Personalized neoantigen vaccine enhances the therapeutic efficacy of bevacizumab and anti-PD-1 antibody in advanced non-small cell lung cancer.

Clinically, a considerable number of non-small cell lung cancer (NSCLC) patients are unable to receive or resist chemotherapy, and the efficacy of non-chemotherapy treatment strategies based on anti-angiogenic agents combined with immune checkpoint blockade is still unsatisfactory. Neoantigen vaccine, based on personalized tumor DNA mutations, could elicit tumor specific T cell infiltration into the tumor site, exerting potent anti-tumor efficacy. Here, we evaluated the feasibility and safety of a new antitumor strategy by adding neoantigen vaccine to the regimen of bevacizumab and anti-PD-1 antibody. Firstly, 7 novel immunogenic neoantigen peptides were identified and developed for neoantigen vaccine (LLCvac), which can elicit strong antitumor immune response in vivo. Then, in orthotopic lung cancer model, LLCvac further combining with bevacizumab and anti-PD-1 antibody exerted a stronger antitumor effect, exhibiting significant decrease of tumor volume without obvious toxicity. Furthermore, tumor immune microenvironment assessment also showed that the proportion of neoantigen-specific T cells in blood could be induced dramatically by the combined therapy. And a large amount of neoantigen-specific Ki67-positive CD8+ T cells were found in tumor tissues, which infiltrated tumor tissues effectively to kill tumor cells expressing identified neoantigens. Overall, these results suggested that this combined therapy could safely induce robust antitumor efficacy, serving as an effective chemotherapy-free strategy for NSCLC treatment.

Personalized neoantigen vaccine prevents postoperative recurrence in hepatocellular carcinoma patients with vascular invasion.

BACKGROUND: Clinically, prophylactic anti-recurrence treatments for hepatocellular carcinoma (HCC) patients after radical surgery are extremely limited. Neoantigen based vaccine can generate robust anti-tumor immune response in several solid tumors but whether it could induce anti-tumor immune response in HCC and serve as a safe and effective prophylactic strategy for preventing postoperative HCC recurrence still remain largely unclear. METHODS: Personalized neoantigen vaccine was designed and immunized for 10 HCC patients with high risk of postoperative recurrence in a prime-boost schedule. The safety and immune response were assessed through adverse events, tissue sequencing, ELISpot, TCR sequencing. The clinical response was evaluated by recurrence-free survival (RFS) and personalized circulating tumor DNA (ctDNA) sequencing. RESULTS: In the 10 enrolled patients, no obvious adverse events were observed during neoantigen vaccinations. Until the deadline of clinical trial, 8 of 10 patients were confirmed with clinical relapse by imaging, the other 2 patients remained relapse-free. From receiving first neoantigen vaccination, the median RFS of 10 patients were 7.4 months. Among 7 patients received all planned neoantigen vaccinations, 5 of them demonstrated neoantigen-induced T cell responses and have significantly longer RFS after radical surgery than other 5 patients without responsive neoantigens or only with prime vaccination and propensity scores matching control patients (p = 0.035). Moreover, tracking personalized neoantigen mutations in ctDNA could provide real-time evaluation of clinical response in HCC patients during neoantigen vaccination and follow up. CONCLUSION: Personalized neoantigen vaccine is proved as a safe, feasible and effective strategy for HCC anti-recurrence, and its progression could be sensitively monitored by corresponding neoantigen mutations in ctDNA, and thus provided solid information for individualized medicine in HCC. TRIAL REGISTRATION: This study was registered at Chinese Clinical Trial Registry; Registration number: ChiCTR1900020990 .

Accurate transcriptome assembly by Nanopore RNA sequencing reveals novel functional transcripts in hepatocellular carcinoma.

