Nucleophagy-Like Ultrastructural Remodeling in 3D Endometrial Carcinoma Spheroids Exposed to Lithium Chloride

Nucleophagy-Like Ultrastructural Remodeling in 3D Endometrial Carcinoma Spheroids Exposed to Lithium Chloride | 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 Nucleophagy-Like Ultrastructural Remodeling in 3D Endometrial Carcinoma Spheroids Exposed to Lithium Chloride Berna Yıldırım, Kudret Kulak, Ayhan Bilir This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7547146/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Nucleophagy, the selective autophagic degradation of nuclear components, remains poorly characterized in mammalian systems, especially in the context of cancer. Lithium chloride (LiCl) has been reported to modulate cellular stress responses and induce mitophagy-like processes; however, its effects on nuclear architecture have not been clearly defined at the ultrastructural level. Here, we used three-dimensional spheroids of Ishikawa endometrial cancer cells exposed to 10 mM or 50 mM LiCl for up to 96 hours. Cell cycle distribution, BrdU incorporation, and viability were evaluated using flow cytometry and immunohistochemistry, while ultrastructural changes were examined by transmission electron microscopy (TEM). High-dose LiCl (50 mM) induced marked G1/G0 arrest and a sustained decrease in BrdU-positive cells, while Annexin V-FITC/PI staining revealed reduced viability without a proportional increase in apoptotic or necrotic fractions. TEM analysis revealed nuclear envelope elongation, double-membraned vesicles, and cytoplasmic lysis, morphological features that are suggestive of nucleophagy-like remodeling. Notably, preliminary data with 10 mM LiCl revealed early nuclear envelope changes and autophagic vacuole formation, supporting a time- and dose-dependent nuclear response to lithium. It should be emphasized that all findings are descriptive and based solely on ultrastructural (TEM) analysis, without direct molecular validation; therefore, these results should be interpreted as suggestive rather than definitive evidence of nucleophagy. The use of a 3D spheroid model offers a physiologically relevant platform to investigate nuclear remodeling under pharmacological stress. Future studies incorporating molecular markers and loss-of-function approaches will be essential to confirm these observations and assess their clinical relevance in endometrial cancer. Nucleophagy Lithium chloride Endometrial cancer 3D spheroid Ultrastructure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Autophagy is an evolutionarily conserved lysosomal degradation mechanism that plays a central role in maintaining cellular homeostasis by removing damaged or superfluous cellular components [ 1 ]. Beyond bulk degradation, autophagy also includes selective pathways that target specific intracellular structures, including not only cytoplasmic organelles but also nuclear material [ 2 , 3 ]. Among these, nucleophagy is a specialized process that selectively degrades the nuclear envelope, nucleoplasmic contents, and lamin proteins, particularly in response to cellular stress [ 2 , 4 – 10 ]. Although nucleophagy has been well-characterized in yeast and certain model organisms, its morphological features and regulatory mechanisms remain poorly understood in mammalian cells, particularly in cancer contexts [ 11 ]. Nevertheless, multiple studies have demonstrated that LC3 can translocate into the nucleus and interact with lamin B, thereby facilitating the autophagic degradation of nuclear material [ 3 , 12 – 14 ]. Morphologically, nucleophagy is classified into two major forms: macronucleophagy, in which the nuclear envelope is sequestered into autophagic vesicles, and micronucleophagy, where nuclear fragments are directly engulfed by lysosomal structures [ 4 – 6 , 15 , 16 ]. Autophagy is regulated by a conserved set of autophagy-related (ATG) genes, with the ATG8/LC3 protein family playing a central role in autophagosome formation and cargo selection [ 17 ]. LC3 is involved not only in membrane elongation and closure but also in selective autophagy, acting as an adaptor molecule through its interactions with cargo receptors such as p62 and NBR1 [ 15 – 17 ]. This dual functionality of LC3 positions it as a key mediator in both mitophagy and nucleophagy pathways [ 5 ]. Lithium chloride (LiCl), although classically recognized as a GSK3β inhibitor, has also been shown to suppress proliferation, induce cell cycle arrest, and activate autophagic responses across various biological systems, including neuroprotective and cancer models [ 18 – 27 ]. Recent ultrastructural analyses, including our previous work in 3D endometrial cancer spheroids, have demonstrated that LiCl can induce mitophagy-like remodeling and mitochondrial degeneration [ 28 ]. These effects have been linked to mitochondrial