Mfn1-mediated imbalanced mitochondrial dynamics promotes ovarian cancer stemness by inducing metabolic reprogramming

Mfn1-mediated imbalanced mitochondrial dynamics promotes ovarian cancer stemness by inducing metabolic reprogramming | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mfn1-mediated imbalanced mitochondrial dynamics promotes ovarian cancer stemness by inducing metabolic reprogramming Rahail Ashraf, Kalpana Tankay, Manita Raina, Athira FNU, Sanjay Kumar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7728954/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Mar, 2026 Read the published version in Journal of Physiology and Biochemistry → Version 1 posted 13 You are reading this latest preprint version Abstract Ovarian cancer is the leading cause of death from reproductive system cancer among women worldwide. Ovarian cancer stem cells (OCSCs) are critically involved in metastasis, tumor recurrence, and chemoresistance, and are a significant bottleneck in the treatment. Several studies demonstrated metabolic rewiring and altered mitochondrial dynamics in CSCs. However, the role of Mfn1-mediated imbalanced mitochondrial dynamics in ovarian cancer stemness remains poorly understood. In this study, quantification of mtDNA indicates that CSCs have increased mitochondrial mass compared to the parental adherent cells. CD133 + enriched cells and cancer stem-like cells (spheroid cultured from OC cells) have higher Mfn1 expression and mitochondrial fusion activity. CSCs have increased oxidative phosphorylation (OXPHOS), ATP, and reduced ROS compared to the parental adherent cells. Disruption of mitochondrial dynamics by depletion of Mfn1 modulates the growth and size of spheroid formation and OC stemness. Seahorse analyzer analysis confirms the functional impact of Mfn1 knockdown on mitochondrial respiration. Overexpression of Mfn1 in OC cells, which has a low level of Mfn1 expression, induces increased mitochondrial respiration. Furthermore, to elucidate the relationship between Mfn1 and OXPHOS complex activities and their role in OC stemness, we treated OC cells with 2-Deoxy-D-glucose (2DG), which induces OXPHOS and modulates the expression of cancer stemness markers of OCSCs through Mfn1. During stemness acquisition, CSCs undergo Mfn1-mediated mitochondrial rearrangement, which could be a potential therapeutic strategy against ovarian cancer. Mitochondrial fusion Mfn1 ETC complexes Metabolic reprogramming Ovarian cancer stem cells CD133+ 2-Deoxy-D-glucose Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Epithelial ovarian cancer (EOC) is the most lethal reproductive system cancer, accounting for more deaths than any other gynecological malignancy worldwide. The factors limiting current therapy's efficacy are the delayed onset of symptoms, poor prognosis, therapeutic resistance, and disease recurrence. Cancer stem cells (CSCs) are a small subgroup of the tumor population with self-renewal properties contributing to cancer progression, disease recurrence, and drug resistance [ 7 , 25 ]. Thus, understanding the precise molecular mechanism underpinning the stemness properties of ovarian cancer stem cells (OCSCs) will be relevant in developing effective strategies to combat ovarian cancer (OC). CSCs have distinct metabolic properties compared to non-CSC tumor cells [ 35 , 39 ]. Aberrant ATP generation by the glycolytic pathway is a well-known feature of non-CSCs, required to support the biosynthetic demands for cell growth and metastasis [ 12 ]. Under particular environmental conditions, CSCs may switch between glycolysis and oxidative phosphorylation (OXPHOS). In CSC, epithelial-to-mesenchymal transition (EMT) induction drives highly glycolytic metabolism, which depends on mitochondrial dynamics [ 26 , 30 ]. Mfn1 knockdown increases mitochondrial fission, induces the expression of glycolytic genes, and increases migration of cells [ 52 ]. Several studies demonstrated the reprogramming of glycolysis to mitochondrial OXPHOS and its crucial role in CSC maintenance [ 46 , 28 , 27 ]. Hypoxia induces CSC-like phenotypes in human OC cells [ 34 ]. Hypoxia acts on mitochondria by changing the protein composition of the electron transport chain (ETC), mitochondrial mass, and mitochondrial morphology. Consequently, it comprises an altered respiration rate by modulating OXPHOS and increasing or decreasing ROS-producing capacity. OXPHOS is adapted to hypoxia by remodeling the ETC and TCA cycle activity. Metabolic alteration in CSCs may contribute to therapeutic resistance. Thus, altered metabolism in CSCs needs to be better understood. In small-cell lung cancer, CSCs preferentially use OXPHOS over glycolysis to fulfill their energy requirements [ 17 ]. The notch-1-AMPK axis enhances mitochondrial OXPHOS to support lung cancer stem-like cell expansion [ 29 ]. Lipogenesis induces OPA1-mediated mitochondrial fusion that maintains stemness in human non-small cell lung cancer [ 28 ]. In liver CSCs, the treatment of mitochondrial fission inhibitor mDivi-1 (inhibitor of Drp1 activity) reduces CD133 and CD44 expression, thus decreasing stemness properties by inhibiting OXPHOS [ 27 ]. CD133, CD44, Oct4, Nanog, and Sox2 are overexpressed in CSCs and are well-established CSC markers [ 54 ]. In pancreatic cancer, CSCs switch from glycolysis to OXPHOS to meet the energy requirements [ 38 ]. MYC and MCL1 confer stemness in drug-resistant breast cancer cells by expanding mitochondrial OXPHOS [ 23 ]. In OC, platinum treatment enriches CSCs by inducing mitochondrial OXPHOS, therapeutic resistance, and disease recurrence [ 42 ]. However, the underlying mechanisms for the deviant mitochondrial metabolism of OC stemness are mainly unknown. Metabolic plasticity correlates with mitochondrial remodeling to maintain cellular homeostasis. Mitochondria are dynamic organelles that reorganize continually to form and degrade their intracellular networks through balanced fusion and fission events, and changes in these networks modulate mitochondrial functions, including energy production [ 37 ]. Mitochondrial morphology varies and changes rapidly through mitochondrial fission and fusion regulators in response to metabolic cues [ 50 ]. Glycolysis and OXPHOS are the two preferred pathways used by EOC to provide sufficient energy to promote cancer progression [ 32 ]. In human pluripotent stem cells, ATP production largely depends on OXPHOS during differentiation [ 44 , 10 ]. Several studies have reported increased OXPHOS linked with cell proliferation, migration, metastasis, maintaining cancer stemness properties, and chemoresistance in OC [ 20 , 10 ]. The mechanism underlying elevated OXPHOS in OCSCs remains undefined. It is widely believed that balanced mitochondrial fission and fusion states may have a decisive role in the control of metabolism, and elongated networks of mitochondria are more bioenergetically efficient [ 18 ]. Mitochondrial fusion favors high mitochondrial metabolism for energy production in CSCs [ 10 ], making it relevant to understand its role in the underlying mechanism for elevated OXPHOS. Mfn1 and 2 are large GTPases located in the outer mitochondrial membrane, and another GTPase, OPA1, in the inner mitochondrial membrane regulates mitochondrial fusion [ 13 ]. Fis1, an outer membrane protein, and other adaptor proteins recruit Drp1, a dynamin-related GTPase protein, and coordinate the mitochondrial fission process. [ 13 ]. A recent study demonstrated that an AKT inhibitor suppresses breast cancer stemness by Mfn1-modulated mitochondrial dynamics and functions [ 16 ]. Mitofusin depletion induces pluripotency through remodeling mitochondrial dynamics and consequent reprogramming of cellular metabolism [ 41 ]. In the Drosophila brain, mitochondrial fusion-induced metabolic reprogramming could be crucial for immortalizing tumor-initiating cells (TICs) [ 9 ]. However, the role of Mfn1-mediated altered mitochondrial dynamics in human OCSCs is unclear. In the present study, we demonstrated that CSCs have increased mitochondrial mass, enhanced Mfn1 expression and mitochondrial fusion, and higher expression of ETC complexes subunits. Downregulation of Mfn1 in CSC-like cells reduced the expression of stemness properties by downregulating the OXPHOS complexes and mitochondrial respiration. Overexpression of Mfn1 in SKOV-3 cells, which has a low level of endogenous Mfn1 expression, increases mitochondrial respiration. Mfn1-mediated imbalanced mitochondrial dynamics promotes OC stemness by inducing OXPHOS. Our findings indicate that ovarian CSCs undergo Mfn1-mediated mitochondrial remodeling and, consequently, mitochondrial functions, which could be a potential therapeutic strategy in ovarian CSCs. 2. Methodology 2.1. Cell culture and 2 DG treatment SKOV-3 and OVCAR-3 cells were purchased from ATCC. A2780 cells were procured from ECACC. SKOV-3 and A2780 cells were grown and maintained in McCoy’s 5A and RPMI 1640 medium with 10% (v/v) FBS (Sigma, USA), 2mM L-glutamine (Sigma, USA), and 100U/ml of pen-strep (Sigma, USA). OVCAR-3 cells were cultured in RPMI 1640 media with 20% FBS and 0.1% bovine insulin (Sigma, USA) and 100U/ml of pen-strep (Sigma, USA). Cells were maintained in a 37°C incubator with 5% CO 2 . Low-passage cells were used in the experiments after confirmation of mycoplasma-negative status by periodically testing using a mycoplasma detection kit (Lonza). 2-Deoxy-D-glucose (2DG) was procured from Sigma-Aldrich and used per the manufacturer's instructions. 2.2. Transfection OC cells were seeded and grown till 40–50% confluence in 6-well plates and used for experiments, including Mfn1 knockdown experiments. Transfection of shRNA Mfn1 (Sigma, USA) and control plasmid was performed using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s recommended protocol. 2.3. Spheroid-forming assay and CD133 + isolation 2.3.1. Spheroid culture For spheroid culture, 2 x 10 6 cells were seeded in the ultra-low attachment culture dishes (Corning) with F12/knockout DMEM medium (Thermo Fisher Scientific) supplemented with 20% knockout serum replacement (Life Technologies), 20 ng/mL EGF, 10 ng/mL bFGF, 1% L-glutamine, and 1% penicillin-streptomycin. The spheroids were cultured for 6–8 days, changed media every three days, and grown at 37°C with 5% CO 2 in the CO 2 incubator. 2.3.2. Spheroid formation assay A2780 cells were transfected with shMfn1 and a control plasmid. After overnight culture, A2780 cells were seeded in low attachment dishes (Corning) containing CSC media and incubated for up to 6 days at 37°C with 5% CO 2 . Fresh media was replaced every 3 days, and the spheroids were imaged daily using an inverted microscope. Spheroids growth was assessed by measuring spheroid size using ImageJ from the obtained microscopic images. 2.3.3. CD133 + isolation Enriched CD133 + cells were isolated using the CD133 MicroBead Kit (Miltenyi Biotec) as per the recommended protocol suggested by the manufacturer and a previously published method [ 4 ]. A2780 cells were grown until confluency and trypsinized. 10 7 total cells were resuspended in MACS buffer, and 20 µL of FcR blocking reagent was added, followed by the addition of 20 µL of CD133 microbeads (Miltenyi Biotec, Germany). After mixing well, the cell suspension was incubated at 4°C on a rotator for 25 minutes, washed twice, and centrifuged at 400g for 15 minutes. The supernatant was aspirated completely, then the cells were resuspended in the buffer. MACS column (Miltenyi Biotec) was placed into the magnetic separator (Miltenyi Biotec), followed by applying the cell suspension onto the MACS LS column (Miltenyi Biotec). CD133 + cells were retained in the column, and the flow-through containing CD133 − cells were collected. Next, the column was removed from the separator, and a buffer was added to flush out the CD133 + cells retained in the column by firmly pushing the plunger onto the column. 2.4. Immunofluorescence and confocal studies For immunofluorescence studies, 2 × 10 4 cells were seeded on coverslips and grown overnight. We transfected OC cells using Mfn1 shRNA and an empty vector and incubated the cells for 24 hrs. Cells were washed thrice and fixed using 4% paraformaldehyde. Next, cells were permeabilized using 0.1% Triton X-100 for 5 minutes, followed by blocking the cells using 5% BSA for 1 hour [ 21 ]. The primary antibodies in suitable dilutions were added and incubated, and after washing thrice, secondary antibodies were added and incubated at room temperature. After washing briefly, coverslips were mounted on slides using DAPI mounting media and imaged using confocal (Leica TCS SP8 and Olympus FV3000) microscopy. Quantitative analysis of the fluorescent intensity of the obtained confocal images was performed using ImageJ software. 2.5. Mitochondrial morphology Spheroids and CD133 + cells were transferred to 35-mm glass-bottom dishes, stained with MitoTracker Deep Red (100 nM in serum-free medium) and kept for 20 minutes at 37°C. After washing cells briefly, confocal microscopy was used to determine mitochondrial morphology by live imaging (Leica TCS SP8). Morphometric properties of mitochondria, including mitochondrial length and aspect ratio, were measured using the Mitochondria Analyzer plugin in ImageJ software. 2.6. Immunoblot analysis For protein extraction, OC cells were lysed in RIPA lysis buffer with freshly added protease, and phosphatase inhibitors and quantified the protein concentration. Lysates were prepared, electrophoresed using SDS-PAGE, and transferred to a PVDF membrane. Next, membranes were blocked using 5% BSA, followed by the addition of primary antibody and incubated overnight at 4°C, followed by the addition of HRP-conjugated secondary antibodies and analyzed using ECL by chemiluminescence imaging system (Amersham Imager, GE Health Bioscience). The western blot data were quantified using ImageJ software and normalized for loading control. The list of resources, such as primary and secondary antibodies, is listed in Supplementary Table S1 . 2.7 RNA isolation and Reverse-transcription q-PCR analysis Total RNA from OC cells was extracted using TRIzol reagent (Invitrogen) as per the manufacturer’s instructions, followed by cDNA generation using iScript cDNA Synthesis kit (BioRad, Cat 1708890). Real-time RT-qPCR was performed using iTaq Universal SYBR green Supermix (BioRad, 725121) in the BioRad CFX Real-Time PCR System. The primers were listed in Supplementary Table S1 . Relative fold change was calculated by ddCT methods using 18S rRNA as an internal control. 