Organoid-Based Transcriptomics Indicate IFIT-Associated Immune Modulation during Cryptotanshinone Treatment in Bladder Cancer

“GO (Gene Ontology)[33] categorizes genes into biological processes, molecular functions, and cellular components, while KEGG[34] (Kyoto Encyclopedia of Genes and Genomes) maps genes to specific pathways.”

“GO functional and KEGG pathway enrichment analyses and visualizations were performed using the R packages clusterProfiler[35], enrichplot, and ggplot2.”

“Subsequent analyses were performed in R (version 4.3.1) [30] using RStudio as the development environment.”

“Chemokines and their receptors coordinate the trafficking and spatial organization of immune cells within tumors[59], whereas MHC molecules are involved in antigen presentation and subsequent T-cell activation, which are essential for antitumor immune responses[58, 60].”

“Importantly, these effects were also observed in a three-dimensional bladder tumor organoid model, which captures key features relevant to the tumor microenvironment[74].”

“If unavailable, predicted structures from the AlphaFold Protein Structure Database[47, 48], linked to UniProt[49] entries, can be used (Table S1).”

“Chemokines and their receptors coordinate the trafficking and spatial organization of immune cells within tumors[59], whereas MHC molecules are involved in antigen presentation and subsequent T-cell activation, which are essential for antitumor immune responses[58, 60].”

“10.2), with further evaluation performed through the cytoNCA plugin[36] to assess the degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC).”

“Functional annotation indicated that many of these genes belong to interferon-stimulated genes (ISGs), which are known to participate in antiviral responses and immune-related processes in cancer[63, 64].”

“Chemokines and their receptors coordinate the trafficking and spatial organization of immune cells within tumors[59], whereas MHC molecules are involved in antigen presentation and subsequent T-cell activation, which are essential for antitumor immune responses[58, 60].”

“Although chemotherapy and radical cystectomy continue to represent standard treatment approaches, their clinical benefits are often constrained by treatment resistance and disease relapse[71].”

“At the mechanistic level, GeneMANIA-based network analysis, together with prior literature, supports the involvement of the cGAS–STING[78] and JAK-STAT[79] signaling pathways play important regulatory roles in the expression of IFIT family genes.”

“…originates from urothelial cells and is characterized by clinical manifestations such as hematuria and urinary abnormalities[1]; in 2022, there were approximately 614,000 new cases and 220,000 deaths worldwide, with significantly higher incidence and mortality rates in men compared to women[2].”

“Functional annotation indicated that many of these genes belong to interferon-stimulated genes (ISGs), which are known to participate in antiviral responses and immune-related processes in cancer[63, 64].”

“Their expression is primarily regulated by interferon-induced JAK-STAT [16, 17] and cGAS-STING[18, 19] signaling pathways.”

“Binding affinity thresholds ( −4.25 kcal/mol for standard binding, −5.0 kcal/mol for good binding, and −7.0 kcal/mol for strong binding) were applied based on established criteria[70].”

“Previous studies[65-69] have demonstrated that cyclic GMP–AMP synthase (cGAS) detects aberrant cytoplasmic double-stranded DNA arising from viral infection, pathogenic invasion, or genomic instability in tumor cells, thereby activating the STING1–TBK1–IRF3/7 signaling cascade and inducing type I…”

“1A), including baicalein (BCA)[52], puerarin (PUE)[53], betulinic acid (BA)[54], tanshinone IIA (Tan-IIA)[55], and cryptotanshinone (CTS)[14], all of which have been reported to exhibit anti-bladder cancer activity — on bladder cancer cell lines 5637 and T24 in vitro.”

“Organoids also support personalized treatment by predicting patient-specific drug responses, helping to identify the most effective therapies and streamline drug discovery[12].”

“Their expression is primarily regulated by interferon-induced JAK-STAT [16, 17] and cGAS-STING[18, 19] signaling pathways.”

“Previous studies have demonstrated that CTS promotes apoptosis in bladder cancer cells via the PTEN/PI3K/AKT signaling pathway[14] 4 and suppresses tumor cell invasiveness through modulation of the mTOR/β-catenin/N-cadherin axis[15].”