stress, mTOR pathway inhibition, and ER-mediated autophagy activation—particularly through modulation of IP₃R-dependent ER stress responses [ 29 ]. However, whether LiCl can also trigger a nucleus-targeted autophagic response, such as nucleophagy, in mammalian cancer cells remains largely unexplored. The present study addresses this gap by providing a detailed ultrastructural evaluation of nuclear remodeling in LiCl-treated endometrial carcinoma spheroids. While LC3/Lamin B interactions were not directly assessed, the observed nuclear envelope invaginations and double-membraned vesicles are morphologically compatible with LC3-associated nucleophagy pathways described in previous reports. However, since no molecular confirmation was performed, these findings should be interpreted as suggestive and descriptive rather than conclusive evidence of nucleophagy. Importantly, our results also link nuclear remodeling to early cell cycle arrest and BrdU suppression, raising the possibility that nucleophagy may contribute to the observed proliferative blockade in lithium-treated cancer cells. Future studies using immunofluorescence, immunogold TEM, or autophagy-deficient models will be essential to confirm the molecular mechanisms underlying this response. Moreover, while the use of a 3D spheroid model provides a physiologically relevant platform to investigate subcellular stress responses, the clinical and translational relevance of these findings remains to be determined. Materials and Methods Three-Dimensional (Spheroid) Cell Culture and LiCl Treatment A multicellular 3D spheroid model was established using a liquid-overlay technique. Ishikawa endometrial adenocarcinoma cells (ATCC) were cultured in DMEM/F12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, USA), 1% L-glutamine, and 1% penicillin–streptomycin at 37°C in a humidified 5% CO₂ incubator. For spheroid formation, semi-confluent monolayers were trypsinized, and cells with 100% viability were seeded at a density of 3 × 10⁵ cells/mL in ultra-low attachment 6-well plates (Corning, USA) pre-coated with 3% Noble agar (Difco, USA), and cultured for 48 h. Mature spheroids (120–300 µm in diameter) were selected and treated with 50 mM lithium chloride (LiCl; Sigma-Aldrich, USA) for 24, 48, 72, or 96 hours. Control groups received vehicle treatment (distilled water). [ 30 – 34 ]. Bromodeoxyuridine (BrdU) Labeling and Immunohistochemistry To assess DNA synthesis, BrdU (5-bromo-2′-deoxyuridine; 20 µM; Roche, Germany) was added to the culture medium 4 hours before fixation. Spheroids were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 µm thickness. Sections were deparaffinized, and antigen retrieval was performed in citrate buffer (pH 6.0). DNA denaturation was carried out using 2N HCl at room temperature for 30 minutes, followed by neutralization in 0.1 M sodium borate (pH 8.5). Sections were incubated with mouse monoclonal anti-BrdU antibody (1:200; Roche) and HRP-conjugated secondary antibody (Dako, USA). Visualization was performed using 3,3'-diaminobenzidine (DAB). BrdU-positive nuclei were counted manually in five randomly selected fields by two blinded histologists. [ 31 ]. Cell Cycle Analysis For flow cytometric cell cycle analysis, spheroids were dissociated into single-cell suspensions using Accutase (Sigma-Aldrich, USA) at 37°C for 15 minutes. Cells were fixed in 70% ethanol at − 20°C overnight. Following fixation, cells were treated with RNase A (100 µg/mL) and stained with propidium iodide (PI; 50 µg/mL) in the dark at room temperature for 30 minutes. DNA content was analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, USA), and cell cycle phases (G₀/G₁, S, and G₂/M) were quantified using ModFit LT software (Verity Software House, USA). All samples were analyzed in triplicate. Annexin V-FITC/PI Apoptosis Assay Apoptotic and necrotic cell populations were assessed using the Annexin V-FITC/PI apoptosis detection kit (BD Pharmingen, USA), following the manufacturer’s protocol. Briefly, 1 × 10⁵ cells were washed with PBS and resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂). Cells were stained with 5 µL Annexin V-FITC and 5 µL PI in 100 µL of binding buffer for 15 minutes at room temperature in the dark. Samples were acquired using a BD FACSCalibur cytometer, and 10,000 events per sample were analyzed with CellQuest and FlowJo software to determine viable, early apoptotic, late apoptotic, and necrotic populations. Transmission Electron Microscopy (TEM) To assess ultrastructural changes, spheroids were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4°C overnight. Post-fixation was carried out with 1% osmium tetroxide in the same buffer for 1 hour at 4°C. Following en bloc staining with 1% uranyl acetate at 4°C for 1 hour, samples were dehydrated in a graded acetone series and embedded in Epon 812 resin. Ultrathin sections (70–90 nm) were cut using a Leica ultramicrotome and collected on copper grids. Sections were stained with 5% uranyl acetate and Reynold’s lead citrate, then examined under a JEOL JEM-1400 Plus transmission electron microscope (JEOL Ltd., Japan) operating at 80 kV. Images were captured at various magnifications. Statistical Analysis All experiments were conducted in biological triplicates (n = 3). Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, USA). One-way ANOVA with Dunnett’s, Sidak’s or Tukey’s post hoc tests was used to compare treated and control groups. Two-way ANOVA was applied to evaluate interaction effects (e.g., treatment and time). A p-value < 0.05 was considered statistically significant. Results 1. LiCl induces G1/G0 arrest in a time-dependent manner To assess the impact of lithium chloride (LiCl) on cell cycle regulation in 3D Ishikawa spheroids, the distribution of cells in G1 + G0, S, and G2 + M phases was quantified at four time points (24, 48, 72, and 96 hours). As illustrated in Fig. 1 , cells treated with 50 mM LiCl showed time-dependent alterations in cell cycle progression. Two-way ANOVA analysis demonstrated a significant effect of treatment condition (column factor: p 0.9999). Notably, LiCl exposure resulted in a pronounced accumulation of cells in the G1 + G0 phase, particularly at 72 and 96 hours, accompanied by a concurrent decline in S-phase populations. These findings indicate that LiCl induces G1-phase arrest in a time-dependent manner and suppresses DNA synthesis activity. Tukey’s post hoc comparisons confirmed that the increase in G1/G0 population and the reduction in S-phase cells under 96h LiCl treatment were statistically significant (p = 0.0007). 2. BrdU incorporation indicates decreased S-phase entry upon LiCl treatment BrdU labeling analysis demonstrated a consistent and significant reduction in proliferative capacity upon LiCl treatment across all time points examined (Fig. 2 ). Quantification of BrdU-positive cells revealed that LiCl (50 mM) significantly decreased S-phase cell percentage at 24h (59.95% ± 1.52 vs. 49.97% ± 1.68), 48h (55.14% ± 2.47 vs. 37.14% ± 1.61), 72h (41.57% ± 1.00 vs. 30.73% ± 1.00), and 96h (50.24% ± 1.55 vs. 39.00% ± 1.59) compared to untreated controls (p < 0.0001 for all). Two-way ANOVA confirmed a significant effect of both time and treatment, with no significant interaction, indicating an overall suppressive effect of LiCl on proliferation independent of exposure duration. 3. LiCl treatment reduces viable cell population without triggering classical apoptotic pathways Flow cytometric Annexin V-FITC/PI analysis demonstrated a progressive reduction in the viable (LL) cell fraction over time following LiCl treatment. Quantitative comparison revealed that the decrease in viability was statistically significant at all time points compared to 0h controls (p < 0.0001, Dunnett’s test), whereas no significant increase was detected in early apoptotic (LR), late apoptotic/necrotic (UR), or necrotic (UL) populations (Fig. 3 ). 4. High-dose LiCl (50 mM) induces pronounced nuclear envelope remodeling consistent with nucleophagy Transmission electron microscopy (TEM) analysis of Ishikawa spheroids treated with 50 mM lithium chloride for 72 hours revealed striking ultrastructural changes, especially involving the nuclear compartment (Figs. 4 B and 5 ). Cells exhibited marked nuclear envelope elongation (NEE) and cytoplasmic lytic areas (LC) in the absence of classical apoptotic morphology. In addition, double-membraned (DM) vesicles were observed in the cytoplasm, some surrounding damaged mitochondria (M), suggestive of mitophagic activity as reported in lithium-related stress contexts. In more advanced sections (Fig. 5 ), the nuclear envelope remodeling became more extensive and spatially distinct, occasionally appearing to surround electron-dense material. These features, together with the presence of autophagic vacuole–like structures, support the occurrence of nucleophagic degradation under high-dose lithium stress. Unlike mitochondrial autophagy, nuclear envelope elongation and selective nuclear compartment alterations observed here represent a less-characterized but morphologically distinct process—consistent with nucleophagy. Discussion 1. Lithium-Induced G1 Arrest and Antiproliferative Effects Our data demonstrate that high-dose lithium chloride (50 mM) induces robust G1/G0 cell cycle arrest in Ishikawa endometrial cancer spheroids, accompanied by a marked reduction in BrdU incorporation over time. These findings are consistent with earlier studies showing that lithium can modulate key cell cycle regulators and suppress DNA synthesis, potentially through inhibition of inositol monophosphatase and related signaling pathways [ 18 , 19 ]. The observed G1 enrichment suggests that LiCl may disrupt proliferative signaling, possibly via mitochondrial or metabolic checkpoints. Notably, this cell cycle blockade was temporally associated with early ultrastructural signs of nuclear stress observed by transmission electron microscopy (Fig. 4 B), including nuclear envelope elongation and cytoplasmic lytic changes. While these features are compatible with the initiation of nucleophagy-like remodeling, it should be emphasized that our interpretations are based exclusively on TEM morphology without molecular confirmation. Thus, a potential link between G1 arrest and nucleophagic remodeling remains suggestive and requires further validation. 