The long reads of Nanopore sequencing permit accurate transcript assembly and ease in discovering novel transcripts with potentially important functions in cancers. The wide adoption of Nanopore sequencing for transcript quantification, however, is largely limited by high costs. To address this issue, we developed a bioinformatics software, NovelQuant, that can specifically quantify long-read-assembled novel transcripts with short-read sequencing data. Nanopore Direct RNA Sequencing was carried out on three hepatocellular carcinoma (HCC) patients' tumor, matched portal vein tumor thrombus, and peritumor to reconstruct the HCC transcriptome. Then, based on the reconstructed transcriptome, NovelQuant was applied on Illumina RNA sequencing data of 59 HCC patients' tumor and paired peritumor to quantify novel transcripts. Our further analysis revealed 361 novel transcripts dysregulated in HCC and that 101 of them were significantly associated with prognosis. There were 19 novel prognostic transcripts predicted to be long noncoding RNAs (lncRNAs), and some of them had regulatory targets that were reported to be associated with HCC. Additionally, 42 novel prognostic transcripts were predicted to be protein-coding mRNAs, and many of them could be involved in xenobiotic metabolism. Moreover, the tumor-suppressive roles of two representative novel prognostic transcripts, CDO1-novel (lncRNA) and CYP2A6-novel (protein-coding mRNA), were further functionally validated during HCC progression. Overall, the current study shows a possibility of combining long- and short-read sequencing to explore functionally important novel transcripts in HCC with accuracy and cost-efficiency, which expands the pool of molecular biomarkers that could enhance our understanding of the molecular mechanisms of HCC.

Profiling of hepatocellular carcinoma neoantigens reveals immune microenvironment and clonal evolution related patterns.

OBJECTIVE: Neoantigens derived from tumor-specific genomic alterations have demonstrated great potential for immunotherapeutic interventions in cancers. However, the comprehensive profile of hepatocellular carcinoma (HCC) neoantigens and their complex interplay with immune microenvironment and tumor evolution have not been fully addressed. METHODS: Here we integrated whole exome sequencing data, transcriptome sequencing data and clinical information of 72 primary HCC patients to characterize the HCC neoantigen profile, and systematically explored its interactions with tumor clonal evolution, driver mutations and immune microenvironments. RESULTS: We observed that higher somatic mutation/neoantigen load was associated with better clinical outcomes and HCC patients could be further divided into two subgroups with distinct prognosis based on their neoantigen expression patterns. HCC subgroup with neoantigen expression probability high (NEP-H) showed more aggressive pathologic features including increased incidence of tumor thrombus (P=0.038), higher recurrence rate (P=0.029), more inclined to lack tumor capsule (P=0.026) and with more microsatellite instability sites (P=0.006). In addition, NEP-H subgroup was also characterized by higher chance to be involved in tumor clonal evolution [odds ratio (OR)=46.7, P<0.001]. Gene set enrichment analysis revealed that upregulation of MYC and its targets could suppress immune responses, leading to elevated neoantigen expression proportion in tumor cells. Furthermore, we discovered an immune escape mechanism that tumors could become more inconspicuous by evolving subclones with less immunogenicity. We observed that smaller clonal mutation clusters with higher immunogenicity in tumor were more likely to involve in clonal evolution. Based on identified neoantigen profiles, we also discovered series of neoantigenic hotspot genes, which could serve as potential actionable targets in future. CONCLUSIONS: Our results revealed the landscape of HCC neoantigens and discovered two clinically relevant subgroups with distinct neoantigen expression patterns, suggesting the neoantigen expression should be fully considered in future immunotherapeutic interventions.

CDK13 RNA Over-Editing Mediated by ADAR1 Associates with Poor Prognosis of Hepatocellular Carcinoma Patients.

BACKGROUND/AIMS: Aberrant RNA editing, mediated by adenosine deaminases acting on RNA (ADAR), serves as a post-transcriptional event participating in tumorigenesis and prognosis. However, the RNA editing profiles during HCC progression and their clinical correlations remain unclear. METHODS: Multiple tissue samples were collected from an advanced HCC patient. RNA-seq was performed to obtain the RNA editing profiles for each sample. Two RNA editing sites from CDK13 were further validated in 60 HCC patients; and their potential regulatory mechanisms were investigated. RESULTS: In-depth analysis of the RNA-seq data revealed a significant number of editing sites (632-816) in coding regions for each tissue sample, showing branched evolution during tumorigenesis and metastasis. Two editing sites (Q103R and K96R) in CDK13 showed significant over-editing in tumor, and these phenomenon were validated in 60 HCC patients. Furthermore, the clinicopathological analysis revealed that these CDK13 over-editing sites were positively associated with TNM, PVTT and poor prognosis. In addition, the editing level of these sites were significantly correlated with the expression of ADAR1. Loss of function assays further proved that these CDK13 over-editing sites were mediated by ADAR1 in HCC cells. CONCLUSIONS: CDK13 RNA over-editing sites mediated by ADAR1 may serve as novel cancer driver events in HCC progression.

