARID1A/NPM1/PD-L1 axis, leading to poor outcome but sensitivity to ICI treatment.