2.8. ROS estimation To assess ROS generation in mitochondria, the spheroids were transferred to 35-mm glass-bottom dishes after spheroid development in low attachment dishes. MitoSOX Red reagent (Invitrogen) (5 µM dissolved in serum-free media) was added for 15 minutes and incubated at 37°C as per the suggested protocol by the manufacturer, and images were captured using confocal microscopy (Leica TCS SP8). Quantitative analysis of the fluorescent MitoSOX Red intensity of the obtained confocal images was performed using ImageJ software. 2.9. ATP determination assay Lysate pellets of adherent and spheroid OC were collected to determine the changes in the ATP levels. The ATP determination assay was performed using the ATP determination Kit (Invitrogen) according to the manufacturer’s instructions. Luminescence was recorded using a Multi-Mode Microplate Reader (BioTek). ATP concentrations were calculated using a standard curve and normalized. 2.10. Mitochondrial DNA quantification Total genomic DNA was extracted from adherent and spheroid OC cells using the GeneJet Genomic DNA purification kit (Thermofisher) per the manufacturer’s guidelines. The concentration of extracted DNA was quantified, and 0.03–0.1 ng/µl was used for mtDNA content measurement using RT-qPCR. RT-qPCR was performed using iTaq Universal SYBR green Supermix (BioRad, 725121) in the BioRad CFX Real-Time PCR System. β-actin was used as an internal control. The primers used are listed in Supplementary Table S1 . The threshold cycle difference in control ND1/control β-actin was used to measure the relative abundance of mitochondrial DNA [ 40 ]. 2.11. Measurement of Oxygen Consumption Rate (OCR) The oxygen consumption rate (OCR) was measured using the Seahorse XFe24 Analyzer (Agilent Technologies, USA) to assess mitochondrial respiration. A2780 and SKOV-3 cells were transfected with the shMfn1 and Mfn1 overexpression plasmid, respectively, and seeded at 20,000 cells per well in Seahorse XFe24 cell culture microplates (Agilent Technologies) and kept overnight. Cells were treated with 2DG, and the culture medium was then replaced with Seahorse XF DMEM assay medium (Agilent Technologies) supplemented with 2 mM L-glutamine, 10 mM D-glucose, and 1 mM sodium pyruvate. According to the manufacturer's instructions, OCR was measured using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, Cat. #103015-100). The sequential injection of oligomycin (1.5 µM), FCCP (0.5 µM), and rotenone/antimycin (0.5 µM) enabled the determination of basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. Data were normalized to cell number by staining with 0.1% crystal violet and analyzed using the Wave software (Agilent Technologies). 2.12. scRNA sequencing data acquisition, preprocessing, and data integration The single-cell RNA sequencing dataset, GSE184880, is obtained from the publicly available NCBI-GEO (Home - GEO - NCBI) database. The scRNA sequencing dataset contains normal and high-grade serous ovarian carcinoma tissue samples. 5 normal tissue samples and 7 cancerous tissue samples from OC were included for the analysis. The gene expression count matrix and associated metadata were loaded into R(v4.5.0), and the preprocessing and downstream analysis were conducted using the Seurat package. We created the Seurat object using the gene expression matrix, and then the Seurat object was normalized, and expression values were scaled using the NormalizeData and ScaleData functions. The FindVariable function was used to identify variable genes. The RunPCA function was employed to conduct principal component analysis (PCA). Clustering was carried out using principal components with FindNeighbors and FindClusters at 3 resolutions. 2.12.1 Identification of cancer stem-like cells, differential gene expression, and OXPHOS correlation We employed the FindAll markers function via Wilcoxon rank-sum tests to identify different genes in the clustered cells. Each cluster was annotated as a specific cell type based on the canonical marker gene expression. To identify ovarian cancer stem-like cells, canonical stemness markers (CD44, ALDH1A1, PROM1, SOX2, NANOG, POU5F1, EPCAM) were visualized using FeaturePlot and Violin plots. Among these, EPCAM expression was used as a functional classifier for stemness. Pathway activities were quantified using gene module scoring. Genes associated with OXPHOS were obtained from the MSigDB Hallmark gene sets via the msigdbr package. An OXPHOS module score was computed and assigned to each cell (AddModuleScore). Spearman’s rank correlation was used to assess the association between MFN1 expression and both OXPHOS scores. Scatter plots with linear fits and annotated correlation statistics were generated. 2.13. Statistical Methods All data presented are mean ± standard deviation. Statistical difference was determined by Student's t-tests for two-group comparisons. The significance levels are shown as follows: ***p < 0.001, **p < 0.01, and *p < 0.05. The significance level is considered significant if the p-value is less than 0.05. 3. Results 3.1. Ovarian cancer stem cells exhibit high mitochondrial fusion activity To understand the role of altered mitochondrial dynamics and function in CSC, we cultured spheroids from OC cells in CSC culture medium under non-adherent conditions and employed them as cancer stem-like cells (CSLCs) (Supplementary Fig. 1A). Cells cultured in adherent states were used as non-CSCs [ 36 ]. The tumor microenvironment is heterogeneous, and the culture of human cancer cells retains phenotypic and functional differences. The stem cell-like properties of ovarian A2780 and SKOV-3 cultured spheroids were validated by assessing the expression of Nestin, Oct4, and Nanog (Supplementary Fig. 1B, C and D). Next, we checked the mtDNA content in spheroids cultured from A2780 and SKOV-3 OC cells. Spheroids from both cell types have higher mtDNA content than adherent parental cells (Fig. 1 A and B). Ovarian CSCs showed more mitochondrial mass than adherent parental cells. Next, the mitochondrial morphology of CSLCs and non-CSCs cultured from OVCAR-3 and A2780 cells was assessed by MitoTracker-Red staining followed by confocal microscopy. Both non-CSCs of OVCAR-3 and A2780 exhibited smaller, rounded mitochondria. In contrast, the spheroid displayed more tubulated and elongated mitochondria (Fig. 1 C and D). The respective graphs adjacent to the representative images display the quantitative analysis of mitochondrial length and aspect ratio, and our study observed significantly higher mitochondrial length and aspect ratio in the spheroids than in adherent cells. Next, to understand the status of mitochondrial dynamics regulator expression, we performed a western blot, and our data showed the increased expression of mitochondrial fusion proteins Mfn1&2 and reduced expression of Drp1 in spheroids cultured from A2780 and SKOV-3 cells compared to parental adherent cells (Fig. 1 E and F). Previous studies have shown that CD133 is a potential marker for CSCs in many solid tumors, including OC [ 14 ]. Next, by employing CD133 as a classical biomarker of CSC, OCSCs (CD133 + ) were isolated from A2780 cells and further confirmed by immunofluorescence using DAPI and CD133-specific antibodies (Fig. 1 G). Further, the mitochondrial morphology of CD133 − and CD133 + cells was assessed by MitoTracker-Red staining followed by confocal microscopy. CD133 + cells displayed more elongated mitochondria than CD133 − cells, which exhibited fragmented mitochondria (Fig. 1 H). The graphs adjacent to the representative images display significantly higher mitochondrial length and aspect ratio in the CD133 + than in CD133 − cells. We also performed a Western blot to determine the expression of mitochondrial dynamics regulator expression in CD133 + and CD133 − cells. CD133 + cells showed higher Mfn1 and reduced Drp1 expression compared to CD133 − cells (Fig. 1 I), further validating the higher mitochondrial fusion activity in CD133 + cells. Furthermore, a robust increment in Mfn1 expression in spheroids cultured from A2780 and SKOV-3 and CD133 + cells indicates their crucial role in OCSCs maintenance. Mitochondrial dynamics are crucial for mitochondrial morphology and mitochondrial functions. Next, we monitored the overall respiratory activity in the spheroids cultured from SKOV-3 OC cells by measuring cellular ATP levels and compared them with those of parent adherent cells. Spheroids have relatively higher ATP than adherent cells (Fig. 1 J). Further, we used MitoSox red reagent to stain and evaluate mitochondrial ROS generation in CD133 + and CD133 − cells. CD133 + cells had reduced mtROS compared to the CD133 − cells (Fig. 1 K). Our findings indicate that spheroids have higher mitochondrial content, increased mitochondrial fusion activity, and Mfn1 expression. 3.2. Ovarian cancer stem cells have higher oxidative phosphorylation Cells yield energy through ATP predominantly through glycolysis and mitochondrial OXPHOS. Energy metabolism is crucial for maintaining stemness in CSCs [ 27 , 28 ]. In the present study, we characterize OXPHOS by measuring the expression of ETC subunits (NDUFB8 for complex I, SDHB for complex II, UQCRC2 for complex III, MTCO2 for complex IV, and ATP5A for complex V) in CSLCs and compared them with non-CSCs. We performed a Western blot to assess ETC complex components in spheroids and compared them with those of the adherent cells. Both A2780 and OVCAR-3 spheroids had increased expression of most of the ETC complex components compared to the adherent cells, and CD133 + cells had increased expression of ETC complex components compared to the CD133 − cells (Fig. 2 A, B, and C), indicating that CSCs preferably use mitochondrial OXPHOS to generate ATP. Next, we measured the COX, ATP5A, and Cyt-C gene expression in spheroids and compared them with non-CSCs adherent cultured cells (Fig. 2Di-iii) and found that OC spheroids are enriched in COX, ATP5A, and Cyt-C gene expression and thus exhibit a more robust expression of OXPHOS machinery than non-CSCs. Next, we used the publicly available single-cell RNA sequencing (scRNA seq) dataset GSE184880. We subsetted the cancer stem cell-like (CSCL) and non-cancer stem cell-like (non-CSCL) population from the entire tumor cell population based on the expression of EPCAM (Supplementary Fig. 1C, D, and E). scRNA sequencing analysis of ovarian patient tumor cells revealed that MFN1 expression is significantly increased in the CSCL OC cells compared to non-CSCL OC cells. Bar plot showing mean log-normalized expression of MFN1 in CSCL vs Non-CSCL cells (Fig. 2 E). This further strengthens the clinical relevance of Mfn1 abundance in the OCSCL cells in ovarian tumors. To assess whether the Mfn1 upregulation is functionally linked to the mitochondrial metabolism, we next evaluated the OXPHOS activity using a module scoring approach. Ovarian CSCL cells show a significant increase in the OXPHOS module score compared to the non-CSCL cells (Fig. 2 F). In addition to that, correlation analysis demonstrates a modest positive correlation (Spearman’s correlation coefficient, r = + 0.23) between Mfn1 expression and OXPHOS activity. Together, these findings suggest that ovarian CSCs show increased Mfn1 expression and OXPHOS activity, suggesting a potential role of Mfn1 in OXPHOS in ovarian CSCL cells. 3.3. Mfn1 is crucial for stemness in ovarian cancer stem cells Further, to assess the functional outcome of higher mitochondrial fusion in OCSCs, we used shMfn1 to knock down Mfn1 in SKOV-3 and A2780 cells and analyzed stemness properties. Two OC cells, A2780 and SKOV-3, were transfected with shMfn1 and a control plasmid and subjected to a Western blot. Cells after knocking down Mfn1 showed a notably reduced stemness by downregulating the stemness markers such as Oct4, CD44, Nanog, and Sox2 in OC cells (Fig. 3 A and B). Subsequently, we transfected A2780 cells with shMfn1 and a control plasmid, grew spheroids for 5 days, and analyzed sphere formation. Cells with reduced Mfn1 expression inhibited sphere formation and reduced the spheroid’s size compared to the control (Fig. 3 C). We performed the immunofluorescence experiments to evaluate the effect of Mfn1 knockdown on cancer stemness using DAPI, Mfn1, and Oct4 antibodies for the staining. We acquired images after staining, and our study suggested that knocking down Mfn1 in A2780 and OVCAR-3 cells decreased the expression of Oct4 (Fig. 3 D & E) compared to the control. This finding supports the critical role of Mfn1 in maintaining the stemness of OC cells. 3.4. Reduced Mfn1 levels impede oxidative phosphorylation and cancer stemness in OCSCs To understand how Mfn1 contributes to OC cell stemness, we knocked down Mfn1 in A2780 and SKOV-3 cells and performed a western blot to evaluate the ETC complexes I-V expression. The Mfn1 expression correlates with the expression of ETC complexes, as Mfn1 knockdown reduces the expression levels of SDHB, UQCRC2, and ATP5A (Fig. 4 A and B). Further, to ascertain the Mfn1 influence over ETC, we performed an immunofluorescence study using DAPI, Mfn1, and ATP5A antibodies for the staining. Reduced Mfn1 levels in SKOV-3 and A2780 cells correlate with reduced ATP5A expression (Fig. 4 C and D). Further, another immunofluorescence study using DAPI, Mfn1, and MTCO2 antibodies for the staining revealed that attenuation of Mfn1 reduced the MTCO2 expression in A2780 and OVCAR-3 OC cells (Fig. 4 E and F). OCSLCs have higher Mfn1 levels that enhance mitochondrial fusion, induce OXPHOS and promote stemness. The SKOV-3 cell line exhibits relatively low basal Mfn1 expression. Therefore, we overexpressed Mfn1 in SKOV-3 cells and measured the OXPHOS markers. Overexpression of Mfn1 increases the OXPHOS markers, MTCO2, and ATP5A compared to the control (Fig. 4 G). To further assess the effect of Mfn1 overexpression on the OXPHOS, we evaluated the OCR using the Seahorse analyzer. The Mfn1 overexpression increases mitochondrial respiration overall compared to the control (Fig. 4 H). Specifically, the OCR parameters, such as the proton leak and maximal respiration, are significantly increased compared to the control (Fig. 4 Hi-iii). 3.5. 2-DG induces oxidative phosphorylation and promotes stemness in ovarian cancer cells through Mfn1 OXPHOS has two critical functions in driving cancer. It satisfies the bioenergetics demands by providing ATP and funnels carbon from glucose for macromolecule synthesis. Mitochondrial matrix enzymes involved in the tricarboxylic acid (TCA) cycle and transmembrane protein complexes of ETC are fundamental to this process. A recent study has shown that 2DG treatment increases stemness by upregulating OXPHOS in liver CSCs [ 27 ]. 