Opposing Roles of Wnt Inhibitors IGFBP-4 and Dkk1 in Cardiac Ischemia by Differential Targeting of LRP5/6 and ß-catenin.

BACKGROUND: Myocardial infarction is one of the leading causes of morbidity and mortality worldwide, triggering irreversible myocardial cell damage and heart failure. The role of low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) as coreceptors of the Wnt/ß-catenin pathway in the adult heart remain unknown. Insulin-like growth factor binding protein 4 and dickkopf-related protein 1 (Dkk1) are 2 secreted LRP5/6 binding proteins that play a crucial role in heart development through preventing Wnt/ß-catenin pathway activation. However, their roles in the adult heart remain unexplored. METHODS: To understand the role of LRP5/6 and ß-catenin in the adult heart, we constructed conditional cardiomyocyte-specific LRP5/6 and ß-catenin knockout mice and induced surgical myocardial infarction. We also directly injected recombinant proteins of insulin-like growth factor binding protein 4 and Dkk1 into the heart immediately following myocardial infarction to further examine the mechanisms through which these proteins regulate LRP5/6 and ß-catenin. RESULTS: Deletion of LRP5/6 promoted cardiac ischemic insults. Conversely, deficiency of ß-catenin, a downstream target of LRP5/6, was beneficial in ischemic injury. It is interesting to note that although both insulin-like growth factor binding protein 4 and Dkk1 are secreted Wnt/ß-catenin pathway inhibitors, insulin-like growth factor binding protein 4 protected the ischemic heart by inhibiting ß-catenin, whereas Dkk1 enhanced the injury response mainly through inducing LRP5/6 endocytosis and degradation. CONCLUSIONS: Our findings reveal previously unidentified dual roles of LRP5/6 involved in the cardiomyocyte response to ischemic injury. These findings suggest new therapeutic strategies in ischemic heart disease by fine-tuning LRP5/6 and ß-catenin signaling within the Wnt/ß-catenin pathway.

TLR3 activation enhances abscopal effect of radiotherapy in HCC by promoting tumor ferroptosis.

Radiotherapy (RT) has been reported to induce abscopal effect in advanced hepatocellular carcinoma (HCC), but such phenomenon was only observed in sporadic cases. Here, we demonstrated that subcutaneous administration of Toll-like receptor 3 (TLR3) agonist poly(I:C) could strengthen the abscopal effect during RT through activating tumor cell ferroptosis signals in bilateral HCC subcutaneous tumor mouse models, which could be significantly abolished by TLR3 knock-out or ferroptosis inhibitor ferrostatin-1. Moreover, poly(I:C) could promote the presentation of tumor neoantigens by dendritic cells to enhance the recruitment of activated CD8+ T cells into distant tumor tissues for inducing tumor cell ferroptosis during RT treatment. Finally, the safety and feasibility of combining poly(I:C) with RT for treating advanced HCC patients were further verified in a prospective clinical trial. Thus, enhancing TLR3 signaling activation during RT could provide a novel strategy for strengthening abscopal effect to improve the clinical benefits of advanced HCC patients.

Identification of tumor mutations in plasma based on mutation variant frequency change (MVFC).