2. Non-Apoptotic Cell Death Under Lithium Exposure Annexin V-FITC/PI analysis revealed a clear reduction in cell viability following LiCl treatment; however, this was not accompanied by a significant increase in apoptotic or necrotic populations. These findings suggest the involvement of non-apoptotic, regulated cell death pathways, possibly including autophagy-related mechanisms. Such results are in line with emerging concepts of non-canonical cell death, including nucleophagy, a form of selective autophagy targeting the nucleus. The absence of classical apoptotic features further supports previous reports in lithium-treated models, where cell death may occur via alternative, caspase-independent mechanisms [ 29 , 35 , 36 ]. It is important to note, however, that our data are descriptive and based solely on ultrastructural and cytometric analyses; thus, the precise molecular identity of the cell death pathway remains to be confirmed. Accordingly, we pursued further ultrastructural evaluation by TEM to explore whether LiCl triggers nuclear-selective degradation compatible with nucleophagy-like remodeling. 3. Ultrastructural Evidence for Lithium-Induced Nucleophagy Transmission electron microscopy revealed pronounced structural changes in LiCl-treated spheroids, most notably involving the nuclear envelope. Cells exhibited nuclear envelope elongation (NEE), double-membraned vesicles (DM), and cytoplasmic lytic regions (LC), all observed in the absence of classical apoptotic morphology. These features are morphologically consistent with nucleophagy-like processes, as described in previous ultrastructural studies in yeast and mammalian models [ 2 , 3 ]. Notably, nuclear envelope invaginations and sequestration of electron-dense nuclear material were frequently observed, suggesting the involvement of nuclear autophagic remodeling. While double-membraned vesicles compatible with mitophagy-like structures were also detected—as previously reported under LiCl-induced stress [ 18 , 32 ], the predominant finding was the spatially distinct reorganization of nuclear architecture. Although mitophagy-related changes have been observed in our prior lithium-treated spheroid experiments, the present results underscore a more pronounced, nucleus-directed autophagic response. It is important to emphasize that these interpretations are based solely on TEM morphology, and molecular markers of nucleophagy or mitophagy were not analyzed. Thus, our findings should be regarded as descriptive and hypothesis-generating, highlighting the potential for LiCl to promote nuclear-selective autophagic remodeling in 3D endometrial cancer spheroids. 4. Time and Dose Dependent Nuclear Stress Responses Our preliminary experiments with 10 mM LiCl revealed early ultrastructural indications of nuclear stress, such as mild nuclear envelope elongation (NEE), cytoplasmic autophagic vacuoles (AV), and alterations in Golgi and ER membranes (see Supplementary Figs. 1 and 2). These features are morphologically compatible with the initial stages of nucleophagy-like remodeling, as described in previous reports, although no molecular markers were evaluated in this study. Importantly, these nuclear changes were observed even under sublethal lithium exposure, indicating that the initiation of nuclear remodeling is not limited to overt cytotoxicity. With 50 mM LiCl and 72-hour exposure, nuclear stress progressed to pronounced envelope elongation, invagination, and autophagic sequestration of electron-dense nuclear material—hallmarks that are consistent with advanced nucleophagic processes described in the literature. This time- and dose-dependent evolution highlights the dynamic progression of lithium-induced nuclear remodeling and reinforces the view that the nucleus can become a primary target of autophagic activity under pharmacological stress. Notably, these ultrastructural alterations were observed in parallel with G1/G0 arrest and BrdU suppression, suggesting a possible association between nuclear envelope remodeling and early proliferative blockade. However, since all observations are based exclusively on transmission electron microscopy, our interpretations remain descriptive and should be validated with molecular assays in future studies. This conclusion is further supported by statistical analysis, demonstrating a significant increase in the G1/G0 fraction and a reduction in S-phase cells in LiCl-treated spheroids (p = 0.0007, two-way ANOVA with Tukey’s post hoc test). 