2DG inhibits glucose-6-phosphate production from glucose by reducing the hexokinase and phosphoglucoisomerase activities and stops glycolysis [ 45 ]. It has also been shown to inhibit protein N-glycosylation selectively [ 3 ]. To detect whether 2DG treatment induces OXPHOS in OC cells, we treated two OC cell types, A2780 and OVCAR-3, with 2DG (0, 1.25. 2.5, and 5 mM) and checked the status of ETC I-IV complexes. The expression of ATP5A, SDHB, UQCRC2, and MTCO2 has increased consistently at 2.5 mM in A2780, and the expression of ATP5A, SDHB, UQCRC2, and NDUFB8 at 2.5 mM of 2DG in OVCAR-3 cells. (Fig. 5 A and B). To confirm that 2DG enhances OXPHOS in OCSC, we isolated CD133 + cells from A2780 and treated them with 2DG (2.5 mM) for 24 hours. The expression of ETC complexes I-IV was then examined. Our findings demonstrated that 2DG treatment increases the expression of ETC complexes in CD133 + A2780 cells compared to 2DG-treated CD133 − cells. These findings suggest that 2DG further enhances the OXPHOS in OCSCs (Fig. 5 C). Futher, we knocked down Mfn1 in two OC cell types, treated them with 2DG (2.5 mM), and checked the expression of ETC complexes. We observed that 2DG treatment in the presence of Mfn1 induces the expression of ETC complexes, whereas knocking down Mfn1 in cells reduces their level. Even after 2DG treatment, it failed to induce the expression of the ETC complexes (Fig. 5 D and E), indicating the crucial role of Mfn1-mediated fusion in inducing OXPHOS. Further, we used Seahorse analyzer to evaluate the functional impact of Mfn1 knockdown on mitochondrial respiration with and without 2DG treatment on A2780 cells. OCR was monitored over time following the sequential addition of oligomycin, FCCP, and rotenone/antimycin (Fig. 5 F). Mfn1 knockdown shows a reduction in the OCR compared to the control group. A significant decrease in the maximal respiration upon Mfn1 knockdown indicates impaired ETC function, suggesting disrupted mitochondrial integrity (Fig. 5 Fiii). Cells treated with 2DG alone show an overall increase in OCR, with a notably significant increase in basal respiration and ATP-linked respiration compared to the control (Fig. 5 Fi and Fiv). This finding demonstrates that the glycolysis inhibitor, 2DG, induces a shift from glycolysis to mitochondrial respiration to meet the cellular energy demand. However, A2780 cells treated with 2DG after Mfn1 knockdown have reduced OCR compared to controls. Thus, 2DG requires Mfn1 to induce mitochondrial respiration and is crucial for the 2DG-induced metabolic shift from glycolysis to OXPHOS. Further, to understand the significance of Mfn1-mediated mitochondrial fusion and metabolic phenotypes in maintaining CSC properties, three OC cell types were treated with 2DG. We treated A2780, SKOV-3, and OVCAR-3 OC cells with 2DG (0, 1.25, 2.5, and 5 mM) for 24 hours and checked the expression of cancer stemness-related genes and Mfn1. 2DG treatment showed induced Mfn1 and abundant stemness markers such as CD133, Nanog, Oct4, and Sox2 at 2.5 mM concentration of 2DG (Fig. 6 A, B and C). To further confirm that 2DG enhances OC stemness in OCSCs, we isolated CD133 + cells from A2780 and treated them with 2DG (2.5 mM) for 24 hours. The immunoblot analysis demonstrated that 2DG treatment further increases the expression of stemness markers and Mfn1 in CD133 + A2780 cells than in CD133 − cells (Fig. 6 D). To determine whether 2DG treatment enhances OC stemness by upregulating Mfn1 expression, A2780 and OVCAR-3 OC cells were transfected with shRNA Mfn1 and the empty vector as a control and examined the stemness properties. The knockdown of Mfn1 reduced the expression of stemness-related genes CD133, Oct4, and Nanog in OVCAR-3 and A2780 cells (Fig. 6 E and F). 2DG treatment induced Mfn1 and stemness markers expression in control cells but failed to induce stemness gene expression in Mfn1-deficient cells (Fig. 6 E and F), indicating the crucial role of increased Mfn1 expression and mitochondrial fusion in OC stemness induced by 2DG treatment. Our data suggest that OCSCs exhibit high Mfn1-mediated mitochondrial fusion activity and induce cancer stemness by increasing OXPHOS. CSCs contribute to drug resistance development in OC. ABC transporters, including ABCG2, and autophagy are known to contribute to drug resistance. To understand the possible role of Mfn1 in modulating the expression of ABCG2 and autophagic markers, first, we determined the ABCG2 expression and autophagic regulators in A2780 CD133 + cells and compared them with A2780 CD133 − cells. Our findings reveal elevated expression of ABCG2, LC3-II, and ATG5 in CD133 + cells compared to CD133 − cells (Fig. 7 A). Next, we defined the expression of ABCG2 and autophagic regulators (LC3-I/II and p62) in A2780 Mfn1-knockdown cells. Reduced expression of ABCG2, LC3-II, and p62 was observed in A2780 cells after Mfn1 knockdown (Fig. 7 B). Together, our results demonstrate that ovarian CSCs exhibit higher expression of ABCG2 and autophagy markers, and knocking down Mfn1 reduces the expression of ABCG2 and autophagic markers. However, further study is required to understand the underlying mechanism of how Mfn1 contributes to chemoresistance. 4. Discussion CSCs are primarily responsible for tumor recurrence and drug resistance. Thus, understanding the features of these cells is crucial to eradicating CSCs and therefore eliminating tumors [ 48 ]. CSCs are metabolically and functionally distinct from non-CSCs [ 8 ] and could be potential targets for cancer treatment. In the present study, we quantified mtDNA from spheroids and adherent cells, and our result indicates that CSCs have increased mitochondrial mass compared to the parental adherent cells. Understanding the mitochondrial abundance in spheroids will provide insight into how cells in cancer adapt to their environment and develop resistance to treatment. Mitochondria produce ATP through OXPHOS, and mitochondrial mass is directly linked to the cell’s OXPHOS capacity and cellular energy demands. Next, using biochemical and microscopic insights, we defined that ovarian CSLCs and CD133 + cells exhibited higher Mfn1 expression and, subsequently, higher mitochondrial fusion. Also, CSCs have increased ATP and reduced ROS levels compared to the parental adherent cells. Further, we showed that CD133 + enriched cells and spheroids exhibited higher expression of ETC subunits (NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A). Increased Mfn1 expression correlates with overexpression of OXPHOS complexes. We also observed that the Mfn1 protein modulates the growth and size of spheroid formation, cancer stemness, and OXPHOS activity, and knocking down Mfn1 reduces the expression of stemness markers such as Oct4, CD44, Nanog, and Sox2 in CD133 + cells and spheroids cultured from OC cells. Furthermore, to elucidate the relationship between Mfn1 and OXPHOS complex and their role in OC stemness, we knocked down Mfn1, followed by treatment with 2DG. 2DG induces OXPHOS and modulates the cancer stemness markers through Mfn1. Mfn1 knockdown correlates with the reduced mitochondrial respiration in OC cells. Restoring balanced mitochondrial dynamics by reducing Mfn1 activity will be a promising therapeutic strategy for drug resistance and relapse. In the present work, we demonstrated that spheroids cultured from OC cells have increased mitochondrial mass compared to the parental adherent cells, and these findings are consistent with previous studies where CSCs from other cancer cell types displayed increased mitochondrial mass [ 51 ]. Considering increased mitochondrial mass in CSCs, we focused on the expression of proteins involved in mitochondrial dynamics and morphology. Our studies determined that CD133 + enriched cells and spheroids cultured from OC cells exhibit increased mitochondrial fusion and higher Mfn1 expression. Mitochondria are dynamic and essential organelles for various cellular activities. Mitochondrial dynamics preserve mitochondrial homeostasis under normal and diseased conditions [ 43 ]. A balanced fission and fusion event regulate mitochondrial dynamics and maintains mitochondrial morphology, which is crucial for maintaining the optimal function of mitochondria [ 1 , 50 ]. Mitochondrial fission eliminates damaged mitochondria, and mitochondrial fusion ensures optimal OXPHOS for high-quality mitochondria [ 2 ]. Mfn1 and 2, and OPA1 coordinate the mitochondrial fusion process, while mitochondrial fission needs Drp1 and adaptor proteins such as Fis1, MFF1, MiD49, and 50 [ 13 ]. Several studies reported that mitochondria are highly fragmented in various cancers, including OC, with higher Drp1 and reduced Mfn1 and 2 expressions [ 53 , 5 , 6 ]. Our data shows that CSCLs have increased ATP and reduced ROS levels in CD133 + cells. Increased mitochondrial fusion supports ATP generation in spheroids cultured from OC cells and CD133 + enriched cells. Hypoxia in tumor cells exhibits an increased glucose metabolism anaerobically, thus producing abundant lactate [ 31 , 11 ]. Due to lower basal lactate levels in CSCs, CSCs show an increased capacity for lactate uptake. CSCs are likely to maintain a lower lactate level by reduced lactate production and by lactate serving as a substrate for increased mitochondrial activities [ 33 ]. Thus, CSCs are metabolically reprogrammed and primarily rely on OXPHOS for energy requirements [ 22 , 38 , 19 ]. Hence, we checked the levels of ETC complexes in CD133 + and spheroids. Our studies demonstrated that CD133 + cells and spheroids have higher Mfn1, ETC complexes I-V at protein levels, and knocking down Mfn1 correlates with reduced expression of ETC complexes I-V. Our study explains how CSCs rely on mitochondria for stemness. Crosstalk between Mfn1-mediated mitochondrial fusion and OXPHOS regulates stemness in ovarian CSCs. Reconfiguring metabolic pathways is a prerequisite for stem cell differentiation and function [ 55 ]. Previous studies have shown that tumor organoids cultured from cholangiocarcinoma (CCA) have increased mitochondrial fusion, and reducing Mfn1 and OPA1 inhibits the fusion process and modulates cell metabolism [ 24 ]. In human NSCLC, enhanced lipogenesis induces OPA1-mediated mitochondrial fusion and cancer stemness [ 28 ]. Mitochondrial fusion supports the OXPHOS process required for cell growth [ 47 , 49 ]. Mfn1 expression correlates with mitochondrial fusion and respiratory capacity of OXPHOS, and knocking down Mfn1 leads to decreased mitochondrial respiration. Our findings also demonstrated that Mfn1 knockdown reduces spheroid formation capacity in OC cells by decreasing the expression of CSC markers such as Oct4, Nanog, CD44, and Sox2. Further, to elucidate the robust relationship between Mfn1, OXPHOS, and OC stemness, we treated cells with 2DG to induce OXPHOS in OC cells. 2DG treatment induces Mfn1 expression and OC stemness by increasing OXPHOS. After the knockdown of Mfn1 in OC cells, 2DG treatment failed to induce OC stemness and OXPHOS, indicating that 2DG treatment enhances OXPHOS and OCSC stemness through Mfn1. 2DG treatment induces cancer stemness properties in liver CSCs by upregulating oxidative stress levels [ 27 ]. The present study defined OCSCs as having a higher expression of Mfn1, supporting the increased OXPHOS activity and maintaining OC stemness. Our results showed that CSCs exhibited altered mitochondrial dynamics, function, and metabolic reprogramming. Mfn1 and mitochondrial fusion are crucial for inducing OXPHOS and maintaining the stemness of OCSCs. Restoration of balanced mitochondrial dynamics, particularly Mfn1 expression, whose depletion decreases the stemness properties of ovarian CSCs. However, the study has some limitations. Using patient-derived organoids (PDOs) will validate the overall concept and support our findings, leading to a possible therapeutic approach for OC patient treatment. Declarations CRediT authorship contribution statement : Rahail Ashraf : Methodology, Formal analysis, Investigation, Data Curation, revising manuscript. Kalpana Tankay : Methodology, Formal analysis, Investigation, Data Curation, revising the manuscript, Manita Raina : Investigation, Data Curation, revising manuscript, Athira : Investigation, Data Curation, Sanjay Kumar : Conceptualization, Methodology, Writing - Original Draft, Supervision, Project administration and Funding acquisition. Acknowledgments : SK thanks DST SERB for awarding the core research grant, DBT for granting the Ramalingaswami Re-entry fellowship, and the Indian Institute of Science Education & Research (IISER) Tirupati for their support. We also acknowledge the Seahorse facility at IISER Tirupati. Funding sources and disclosure of conflicts of interest : This work was supported by DST-SERB (CRG/2019/002104), Ramalingaswami re-entry fellowship, DBT (BT/RLF/Re-entry/13/2016), and IISER Tirupati funds to SK. RA, MR, and Athira are thankful to IISER Tirupati and KT is thankful to CSIR for the fellowship. Declaration of competing interests : The authors declare they have no commercial or other competing interests to disclose. Data availability statement : The data supporting this study’s findings are available upon request from the corresponding author. Clinical trial number: not applicable. References Abrisch RG, Gumbin SC, Wisniewski BT, Lackner LL, Voeltz GK (2020) Fission and Fusion machineries converge at ER contact sites to regulate mitochondrial morphology. J Cell Biol 219:e201911122. https://doi.org/10.1083/jcb.201911122 Adebayo M, Singh S, Singh AP, Dasgupta S (2021) Mitochondrial fusion and fission: The fine-tune balance for cellular homeostasis. FASEB J 35:e21620. https://doi.org/10.1096/fj.202100067R Ahadova A, Gebert J, von Knebel Doeberitz M, Kopitz J, Kloor M (2015) Dose-dependent effect of 2-deoxy-D-glucose on glycoprotein mannosylation in cancer cells. 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(A& B) A2780 and SKOV-3 cells were cultured in adherent states and used as non-cancer stem cells (CSC) and spheroids were cultured from A2780 and SKOV-3 cells under non-adherent conditions and used as CSCs. (C, D & E) Spheroids are characterized by assessing the expression of Oct4, Sox2, Nanog, and CD44 using qRT-PCR and compared to adherent cells. RNA is isolated from adherent and spheroid cells, converted into cDNA, measured relative gene expression using SYBR green-based qRT-PCR, and analyzed by delta-delta-CT (ddCT) methods using 18S rRNA as an internal control. Error bars indicate mean ± SD in spheroid and adherent cells. * P <0.05, ** P <0.001. (E) UMAP projection scRNA sequencing transcriptomes showing 26 different clusters. (F) Classification of cells into CSC-like (green) and non-CSC-like (grey) populations based on EPCAM expression. (G) Feature plot showing the canonical stemness-associated markers CD44, ALDH1A1, PROM1, SOX2, NANOG, POU5F1, and EPCAM across the UMAP. 