To overcome the dependency of strategies utilizing cell-free DNA (cfDNA) on tissue sampling, the emergence of sequencing panels for non-invasive mutation screening was promoted. However, cfDNA sequencing with panels still suffers from either inaccuracy or omission, and novel approaches for accurately screening tumor mutations solely based on plasma without gene panel restriction are urgently needed. We performed unique molecular identifier (UMI) target sequencing on plasma samples and peripheral blood mononuclear cells (PBMCs) from 85 hepatocellular carcinoma (HCC) patients receiving surgical resection, which were divided into an exploration dataset (20 patients) or an evaluation dataset (65 patients). Plasma mutations were identified in pre-operative plasma, and the mutation variant frequency change (MVFC) between post- and pre-operative plasma was then calculated. In the exploration dataset, we observed that plasma mutations with MVFC < 0.2 were enriched for tumor mutations identified in tumor tissues and had frequency changes that correlated with tumor burden; these plasma mutations were therefore defined as MVFC-identified tumor mutations. The presence of MVFC-identified tumor mutations after surgery was related to shorter relapse-free survival (RFS) in both datasets and thus indicated minimum residual disease (MRD). The combination of MVFC-identified tumor mutations and Alpha Fetoprotein (AFP) could further improve MRD detection (P < 0.0001). Identification of tumor mutations based on MVFC was also confirmed to be applicable with a different gene panel. Overall, we proposed a novel strategy for non-invasive tumor mutation screening using solely plasma that could be utilized in HCC tumor-burden monitoring and MRD detection.

Tracking Cell Viability for Adipose-Derived Mesenchymal Stem Cell-Based Therapy by Quantitative Fluorescence Imaging in the Second Near-Infrared Window.

Cell survival rate determines engraftment efficiency in adipose-derived mesenchymal stem cell (ADSC)-based regenerative medicine. In vivo monitoring of ADSC viability to achieve effective tissue regeneration is a major challenge for ADSC therapy. Here, we developed an activated near-infrared II (NIR-II) fluorescent nanoparticle consisting of lanthanide-based down-conversion nanoparticles (DCNPs) and IR786s (DCNP@IR786s) for cell labeling and real-time tracking of ADSC viability in vivo. In dying ADSCs due to excessive ROS generation, absorption competition-induced emission of IR786s was destroyed, which could turn on the NIR-II fluorescent intensity of DCNPs at 1550 nm by 808 nm laser excitation. In contrast, the NIR-II fluorescent intensity of DCNPs was stable at 1550 nm by 980 nm laser excitation. This ratiometric fluorescent signal was precise and sensitive for tracking ADSC viability in vivo. Significantly, the nanoparticle could be applied to quantitively evaluate stem cell viability in real-time in vivo. Using this method, we successfully sought two small molecules including glutathione and dexamethasone that could improve stem cell engraftment efficiency and enhance ADSC therapy in a liver fibrotic mouse model. Therefore, we provide a potential strategy for real-time in vivo quantitative tracking of stem cell viability in ADSC therapy.

Dysregulation of global circular RNA abundance regulated by spliceosomes predicts prognosis in hepatocellular carcinoma.

CircRNAs have been reported to play crucial roles in tumor progression and recurrence, showing potential as biomarkers in cancer. However, the global abundance of circRNA and their involvement in hepatocellular carcinoma (HCC) development have not been fully explored. Whole transcriptome sequencing was performed on tumor and peritumor from 60 patients with HCC to quantify the expression of circRNAs, and the global circRNA abundance was calculated by circRNA index (CRI). Gene-set enrichment analysis and weighted gene co-expression network analysis were used to reveal the biological signaling pathways associated with the global circRNA abundance. The correlation between the global circRNA abundance and the infiltration level of CD8+ T cells was explored by immunohistochemical assays. Small interfering RNA was used to knock down the pre-messenger RNA spliceosome in HCC cell lines to verify the regulation of spliceosome in global circRNA abundance. We found that dysregulation of global circRNA abundance in both tumor and peritumor could lead to worse prognosis. The immunohistochemical assay further revealed that the dysregulation of global circRNA abundance in both tumor and peritumor would obstruct the CD8+ T cells from invading into the tumor, which might explain its correlation with HCC prognosis. We also demonstrated that the spliceosome genes were the main factors to regulate the global circRNA abundance in HCC, and these results were also confirmed by knockdown experiments. Conclusion: This study revealed the association between the global circRNA abundance and patients' prognosis and its underlying mechanism.

Personalized neoantigen vaccine combined with PD-1 blockade increases CD8+ tissue-resident memory T-cell infiltration in preclinical hepatocellular carcinoma models.