5. Mechanistic Considerations and Future Directions While direct molecular confirmation of LC3 or Lamin B involvement was not performed in the present study, the observed double-membraned vesicles and nuclear envelope elongation are morphologically consistent with LC3-associated nucleophagy-like processes described in prior reports [ 3 ]. Previous studies have indicated that LC3 can translocate into the nucleus and interact with Lamin B, thereby mediating the selective autophagic degradation of nuclear components. The double membranes identified by our TEM analysis may reflect isolation membranes originating from the endoplasmic reticulum (ER) or nuclear envelope extensions, potentially regulated by the Atg8/LC3 lipidation system [ 12 ]. However, these interpretations are based exclusively on ultrastructural criteria and should be regarded as descriptive rather than definitive. To conclusively validate these structures as nucleophagosomes, future studies should employ immunogold TEM, LC3/Lamin B co-localization by immunofluorescence, and autophagy-deficient (e.g., Atg5/Atg7 knockout) spheroid models. These approaches will also clarify the contribution of upstream regulators, such as mTOR inhibition or ER stress signaling, in orchestrating nuclear autophagic responses under lithium exposure [ 29 ]. Conclusion In summary, our findings reveal that high-dose lithium chloride induces nuclear envelope remodeling and autophagy-related features in 3D endometrial cancer spheroids, independent of classical apoptosis. Transmission electron microscopy uncovered hallmark features morphologically consistent with nucleophagy-like remodeling, such as nuclear envelope elongation and double-membraned vesicles, in a time- and dose-dependent manner. It is important to emphasize that all interpretations are based exclusively on ultrastructural findings (TEM), without direct molecular validation; therefore, these results should be regarded as descriptive rather than definitive. While LC3/Lamin B interactions were not directly assessed, the observed double-membraned structures and nuclear envelope changes are compatible with LC3-associated nucleophagy pathways reported in previous literature. Future studies incorporating nuclear-specific autophagy markers, immunofluorescence, genetic loss-of-function models, and physiologically relevant LiCl concentrations, ideally in vivo , are warranted to confirm the mechanistic basis and translational impact of these observations. Importantly, the use of a 3D spheroid model provides a physiologically relevant platform to investigate the spatial and temporal dynamics of nuclear remodeling under pharmacological stress, more accurately recapitulating in vivo tumor architecture than monolayer cultures. However, it should be noted that the highest LiCl concentration tested (50 mM) exceeds physiological and therapeutic ranges, which may limit the direct clinical relevance of these findings. Nevertheless, these results expand our understanding of lithium’s intracellular effects beyond mitochondrial targets, positioning the nucleus as a potential site of autophagy-related regulation in cancer cells. As lithium salts continue to be explored for repurposing in oncology, characterizing such non-classical stress responses may uncover new therapeutic vulnerabilities, especially in tumors with aberrant nuclear architecture, and may inform the development of targeted autophagy modulators for endometrial and other nuclear-dysregulated cancers. Declarations Competing Interests The authors declare no competing interests. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution A.B. designed and supervised the study, conducted all experimental procedures including cell culture, immunohistochemistry, flow cytometry, and transmission electron microscopy, and contributed to data analysis. B.Y. performed data interpretation, prepared the figures, and wrote the initial draft of the manuscript. K.K. provided technical assistance and critical input during manuscript revision. 