biostudies-arrayexpressMária DubišováMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15669Cancer cells with severe defects in mitochondrial DNA (mtDNA) can import mitochondria via horizontal mitochondrial transfer (HMT) to restore respiration. Mitochondrial respiration is necessary for the activity of dihydroorotate dehydrogenase (DHODH), an enzyme of the inner mitochondrial membrane that catalyzes the fourth step of de novo pyrimidine synthesis. Here, we investigated the role of de novo synthesis of pyrimidines in driving tumor growth in mtDNA-deficient (ρ0) cells. While ρ0 cells grafted in mice readily acquired mtDNA, this process was delayed in cells transfected with alternative oxidase (AOX), which combines the functions of mitochondrial respiratory complexes III and IV. The ρ0 AOX cells were glycolytic but maintained normal DHODH activity and pyrimidine production. Deletion of DHODH in a panel of tumor cells completely blocked or delayed tumor growth. The grafted ρ0 cells rapidly recruited tumor-promoting/stabilizing cells of the innate immune system, including pro-tumor M2 macrophages, neutrophils, eosinophils, and mesenchymal stromal cells (MSCs). The ρ0 cells recruited MSCs early after grafting, which were potential mitochondrial donors. Grafting MSCs together with ρ0 cancer cells into mice resulted in mitochondrial transfer from MSCs to cancer cells. Overall, these findings indicate that cancer cells with compromised mitochondrial function readily acquire mtDNA from other cells in the tumor microenvironment to restore DHODH-dependent respiration and de novo pyrimidine synthesis. The inhibition of tumor growth induced by blocking DHODH supports targeting pyrimidine synthesis as a potential widely applicable therapeutic approach.biostudies-arrayexpressNucleic Acid Extraction - Total RNA was extracted using the Total RNA Purification Kit (NorgenBiotech, #17200) according to the manufacturer’s protocol. Total RNA was diluted in 20 μL of elution buffer (included in the kit). RNA Clean & Concentrator-5 (Zymo Research, #R1013) was used to remove contaminating genomic DNA. DNase I treatment was done following the manufacturer’s instructions (Appendix - In-column DNase I treatment). The final total RNA was diluted in 20 μL of DNase/RNase-free water (included in the kit). Concentration of total RNA was assessed using a spectrophotometer (Nanodrop 2000, ThermoFisher Scientific), and the quality of the RNA was assessed using Fragment Analyzer 5200 (Agilent, RNA 15 nt Kit, #DNF-471). The RNA quality number (RQN) was above 8 for all samples.Sample Collection - Quantitative real-time reverse transcription PCR (qRT-PCR) Total RNA was extracted from 4T1, 4T1 ρ0 and 4T1 ρ0 AOX cells in triplicates using RNAzol (Molecular Research Center), and its purity and concentration were assessed using NanoDrop (ThermoFisher Scientific).Library Construction - Total RNA (100 ng) was used for the library preparation using the QuantSeq 3’ mRNA-Seq V2 Library Prep Kit with UDI (Lexogen, #192.24) according to the manufacturer’s protocol. Library qualities were assessed using the Fragment Analyzer 5200 (Agilent, High Sensitivity NGS Fragment Kit, #DNF-474), and concentrations were determined by the Qubit 4 Fluorometer (ThermoFisher Scientific, dsDNA Quantification Assay Kit, #Q32854). Libraries were sequenced using NextSeq500 Mid Output 150 cycles (Illumina) with 144 cycles.Sequencing - Reads were filtered for low-quality reads and adaptor sequences using Cutadapt (v. 3.5). The cleaned reads were then aligned using Hisat2 (v. 2.2.1) to the Mouse genome version GRCm39 and annotation version GCF_000001635.27. A count table was then generated using FeatureCounts (v. 2.0.3). The counts were normalized and analyzed using pyDESeq2 (v. 0.3.5) in Python (v. 3.11). Differential gene expression (DEG) was assessed across all conditions for each experiment.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Reads were filtered for low-quality sequences and adaptor contamination using Cutadapt (v. 3.5). The cleaned reads were aligned to the Mouse genome GRCm39 using Hisat2 (v. 2.2.1). Gene-level counts were generated using FeatureCounts (v. 2.0.3). Counts were normalized and analyzed using pyDESeq2 (v. 0.3.5) in Python (v. 3.11). Differential gene expression was assessed across all experimental conditions.UnknownTranscriptomicsGenomicsProteomicsNextSeq 500RNA-seq of coding RNAMus musculusMária DubišováfalseMitochondrial Transfer Rescues Respiration to Support De Novo Pyrimidine Biosynthesis and Tumor ProgressionCancer cells with severe defects in mitochondrial DNA (mtDNA) can import mitochondria via horizontal mitochondrial transfer (HMT) to restore respiration. Mitochondrial respiration is necessary for the activity of dihydroorotate dehydrogenase (DHODH), an enzyme of the inner mitochondrial membrane that catalyzes the fourth step of de novo pyrimidine synthesis. Here, we investigated the role of de novo synthesis of pyrimidines in driving tumor growth in mtDNA-deficient (ρ0) cells. While ρ0 cells grafted in mice readily acquired mtDNA, this process was delayed in cells transfected with alternative oxidase (AOX), which combines the functions of mitochondrial respiratory complexes III and IV. The ρ0 AOX cells were glycolytic but maintained normal DHODH activity and pyrimidine production. Deletion of DHODH in a panel of tumor cells completely blocked or delayed tumor growth. The grafted ρ0 cells rapidly recruited tumor-promoting/stabilizing cells of the innate immune system, including pro-tumor M2 macrophages, neutrophils, eosinophils, and mesenchymal stromal cells (MSCs). The ρ0 cells recruited MSCs early after grafting, which were potential mitochondrial donors. Grafting MSCs together with ρ0 cancer cells into mice resulted in mitochondrial transfer from MSCs to cancer cells. Overall, these findings indicate that cancer cells with compromised mitochondrial function readily acquire mtDNA from other cells in the tumor microenvironment to restore DHODH-dependent respiration and de novo pyrimidine synthesis. The inhibition of tumor growth induced by blocking DHODH supports targeting pyrimidine synthesis as a potential widely applicable therapeutic approach.2025-10-14T00:00:00Z2025-10-14T09:21:31.306Z2025-10-13T14:30:49.004ZE-MTAB-15669ERP181107EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184