BACKGROUND: Personalized neoantigen vaccine could induce a robust antitumor immune response in multiple cancers, whose efficacy could be further enhanced by combining with programmed cell death 1 blockade (α-PD-1). However, the corresponding immune response and synergistic mechanisms remain largely unclear. Here, we aimed to develop clinically available combinational therapeutic strategy and further explore its potential antitumor mechanisms in hepatocellular carcinoma (HCC). METHODS: Neoantigen peptide vaccine (NeoVAC) for murine HCC cell line Hepa1-6 was developed and optimized by neoantigen screening and adjuvant optimization. Then the synergistic efficacy and related molecular mechanisms of NeoVAC combined with α-PD-1 in HCC were evaluated by orthotopic HCC mouse model, single-cell RNA sequencing, tetramer flow cytometry, immunofluorescence, etc. The tumor-killing capacity of CD8+ tissue-resident memory T cells (CD8+ TRMs) was assessed by orthotopic HCC mouse model, and autologous patient-derived cells. RESULTS: NeoVAC, which consisted of seven high immunogenic neoantigen peptides and clinical-grade Poly(I:C), could generate a strong antitumor immune response in HCC mouse models. Significantly, its efficacy could be further improved by combining with α-PD-1, with 80% of durable tumor regression and long-term immune memory in orthotopic HCC models. Moreover, in-depth analysis of the tumor immune microenvironment showed that the percentage of CD8+ TRMs was remarkedly increased in NeoVAC plus α-PD-1 treatment group, and positively associated with the antitumor efficacy. In vitro and in vivo T-cell cytotoxicity assay further confirmed the strong tumor-killing capacity of CD8+ TRMs sorting from orthotopic mouse HCC or patient's HCC tissue. CONCLUSIONS: This study showed that NeoVAC plus α-PD-1 could induce a strong antitumor response and long-term tumor-specific immune memory in HCC by increasing CD8+ TRMs infiltration, which might serve as a potential immune-therapeutic target for HCC.

Copy number profiling of circulating free DNA predicts transarterial chemoembolization response in advanced hepatocellular carcinoma.

Transarterial chemoembolization (TACE) is the most commonly used treatment for advanced hepatocellular carcinoma (HCC), but still lacks accurate real-time biomarkers for monitoring its therapeutic efficacy. Here, we explored whether copy number profiling of circulating free DNA (cfDNA) could be utilized to predict responses and prognosis in HCC patients with TACE treatment. In total, 266 plasma cfDNA samples were collected from 64 HCC patients, 57 liver cirrhosis (LC) patients and 32 healthy volunteers. We performed low-depth whole-genome sequencing (LD-WGS) on cfDNA samples to conduct copy number variant (CNV) analysis and tumour fraction (TFx) quantification. Then, the correlation between TFx/CNVs and therapeutic efficacy, treatment outcomes and lipiodol deposition were explored. The change in TFx during TACE treatment was associated with patients' tumour burden, and could accurately and earlier predict treatment response and prognosis, providing an alternative strategy other than mRECIST. Meanwhile, the chromosomal 16q/NQO1 amplification indicated worse therapeutic response; in patients who underwent multiple TACE sessions, TFx change during their first TACE treatment reflected the long-term survival; additionally, the copy number amplification of chromosome 1q, 3p, 6p, 8q, 10p, 12q, 18p or 18q affected lipiodol deposition. Overall, we have provided a new liquid biopsy approach for future TACE management of HCC patients.

Circular RNA CircEPB41L2 Functions as Tumor Suppressor in Hepatocellular Carcinoma Through Sponging miR-590-5p.