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Kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYJACAwYGGx42GBMEDoAINvxa0iBaDoC1MBPWAgSHkUwHasEL5NvPHij42XZeho//8LHPHwruJW5nP3/wAEONHQOfdAN2R53JSzDsbbvNwyaRljzjgEFx4s6eZKBtx5IZ2GQO4PBHjoEBL1gLjzHQLwmJGw6AtLABkUQCdof1vzEw/Nt2joeN//xniJbzj4Fa/uHWwnAjx8CYt+0AMMRymCFabgBtYWzDrcXgxhsDY5lzySC/GDOcMUgw3jnjscGBxD6QCC6H5ZgZvimzs5fvP/yYoeJPgux2/sTHHz58s5OTn4HDYcAoM2BEjjVwbAIV8+BSDwTMDxj+oGsZBaNgFIyCUYAEAC9HWsj97w8YAAAAAElFTkSuQmCC","orcid":"","institution":"Indian Institute of Science Education and Research (IISER) Tirupati","correspondingAuthor":true,"prefix":"","firstName":"Sanjay","middleName":"","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2025-09-27 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17:59:11","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186448,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/77490b0b99508cff5ebd278e.html"},{"id":93710784,"identity":"2afef53a-d18b-4799-9365-f00a9dfec805","added_by":"auto","created_at":"2025-10-16 17:59:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":497981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOvarian cancer stem cells exhibit high mitochondrial fusion. \u003c/strong\u003e(\u003cstrong\u003eA \u0026amp; B\u003c/strong\u003e) Adherent and spheroid cells were cultured from A2780 and SKOV-3, and mtDNA content was estimated using qRT-PCR with ND1: β-actin gene-specific primers; mtDNA gene ND1 was normalized to genomic DNA β-actin. (\u003cstrong\u003eC \u0026amp; D\u003c/strong\u003e) Adherent and spheroid cells of OVCAR-3 and A2780 were stained with MitoTracker-Red for 30 minutes, and mitochondrial morphology was observed using confocal microscopy. Spheroids have shown increased mitochondrial fusion. Quantitative mitochondrial length and aspect ratio analysis is presented adjacent to the representative images. \u0026nbsp;Scale bar: 8 µm. (\u003cstrong\u003eE \u0026amp; F\u003c/strong\u003e) Immunoblot analysis of mitochondrial fusion regulators Mfn1 and 2, and fission regulator Drp1 proteins in spheroid and adherent cell cultures from A2780 and SKOV-3 cells. Spheroids have notably upregulated Mfn1 expression, slightly increase in Mfn2, and reduced Drp1 expression. The immunoblot data were quantified using ImageJ software and normalized with the loading control. (\u003cstrong\u003eG\u003c/strong\u003e) A2780 cells were stained with DAPI and CD133 antibody after isolation of CD133\u003csup\u003e+\u003c/sup\u003e enriched cells. Scale bar: 10 µm. The adjacent graph shows the mean CD133 intensity measured using ImageJ software. (\u003cstrong\u003eH\u003c/strong\u003e) CD133\u003csup\u003e-\u003c/sup\u003e and CD133\u003csup\u003e+\u003c/sup\u003e cells were stained with MitoTracker-Red for 30 minutes to visualize mitochondrial morphology using confocal microscopy. CD133\u003csup\u003e+\u003c/sup\u003e enriched cells have increased mitochondrial fusion. The graph shows the quantitative analysis of mitochondrial length and aspect ratio. Scale bar: 10 µm. (\u003cstrong\u003eI\u003c/strong\u003e) Immunoblot analysis of Mfn1 and 2, and Drp1 proteins in CD133\u003csup\u003e-\u003c/sup\u003e and CD133\u003csup\u003e+\u003c/sup\u003e cells isolated from A2780 cells. CD133\u003csup\u003e+\u003c/sup\u003e cells have significantly upregulated Mfn1 expression, slightly increase in Mfn2, and reduced Drp1 expression. The immunoblot data were quantified using ImageJ software and normalized to the loading control. (\u003cstrong\u003eJ\u003c/strong\u003e) The graph represents normalized ATP concentrations in spheroids and adherent cells. Spheroids cultured from A2780 and SKOV-3 cells have higher ATP content compared to the adherent culture in spheroids and adherent cells. (\u003cstrong\u003eK\u003c/strong\u003e) Confocal microscopic analysis of mitochondrial ROS generation in (A2780) CD133\u003csup\u003e-\u003c/sup\u003e and CD133\u003csup\u003e+ \u003c/sup\u003ecells. A2780 CD133\u003csup\u003e+\u003c/sup\u003e cells have reduced ROS generation compared to CD133\u003csup\u003e-\u003c/sup\u003e cells. The graph shows the mean MitoSOX intensity between CD133\u003csup\u003e- \u003c/sup\u003eand CD133\u003csup\u003e+\u003c/sup\u003e A2780 cells. Scale bar: 10 µm. The data represent the mean ± SD (n=3) *P\u0026lt;0.05, **P\u0026lt;0.01, ***P ≤ 0.001, ns: non-significant.\u003c/p\u003e","description":"","filename":"OnlineRevisedFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/3ea2b14dc6abb22ab4791139.png"},{"id":93710783,"identity":"bcb504b1-be59-4052-80cf-5b2d465a2b0f","added_by":"auto","created_at":"2025-10-16 17:59:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":239266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOvarian cancer stem cells have higher oxidative phosphorylation. \u003c/strong\u003e(\u003cstrong\u003eA \u0026amp; B\u003c/strong\u003e) Immunoblot analysis of Mfn1, ETC I-V complex proteins in spheroid and adherent cell cultures from A2780 and OVCAR-3 cells. Spheroids cultured from A2780 and OVCAR-3 cells have increased expression of Mfn1, SDHB, UQCRC2, ATP5A, and MTCO2. The immunoblot data were quantified using ImageJ software, normalized to the loading control and displayed as a graph adjacent to the panel. \u0026nbsp;(\u003cstrong\u003eC\u003c/strong\u003e) Immunoblot analysis of ETC complexes in CD133\u003csup\u003e- \u003c/sup\u003eand CD133\u003csup\u003e+\u003c/sup\u003e cells isolated from A2780 cells. CD133\u003csup\u003e+\u003c/sup\u003e enriched cells have increased expression of UQCRC2, ATP5A, and MTCO2. The immunoblot data were quantified using ImageJ software, normalized to the loading control and displayed as a graph adjacent to the panel. (\u003cstrong\u003eD\u003c/strong\u003e) Graph showing qRT-PCR analysis of (\u003cstrong\u003eDi\u003c/strong\u003e) COX, (\u003cstrong\u003eDii\u003c/strong\u003e) ATP5A, (\u003cstrong\u003eDiii\u003c/strong\u003e) CytC genes, indicating mRNA expression in A2780 spheroid compared to adherent cells. RNA is isolated from adherent and spheroid cells, converted into cDNA and measured relative gene expression using SYBR-green based qRT-PCR and analyzed by delta-delta-CT (ddCT) methods using 18S rRNA as an internal control. Fold changes in spheroids were calculated by setting the mean fractions of adherent cells as one. The data represent the mean ± SD (n=3) *P\u0026lt;0.05, **P\u0026lt;0.01, ***P ≤ 0.001, ns: non-significant.\u003cstrong\u003e (E) \u003c/strong\u003eThe bar graph shows the mean log-normalized expression of Mfn1 was markedly higher in CSC-like (CSCL) cells, and Wilcoxon rank-sum testing confirmed a highly significant difference (p \u0026lt; 0.001). (\u003cstrong\u003eF\u003c/strong\u003e) Violin plot showing OXPHOS module score in CSC-like (green) vs Non-CSC-like (gray) ovarian cancer cells. OXPHOS score was calculated using Seurat’s AddModuleScore() with Hallmark OXPHOS genes. CSC-like cells show significantly higher OXPHOS activity (Wilcoxon test, p \u0026lt; 2e-16). The red line represents a linear regression fit with 95% confidence interval. (\u003cstrong\u003eG\u003c/strong\u003e) Scatter plot showing the correlation between Mfn1 expression (x-axis) and OXPHOS module score (y-axis) in single cells (Spearman’s r = 0.23, p \u0026lt; 2.2e-16).\u003c/p\u003e","description":"","filename":"OnlineRevisedFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/fa5b0cba00f4695fa921f315.png"},{"id":93710786,"identity":"02cf1e49-c2da-4867-ba00-f77230bb808f","added_by":"auto","created_at":"2025-10-16 17:59:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":735301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMfn1-mediated mitochondrial fusion is required for ovarian cancer stemness. \u003c/strong\u003e(\u003cstrong\u003eA \u0026amp; B\u003c/strong\u003e). Western blot analysis of Mfn1, Oct4, CD44, Nanog, Sox2, and β-actin in control and transient transfection with shMfn1 in A2780 and SKOV3 cells. Mfn1-deficient cells have reduced expression of Oct4, CD44, Nanog, and Sox2 compared to controls. The immunoblot data were quantified using ImageJ software and normalized to the loading control and displayed as a graph adjacent to the panel. (\u003cstrong\u003eC\u003c/strong\u003e) Microscopic images of spheroids cultured from A2780 cells in control or after Mfn1 knockdown from days 0 to 5 of spheroid culture. Images were taken at 10X magnification. The graph shows the normalized spheroid size from day 0 to day 5 between A2780 control and shMfn1 cells. (\u003cstrong\u003eD \u0026amp; E\u003c/strong\u003e) Immunofluorescence study confirmed that reduced Mfn1 expression decreases Oct4 expression in OVCAR-3 and A2780 cells. Cells after knockdown of Mfn1 and control cells were stained with DAPI, and antibodies against Mfn1 and Oct4 in OVCAR-3 and A2780 cells. Representative images were taken by confocal microscopy for both groups. The graph showing the relative intensity of Mfn1 and Oct4 between control and shMfn1 groups is presented adjacent to the images. Scale bar: 20 µm. The data represent the mean ± SD (n=3) *P\u0026lt;0.05, **P\u0026lt;0.01, ***P ≤ 0.001, ns: non-significant.\u003c/p\u003e","description":"","filename":"OnlineRevisedFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/e458dc1c6b470d75e94be91c.png"},{"id":93710788,"identity":"1ee71f2f-e7e8-40cf-9398-fbc59e5b52ee","added_by":"auto","created_at":"2025-10-16 17:59:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":564228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMfn1 expression modulates oxidative phosphorylation. \u003c/strong\u003e(\u003cstrong\u003eA \u0026amp; B\u003c/strong\u003e) Western blot analysis of Mfn1, ETC complexes after knockdown of Mfn1 in A2780 and SKOV-3 cells. The immunoblot data were quantified using ImageJ software, and normalized to the loading control and displayed as a graph adjacent to the panel. (\u003cstrong\u003eC \u0026amp; D\u003c/strong\u003e) Immunofluorescence study confirmed that reduced Mfn1 expression decreases ATP5A expression in A2780 and SKOV-3 cells, respectively. After knockdown of Mfn1 and control cells were stained with DAPI, Mfn1, and ATP5A expression in SKOV-3 and A2780 cells. Representative images were taken by confocal microscopy in both groups. The graph showing the relative intensity of Mfn1 and ATP5A between the control and shMfn1 groups is presented adjacent to the images. Scale bar: 10 µm. (\u003cstrong\u003eE \u0026amp; F\u003c/strong\u003e) Immunofluorescence study confirmed that reduced Mfn1 expression decreases MTCO2 expression in OVCAR-3 and A2780 cells. After knockdown of Mfn1 and control cells were stained with DAPI, Mfn1, and MTCO2 expression in OVCAR-3 and A2780 cells. Representative images were taken by confocal microscopy in both groups. The graph showing the relative intensity of Mfn1 and ATP5A between the control and shMfn1 groups is presented adjacent to the images. Scale bar: 10 µm. The data represent the mean ± SD (n=3) *P\u0026lt;0.05, **P\u0026lt;0.01, ***P ≤ 0.001, ns: non-significant. (\u003cstrong\u003eG\u003c/strong\u003e) Western blot images show the expression of Mfn1 and OXPHOS markers in control and Mfn1-overexpressed SKOV-3 cells. (\u003cstrong\u003eH-Hiii\u003c/strong\u003e) The graphs represent the OCR measurement, basal respiration, proton leak, and maximal respiration of control and Mfn1 overexpression in SKOV3 cells.\u003c/p\u003e","description":"","filename":"OnlineRevisedFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/3f63caaef49ca37d33b3f965.png"},{"id":93710921,"identity":"152f12f7-c87e-4f3d-8b2c-ebcd40125bfa","added_by":"auto","created_at":"2025-10-16 18:07:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":419044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2DG treatment induces oxidative phosphorylation through Mfn1. \u003c/strong\u003e(\u003cstrong\u003eA \u0026amp; B\u003c/strong\u003e) Western blot analysis showing the dose-dependent effect of 2DG in ATP5A, SDHB, UQCRC2, NDUFB8, and MTCO2 expression in A2780 and OVCAR-3 cells. The immunoblot data were quantified using ImageJ software, normalized to the loading control and displayed as a graph adjacent to the panel. (\u003cstrong\u003eC\u003c/strong\u003e) Western blot analysis of Mfn1, ATP5A, SDHB, UQCRC2, NDUFB8, and MTCO2 expression after 2DG treatment in CD133\u003csup\u003e- \u003c/sup\u003eand CD133\u003csup\u003e+\u003c/sup\u003e cells isolated from A2780 cells. CD133\u003csup\u003e+\u003c/sup\u003e enriched cells have increased expression of ETC complexes after 2DG treatment. The immunoblot data were quantified using ImageJ software, normalized to the loading control and displayed as a graph adjacent to the panel. (\u003cstrong\u003eD \u0026amp; E\u003c/strong\u003e) Western blot analysis of Mfn1, MTCO2, UQCRC2, NDUFB8, and ATP5A in control and cells after Mfn1 knockdown using shMfn1 and 2DG treatment at different doses in control cells. 2DG treatment at 2.5mM for 24 h induces Mfn1, MTCO2, UQCRC2, NDUFB8, and ATP5A expression compared to cells without 2DG treatment. 2DG treatment failed to induce Mfn1, MTCO2, UQCRC2, NDUFB8, and ATP5A expression in A2780 and OVCAR-3 after Mfn1 knockdown. The western blot data were quantified using ImageJ software, normalized to the loading control and displayed as a graph adjacent to the panel. (\u003cstrong\u003eF\u003c/strong\u003e) Mitochondrial respiration, depicted as oxygen consumption rate (OCR), is measured between control and shMfn1 groups with or without 2DG treatment using Seahorse analyzer. Graphs showing (\u003cstrong\u003eFi\u003c/strong\u003e) Basal respiration, (\u003cstrong\u003eFii\u003c/strong\u003e) proton leak, (\u003cstrong\u003eFiii\u003c/strong\u003e) Maximal respiration, and (\u003cstrong\u003eFiv\u003c/strong\u003e) ATP-linked respiration derived during a Seahorse analyzer assay. The data represent the mean ± SD (n=3) *P\u0026lt;0.05, **P\u0026lt;0.01, ***P ≤ 0.001, ns: non-significant.