Cancer remains a leading global cause of mortality, making early detection crucial for improving survival outcomes. The study aims to develop a machine learning-enabled blood-derived exosomal RNA profiling platform for multi-cancer detection and localization. In this multi-phase, multi-center study, we analyzed RNA from exosomes derived from peripheral blood plasma in 818 participants across eight cancer types during the discovery phase. Machine learning techniques were applied to identify potential pan-cancer biomarkers. During the screening and model validation phases, the sample size was progressively expanded to 1,385 participants in two steps, while the candidate biomarkers were refined into a set of 12 exosomal tumor RNA signatures (ETR.sig). In the subsequent model construction phase, diagnostic models were developed using the expanded cohort and ETR.sig. Statistical analyses included the calculation of receiver operating characteristic (ROC) curves and AUC values to assess the models' ability to distinguish cancer cases from controls and determine tumor origins. To further validate and explore the biological relevance of the identified biomarkers, we integrated tissue RNA-seq, single-cell data, and clinical information. Machine learning analysis initially identified 33 candidate biomarkers, which were narrowed down to 20 ETR.sig in the screening phase and 12 ETR.sig in the validation phase. In the model construction phase, a diagnostic model based on ETR.sig, built using the Random Forest (RF) algorithm, showed excellent performance with an AUC of 0.915 for distinguishing pan-cancer from controls. The multi-class classification model also demonstrated strong classification power, with macro-average and micro-average AUCs of 0.983 and 0.985, respectively, for differentiating between eight cancer types. Additionally, tumor origin classification using the RF-based diagnostic models achieved high AUC values: BRCA 0.976, COAD 0.98, KIRC 0.947, LIHC 0.967, LUAD 0.853, OV 0.972, PAAD 0.977, and PRAD 0.898. Integration of tissue RNA-seq, single-cell data, and clinical information revealed key associations between ETR.sig-related genes and tumor development. The study demonstrates the robust potential of exosomal RNA as a minimally invasive biomarker resource for cancer detection. The developed ETR.sig platform offers a promising tool for precision oncology and broad-spectrum cancer screening, integrating advanced computational models with nanoscale vesicle biology for accurate and rapid diagnosis.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the biosynthesis of nicotinamide adenine dinucleotide (NAD+), making it a potential target for cancer therapy. Two challenges hinder its translation in the clinic: targeting the extracellular form of NAMPT (eNAMPT) remains insufficient, and side effects are observed in normal tissues. We previously utilized proteolysis-targeting chimera (PROTAC) to develop two compounds capable of simultaneously degrading iNAMPT and eNAMPT. Unfortunately, the pharmacokinetic properties were inadequate, and toxicities similar to those associated with traditional inhibitors arose. We have developed a next-generation PROTAC molecule 632005 to address these challenges, demonstrating exceptional target selectivity and bioavailability, improved in vivo exposure, extended half-life, and reduced clearance rate. When combined with nicotinic acid, 632005 exhibits safety and robust efficacy in treating NAPRT-deficient pan-cancers, including xenograft models with hematologic malignancy and prostate cancer and patient-derived xenograft (PDX) models with liver cancer. Our findings provide clinical references for patient selection and treatment strategies involving NAMPT-targeting PROTACs.

Background:Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, but effective early detection and prognostication methods are lacking.Methods:The Cox regression model was built to stratify the HCC patients. The single-cell RNA sequencing data analysis and gene set enrichment analysis were employed to investigate the biological function of identified markers. PLCB1 gain- or loss-of-function experiments were performed, and obtained HCC samples were analyzed using quantitative real-time PCR and immunohistochemistry assay to validate the biological function of identified markers.Results:In this study, we developed a model using optimized markers for HCC recurrence prediction. Specifically, we screened out 8 genes through a series of data analyses, and built a multivariable Cox model based on their expression. The risk stratifications using the Eight-Gene Cox (EGC) model were closely associated with the recurrence-free survivals (RFS) in both training and three validation cohorts. We further demonstrated that this risk stratification could serve as an independent predictor in predicting HCC recurrence, and that the EGC model could outperform other models. Moreover, we also investigated the cell-type-specific expression patterns of the eight recurrence-related genes in tumor microenvironment using single-cell RNA sequencing data, and interpreted their functional roles from correlation and gene set enrichment analyses, in vitro and in vivo experiments. Particularly, PLCB1 and SLC22A7 were predominantly expressed in malignant cells, and they were predicted to promote angiogenesis and to help maintain normal metabolism in liver, respectively. In contrast, both FASLG and IL2RB were specifically expressed in T cells, and were highly correlated with T cell marker genes, suggesting that these two genes might assist in maintaining normal function of T cell-mediated immune response in tumor tissues.Conclusion:In conclusion, the EGC model and eight identified marker genes could not only facilitate the accurate prediction of HCC recurrence, but also improve our understanding of the mechanisms behind HCC recurrence.