BACKGROUND: Circular RNAs (circRNAs) could interact with miRNAs to regulate gene expression, participating in hepatocellular carcinoma (HCC) initiation and development. This work aimed to determine the potential function and molecular mechanism of circEPB41L2 (hsa_circ_0077837) during HCC progression. MATERIALS AND METHODS: The expression of circEPB41L2 in HCC tissues and HCC cell lines was quantified using real-time quantitative PCR (qRT-PCR). CCK-8 assays and colony formation assays were utilized to detect the proliferation of HCC cells. Wound healing assay and transwell assay were performed to determine the capability of migration and invasion for HCC cells. Western blot was conducted to determine gene expression on protein levels. The effect of circEPB41L2 on HCC in vivo was investigated via xenograft experiment. Interaction between circEPB41L2 and miR-590-5p was predicted through bioinformatics methods and confirmed via luciferase reporter assay. RESULTS: Extensive analysis of circRNA profiles in tumor and matched para-tumor tissues collected from 61 HCC patients identified that circEPB41L2 was significantly down-regulated in HCC, which was further confirmed in another HCC group by qRT-PCR analysis. The clinicopathological analysis revealed that down-regulation of circEPB41L2 was negatively associated with tumor size, vascular invasion and alpha-fetoprotein, while positively correlated with HCC prognosis. The biological function experiments showed that overexpression of circEPB41L2 could obviously inhibit the proliferation and metastasis of HCC cells in vitro, while knockdown of circEPB41L2 induced opposite results. Moreover, we also found that circEPB41L2 inhibited HCC migration and invasion though EMT signaling pathway. Similarly, overexpression of circEPB41L2 can also significantly inhibit the proliferation of HCC cells in vivo. Bioinformatic analysis and luciferase reporter assay revealed that circEPB41L2 interacts directly with miR-590-5p and the corresponding biological functions were also verified in miRNA rescue experiments. CONCLUSION: Our results suggest that circEPB41L2 might function as a tumor suppressor during HCC progression by sponging miR-590-5p.

Personalized neoantigen-based immunotherapy for advanced collecting duct carcinoma: case report.

BACKGROUND: Collecting duct carcinoma (CDC) of the kidney is a rare and highly aggressive malignant tumor with the worst prognosis among all renal cancers. Nevertheless, the first-line treatments, including chemotherapy and target therapy, usually show poor response to CDC. Recent studies have suggested that immunotherapy targeting personal tumor-specific neoantigens could be a promising strategy for several solid cancers. However, whether it has therapeutic potential in CDC remains unclear. CASE PRESENTATION: Here, we report a case of an Asian patient who underwent personalized neoantigen-based immunotherapy. The patient was diagnosed with metastatic CDC and suffered extensive tumor progression following sorafenib treatment. Based on the patient's own somatic mutational profile, a total of 13 neoantigens were identified and corresponding long-peptide vaccine and neoantigen-reactive T cells (NRTs) were prepared. After six cycles of neoantigen-based vaccination and T-cell immunotherapy, the patient was reported with stable disease status in tumor burden and significant alleviation of bone pain. Ex vivo interferon-γ enzyme-linked immunospot assay proved the reactivity to 12 of 13 neoantigens in peripheral blood mononuclear cells collected after immunotherapy, and the preferential reactivity to mutant peptides compared with corresponding wild-type peptides was also observed for 3 of the neoantigens. Surprisingly, biopsy sample collected from CDC sites after 3 months of immunotherapy showed decreased mutant allele frequency corresponding to 92% (12/13) of the neoantigens, indicating the elimination of tumor cells carrying these neoantigens. CONCLUSIONS: Our case report demonstrated that the combined therapy of neoantigen peptide vaccination and NRT cell infusion showed certain efficacy in this CDC case, even when the patient carried only a relatively low tumor mutation burden. These results indicated that the personalized neoantigen-based immunotherapy was a promising new strategy for advanced CDC. TRIAL REGISTRATION NUMBER: ChiCTR1800017836.

An Isothermal Method for Sensitive Detection of Mycobacterium tuberculosis Complex Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a Cis and Trans Cleavage.

Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA based on conserved sequences of MTB from designed guide RNA library. Then, we proposed a novel detection method based on recombinase polymerase amplification and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay, based on fluorescence detection, showed 4.48 fmol/L of limit of detection and good linear correlation of concentration with fluorescence value (R2 = 0.9775). It also showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% CI, compared with culture method. Taken together, the CRISPR/Cas12a system showed good specificity, excellent sensitivity, and excellent accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

Genomic and transcriptional Profiling of tumor infiltrated CD8+ T cells revealed functional heterogeneity of antitumor immunity in hepatocellular carcinoma.

