Background:Brain metastasis (BrM) has been a challenge for lung cancer treatment, but the mechanisms underlying lung cancer BrM remain elusive. This study aims to dissect cellular components and their spatial distribution in human BrM tumors of lung adenocarcinoma (LUAD) and identify potential therapeutic targets. Methods:We performed single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) on three LUAD BrMs, and validated our findings using public scRNA-seq data of 10 LUAD BrMs. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) and functional experiments were employed for experimental studies. Results:By combining scRNA-seq and ST, our analysis revealed the inter- and intra-tumoral heterogeneity of cellular components and their spatial localization within LUAD BrMs. Through RNA velocity and transcription factor (TF) regulatory activity analyses, we identified ATF3 as a potential regulator of the mesenchymal-epithelial transition (MET) program, which plays crucial roles in the colonization of tumor cells at metastatic sites. Furthermore, we demonstrated that knockdown of ATF3 significantly inhibited cancer cell proliferation while promoting cancer cell migration. Mechanistically, ATF3 knockdown could reverse the MET program. Additionally, we revealed that LGALS3/ANXA2-mediated cell-cell interaction between macrophage and tumor cells may also promote the MET program. Conclusions:Our study provides a single-cell atlas of the cellular composition in BrM of LUAD and identifies ATF3 as a potential therapeutic target for BrM treatment.

Purpose Neuroblastoma (NB) originates from differentiation arrest of sympathoadrenal progenitors in the neural crest. It is necessary to reveal the differentiation mechanism of NB. Previously, we reported that Purkinje cell protein 4 (PCP4) is a well-differentiated marker of NB tissues. Herein, we explored the underlying mechanism of PCP4 induced differentiation in order to find better treatment options for patients. Methods We screened the interacting proteins of PCP4 by co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometry (LC-MS/MS). Then we investigated the relevance between expression of calmodulin-dependent protein kinase II gamma (CAMK2G) and clinical features using R2 platform. We also explored the function of CAMK2G in NB cells by knockdown and RNA sequencing. Results Here, we verified the binding of PCP4 and calmodulin (CaM) by Co-IP and identified a target kinase of CaM, CAMK2G by LC-MS/MS. PCP4 overexpression activates the autophosphorylation of CAMK2G. Patients with high CAMK2G expression had better survival while low CAMK2G was associated with unfavorable clinical features including MYCN-amplification, unfavorable histology, progression and high INSS stage. CAMK2G knockdown inhibited neurite outgrowth and down-regulated neuronal differentiation markers (NF-H, MAP2), yet promoted migration, invasion and proliferation. Gene Ontology (GO) analysis showed that knockdown of CAMK2G downregulated the expression of neuronal differentiation-related genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that knockdown of CAMK2G upregulated the expression of migration-related genes. Conclusion These findings indicate that CAMK2G activated by PCP4/CaM complex promotes differentiation and inhibits migration in NB cells. Level of evidence Not applicable

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Long non-coding RNA LINC01296 has been shown to predict the invasiveness and poor outcomes of patients with NB. Our study validated its prognostic value and investigated the biological function and potential mechanism of LINC01296 regulating NB. Results illuminated that LINC01296 expression was significantly correlated with unfavorable prognosis and malignant clinical features according to the public NB database. We identified that silencing LINC01296 repressed NB cell proliferation and migration and promoted apoptosis. Moreover, LINC01296 knockdown inhibited tumor growth in vivo. The opposite results were observed through the dCas9 Synergistic Activation Mediator System (dCas9/SAM) activating LINC01296. Mechanistically, we revealed that LINC01296 could directly bind to nucleolin (NCL), forming a complex that activated SRY-box transcription factor 11 (SOX11) gene transcription and accelerated tumor progression. In conclusion, our findings uncover a crucial role of the LINC01296-NCL-SOX11 complex in NB tumorigenesis and may serve as a prognostic biomarker and effective therapeutic target for NB.