\u003c/p\u003e","description":"","filename":"OnlineRevisedFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/18f7ad82ff8be9696c40738a.png"},{"id":93710789,"identity":"07b3ba45-a064-4dbb-b958-01086c8a4abc","added_by":"auto","created_at":"2025-10-16 17:59:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":292014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2DG treatment induces ovarian cancer cells' stemness through Mfn1. \u003c/strong\u003e(\u003cstrong\u003eA, B \u0026amp; C\u003c/strong\u003e) Western blot analysis shows the dose-dependent effect of 2DG in Mfn1, CD133, Nanog, and Oct4 expression in A2780, CD133 and Nanog in SKOV-3, and Oct4 in OVCAR-3 cells. The immunoblot data were quantified using ImageJ software, normalized to the loading control and displayed as a graph adjacent to the panel. (\u003cstrong\u003eD\u003c/strong\u003e) Immunoblot analysis of Oct4, Nanog, and Sox2 after 2DG treatment in CD133\u003csup\u003e-\u003c/sup\u003e and CD133\u003csup\u003e+\u003c/sup\u003e cells isolated from A2780 cells. CD133\u003csup\u003e+\u003c/sup\u003e enriched cells have increased expression of Oct4, Nanog, and Sox2 after 2DG treatment. The immunoblot data were quantified using ImageJ software, normalized to the loading control and displayed as a graph adjacent to the panel. (\u003cstrong\u003eE \u0026amp; F\u003c/strong\u003e) Immunoblot analysis of Mfn1, CD133, Oct4, and Nanog in A2780 and OVCAR-3 cells after Mfn1 knockdown using shMfn1 and control cells and 2DG treatment (2.5mM for 24 h). 2DG treatment failed to induce Mfn1, CD133, Oct4 and Nanog expression in A2780 and OVCAR-3 after Mfn1 knockdown. The immunoblot data were quantified using image J software and normalized to the loading control and displayed as a graph adjacent to the panel. In control, 2DG treatment induces Oct4, Nanog, and CD133 expression compared to cells without 2DG treatment. The data represent the mean ± SD (n=3) *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e ≤ 0.001, ns: non-significant.\u003c/p\u003e","description":"","filename":"OnlineRevisedFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/b9675df6b1e74f154526bc94.png"},{"id":93711324,"identity":"2ab9b9f8-f9a7-4bff-8464-56c27acc2697","added_by":"auto","created_at":"2025-10-16 18:15:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":128878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD133\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e enriched cells have increased autophagy and ABCG2 expression. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Western blot analysis shows increased expression of autophagic markers LC3-I/II, ATG5, and ABCG2 expression in CD133\u003csup\u003e+\u003c/sup\u003e enriched cells compared to CD133\u003csup\u003e-. \u003c/sup\u003eThe immunoblot data were quantified using ImageJ software, normalized to the loading control and displayed as a graph adjacent to the panel. (\u003cstrong\u003eB\u003c/strong\u003e) Western blot analysis shows Mfn1, p62, LC3-I/II, and ABCG2 expression after Mfn1 knockdown in A2780 cells. Mfn1-deficient cells have reduced expression of Mfn1, p62, LC3-I/II and ABCG2 compared to the control. The immunoblot data were quantified using ImageJ software, normalized to the loading control and displayed as a graph adjacent to the panel. The data represent the mean ± SD (n=3) *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e ≤ 0.001, ns: non-significant.\u003c/p\u003e","description":"","filename":"OnlineRevisedFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/ef188a1d46d2603f1a696753.png"},{"id":105224754,"identity":"0ef02f07-9103-4883-abf9-070795a63588","added_by":"auto","created_at":"2026-03-23 16:16:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5209387,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/4538f403-529b-4bd8-bc74-d582e85ca554.pdf"},{"id":93710815,"identity":"85cd8019-4165-4c4f-8354-02a747dd44a6","added_by":"auto","created_at":"2025-10-16 17:59:12","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":41575130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1. (A\u0026amp; B) \u003c/strong\u003eA2780 and SKOV-3\u003cstrong\u003e \u003c/strong\u003ecells were cultured in adherent states and used as non-cancer stem cells (CSC) and spheroids were cultured from A2780 and SKOV-3 cells under non-adherent conditions and used as CSCs. (\u003cstrong\u003eC, D \u0026amp; E\u003c/strong\u003e) Spheroids are characterized by assessing the expression of Oct4, Sox2, Nanog, and CD44 using qRT-PCR and compared to adherent cells. RNA is isolated from adherent and spheroid cells, converted into cDNA, measured relative gene expression using SYBR green-based qRT-PCR, and analyzed by delta-delta-CT (ddCT) methods using 18S rRNA as an internal control. Error bars indicate mean ± SD in spheroid and adherent cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. \u0026nbsp;(\u003cstrong\u003eE) \u003c/strong\u003eUMAP projection scRNA sequencing transcriptomes showing 26 different clusters. (\u003cstrong\u003eF\u003c/strong\u003e) Classification of cells into CSC-like (green) and non-CSC-like (grey) populations based on EPCAM expression. (\u003cstrong\u003eG\u003c/strong\u003e) Feature plot showing the canonical stemness-associated markers CD44, ALDH1A1, PROM1, SOX2, NANOG, POU5F1, and EPCAM across the UMAP.\u003c/p\u003e","description":"","filename":"RevisedSupplimentaryFigure1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/679f3a2213ab3377f970b256.tiff"},{"id":93711323,"identity":"b27c688e-dea1-4bc3-88e6-01628d5e3f84","added_by":"auto","created_at":"2025-10-16 18:15:11","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23699,"visible":true,"origin":"","legend":"","description":"","filename":"TableMfn1.docxF.docx","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/ffdd64435067fbefa419e571.docx"},{"id":93710926,"identity":"dacf810d-174a-4c9d-bb13-61e3b24dc06a","added_by":"auto","created_at":"2025-10-16 18:07:11","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7517390,"visible":true,"origin":"","legend":"","description":"","filename":"Supplimentaryfilerawwesternblotimages.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7728954/v1/e657574d9d1c3ceb71482cb6.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mfn1-mediated imbalanced mitochondrial dynamics promotes ovarian cancer stemness by inducing metabolic reprogramming","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEpithelial ovarian cancer (EOC) is the most lethal reproductive system cancer, accounting for more deaths than any other gynecological malignancy worldwide. The factors limiting current therapy's efficacy are the delayed onset of symptoms, poor prognosis, therapeutic resistance, and disease recurrence. Cancer stem cells (CSCs) are a small subgroup of the tumor population with self-renewal properties contributing to cancer progression, disease recurrence, and drug resistance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Thus, understanding the precise molecular mechanism underpinning the stemness properties of ovarian cancer stem cells (OCSCs) will be relevant in developing effective strategies to combat ovarian cancer (OC).\u003c/p\u003e\u003cp\u003eCSCs have distinct metabolic properties compared to non-CSC tumor cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Aberrant ATP generation by the glycolytic pathway is a well-known feature of non-CSCs, required to support the biosynthetic demands for cell growth and metastasis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Under particular environmental conditions, CSCs may switch between glycolysis and oxidative phosphorylation (OXPHOS). In CSC, epithelial-to-mesenchymal transition (EMT) induction drives highly glycolytic metabolism, which depends on mitochondrial dynamics [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Mfn1 knockdown increases mitochondrial fission, induces the expression of glycolytic genes, and increases migration of cells [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Several studies demonstrated the reprogramming of glycolysis to mitochondrial OXPHOS and its crucial role in CSC maintenance [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Hypoxia induces CSC-like phenotypes in human OC cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Hypoxia acts on mitochondria by changing the protein composition of the electron transport chain (ETC), mitochondrial mass, and mitochondrial morphology. Consequently, it comprises an altered respiration rate by modulating OXPHOS and increasing or decreasing ROS-producing capacity. OXPHOS is adapted to hypoxia by remodeling the ETC and TCA cycle activity. Metabolic alteration in CSCs may contribute to therapeutic resistance. Thus, altered metabolism in CSCs needs to be better understood. In small-cell lung cancer, CSCs preferentially use OXPHOS over glycolysis to fulfill their energy requirements [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The notch-1-AMPK axis enhances mitochondrial OXPHOS to support lung cancer stem-like cell expansion [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Lipogenesis induces OPA1-mediated mitochondrial fusion that maintains stemness in human non-small cell lung cancer [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In liver CSCs, the treatment of mitochondrial fission inhibitor mDivi-1 (inhibitor of Drp1 activity) reduces CD133 and CD44 expression, thus decreasing stemness properties by inhibiting OXPHOS [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. CD133, CD44, Oct4, Nanog, and Sox2 are overexpressed in CSCs and are well-established CSC markers [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In pancreatic cancer, CSCs switch from glycolysis to OXPHOS to meet the energy requirements [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. MYC and MCL1 confer stemness in drug-resistant breast cancer cells by expanding mitochondrial OXPHOS [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In OC, platinum treatment enriches CSCs by inducing mitochondrial OXPHOS, therapeutic resistance, and disease recurrence [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, the underlying mechanisms for the deviant mitochondrial metabolism of OC stemness are mainly unknown.\u003c/p\u003e\u003cp\u003eMetabolic plasticity correlates with mitochondrial remodeling to maintain cellular homeostasis. Mitochondria are dynamic organelles that reorganize continually to form and degrade their intracellular networks through balanced fusion and fission events, and changes in these networks modulate mitochondrial functions, including energy production [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Mitochondrial morphology varies and changes rapidly through mitochondrial fission and fusion regulators in response to metabolic cues [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Glycolysis and OXPHOS are the two preferred pathways used by EOC to provide sufficient energy to promote cancer progression [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In human pluripotent stem cells, ATP production largely depends on OXPHOS during differentiation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Several studies have reported increased OXPHOS linked with cell proliferation, migration, metastasis, maintaining cancer stemness properties, and chemoresistance in OC [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The mechanism underlying elevated OXPHOS in OCSCs remains undefined.\u003c/p\u003e\u003cp\u003eIt is widely believed that balanced mitochondrial fission and fusion states may have a decisive role in the control of metabolism, and elongated networks of mitochondria are more bioenergetically efficient [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Mitochondrial fusion favors high mitochondrial metabolism for energy production in CSCs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], making it relevant to understand its role in the underlying mechanism for elevated OXPHOS. Mfn1 and 2 are large GTPases located in the outer mitochondrial membrane, and another GTPase, OPA1, in the inner mitochondrial membrane regulates mitochondrial fusion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Fis1, an outer membrane protein, and other adaptor proteins recruit Drp1, a dynamin-related GTPase protein, and coordinate the mitochondrial fission process. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A recent study demonstrated that an AKT inhibitor suppresses breast cancer stemness by Mfn1-modulated mitochondrial dynamics and functions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Mitofusin depletion induces pluripotency through remodeling mitochondrial dynamics and consequent reprogramming of cellular metabolism [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the Drosophila brain, mitochondrial fusion-induced metabolic reprogramming could be crucial for immortalizing tumor-initiating cells (TICs) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the role of Mfn1-mediated altered mitochondrial dynamics in human OCSCs is unclear.\u003c/p\u003e\u003cp\u003eIn the present study, we demonstrated that CSCs have increased mitochondrial mass, enhanced Mfn1 expression and mitochondrial fusion, and higher expression of ETC complexes subunits. Downregulation of Mfn1 in CSC-like cells reduced the expression of stemness properties by downregulating the OXPHOS complexes and mitochondrial respiration. Overexpression of Mfn1 in SKOV-3 cells, which has a low level of endogenous Mfn1 expression, increases mitochondrial respiration. Mfn1-mediated imbalanced mitochondrial dynamics promotes OC stemness by inducing OXPHOS. Our findings indicate that ovarian CSCs undergo Mfn1-mediated mitochondrial remodeling and, consequently, mitochondrial functions, which could be a potential therapeutic strategy in ovarian CSCs.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cell culture and 2 DG treatment\u003c/h2\u003e\u003cp\u003eSKOV-3 and OVCAR-3 cells were purchased from ATCC. A2780 cells were procured from ECACC. SKOV-3 and A2780 cells were grown and maintained in McCoy\u0026rsquo;s 5A and RPMI 1640 medium with 10% (v/v) FBS (Sigma, USA), 2mM L-glutamine (Sigma, USA), and 100U/ml of pen-strep (Sigma, USA). OVCAR-3 cells were cultured in RPMI 1640 media with 20% FBS and 0.1% bovine insulin (Sigma, USA) and 100U/ml of pen-strep (Sigma, USA). Cells were maintained in a 37\u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Low-passage cells were used in the experiments after confirmation of mycoplasma-negative status by periodically testing using a mycoplasma detection kit (Lonza). 