The transcriptional repressor Zinc Finger and BTB domain-containing protein 1 (ZBTB1) is an essential member of the BTB-ZF family and plays an important role in the development of the immune system and DNA repair.1 The mechanisms by which ZBTB1 regulates multiple immune-related pathways involved in cancer progression have been investigated.2 Although ZBTB1 has been indicated to be a tumour suppressor in breast cancer, its biological functions and clinical significance in other malignant tumours remain unclear.3 Pancreatic carcinoma is a fatal malignant tumour with an increasing incidence. Due to difficulty in early diagnosis and poor prognosis, the 5-year survival rate is only 2%–9%.4 Therefore, it is very important to better understand the associated risk genes and prognostic indicators of pancreatic carcinoma. To our knowledge, bioinformatics analysis has not been applied to explore the role of ZBTB1 in pancreatic carcinoma. Our study revealed a candidate driver of pancreatic carcinoma that may be a potential target for precision treatment. To evaluate the clinical significance of ZBTB1 in pancreatic carcinoma, we used human tissue microarray (TMA) technology and immunohistochemistry (IHC) with an anti-ZBTB1 monoclonal antibody to examine 60 tumour tissue samples and 47 matched and unmatched non-neoplastic tissue samples. The IHC results were assigned an average score. Brown or brownish-yellow particles were defined as positive cells. First, the staining intensity was characterized as follows: no staining, 0 points; pale yellow staining, 1 point; brownish-brown staining, 2 points; and brown staining, 3 points. Then, the percentage of stained cells relative to the total cells was classified as follows: ≤5%, 0 points; 6%–25%, 1 point; 26%–50%, 2 points; 51%–75%, 3 points; and ≥76%, 4 points. The score of each sample was calculated as the sum of the scores of (1) and (2). A score ≥8 was regarded as high expression, and a score <8 was regarded as low expression.3, 5 In this study, the mRNA expression of ZBTB1 between pancreatic carcinoma and pancreatic tissue was compared by GEPIA (Gene Expression Profiling Interactive Analysis) (http://gepia.cancer-pku.cn/). The results indicated that the expression levels of ZBTB1 were higher in pancreatic carcinoma tissues than in normal tissues, and the expression of ZBTB1 did not significantly differ based on the tumour stage of pancreatic carcinoma. The Kaplan-Meier curve and log rank test analyses revealed that the increased ZBTB1 mRNA levels were not significantly associated with the overall survival (OS) of all of the patients with pancreatic carcinoma (p = 0.35) (Figure 1A). To validate the above conclusions, we next used the Oncomine Gene Expression Array database (https://www.oncomine.org/) to analyse the ZBTB1 mRNA expression level and prognosis of patients with pancreatic carcinoma.6 In 3 data sets with a total sample size of 147, ZBTB1 was overexpressed to some extent in pancreatic carcinoma tissues compared with normal tissues (Figure 1B). ZBTB1 was found to be more highly expressed in pancreatic carcinoma (p = 0.008), pancreatic ductal adenocarcinoma (p = 2.49E-7) and pancreatic carcinoma (p = 0.007) samples than in normal samples. Remarkably, using clinical samples in Collision's datasets to analyse OS, we did not come to a similar conclusion. The mRNA level of ZBTB1 was significantly correlated with OS, and the mortality of patients with high expression was higher (p = 0.0141)(Figure 1C). The effect of ZBTB1 on the survival of pancreatic carcinoma patients was further explored by the Kaplan-Meier Plotter tools (https://kmplot.com/analysis/). Kaplan-Meier curve and log rank test analyses revealed that increased ZBTB1 mRNA levels were significantly associated with relapse-free survival (RFS) (p = 0.043) but not overall survival (OS) (p = 0.12) in all of the patients with pancreatic carcinoma (Figure 1D). Figure 1E-F shows that ZBTB1 was aberrantly expressed in pancreatic carcinoma and positive in lymph node metastasis (Figure 1E-F). The IHC score results showed that ZBTB1 protein expression in the positive lymph node metastasis group was significantly higher than that in the negative lymph node metastasis group (Figure 1G); the expression level in pancreatic carcinoma tissue was significantly higher than that in adjacent normal tissues (Figure 1H). This indicates that ZBTB1 is necessary to maintain the malignant phenotype and for tumour metastasis. Meanwhile, the survival curve results showed that high ZBTB1 expression was not significantly associated with overall survival (OS) (p = 