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16:19:49","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80580,"visible":true,"origin":"","legend":"","description":"","filename":"b0722fd4256f4f04b58ea4670142f6091structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/46b0c94359c9cf7b4fbafac4.xml"},{"id":98246570,"identity":"0cd4d286-26c6-44ac-81f2-6ac13620769c","added_by":"auto","created_at":"2025-12-15 16:19:49","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86675,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/1218080ad8ffdb48ecba771c.html"},{"id":98434843,"identity":"2f4bf055-0521-41c6-9381-66caeb9ac165","added_by":"auto","created_at":"2025-12-17 16:52:40","extension":"tiff","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":315159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell cycle analysis of Ishikawa spheroids after LiCl treatment. \u003c/strong\u003eCells were treated with 50 mM lithium chloride or vehicle (control) and analyzed at 24, 48, 72, and 96 hours. Cell cycle distribution (G1/G0, S, G2/M phases) was determined from BrdU-stained sections using image-based quantification. LiCl treatment led to a time-dependent increase in the G1/G0 fraction and a decrease in S-phase cells. Data represent mean values from three independent experiments; statistical significance was observed at 96 hours (p = 0.0007, two-way ANOVA with Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"Figure1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/b76dd9e0c0da6ea7479b40c9.tiff"},{"id":98434397,"identity":"14778426-0be3-4c0a-9775-c7110e3db525","added_by":"auto","created_at":"2025-12-17 16:52:03","extension":"tiff","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":183922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of LiCl on BrdU incorporation in 3D Ishikawa spheroids. \u003c/strong\u003eSpheroids were treated with 50 mM lithium chloride or vehicle (control) and analyzed for BrdU incorporation at 24, 48, 72, and 96 hours. LiCl exposure resulted in a marked, time-dependent reduction in BrdU-positive nuclei compared to controls. Data are presented as mean ± SD (n = 30). Statistical analysis was performed using two-way ANOVA with Sidak’s post hoc test (****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/ac69c684f9c9efea5e30834a.tiff"},{"id":98433001,"identity":"a6d19dae-a305-4ad8-a7a8-18710fec0e7d","added_by":"auto","created_at":"2025-12-17 16:50:11","extension":"tiff","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":321644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometric analysis of cell viability in LiCl-treated Ishikawa spheroids. \u003c/strong\u003eSingle-cell suspensions were prepared from spheroids treated with 50 mM lithium chloride or vehicle (control) and analyzed at 0, 24, 48, 72, and 96 hours using Annexin V-FITC and propidium iodide staining. Viable, apoptotic, and necrotic cell populations were quantified by quadrant-based gating. LiCl treatment resulted in a significant time-dependent reduction in viable cells, without a corresponding increase in apoptotic or necrotic fractions. Data are shown as mean ± SD (n = 4 per group). Statistical analysis was performed using two-way ANOVA with Dunnett’s post hoc test (****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/ec6498a8f700446d8665f5f1.tiff"},{"id":98434437,"identity":"3013cffb-1147-4b6d-9ca2-0200eab6272d","added_by":"auto","created_at":"2025-12-17 16:52:06","extension":"tiff","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":980518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltrastructural analysis of Ishikawa spheroids after high-dose LiCl exposure. \u003c/strong\u003e(A) Control spheroids at 72 hours display intact plasma membranes, continuous nuclear envelope, and preserved mitochondrial cristae, with no ultrastructural signs of autophagy. (B) Spheroids treated with 50 mM lithium chloride for 72 hours exhibit cytoplasmic degeneration, nuclear envelope elongation, and double-membraned structures, consistent with early-stage nucleophagy and mitophagy. Mitochondrial swelling and mild endoplasmic reticulum dilation are also observed. Scale bars: 500 nm. Abbreviations: PM, plasma membrane; NM, nuclear membrane; N, nucleus; M, mitochondrion; Cr, cristae; LC, lytic cytoplasm; NEE, nuclear envelope elongation; DM, double membrane; ER, endoplasmic reticulum.\u003c/p\u003e","description":"","filename":"figure4.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/85f872b2f8df6f0bca4bbcfb.tiff"},{"id":98246558,"identity":"b5bd4507-d4a1-426d-adff-d13381070bc8","added_by":"auto","created_at":"2025-12-15 16:19:49","extension":"tiff","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":608883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltrastructural evidence of nucleophagy and mitophagy in LiCl-treated spheroids. \u003c/strong\u003eTransmission electron microscopy of spheroids treated with 50 mM lithium chloride for 72 hours shows marked nuclear envelope elongation and multilayered double-membraned vesicles, consistent with autophagic isolation membranes. Some double-membraned structures partially envelop nuclear regions, distinguishing them morphologically from typical mitophagic vesicles. Scale bar: 500 nm. Abbreviations: NEE, nuclear envelope elongation; DM, double membrane; M, mitochondrion; ER, endoplasmic reticulum.\u003c/p\u003e","description":"","filename":"Figure5.