Non-small cell lung cancer (NSCLC), accounting for 85% of all lung cancer, is one of the leading causes of cancer-related death worldwide. Previously, we demonstrated that MPZL1 gene amplification promotes liver cancer metastasis through activating Src/Cortactin pathway. However, the clinical relevance and biological roles of the MPZL1 gene in lung cancer are still unknown. Here, we found that MPZL1 expression upregulates in human NSCLC, which is partly due to the copy number amplification of this gene. Next, we observed that high MPZL1 expression correlates with unfavorable prognosis of NSCLC patients. We further demonstrated that ectopic MPZL1 overexpression promotes in vitro migratory but not proliferation and colony formation abilities of both H1299 and H460 cells. Consistently, we found that MPZL1 knockdown impairs the migratory abilities of A549 and H1775 cells. Moreover, we found that MPZL1 knockdown inhibits in vivo metastatic but not tumor growth abilities of the A549 cells. Additionally, a total of 297 differentially expressed genes (DEGs) were identified by RNA sequencing in A549 cells upon MPZL1 knockdown. By integrative analysis of DEGs regulated by MPZL1 in A549 cells and human NSCLC tissues, we revealed that COL11A1 is the potential effector gene that positively regulated by MPZL1 and correlates with poor prognosis of NSCLC patients. In conclusion, our work indicates that one of the mechanisms by which MPZL1 promotes NSCLC metastasis is through upregulating the COL11A1, and MPZL1 can be used as a biomarker to predict the prognosis of NSCLC patients.

Neuroblastoma is the most common and deadliest tumor in infancy. WDR5 (WD Repeat Domain 5), a critical factor supporting an N-myc transcriptional complex via its WBM site and interacting with chromosome via its WIN site, promotes the progression of neuroblastoma, thus making it a potential anti-neuroblastoma drug target. So far, a few WIN site inhibitors have been reported, and the WBM site disruptors are rare to see. In this study we conducted virtual screening to identify candidate hit compounds targeting the WBM site of WDR5. As a result, 60 compounds were selected as candidate WBM site inhibitors. Cell proliferation assay demonstrated 6 structurally distinct WBM site inhibitors, numbering as compounds 4, 7, 11, 13, 19 and 22, which potently suppressed 3 neuroblastoma cell lines (MYCN-amplified IMR32 and LAN5 cell lines, and MYCN-unamplified SK-N-AS cell line). Among them, compound 19 suppressed the proliferation of IMR32 and LAN5 cells with EC50 values of 12.34 and 14.89 μM, respectively, and exerted a moderate inhibition on SK-N-AS cells, without affecting HEK293T cells at 20 μM. Analysis of high-resolution crystal complex structure of compound 19 against WDR5 revealed that it competitively occupied the hydrophobic pocket where V264 was located, which might disrupt the interaction of MYC with WDR5 and further MYC-medicated gene transcription. By performing RNA-seq analysis we demonstrated the differences in molecular action mechanisms of the compound 19 and a WIN site inhibitor OICR-9429. Most interestingly, we established the particularly high synergy rate by combining WBM site inhibitor 19 and the WIN site inhibitor OICR-9429, providing a novel therapeutic avenue for neuroblastoma.

OBJECTIVE:The roles of circRNAs in neuroblastoma (NB) are unclear. We used next-generation sequencing to detect the circRNA expression profiles in NB to identify the key circRNAs and analyzed the relationships between the circRNAs and clinical features.METHODS:Five paired neuroblastoma tumor and adjacent normal fetal adrenal medulla samples were collected for high-throughput RNA sequencing. Bioinformatics analysis was performed for functional annotation of the host genes of differentially expressed circRNAs. Validation of dysregulated circRNAs was performed by real-time quantitative reverse transcription polymerase chain reaction. The relationships between the key circRNAs and clinical features were analyzed. In addition, overexpression of key circRNAs in an NB cell line, as well as cell proliferation assays, colony formation assays and cell migration assays, was conducted to investigate the biological functions of key circRNAs.RESULTS:A total of 4704 differentially expressed circRNAs were found, including 2462 up-regulated and 2242 down-regulated circRNAs. According to our previous studies, the predicted target circRNAs of miR-21 involved in tumorigenic signaling pathways were selected, including circRNA-TBC1D4, circRNA-NAALAD2 and circRNA-TGFBR3. These circRNAs were associated with clinical features, and the circRNA expression was significantly lower (P < 0.05) in the NB tissues than in normal adrenal tissues. Overexpression of circRNA-TBC1D4 promotes NB cell migration, but not proliferation and colony-formation in vitro.CONCLUSION:We suggest that circRNA-TBC1D4, circRNA-NAALAD2 and circRNA-TGFBR3 may be cancer suppressor genes, which act by sponging miR-21 in NB. Further investigations are needed to elucidate the underlying mechanism.