0.0758) (Figure 1I). Overall, in a previous study, we found that ZBTB1 was highly expressed in pancreatic carcinoma and significantly associated with patient RFS but not OS. Obviously, the expression of ZBTB1 is clearly related to the occurrence of pancreatic carcinoma. We decided to use the cBioPortal online tool (cBioPortal for Cancer Genomics) (http://www.cbioportal.org/) to analyse the alterations and correlations of ZBTB1 to explore its potential mechanism in tumour cells.7 ZBTB1 was altered in 3 samples of 1033 patients with pancreatic carcinoma (0.7%) (Figure 1K). Coexpression analysis of Segara's datasets showed that SLC4A7 expression was highly correlated with ZBTB1 in pancreatic carcinoma (Figure 1J). SLC4A7 has been shown to be expressed at significantly higher levels in human pancreatic ductal carcinoma, and silencing SLC4A7 in mice slows pancreatic tumour growth.8 Clearly, ZBTB1 did not appear to be prone to mutations in pancreatic carcinoma that resulted in effects on its neighbouring genes. Figure 1L shows 38 proteins with direct or indirect interactions with ZBTB1 (string-DB database) (Figure 1L). ARMCX2 was also identified by Xu et al.9 to be a very important regulator in gastric cancer9; and BTBD1 localized in cytoplasmic bodies is increased in cancer cells.10 ZBTB1 may contribute to tumour cell proliferation and apoptosis by regulating these proteins. The functions of ZBTB1 and the genes significantly associated with ZBTB1 were predicted by analysing gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) with the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). Figure 1M shows that proteasomal protein catabolic processes, ubiquitin ligase complexes and ubiquitin-like protein transferase activities in pancreatic cancer are significantly regulated by ZBTB1 and related genes (Figure 1M). It is well known that the ubiquitination proteasome pathway is inextricably linked to cancer initiation, progression and prognosis. Twenty-five pathways related to ZBTB1 and its associated genes in pancreatic carcinoma were identified by KEGG analysis (Figure 1N). In these pathways, autophagy, ubiquitin-mediated proteolysis and endocytosis are involved in the development and pathogenesis of pancreatic carcinoma. In summary, this study is the first to explore the expression and prognostic value of ZBTB1 in pancreatic carcinoma tissue with tissue microarray (TMA) technology, immunohistochemistry (IHC), GEPIA, Oncomine, Kaplan-Meier Plotter, cBioPortal, String-DB, DAVID and other databases. Our results suggest that the enhanced expression of ZBTB1 contributes significantly to pancreatic carcinogenesis. ZBTB1 may be a potential new candidate biomarker to identify additional therapeutic targets in the battle against. This work was supported by the National Natural Science Foundation of China (81760287,31700763), the Science and Technology Development Program of Jilin Province (20180201040NY, 20190301042NY, 20200402041NC), and the Science and Technology Project of the Education Department of Jilin Province during the 13th Five-year Plan (JJKH20200360KJ). The authors confirm that there are no conflicts of interest. Ming-yang Cheng: Formal analysis (lead); Investigation (lead); Methodology (equal); Project administration (equal); Writing-original draft (equal). Yan Zeng: Data curation (equal); Investigation (equal); Methodology (equal); Validation (equal). Yu Sun: Investigation (supporting); Methodology (supporting). Chun-wei Shi: Investigation (supporting); Methodology (supporting). Jun-hong Wang: Investigation (supporting); Methodology (supporting). Feng-di LI: Investigation (supporting); Methodology (supporting). Yi-yuan Lu: Investigation (supporting); Methodology (supporting). Jing-ying Wang: Investigation (supporting); Methodology (supporting). Ru-yu Wang: Investigation (supporting); Methodology (supporting). Xin-yang Li: Investigation (supporting); Methodology (supporting). Xiao-xu Li: Investigation (supporting); Methodology (supporting). Shu-hui Fan: Investigation (supporting); Methodology (supporting). Gui-lian Yang: Project administration (equal); Resources (equal); Supervision (equal). Xin Cao: Conceptualization (equal); Funding acquisition (equal); Project administration (equal); Supervision (equal); Writing-original draft (lead); Writing-review & editing (equal). Bin Xu: Conceptualization (equal); Investigation (equal); Methodology (lead); Project administration (equal); Writing-review & editing (equal). Chunfeng Wang: Conceptualization (lead); Funding acquisition (lead); Supervision (lead); Writing-review & editing (equal).