2-Deoxy-D-glucose (2DG) was procured from Sigma-Aldrich and used per the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Transfection\u003c/h2\u003e\u003cp\u003eOC cells were seeded and grown till 40\u0026ndash;50% confluence in 6-well plates and used for experiments, including Mfn1 knockdown experiments. Transfection of shRNA Mfn1 (Sigma, USA) and control plasmid was performed using Lipofectamine 2000 (Invitrogen) as per the manufacturer\u0026rsquo;s recommended protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Spheroid-forming assay and CD133\u003csup\u003e+\u003c/sup\u003e isolation\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Spheroid culture\u003c/h2\u003e\u003cp\u003eFor spheroid culture, 2 x 10\u003csup\u003e6\u003c/sup\u003e cells were seeded in the ultra-low attachment culture dishes (Corning) with F12/knockout DMEM medium (Thermo Fisher Scientific) supplemented with 20% knockout serum replacement (Life Technologies), 20 ng/mL EGF, 10 ng/mL bFGF, 1% L-glutamine, and 1% penicillin-streptomycin. The spheroids were cultured for 6\u0026ndash;8 days, changed media every three days, and grown at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in the CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Spheroid formation assay\u003c/h2\u003e\u003cp\u003eA2780 cells were transfected with shMfn1 and a control plasmid. After overnight culture, A2780 cells were seeded in low attachment dishes (Corning) containing CSC media and incubated for up to 6 days at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Fresh media was replaced every 3 days, and the spheroids were imaged daily using an inverted microscope. Spheroids growth was assessed by measuring spheroid size using ImageJ from the obtained microscopic images.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3. CD133\u003csup\u003e+\u003c/sup\u003e isolation\u003c/h2\u003e\u003cp\u003eEnriched CD133\u003csup\u003e+\u003c/sup\u003e cells were isolated using the CD133 MicroBead Kit (Miltenyi Biotec) as per the recommended protocol suggested by the manufacturer and a previously published method [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. A2780 cells were grown until confluency and trypsinized. 10\u003csup\u003e7\u003c/sup\u003e total cells were resuspended in MACS buffer, and 20 \u0026micro;L of FcR blocking reagent was added, followed by the addition of 20 \u0026micro;L of CD133 microbeads (Miltenyi Biotec, Germany). After mixing well, the cell suspension was incubated at 4\u0026deg;C on a rotator for 25 minutes, washed twice, and centrifuged at 400g for 15 minutes. The supernatant was aspirated completely, then the cells were resuspended in the buffer. MACS column (Miltenyi Biotec) was placed into the magnetic separator (Miltenyi Biotec), followed by applying the cell suspension onto the MACS LS column (Miltenyi Biotec). CD133\u003csup\u003e+\u003c/sup\u003e cells were retained in the column, and the flow-through containing CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells were collected. Next, the column was removed from the separator, and a buffer was added to flush out the CD133\u003csup\u003e+\u003c/sup\u003e cells retained in the column by firmly pushing the plunger onto the column.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Immunofluorescence and confocal studies\u003c/h2\u003e\u003cp\u003eFor immunofluorescence studies, 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were seeded on coverslips and grown overnight. We transfected OC cells using Mfn1 shRNA and an empty vector and incubated the cells for 24 hrs. Cells were washed thrice and fixed using 4% paraformaldehyde. Next, cells were permeabilized using 0.1% Triton X-100 for 5 minutes, followed by blocking the cells using 5% BSA for 1 hour [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The primary antibodies in suitable dilutions were added and incubated, and after washing thrice, secondary antibodies were added and incubated at room temperature. After washing briefly, coverslips were mounted on slides using DAPI mounting media and imaged using confocal (Leica TCS SP8 and Olympus FV3000) microscopy. Quantitative analysis of the fluorescent intensity of the obtained confocal images was performed using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Mitochondrial morphology\u003c/h2\u003e\u003cp\u003eSpheroids and CD133\u003csup\u003e+\u003c/sup\u003e cells were transferred to 35-mm glass-bottom dishes, stained with MitoTracker Deep Red (100 nM in serum-free medium) and kept for 20 minutes at 37\u0026deg;C. After washing cells briefly, confocal microscopy was used to determine mitochondrial morphology by live imaging (Leica TCS SP8). Morphometric properties of mitochondria, including mitochondrial length and aspect ratio, were measured using the Mitochondria Analyzer plugin in ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Immunoblot analysis\u003c/h2\u003e\u003cp\u003eFor protein extraction, OC cells were lysed in RIPA lysis buffer with freshly added protease, and phosphatase inhibitors and quantified the protein concentration. Lysates were prepared, electrophoresed using SDS-PAGE, and transferred to a PVDF membrane. Next, membranes were blocked using 5% BSA, followed by the addition of primary antibody and incubated overnight at 4\u0026deg;C, followed by the addition of HRP-conjugated secondary antibodies and analyzed using ECL by chemiluminescence imaging system (Amersham Imager, GE Health Bioscience). The western blot data were quantified using ImageJ software and normalized for loading control. The list of resources, such as primary and secondary antibodies, is listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.7 RNA isolation and Reverse-transcription q-PCR analysis\u003c/h2\u003e\u003cp\u003eTotal RNA from OC cells was extracted using TRIzol reagent (Invitrogen) as per the manufacturer\u0026rsquo;s instructions, followed by cDNA generation using iScript cDNA Synthesis kit (BioRad, Cat 1708890). Real-time RT-qPCR was performed using iTaq Universal SYBR green Supermix (BioRad, 725121) in the BioRad CFX Real-Time PCR System. The primers were listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Relative fold change was calculated by ddCT methods using 18S rRNA as an internal control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.8. ROS estimation\u003c/h2\u003e\u003cp\u003eTo assess ROS generation in mitochondria, the spheroids were transferred to 35-mm glass-bottom dishes after spheroid development in low attachment dishes. MitoSOX Red reagent (Invitrogen) (5 \u0026micro;M dissolved in serum-free media) was added for 15 minutes and incubated at 37\u0026deg;C as per the suggested protocol by the manufacturer, and images were captured using confocal microscopy (Leica TCS SP8). Quantitative analysis of the fluorescent MitoSOX Red intensity of the obtained confocal images was performed using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.9. ATP determination assay\u003c/h2\u003e\u003cp\u003eLysate pellets of adherent and spheroid OC were collected to determine the changes in the ATP levels. The ATP determination assay was performed using the ATP determination Kit (Invitrogen) according to the manufacturer\u0026rsquo;s instructions. Luminescence was recorded using a Multi-Mode Microplate Reader (BioTek). ATP concentrations were calculated using a standard curve and normalized.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Mitochondrial DNA quantification\u003c/h2\u003e\u003cp\u003eTotal genomic DNA was extracted from adherent and spheroid OC cells using the GeneJet Genomic DNA purification kit (Thermofisher) per the manufacturer\u0026rsquo;s guidelines. The concentration of extracted DNA was quantified, and 0.03\u0026ndash;0.1 ng/\u0026micro;l was used for mtDNA content measurement using RT-qPCR. RT-qPCR was performed using iTaq Universal SYBR green Supermix (BioRad, 725121) in the BioRad CFX Real-Time PCR System. β-actin was used as an internal control. The primers used are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The threshold cycle difference in control ND1/control β-actin was used to measure the relative abundance of mitochondrial DNA [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Measurement of Oxygen Consumption Rate (OCR)\u003c/h2\u003e\u003cp\u003eThe oxygen consumption rate (OCR) was measured using the Seahorse XFe24 Analyzer (Agilent Technologies, USA) to assess mitochondrial respiration. A2780 and SKOV-3 cells were transfected with the shMfn1 and Mfn1 overexpression plasmid, respectively, and seeded at 20,000 cells per well in Seahorse XFe24 cell culture microplates (Agilent Technologies) and kept overnight. Cells were treated with 2DG, and the culture medium was then replaced with Seahorse XF DMEM assay medium (Agilent Technologies) supplemented with 2 mM L-glutamine, 10 mM D-glucose, and 1 mM sodium pyruvate. According to the manufacturer's instructions, OCR was measured using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, Cat. #103015-100). The sequential injection of oligomycin (1.5 \u0026micro;M), FCCP (0.5 \u0026micro;M), and rotenone/antimycin (0.5 \u0026micro;M) enabled the determination of basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. Data were normalized to cell number by staining with 0.1% crystal violet and analyzed using the Wave software (Agilent Technologies).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.12. scRNA sequencing data acquisition, preprocessing, and data integration\u003c/h2\u003e\u003cp\u003eThe single-cell RNA sequencing dataset, GSE184880, is obtained from the publicly available NCBI-GEO (Home - GEO - NCBI) database. The scRNA sequencing dataset contains normal and high-grade serous ovarian carcinoma tissue samples. 5 normal tissue samples and 7 cancerous tissue samples from OC were included for the analysis. The gene expression count matrix and associated metadata were loaded into R(v4.5.0), and the preprocessing and downstream analysis were conducted using the Seurat package. We created the Seurat object using the gene expression matrix, and then the Seurat object was normalized, and expression values were scaled using the NormalizeData and ScaleData functions. The FindVariable function was used to identify variable genes. The RunPCA function was employed to conduct principal component analysis (PCA). Clustering was carried out using principal components with FindNeighbors and FindClusters at 3 resolutions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.12.1 Identification of cancer stem-like cells, differential gene expression, and OXPHOS correlation\u003c/h2\u003e\u003cp\u003eWe employed the FindAll markers function via Wilcoxon rank-sum tests to identify different genes in the clustered cells. Each cluster was annotated as a specific cell type based on the canonical marker gene expression. To identify ovarian cancer stem-like cells, canonical stemness markers (CD44, ALDH1A1, PROM1, SOX2, NANOG, POU5F1, EPCAM) were visualized using FeaturePlot and Violin plots. Among these, EPCAM expression was used as a functional classifier for stemness. Pathway activities were quantified using gene module scoring. Genes associated with OXPHOS were obtained from the MSigDB Hallmark gene sets via the msigdbr package. An OXPHOS module score was computed and assigned to each cell (AddModuleScore). Spearman\u0026rsquo;s rank correlation was used to assess the association between MFN1 expression and both OXPHOS scores. Scatter plots with linear fits and annotated correlation statistics were generated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Statistical Methods\u003c/h2\u003e\u003cp\u003eAll data presented are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical difference was determined by Student's t-tests for two-group comparisons. The significance levels are shown as follows: ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The significance level is considered significant if the p-value is less than 0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Ovarian cancer stem cells exhibit high mitochondrial fusion activity\u003c/h2\u003e\u003cp\u003eTo understand the role of altered mitochondrial dynamics and function in CSC, we cultured spheroids from OC cells in CSC culture medium under non-adherent conditions and employed them as cancer stem-like cells (CSLCs) (Supplementary Fig.\u0026nbsp;1A). Cells cultured in adherent states were used as non-CSCs [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The tumor microenvironment is heterogeneous, and the culture of human cancer cells retains phenotypic and functional differences. The stem cell-like properties of ovarian A2780 and SKOV-3 cultured spheroids were validated by assessing the expression of Nestin, Oct4, and Nanog (Supplementary Fig.\u0026nbsp;1B, C and D). Next, we checked the mtDNA content in spheroids cultured from A2780 and SKOV-3 OC cells. Spheroids from both cell types have higher mtDNA content than adherent parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). Ovarian CSCs showed more mitochondrial mass than adherent parental cells. Next, the mitochondrial morphology of CSLCs and non-CSCs cultured from OVCAR-3 and A2780 cells was assessed by MitoTracker-Red staining followed by confocal microscopy. Both non-CSCs of OVCAR-3 and A2780 exhibited smaller, rounded mitochondria. In contrast, the spheroid displayed more tubulated and elongated mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D). The respective graphs adjacent to the representative images display the quantitative analysis of mitochondrial length and aspect ratio, and our study observed significantly higher mitochondrial length and aspect ratio in the spheroids than in adherent cells. Next, to understand the status of mitochondrial dynamics regulator expression, we performed a western blot, and our data showed the increased expression of mitochondrial fusion proteins Mfn1\u0026amp;2 and reduced expression of Drp1 in spheroids cultured from A2780 and SKOV-3 cells compared to parental adherent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrevious studies have shown that CD133 is a potential marker for CSCs in many solid tumors, including OC [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Next, by employing CD133 as a classical biomarker of CSC, OCSCs (CD133\u003csup\u003e+\u003c/sup\u003e) were isolated from A2780 cells and further confirmed by immunofluorescence using DAPI and CD133-specific antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Further, the mitochondrial morphology of CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e and CD133\u003csup\u003e+\u003c/sup\u003e cells was assessed by MitoTracker-Red staining followed by confocal microscopy. CD133\u003csup\u003e+\u003c/sup\u003e cells displayed more elongated mitochondria than CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells, which exhibited fragmented mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). The graphs adjacent to the representative images display significantly higher mitochondrial length and aspect ratio in the CD133\u003csup\u003e+\u003c/sup\u003e than in CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells. We also performed a Western blot to determine the expression of mitochondrial dynamics regulator expression in CD133\u003csup\u003e+\u003c/sup\u003e and CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells. CD133\u003csup\u003e+\u003c/sup\u003e cells showed higher Mfn1 and reduced Drp1 expression compared to CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), further validating the higher mitochondrial fusion activity in CD133\u003csup\u003e+\u003c/sup\u003e cells. Furthermore, a robust increment in Mfn1 expression in spheroids cultured from A2780 and SKOV-3 and CD133\u003csup\u003e+\u003c/sup\u003e cells indicates their crucial role in OCSCs maintenance.