Objective:To further classify liver hemangiomas to examine whether or not a continuum existed between multifocal versus diffuse liver hemangioma.Methods:Between January 2000 and January 2020, a total of 43 children with a diagnosis of multifocal or diffuse hepatic hemangioma at Children's Hospital of Fudan University were analyzed retrospectively. They were divided into multifocal-type (n=34) and diffuse-type (n=9) groups according to radiological features. And multifocal-type group was further divided into countable (MC)(n=6) or uncountable (MU)(n=28) subgroups according to whether lesions were countable or not. Clinical features between each group were compared.Results:For MC, MU and diffuse-type groups, the incidence of cutaneous hemangioma was 66.7%(4/6), 50.0%(14/28) and 44.4%(4/9); the incidence of hepatomegaly 0, 46.4%(13/28) and 100%(9/9); the incidence of heart failure 0, 28.6%(8/28) and 44.4%(4/9); the incidence of dyspnea 0, 25.0%(7/28) and 66.7%(6/9); the incidence of hypothyroidism 0, 21.4%(6/28) and 77.8%(7/9). The incidence of hepatomegaly, dyspnea and hypothyroidism was higher in diffuse-type group with statistical significance than that in MC group ( P<0.001; P=0.017; P=0.006) and that in MU group ( P=0.004; P=0.032; P=0.004). And the incidence of hepatomegaly was higher in MU group than that in MC group ( P=0.040). Among 34 cases in multifocal-type group, 16 cases (47.1%) were observed and 18 cases (52.9%) received medical treatment or medical treatment plus interventional measures. Particularly, 6 cases (100%) in MC group were observed; 10/28 cases (35.7%) in MU group were observed and 18 cases (64.3%) received medical treatment or medical treatment plus interventional measures. Nine cases (100%) in diffuse-type group received medical treatment and 3 cases (33.3%) received medical treatment plus interventional measures. The proportion of children receiving medical treatment or medical treatment plus interventional measures in diffuse-type group was higher than those in MC/MU/multifocal-type group with statistical significance ( P=0.027; P=0.038; P<0.001). And the proportion of those receiving medical treatment or medical treatment plus interventional measures was also higher in MU group than that in MC group with statistical significance ( P=0.006). During a follow-up period of over 6 months, all children in multifocal-type group finally survived with stable conditions. However, 2 cases (22.2%) in diffuse-type group died of severe heart failure after medical treatment plus hepatic arteriovenous fistula embolization. In contrast, the rate of complete remission was 83.3%(5/6) in MC group, 50.0%(14/28) in MU group and 33.3%(3/9) in diffuse-type group. It decreased gradually. Conclusions:MU group is a subgroup of multifocal hepatic hemangioma with too-numerous-to-count lesions on transverse sections of CT/MRI. With similar clinical features to diffuse hepatic hemangioma, there may be a continuum between multifocal and diffuse hepatic hemangiomas. Timely treatment is needed.

Background: Neuroblastoma (NB) is the most common malignant solid tumor in children. This study aimed to investigate investigate the prognostic value of pretreatment plasma D-dimer levels, and its relationship with clinical features in patients with NB. Methods: A total of 136 patients were included in this retrospective study. Information of diagnosis, laboratory examination, imaging examination and treatment in medical records was collected. The correlation between plasma D-dimer levels and other clinical characteristics and outcomes was analyzed statistically. Results: Pretreatment plasma D-dimer levels were significantly associated with unfavorable clinical characteristics including INSS III and IV stage, high-risk disease, metastatic disease, MYCN-amplification, poor histologic type, large tumor size, and elevated levels of lactate dehydrogenase (LDH), neuron-specific enolase (NSE) and serum ferritin (SF). Compared to levels before treatment, levels of plasma D-dimer significantly decreased after 2 and 4 cycles of chemotherapy. The patients who had higher D-dimer levels had worse overall survival (3-year OS rate, high vs. middle vs. low D-dimer group, 25.8% ± 7.8% vs. 57.1% ± 10.8% vs. 86.8% ± 4.4%, P< .001) and progression-free survival (3-year PFS rate, high vs. middle vs. low D-dimer group, 17.2% ± 7.2% vs. 43.7% ± 11.6% vs. 70.9% ± 5.9%, P< .001). According to multivariate analysis, high D-dimer level was confirmed to be an independent prognostic factor for OS (high vs. low, HR, 4.715; 95% CI, 1.143-19.452; P= .032). Conclusions: High pretreatment plasma D-dimer levels were remarkably associated with advanced disease and could serve as a novel biomarker predicting the outcomes of NB patients.Funding Statement: This work was sponsored by Personnel training program for distinguished medical young scholar (2017 Kai Li), New hundred people plan of Shanghai health and Family Planning Commission (2017 BR052 Kai Li).Declaration of Interests: All the authors had nothing to disclose.Ethics Approval Statement: This retrospective study was approved by the Ethics Committee of Children’s Hospital of Fudan University. The written informed consents were received from the patients' parents before treatment.