As key players in HCC antitumor response, the functions of tumor infiltrated CD8+ T cells are significantly affected by surrounding microenvironment. A detailed profiling of their genomic and transcriptional changes could provide valuable insights for both future immunotherapy development and prognosis evaluation. We performed whole exome and transcriptome sequencing on tumor infiltrated CD8+ T cells and CD8+ T cells isolated from other tissue origins (peritumor tissues and corresponding PBMCs) in eight treatment-naive HCC patients. The results demonstrated that transcriptional changes, rather than genomic alterations were the main contributors to the functional alterations of CD8+ T cells in the process of tumor progression. The origins of CD8+ T cells defined their transcriptional landscape, while the tumor infiltrated CD8+ T cells shared more similarity with peritumor-derived CD8+ T cells compared with those CD8+ T cells in blood. In addition, tumor infiltrated CD8+ T cells also showed larger transcriptional heterogeneity among individuals, which was modulated by clinical features such as HBV levels, preoperative anti-viral treatment and the degree of T cell infiltration. We also identified multiple inter-connected pathways involved in the activation and exhaustion of tumor infiltrated CD8+ T cells, among which IL-12 mediated pathway could dynamically reflect the functional status of CD8+ TILs and activation of this pathway indicated a better prognosis. Our results presented an overview picture of CD8+ TILs' genomic and transcriptional landscape and features, as well as how the functional status of CD8+ TILs correlated with patients' clinical course.

Circular RNA profiling identifies circADAMTS13 as a miR-484 sponge which suppresses cell proliferation in hepatocellular carcinoma.

Circular RNA (circRNA) can participate in various biological processes, including tumorigenesis, through their microRNA response elements. Alterations in circRNA profiles during hepatocellular carcinoma (HCC) progression and their clinical significance remain unclear. Here, we present extensive analysis of circRNA profiles in tumor and matched peritumor tissues collected from 10 HCC patients, conducted to identify circRNA related to HCC progression. A total of 42 dysregulated circRNA (38 down-regulated and 4 up-regulated) were identified in HCC tumor tissues compared with matched peritumor tissues, revealing the heterogeneity of circRNA profiles in HCC. CircADAMTS13, derived from Exon 13-14 of the ADAMTS13 gene, was significantly downregulated in HCC tumor tissues. Furthermore, clinicopathological analysis revealed that up-regulation of circADAMTS13 was negatively associated with tumor size but positively associated with prognosis. In addition, overexpression of circADAMTS13 could markedly inhibit HCC cell proliferation in vitro. Bioinformatic analysis and luciferase reporter assays further revealed that circADAMTS13 directly interacts with microRNA (miR)-484. Rescue experiments showed that miR-484 mimics can reverse the tumor-suppressing roles of circADAMTS13 in HCC. Therefore, our results demonstrated that circADAMTS13 can serve as a tumor suppressor during HCC progression via the functional pathway of sponging miR-484.

EBF1-Mediated Upregulation of Ribosome Assembly Factor PNO1 Contributes to Cancer Progression by Negatively Regulating the p53 Signaling Pathway.

The RNA-binding protein PNO1 is critical for ribosome biogenesis, but its potential role in cancer remains unknown. In this study, online data mining, cDNA, and tissue microarrays indicated that PNO1 expression was higher in colorectal cancer tissue than in noncancerous tissue, and its overexpression was associated with worse patient survival. Gain-of-function and loss-of-function studies demonstrated that PNO1 knockdown suppressed growth of colorectal cancer cells in vitro and in vivo, while PNO1 overexpression promoted colorectal cancer cell proliferation in vitro. In colorectal cancer cells expressing wild-type p53, PNO1 knockdown enhanced expression of p53 and its downstream gene p21, and reduced cell viability; these effects were prevented by p53 knockout and attenuated by the p53 inhibitor PFT-α. Moreover, PNO1 knockdown in HCT116 cells decreased levels of 18S rRNA, of 40S and 60S ribosomal subunits, and of the 80S ribosome. It also reduced global protein synthesis, increasing nuclear stress and inhibiting MDM2-mediated ubiquitination and p53 degradation. Overexpressing EBF1 suppressed PNO1 promoter activity and decreased PNO1 mRNA and protein, inhibiting cell proliferation and inducing cell apoptosis through the p53/p21 pathway. In colorectal cancer tissues, the expression of EBF1 correlated inversely with PNO1. Data mining of online breast and lung cancer databases showed increased PNO1 expression and association with poor patient survival; PNO1 knockdown reduced cell viability of cultured breast and lung cancer cells. Taken together, these findings indicate that PNO1 is overexpressed in colorectal cancer and correlates with poor patient survival, and that PNO1 exerts oncogenic effects, at least, in part, by altering ribosome biogenesis. SIGNIFICANCE: This study identifies the ribosome assembly factor PNO1 as a potential oncogene involved in tumor growth and progression of colorectal cancer.