BACKGROUND:Exosomes play crucial roles in regulating the crosstalk between normal and cancer cells in the tumor microenvironment, and in regulating cancer proliferation, migration and invasion through their cargo molecules.METHODS:We analyzed the pro-invasiveness of exosomal circRNA-100,338 in HCC using the transwell invasion assay. The co-culture of human umbilical vein endothelial cells (HUVEC) and exosomes derived from HCC cell lines were used to evaluate the impact of HCC derived exosomes on HUVEC. Nude mice models were used to validate the findings in vitro. Clinically, quantitative RT-PCR was used to quantify the expression of serum exosomal circRNA-100,338 in HCC patients at both pre-surgery within one week and post-surgery within three weeks.RESULTS:We aim to investigate the pro-invasive role of exosomal circRNA-100,338 in HCC metastasis. We for the first time demonstrated that circRNA-100,338 was highly expressed in both highly metastatic HCC cells and their secreted exosomes. The transwell invasion assay showed that the overexpression or knockdown of exosomal circRNA-100,338 significantly enhanced or reduced the invasive abilities of HCC cells. Subsequently, in vitro and in vivo assays showed that exosomal circRNA-100,338 affected the cell proliferation, angiogenesis, permeability, and vasculogenic mimicry (VM) formation ability of human umbilical vein endothelial cells (HUVEC), and tumor metastasis. Furthermore, we also observed that the persistent high expression of exosomal circRNA-100,338 in serum of HCC patients who underwent curative hepatectomy may be a risk indicator of pulmonary metastasis and poor survival.CONCLUSIONS:Our findings indicated that metastatic ability of HCC cells could be enhanced by transferring exosomal circRNA-100,338 to recipient HUVECs, which could affect proangiogenic activity by regulating angiogenesis.

Background: Increasing amounts of evidence indicate that Missing in metastasis B (MIM-B) promotes cancer metastasis. Here, we sought to better understand the mechanism through which MIM-B promotes tumor metastasis in hepatocellular carcinoma (HCC).Methods: We performed confocal microscopy analysis to determine the distributions of MIM-B and caveolin-1 and conducted co-immunoprecipitation assays to detect the interactions between MIM-B and caveolin-1 in vitro.We performed transwell assays to analyze the invasive ability of HCC cells.Changes in the expression levels of key genes and some molecular makers were detected by immunohistochemistry and western blotting in HCC tissue samples.Results: We found that MIM-B co-localizes with caveolin-1 and demonstrated that MIM-B and caveolin-1 interact in vitro.Repressing MIM-B and caveolin-1 expression inhibited the epidermal growth factor receptor signaling pathway.We overexpressed MIM-B and caveolin-1 in Hep3B cells, which enhanced Hep3B cell invasiveness.Furthermore, MHCC97H cell invasiveness was significantly decreased in cells in which MIM-B and caveolin-1 expression was inhibited.Additionally, we found that MIM-B and caveolin-1 were expressed at higher levels in HCC tissues than in paired normal tissues.Moreover, HCC patients with MIM-B and caveolin-1 up-regulation experienced significantly worse outcomes than controls (P < 0.001), and HCC patients with high MIM-B and caveolin-1 expression levels often developed pulmonary metastasis (P < 0.001).Conclusions: MIM-B combined with caveolin-1 promotes metastasis of HCC, and elevated MIM-B and caveolin-1 expression levels are associated with a poor prognosis in HCC patients; therefore, MIM-B and caveolin-1 may represent novel targets for the diagnosis and treatment of HCC.

Objective To investigate the antitumor activity of the oncolytic adenovirus expressing lipocalin 2 gene for colorectal cancer in vivo.Methods BALB/C nude mice subcutaneously inoculated by SW620 cells and grown tumors were treated with injection of ZD-55 virus, Ad-lipocalin 2 virus and ZD55- lipocalin 2 virus respectively.The weight of implanted tumors and the tumor inhibition rate were calculated to evaluate the anti-tumor effect.Cell apoptosis was determined by TUNEL and the protein expression of VEGF and MVD were determined with immunohistochemistry.Results ZD55-lipocalin 2 inhibited the growth of transplanted tumor more significantly than ZD-55 virus and Ad-lipocalin 2 virus ( P ＜ 0.05 ).Tumor cell apoptosis was upregulated and the MVD reduced significantly in ZD55-lipocalin 2 group in contrast to the other two groups (P ＜0.05).Conclusions ZD55-lipocalin 2 induces apoptosis of colorectal tumor cells and inhibits tumor microvascular formation, slowing down the growth of transplantation tumors.