\u003c/p\u003e\u003cp\u003eMitochondrial dynamics are crucial for mitochondrial morphology and mitochondrial functions. Next, we monitored the overall respiratory activity in the spheroids cultured from SKOV-3 OC cells by measuring cellular ATP levels and compared them with those of parent adherent cells. Spheroids have relatively higher ATP than adherent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Further, we used MitoSox red reagent to stain and evaluate mitochondrial ROS generation in CD133\u003csup\u003e+\u003c/sup\u003e and CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells. CD133\u003csup\u003e+\u003c/sup\u003e cells had reduced mtROS compared to the CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Our findings indicate that spheroids have higher mitochondrial content, increased mitochondrial fusion activity, and Mfn1 expression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Ovarian cancer stem cells have higher oxidative phosphorylation\u003c/h2\u003e\u003cp\u003eCells yield energy through ATP predominantly through glycolysis and mitochondrial OXPHOS. Energy metabolism is crucial for maintaining stemness in CSCs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the present study, we characterize OXPHOS by measuring the expression of ETC subunits (NDUFB8 for complex I, SDHB for complex II, UQCRC2 for complex III, MTCO2 for complex IV, and ATP5A for complex V) in CSLCs and compared them with non-CSCs. We performed a Western blot to assess ETC complex components in spheroids and compared them with those of the adherent cells. Both A2780 and OVCAR-3 spheroids had increased expression of most of the ETC complex components compared to the adherent cells, and CD133\u003csup\u003e+\u003c/sup\u003e cells had increased expression of ETC complex components compared to the CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, and C), indicating that CSCs preferably use mitochondrial OXPHOS to generate ATP. Next, we measured the COX, ATP5A, and Cyt-C gene expression in spheroids and compared them with non-CSCs adherent cultured cells (Fig.\u0026nbsp;2Di-iii) and found that OC spheroids are enriched in COX, ATP5A, and Cyt-C gene expression and thus exhibit a more robust expression of OXPHOS machinery than non-CSCs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we used the publicly available single-cell RNA sequencing (scRNA seq) dataset GSE184880. We subsetted the cancer stem cell-like (CSCL) and non-cancer stem cell-like (non-CSCL) population from the entire tumor cell population based on the expression of EPCAM (Supplementary Fig.\u0026nbsp;1C, D, and E). scRNA sequencing analysis of ovarian patient tumor cells revealed that MFN1 expression is significantly increased in the CSCL OC cells compared to non-CSCL OC cells. Bar plot showing mean log-normalized expression of MFN1 in CSCL vs Non-CSCL cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This further strengthens the clinical relevance of Mfn1 abundance in the OCSCL cells in ovarian tumors. To assess whether the Mfn1 upregulation is functionally linked to the mitochondrial metabolism, we next evaluated the OXPHOS activity using a module scoring approach. Ovarian CSCL cells show a significant increase in the OXPHOS module score compared to the non-CSCL cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In addition to that, correlation analysis demonstrates a modest positive correlation (Spearman\u0026rsquo;s correlation coefficient, r\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.23) between Mfn1 expression and OXPHOS activity. Together, these findings suggest that ovarian CSCs show increased Mfn1 expression and OXPHOS activity, suggesting a potential role of Mfn1 in OXPHOS in ovarian CSCL cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Mfn1 is crucial for stemness in ovarian cancer stem cells\u003c/h2\u003e\u003cp\u003eFurther, to assess the functional outcome of higher mitochondrial fusion in OCSCs, we used shMfn1 to knock down Mfn1 in SKOV-3 and A2780 cells and analyzed stemness properties. Two OC cells, A2780 and SKOV-3, were transfected with shMfn1 and a control plasmid and subjected to a Western blot. Cells after knocking down Mfn1 showed a notably reduced stemness by downregulating the stemness markers such as Oct4, CD44, Nanog, and Sox2 in OC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Subsequently, we transfected A2780 cells with shMfn1 and a control plasmid, grew spheroids for 5 days, and analyzed sphere formation. Cells with reduced Mfn1 expression inhibited sphere formation and reduced the spheroid\u0026rsquo;s size compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We performed the immunofluorescence experiments to evaluate the effect of Mfn1 knockdown on cancer stemness using DAPI, Mfn1, and Oct4 antibodies for the staining. We acquired images after staining, and our study suggested that knocking down Mfn1 in A2780 and OVCAR-3 cells decreased the expression of Oct4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD \u0026amp; E) compared to the control. This finding supports the critical role of Mfn1 in maintaining the stemness of OC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Reduced Mfn1 levels impede oxidative phosphorylation and cancer stemness in OCSCs\u003c/h2\u003e\u003cp\u003eTo understand how Mfn1 contributes to OC cell stemness, we knocked down Mfn1 in A2780 and SKOV-3 cells and performed a western blot to evaluate the ETC complexes I-V expression. The Mfn1 expression correlates with the expression of ETC complexes, as Mfn1 knockdown reduces the expression levels of SDHB, UQCRC2, and ATP5A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Further, to ascertain the Mfn1 influence over ETC, we performed an immunofluorescence study using DAPI, Mfn1, and ATP5A antibodies for the staining. Reduced Mfn1 levels in SKOV-3 and A2780 cells correlate with reduced ATP5A expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Further, another immunofluorescence study using DAPI, Mfn1, and MTCO2 antibodies for the staining revealed that attenuation of Mfn1 reduced the MTCO2 expression in A2780 and OVCAR-3 OC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and F). OCSLCs have higher Mfn1 levels that enhance mitochondrial fusion, induce OXPHOS and promote stemness.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe SKOV-3 cell line exhibits relatively low basal Mfn1 expression. Therefore, we overexpressed Mfn1 in SKOV-3 cells and measured the OXPHOS markers. Overexpression of Mfn1 increases the OXPHOS markers, MTCO2, and ATP5A compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). To further assess the effect of Mfn1 overexpression on the OXPHOS, we evaluated the OCR using the Seahorse analyzer. The Mfn1 overexpression increases mitochondrial respiration overall compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Specifically, the OCR parameters, such as the proton leak and maximal respiration, are significantly increased compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e Hi-iii).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.5. 2-DG induces oxidative phosphorylation and promotes stemness in ovarian cancer cells through Mfn1\u003c/h2\u003e\u003cp\u003eOXPHOS has two critical functions in driving cancer. It satisfies the bioenergetics demands by providing ATP and funnels carbon from glucose for macromolecule synthesis. Mitochondrial matrix enzymes involved in the tricarboxylic acid (TCA) cycle and transmembrane protein complexes of ETC are fundamental to this process. A recent study has shown that 2DG treatment increases stemness by upregulating OXPHOS in liver CSCs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 2DG inhibits glucose-6-phosphate production from glucose by reducing the hexokinase and phosphoglucoisomerase activities and stops glycolysis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. It has also been shown to inhibit protein N-glycosylation selectively [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To detect whether 2DG treatment induces OXPHOS in OC cells, we treated two OC cell types, A2780 and OVCAR-3, with 2DG (0, 1.25. 2.5, and 5 mM) and checked the status of ETC I-IV complexes. The expression of ATP5A, SDHB, UQCRC2, and MTCO2 has increased consistently at 2.5 mM in A2780, and the expression of ATP5A, SDHB, UQCRC2, and NDUFB8 at 2.5 mM of 2DG in OVCAR-3 cells. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). To confirm that 2DG enhances OXPHOS in OCSC, we isolated CD133\u003csup\u003e+\u003c/sup\u003e cells from A2780 and treated them with 2DG (2.5 mM) for 24 hours. The expression of ETC complexes I-IV was then examined. Our findings demonstrated that 2DG treatment increases the expression of ETC complexes in CD133\u003csup\u003e+\u003c/sup\u003e A2780 cells compared to 2DG-treated CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells. These findings suggest that 2DG further enhances the OXPHOS in OCSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFuther, we knocked down Mfn1 in two OC cell types, treated them with 2DG (2.5 mM), and checked the expression of ETC complexes. We observed that 2DG treatment in the presence of Mfn1 induces the expression of ETC complexes, whereas knocking down Mfn1 in cells reduces their level. Even after 2DG treatment, it failed to induce the expression of the ETC complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and E), indicating the crucial role of Mfn1-mediated fusion in inducing OXPHOS. Further, we used Seahorse analyzer to evaluate the functional impact of Mfn1 knockdown on mitochondrial respiration with and without 2DG treatment on A2780 cells. OCR was monitored over time following the sequential addition of oligomycin, FCCP, and rotenone/antimycin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Mfn1 knockdown shows a reduction in the OCR compared to the control group. A significant decrease in the maximal respiration upon Mfn1 knockdown indicates impaired ETC function, suggesting disrupted mitochondrial integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eFiii). Cells treated with 2DG alone show an overall increase in OCR, with a notably significant increase in basal respiration and ATP-linked respiration compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eFi and Fiv). This finding demonstrates that the glycolysis inhibitor, 2DG, induces a shift from glycolysis to mitochondrial respiration to meet the cellular energy demand. However, A2780 cells treated with 2DG after Mfn1 knockdown have reduced OCR compared to controls. Thus, 2DG requires Mfn1 to induce mitochondrial respiration and is crucial for the 2DG-induced metabolic shift from glycolysis to OXPHOS.\u003c/p\u003e\u003cp\u003eFurther, to understand the significance of Mfn1-mediated mitochondrial fusion and metabolic phenotypes in maintaining CSC properties, three OC cell types were treated with 2DG. We treated A2780, SKOV-3, and OVCAR-3 OC cells with 2DG (0, 1.25, 2.5, and 5 mM) for 24 hours and checked the expression of cancer stemness-related genes and Mfn1. 2DG treatment showed induced Mfn1 and abundant stemness markers such as CD133, Nanog, Oct4, and Sox2 at 2.5 mM concentration of 2DG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B and C). To further confirm that 2DG enhances OC stemness in OCSCs, we isolated CD133\u003csup\u003e+\u003c/sup\u003e cells from A2780 and treated them with 2DG (2.5 mM) for 24 hours. The immunoblot analysis demonstrated that 2DG treatment further increases the expression of stemness markers and Mfn1 in CD133\u003csup\u003e+\u003c/sup\u003e A2780 cells than in CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether 2DG treatment enhances OC stemness by upregulating Mfn1 expression, A2780 and OVCAR-3 OC cells were transfected with shRNA Mfn1 and the empty vector as a control and examined the stemness properties. The knockdown of Mfn1 reduced the expression of stemness-related genes CD133, Oct4, and Nanog in OVCAR-3 and A2780 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F). 2DG treatment induced Mfn1 and stemness markers expression in control cells but failed to induce stemness gene expression in Mfn1-deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F), indicating the crucial role of increased Mfn1 expression and mitochondrial fusion in OC stemness induced by 2DG treatment. Our data suggest that OCSCs exhibit high Mfn1-mediated mitochondrial fusion activity and induce cancer stemness by increasing OXPHOS.\u003c/p\u003e\u003cp\u003eCSCs contribute to drug resistance development in OC. ABC transporters, including ABCG2, and autophagy are known to contribute to drug resistance. To understand the possible role of Mfn1 in modulating the expression of ABCG2 and autophagic markers, first, we determined the ABCG2 expression and autophagic regulators in A2780 CD133\u003csup\u003e+\u003c/sup\u003e cells and compared them with A2780 CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells. Our findings reveal elevated expression of ABCG2, LC3-II, and ATG5 in CD133\u003csup\u003e+\u003c/sup\u003e cells compared to CD133\u003csup\u003e\u0026minus;\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Next, we defined the expression of ABCG2 and autophagic regulators (LC3-I/II and p62) in A2780 Mfn1-knockdown cells. Reduced expression of ABCG2, LC3-II, and p62 was observed in A2780 cells after Mfn1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Together, our results demonstrate that ovarian CSCs exhibit higher expression of ABCG2 and autophagy markers, and knocking down Mfn1 reduces the expression of ABCG2 and autophagic markers. However, further study is required to understand the underlying mechanism of how Mfn1 contributes to chemoresistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCSCs are primarily responsible for tumor recurrence and drug resistance. Thus, understanding the features of these cells is crucial to eradicating CSCs and therefore eliminating tumors [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. CSCs are metabolically and functionally distinct from non-CSCs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and could be potential targets for cancer treatment. In the present study, we quantified mtDNA from spheroids and adherent cells, and our result indicates that CSCs have increased mitochondrial mass compared to the parental adherent cells. Understanding the mitochondrial abundance in spheroids will provide insight into how cells in cancer adapt to their environment and develop resistance to treatment. Mitochondria produce ATP through OXPHOS, and mitochondrial mass is directly linked to the cell\u0026rsquo;s OXPHOS capacity and cellular energy demands. Next, using biochemical and microscopic insights, we defined that ovarian CSLCs and CD133\u003csup\u003e+\u003c/sup\u003e cells exhibited higher Mfn1 expression and, subsequently, higher mitochondrial fusion. Also, CSCs have increased ATP and reduced ROS levels compared to the parental adherent cells. Further, we showed that CD133\u003csup\u003e+\u003c/sup\u003e enriched cells and spheroids exhibited higher expression of ETC subunits (NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A). Increased Mfn1 expression correlates with overexpression of OXPHOS complexes. We also observed that the Mfn1 protein modulates the growth and size of spheroid formation, cancer stemness, and OXPHOS activity, and knocking down Mfn1 reduces the expression of stemness markers such as Oct4, CD44, Nanog, and Sox2 in CD133\u003csup\u003e+\u003c/sup\u003e cells and spheroids cultured from OC cells.