Comprehensive Liquid Profiling of Circulating Tumor DNA and Protein Biomarkers in Long-Term Follow-Up Patients with Hepatocellular Carcinoma.

PURPOSE: Circulating tumor DNA (ctDNA) provides a novel approach for detecting tumor burden and predicting clinical outcomes of hepatocellular carcinoma (HCC). Here, we performed a thorough evaluation of HCC circulating genetic features and further fully integrated them to build a robust strategy for HCC monitoring and prognostic outcome assessment. EXPERIMENTAL DESIGN: We performed target sequencing and low-coverage whole-genome sequencing on plasma samples collected from 34 long-term follow-up patients with HCC to capture tumor somatic SNVs and CNVs, respectively. Clinical information was also obtained to evaluate the prognostic performance of ctDNA comparing with clinically applied protein biomarkers. RESULTS: All plasma samples before surgery showed somatic genetic variations resembling corresponding tumor tissues. During follow-up, SNVs and CNVs dynamically changed correlating to patients' tumor burden. We integrated the comprehensive ctDNA mutation profiles to provide a robust strategy to accurately assess patients' tumor burden with high consistence comparing with imaging results. This strategy could discover tumor occurrence in advance of imaging for an average of 4.6 months, and showed superior performance than serum biomarkers AFP, AFP-L3%, and Des-Gamma-Carboxy Prothrombin (DCP). Furthermore, our strategy could precisely detect minimal residual disease (MRD) in advance and predict patients' prognostic outcomes for both relapse-free survival (P = 0.001) and overall survival (P = 0.001); further combining ctDNA with DCP could increase the sensitivity for MRD detection. CONCLUSIONS: We demonstrated that plasma CNV and SNV levels dynamically correlated with patients' tumor burden in HCC. Our strategy of comprehensive mutation profile integration could accurately and better evaluate patients' prognostic risk and detect tumor occurrence in advance than traditional strategies.

Baicalin alleviates H2O2­induced injury of H9c2 cardiomyocytes through suppression of the Wnt/ß­catenin signaling pathway.

Baicalin is one of the active ingredients extracted from the dry root of Scutellaria baicalensis Georgi, which has been reported to be effective in preventing myocardial ischemia reperfusion injury. However, the mechanisms underlying its cardioprotective activities remain to be elucidated. In the present study, H2O2­treated cardiomyocyte H9c2 cell line served as an in vitro model of oxidation­damaged cardiomyocytes to evaluate the effects of baicalin on the cardiac injury, and to investigate the underlying molecular mechanism. The results of the TOPFlash/Renilla reporter gene assay indicated that baicalin significantly suppressed the activation of proto­oncogene Wnt­1 (Wnt)/ß­catenin in 293 cells, in a dose dependent manner. In addition, baicalin significantly inhibited H2O2­induced loss of H9c2 cell viability in MTT assay. Furthermore, western blotting analysis demonstrated that baicalin markedly attenuated H2O2­induced cell apoptosis, as demonstrated by the down­regulation of cleaved caspase­3 and the increase in the apoptosis regulator Bcl­2 (Bcl­2)/apoptosis regulator BAX (Bax) ratio following baicalin treatment in H2O2­treated H9c2 cells. Furthermore, baicalin markedly decreased the expression of ß­catenin and downstream Axin­2 and myc proto­oncogene protein in H2O2­treated H9c2 cells. Knockdown of ß­catenin expression inhibited H2O2­induced cell apoptosis. Finally, LiCl (a ß­catenin stabilizer) induced apoptosis of H9c2 cells by upregulating the expression of ß­catenin, which was significantly neutralized by the treatment with baicalin. Taken together, it is hypothesized that baicalin exerts cardioprotective effects via suppression of the Wnt/ß­catenin signaling pathway.