Esophageal squamous cell carcinoma (ESCC) is a lethal disease due to its high invasiveness and metastatic potential, but the mechanisms underlying its progression remain unclear. Although high levels of Toll-like receptor 9 (TLR9) are correlated with poor survival in ESCC, the function and regulation of TLR9 in ESCC cells remain unclear. In this study, we analyzed an ESCC tissue cohort and the TCGA_ESCC database and used mass spectrometry and RNA sequencing to explore mechanisms underlying TLR9 function, which were confirmed by in vitro and in vivo experiments. We found that TLR9 promoted cell motility and conferred resistance to cisplatin in ESCC cells. Mechanistically, TLR9 led to activation of epithelial–mesenchymal transition (EMT) via the NF-κB signaling pathway. Proteomic analysis showed that TLR9 agonist treatment mimicked the effects of TLR9 overexpression on EMT in ESCC cells, whereas TLR9 inhibitor suppressed TLR9-induced activation of EMT. Analyses on the TCGA_ESCC database and multiple miRNA databases revealed positive and negative regulation of TLR9 mRNA levels by SPI1 and miR-574-5p, respectively, which revealed two novel regulation routes for TLR9: one is SPI1-mediated positive regulation, and the other is cisplatin/IRF1/miR-574-5p-mediated negative regulation. The combination of cisplatin with a TLR9 inhibitor significantly improved survival in tumor-bearing mice. Our work provided novel insights into TLR9 in ESCC, suggesting that TLR9 could be a biomarker to predict the prognosis of ESCC patients as well as evaluate the response of ESCC patients to cisplatin treatment. Therefore, targeting TLR9 might represent a potential therapeutic strategy for ESCC treatment.

The mechanical environment is crucial for intervertebral disc degeneration (IDD). However, the mechanisms underlying the regulation of cartilage endplate (CEP) calcification by altered matrix stiffness remain unclear. In this study, we found that matrix stiffness of CEP was positively correlated with the degree of IDD, and stiff matrix, which mimicked the severe degeneration of CEP, promoted inorganic phosphate-induced calcification in CEP chondrocytes. Co-expression analysis of the miRNA and mRNA profiles showed that increasing stiffness resulted in up-regulation of miR-20a and down-regulation of decreased ankylosis protein homolog (ANKH) during inorganic phosphate-induced calcification in CEP chondrocytes. Through a dual luciferase reporter assay, we confirmed that miR-20a directly targets 3'-untranslated regions of ANKH. The inhibition of miR-20a attenuated the calcium deposition and calcification-related gene expression, whereas the overexpression of miR-20a enhanced calcification in CEP chondrocytes on stiff matrix. The rescue of ANKH expression restored the decreased pyrophosphate efflux and inhibited calcification. In clinical samples, the levels of ANKH expression were inversely associated with the degeneration degree of CEP. Thus, our findings demonstrate that the miR-20a/ANKH axis mediates the stiff matrix- promoted CEP calcification, suggesting that miR-20a and ANKH are potential targets in restraining the progression of IDD.

Radiotherapy is one of the standard therapeutic regimens for medulloblastoma (MB). Tumor cells utilize DNA damage repair (DDR) mechanisms to survive and develop resistance during radiotherapy. It has been found that targeting DDR sensitizes tumor cells to radiotherapy in several types of cancer, but whether and how DDR pathways are involved in the MB radiotherapy response remain to be determined. Single-cell RNA sequencing was carried out on 38 MB tissues, followed by expression enrichment assays. Fanconi anemia group D2 gene (FANCD2) expression was evaluated in MB samples and public MB databases. The function of FANCD2 in MB cells was examined using cell counting assays (CCK-8), clone formation, lactate dehydrogenase activity, and in mouse orthotopic models. The FANCD2-related signaling pathway was investigated using assays of peroxidation, a malondialdehyde assay, a reduced glutathione assay, and using FerroOrange to assess intracellular iron ions (Fe2+ ). Here, we report that FANCD2 was highly expressed in the malignant sonic hedgehog (SHH) MB subtype (SHH-MB). FANCD2 played an oncogenic role and predicted worse prognosis in SHH-MB patients. Moreover, FANCD2 knockdown markedly suppressed viability, mobility, and growth of SHH-MB cells and sensitized SHH-MB cells to irradiation. Mechanistically, FANCD2 deficiency led to an accumulation of Fe2+ due to increased divalent metal transporter 1 expression and impaired glutathione peroxidase 4 activity, which further activated ferroptosis and reduced proliferation of SHH-MB cells. Using an orthotopic mouse model, we observed that radiotherapy combined with silencing FANCD2 significantly inhibited the growth of SHH-MB cell-derived tumors in vivo. Our study revealed FANCD2 as a potential therapeutic target in SHH-MB and silencing FANCD2 could sensitize SHH-MB cells to radiotherapy via inducing ferroptosis. © 2024 The Pathological Society of Great Britain and Ireland.