\u003c/p\u003e\u003cp\u003eFurthermore, to elucidate the relationship between Mfn1 and OXPHOS complex and their role in OC stemness, we knocked down Mfn1, followed by treatment with 2DG. 2DG induces OXPHOS and modulates the cancer stemness markers through Mfn1. Mfn1 knockdown correlates with the reduced mitochondrial respiration in OC cells. Restoring balanced mitochondrial dynamics by reducing Mfn1 activity will be a promising therapeutic strategy for drug resistance and relapse.\u003c/p\u003e\u003cp\u003eIn the present work, we demonstrated that spheroids cultured from OC cells have increased mitochondrial mass compared to the parental adherent cells, and these findings are consistent with previous studies where CSCs from other cancer cell types displayed increased mitochondrial mass [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Considering increased mitochondrial mass in CSCs, we focused on the expression of proteins involved in mitochondrial dynamics and morphology. Our studies determined that CD133\u003csup\u003e+\u003c/sup\u003e enriched cells and spheroids cultured from OC cells exhibit increased mitochondrial fusion and higher Mfn1 expression. Mitochondria are dynamic and essential organelles for various cellular activities. Mitochondrial dynamics preserve mitochondrial homeostasis under normal and diseased conditions [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A balanced fission and fusion event regulate mitochondrial dynamics and maintains mitochondrial morphology, which is crucial for maintaining the optimal function of mitochondria [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Mitochondrial fission eliminates damaged mitochondria, and mitochondrial fusion ensures optimal OXPHOS for high-quality mitochondria [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mfn1 and 2, and OPA1 coordinate the mitochondrial fusion process, while mitochondrial fission needs Drp1 and adaptor proteins such as Fis1, MFF1, MiD49, and 50 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Several studies reported that mitochondria are highly fragmented in various cancers, including OC, with higher Drp1 and reduced Mfn1 and 2 expressions [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Our data shows that CSCLs have increased ATP and reduced ROS levels in CD133\u003csup\u003e+\u003c/sup\u003e cells. Increased mitochondrial fusion supports ATP generation in spheroids cultured from OC cells and CD133\u003csup\u003e+\u003c/sup\u003e enriched cells.\u003c/p\u003e\u003cp\u003eHypoxia in tumor cells exhibits an increased glucose metabolism anaerobically, thus producing abundant lactate [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Due to lower basal lactate levels in CSCs, CSCs show an increased capacity for lactate uptake. CSCs are likely to maintain a lower lactate level by reduced lactate production and by lactate serving as a substrate for increased mitochondrial activities [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThus, CSCs are metabolically reprogrammed and primarily rely on OXPHOS for energy requirements [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Hence, we checked the levels of ETC complexes in CD133\u003csup\u003e+\u003c/sup\u003e and spheroids. Our studies demonstrated that CD133\u003csup\u003e+\u003c/sup\u003e cells and spheroids have higher Mfn1, ETC complexes I-V at protein levels, and knocking down Mfn1 correlates with reduced expression of ETC complexes I-V. Our study explains how CSCs rely on mitochondria for stemness. Crosstalk between Mfn1-mediated mitochondrial fusion and OXPHOS regulates stemness in ovarian CSCs.\u003c/p\u003e\u003cp\u003eReconfiguring metabolic pathways is a prerequisite for stem cell differentiation and function [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Previous studies have shown that tumor organoids cultured from cholangiocarcinoma (CCA) have increased mitochondrial fusion, and reducing Mfn1 and OPA1 inhibits the fusion process and modulates cell metabolism [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In human NSCLC, enhanced lipogenesis induces OPA1-mediated mitochondrial fusion and cancer stemness [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Mitochondrial fusion supports the OXPHOS process required for cell growth [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Mfn1 expression correlates with mitochondrial fusion and respiratory capacity of OXPHOS, and knocking down Mfn1 leads to decreased mitochondrial respiration. Our findings also demonstrated that Mfn1 knockdown reduces spheroid formation capacity in OC cells by decreasing the expression of CSC markers such as Oct4, Nanog, CD44, and Sox2. Further, to elucidate the robust relationship between Mfn1, OXPHOS, and OC stemness, we treated cells with 2DG to induce OXPHOS in OC cells. 2DG treatment induces Mfn1 expression and OC stemness by increasing OXPHOS. After the knockdown of Mfn1 in OC cells, 2DG treatment failed to induce OC stemness and OXPHOS, indicating that 2DG treatment enhances OXPHOS and OCSC stemness through Mfn1. 2DG treatment induces cancer stemness properties in liver CSCs by upregulating oxidative stress levels [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The present study defined OCSCs as having a higher expression of Mfn1, supporting the increased OXPHOS activity and maintaining OC stemness. Our results showed that CSCs exhibited altered mitochondrial dynamics, function, and metabolic reprogramming. Mfn1 and mitochondrial fusion are crucial for inducing OXPHOS and maintaining the stemness of OCSCs. Restoration of balanced mitochondrial dynamics, particularly Mfn1 expression, whose depletion decreases the stemness properties of ovarian CSCs. However, the study has some limitations. Using patient-derived organoids (PDOs) will validate the overall concept and support our findings, leading to a possible therapeutic approach for OC patient treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e: \u003cstrong\u003eRahail Ashraf\u003c/strong\u003e: Methodology, Formal analysis, Investigation, Data Curation, revising manuscript. \u003cstrong\u003eKalpana Tankay\u003c/strong\u003e: Methodology, Formal analysis, Investigation, Data Curation, revising the manuscript, \u003cstrong\u003eManita Raina\u003c/strong\u003e: Investigation, Data Curation, revising manuscript, \u003cstrong\u003eAthira\u003c/strong\u003e: Investigation, Data Curation, \u003cstrong\u003eSanjay Kumar\u003c/strong\u003e: Conceptualization, Methodology, Writing - Original Draft, Supervision, Project administration and Funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e: SK thanks DST SERB for awarding the core research grant, DBT for granting the Ramalingaswami Re-entry fellowship, and the Indian Institute of Science Education \u0026amp; Research (IISER) Tirupati for their support. We also acknowledge the Seahorse facility at IISER Tirupati.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources and disclosure of conflicts of interest\u003c/strong\u003e: This work was supported by DST-SERB (CRG/2019/002104), Ramalingaswami re-entry fellowship, DBT (BT/RLF/Re-entry/13/2016), and IISER Tirupati funds to SK. RA, MR, and Athira are thankful to IISER Tirupati and KT is thankful to CSIR for the fellowship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e: The authors declare they have no commercial or other competing interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e: The data supporting this study\u0026rsquo;s findings are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbrisch RG, Gumbin SC, Wisniewski BT, Lackner LL, Voeltz GK (2020) Fission and Fusion machineries converge at ER contact sites to regulate mitochondrial morphology. 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EMBO J 31:2103\u0026ndash;2116. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://10.1038/emboj.2012.71\u003c/span\u003e\u003cspan address=\"https://10.1038/emboj.2012.71\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCRediT authorship contribution statement Rahail Ashraf: Methodology, Formal analysis, Investigation, Data Curation, revising manuscript. Kalpana Tankay: Methodology, Formal analysis, Investigation, Data Curation, revising the manuscript, Manita Raina: Investigation, Data Curation, revising manuscript, Athira: Investigation, Data Curation, Sanjay Kumar: Conceptualization, Methodology, Writing - Original Draft, Supervision, Project administration and Funding acquisition\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpby","sideBox":"Learn more about [Journal of Physiology and Biochemistry](http://link.springer.com/journal/13105)","snPcode":"13105","submissionUrl":"https://submission.nature.com/new-submission/13105/3","title":"Journal of Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mitochondrial fusion, Mfn1, ETC complexes, Metabolic reprogramming, Ovarian cancer stem cells, CD133+, 2-Deoxy-D-glucose","lastPublishedDoi":"10.21203/rs.3.rs-7728954/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7728954/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOvarian cancer is the leading cause of death from reproductive system cancer among women worldwide. Ovarian cancer stem cells (OCSCs) are critically involved in metastasis, tumor recurrence, and chemoresistance, and are a significant bottleneck in the treatment. Several studies demonstrated metabolic rewiring and altered mitochondrial dynamics in CSCs. However, the role of Mfn1-mediated imbalanced mitochondrial dynamics in ovarian cancer stemness remains poorly understood. In this study, quantification of mtDNA indicates that CSCs have increased mitochondrial mass compared to the parental adherent cells. CD133\u003csup\u003e+\u003c/sup\u003e enriched cells and cancer stem-like cells (spheroid cultured from OC cells) have higher Mfn1 expression and mitochondrial fusion activity. CSCs have increased oxidative phosphorylation (OXPHOS), ATP, and reduced ROS compared to the parental adherent cells. Disruption of mitochondrial dynamics by depletion of Mfn1 modulates the growth and size of spheroid formation and OC stemness. Seahorse analyzer analysis confirms the functional impact of Mfn1 knockdown on mitochondrial respiration. Overexpression of Mfn1 in OC cells, which has a low level of Mfn1 expression, induces increased mitochondrial respiration. Furthermore, to elucidate the relationship between Mfn1 and OXPHOS complex activities and their role in OC stemness, we treated OC cells with 2-Deoxy-D-glucose (2DG), which induces OXPHOS and modulates the expression of cancer stemness markers of OCSCs through Mfn1. During stemness acquisition, CSCs undergo Mfn1-mediated mitochondrial rearrangement, which could be a potential therapeutic strategy against ovarian cancer.\u003c/p\u003e","manuscriptTitle":"Mfn1-mediated imbalanced mitochondrial dynamics promotes ovarian cancer stemness by inducing metabolic reprogramming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 17:59:06","doi":"10.21203/rs.3.rs-7728954/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-05T12:45:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-29T08:13:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65951590470364746943501215980249569363","date":"2025-12-22T01:10:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-08T06:49:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T22:15:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30698780122722002236207500932920399365","date":"2025-11-17T15:31:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325078844898961758351650492589465195285","date":"2025-10-30T13:53:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46351785680224835517143626648057486436","date":"2025-10-28T16:25:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73700365435409223687037800918279371207","date":"2025-10-05T16:00:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-05T11:33:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T11:34:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-01T11:23:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Physiology and Biochemistry","date":"2025-09-27T13:59:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpby","sideBox":"Learn more about [Journal of Physiology and Biochemistry](http://link.springer.com/journal/13105)","snPcode":"13105","submissionUrl":"https://submission.nature.com/new-submission/13105/3","title":"Journal of Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"39b99a69-0bc7-468f-8e9f-3c3c16ed1ee7","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:14:08+00:00","versionOfRecord":{"articleIdentity":"rs-7728954","link":"https://doi.org/10.1007/s13105-026-01172-4","journal":{"identity":"journal-of-physiology-and-biochemistry","isVorOnly":false,"title":"Journal of Physiology and Biochemistry"},"publishedOn":"2026-03-18 15:57:36","publishedOnDateReadable":"March 18th, 2026"},"versionCreatedAt":"2025-10-16 17:59:06","video":"","vorDoi":"10.1007/s13105-026-01172-4","vorDoiUrl":"https://doi.org/10.1007/s13105-026-01172-4","workflowStages":[]},"version":"v1","identity":"rs-7728954","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7728954","identity":"rs-7728954","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}