PDF file - 169K, Supplementary Table S1. The clinical features of the glioma specimens used in this study. Supplementary Table S2. Primers used for qRT-PCT, miRNA cloning and luciferase plasmid construction. Supplementary Table S3. Top 20 downregulated miRNAs in tumor tissue (GBM-T) versus adjacent normal tissue (GBM-N). Supplementary Table S4. Top 20 upregulated miRNAs in tumor tissue (GBM-T) versus adjacent normal tissue (GBM-N). Supplementary Table S5. The correlation between miR-663 expression and clinicopathological factors of patients with astrocytoma (Grade I and II). Supplementary Table S6. The correlation between miR-663 expression and clinicopathological factors of patients with anaplastic astrocytoma (Grade III). Supplementary Table S7. The correlation between miR-663 expression and clinicopathological factors of patients with GBM (Grade IV). Supplementary Table S8. Univariate analysis for disease-free survival and overall survival in GBM patients. Supplementary Table S9. Top 30 overlapped predicted targets of miR-663 and their degrees based on signal transduction network.

Supplementary tables from S1-S6.

PDF file - 546K, Supplementary Fig. S3. Identification of PIK3CD as a target by miR-663.

The effect of ATRA on the sensitivity to NK cell killing, survival, and lung metastasis of BCSCs

PDF file - 102K, Supplementary Figures S4: A series of constructs containing that luciferase expression was driven by promoters of RPRM, CDKN1A,GADD45A andGADD45B.

Detecting low-abundance mutations is of particular interest in the fields of biology and medical science. However, most currently available molecular assays have limited sensitivity for the detection of low-abundance mutations. Here, we established a platform for detecting low-level DNA mutations with high sensitivity and accuracy by combining enhanced- ice -COLD-PCR (E- ice -COLD-PCR) and pyrosequencing with di-base addition (PDBA). The PDBA assay was performed by selectively adding one di-base (AG, CT, AC, GT, AT, or GC) instead of one base (A, T, C, or G) into the reaction at a time during sequencing primer extension and thus enabling to increase the sequencing intensity. A specific E- ice -COLD-PCR/PDBA assay was developed for the detection of the most frequent BRAF V600E mutation to verify the feasibility of our method. E- ice -COLD-PCR/PDBA assay permitted the reliable detection of down to 0.007% of mutant alleles in a wild-type background. Furthermore, it required only a small amount of starting material (20 pg) to sensitively detect and identify low-abundance mutations, thus increasing the screening capabilities in limited DNA material. The E- ice -COLD-PCR/PDBA assay was applied in the current study to clinical formalin-fixed paraffin-embedded (FFPE) and plasma samples, and it enabled the detection of BRAF V600E mutations in samples that appeared as a wild type using PCR/conventional pyrosequencing (CP) and E- ice -COLD-PCR/CP. E- ice -COLD-PCR/PDBA assay is a rapid, cost-effective, and highly sensitive method that could improve the detection of low-abundance mutations in routine clinical use.

PDF file - 747K, Supplementary Fig. S2. The effects of miR-663 on GBM cells.

PDF file - 455K, Supplementary Fig. S1. The prognostic values of miR-663 in GBM patients stratified by IDH1/ATRX status.

Knockdown of MICA or MICB in non-BCSCs.

Malignant gliomas are largely refractory to immune checkpoint blockade (ICB) therapy. To explore the under-lying immune regulators, we examine the microenvironment in glioma and find that tumor-infiltrating T cells are mainly confined to the perivascular cuffs and express high levels of CCR5, CXCR3, and programmed cell death protein 1 (PD-1). Combined analysis of T cell clustering with T cell receptor (TCR) clone expansion shows that potential tumor-killing T cells are mainly categorized into pre-exhausted/exhausted and effector CD8+ T subsets, as well as cytotoxic CD4+ T subsets. Notably, a distinct subpopulation of CD4+ T cells ex-hibits innate-like features with preferential interleukin-8 (IL-8) expression. With IL-8-humanized mouse strain, we demonstrate that IL-8-producing CD4+ T, myeloid, and tumor cells orchestrate myeloid-derived suppres-sor cell infiltration and angiogenesis, which results in enhanced tumor growth but reduced ICB efficacy. Antibody-mediated IL-8 blockade or the inhibition of its receptor, CXCR1/2, unleashes anti-PD-1-mediated antitumor immunity. Our findings thus highlight IL-8 as a combinational immunotherapy target for glioma.

Supplementary Text including Supplementary Methods and Supplementary Figure Legends

Suppression of MICA/B by overexpressing miR-20a in BCSCs.

<p>PDF file - 637K, Supplementary Figures S5: The representative flow plots for cell cycle analysis.</p>

Supplementary Figure S2. Doxycycline treatment induces miR-663 expression in GBM cells transfected with miR-663 inducible lentiviral vectors.

<p>PDF file - 637K, Supplementary Figures S5: The representative flow plots for cell cycle analysis.</p>

PDF file - 195K, Supplementary Figures S1: Expression of MEF2D and miR-122 in HCC cell lines and patient-derived primary HCC cultures of tumor cells.