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/7a6a5372481b99a4809ae229.tiff"},{"id":100360136,"identity":"4bae94d6-a853-4a5d-b8b0-21a97b908476","added_by":"auto","created_at":"2026-01-16 07:37:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5332636,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/8a5441a1-8b25-4ff9-bf40-3444cd4d9308.pdf"},{"id":98434540,"identity":"033cdc53-7afc-4f2a-bee0-d506ca13ad47","added_by":"auto","created_at":"2025-12-17 16:52:16","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1158486,"visible":true,"origin":"","legend":"","description":"","filename":"suppfigure1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/515754359faae8a1f3fa730d.tiff"},{"id":98434559,"identity":"e10b780e-6674-4c3e-a2a7-1f4ddbbd388b","added_by":"auto","created_at":"2025-12-17 16:52:19","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1017144,"visible":true,"origin":"","legend":"","description":"","filename":"suppfigure2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7547146/v2/e85e0330d06d6644f1f7e6f9.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nucleophagy-Like Ultrastructural Remodeling in 3D Endometrial Carcinoma Spheroids Exposed to Lithium Chloride","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutophagy is an evolutionarily conserved lysosomal degradation mechanism that plays a central role in maintaining cellular homeostasis by removing damaged or superfluous cellular components [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Beyond bulk degradation, autophagy also includes selective pathways that target specific intracellular structures, including not only cytoplasmic organelles but also nuclear material [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among these, nucleophagy is a specialized process that selectively degrades the nuclear envelope, nucleoplasmic contents, and lamin proteins, particularly in response to cellular stress [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough nucleophagy has been well-characterized in yeast and certain model organisms, its morphological features and regulatory mechanisms remain poorly understood in mammalian cells, particularly in cancer contexts [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Nevertheless, multiple studies have demonstrated that LC3 can translocate into the nucleus and interact with lamin B, thereby facilitating the autophagic degradation of nuclear material [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Morphologically, nucleophagy is classified into two major forms: macronucleophagy, in which the nuclear envelope is sequestered into autophagic vesicles, and micronucleophagy, where nuclear fragments are directly engulfed by lysosomal structures [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAutophagy is regulated by a conserved set of autophagy-related (ATG) genes, with the ATG8/LC3 protein family playing a central role in autophagosome formation and cargo selection [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. LC3 is involved not only in membrane elongation and closure but also in selective autophagy, acting as an adaptor molecule through its interactions with cargo receptors such as p62 and NBR1 [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This dual functionality of LC3 positions it as a key mediator in both mitophagy and nucleophagy pathways [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLithium chloride (LiCl), although classically recognized as a GSK3β inhibitor, has also been shown to suppress proliferation, induce cell cycle arrest, and activate autophagic responses across various biological systems, including neuroprotective and cancer models [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Recent ultrastructural analyses, including our previous work in 3D endometrial cancer spheroids, have demonstrated that LiCl can induce mitophagy-like remodeling and mitochondrial degeneration [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These effects have been linked to mitochondrial stress, mTOR pathway inhibition, and ER-mediated autophagy activation\u0026mdash;particularly through modulation of IP₃R-dependent ER stress responses [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, whether LiCl can also trigger a nucleus-targeted autophagic response, such as nucleophagy, in mammalian cancer cells remains largely unexplored.\u003c/p\u003e\u003cp\u003eThe present study addresses this gap by providing a detailed ultrastructural evaluation of nuclear remodeling in LiCl-treated endometrial carcinoma spheroids. While LC3/Lamin B interactions were not directly assessed, the observed nuclear envelope invaginations and double-membraned vesicles are morphologically compatible with LC3-associated nucleophagy pathways described in previous reports. However, since no molecular confirmation was performed, these findings should be interpreted as suggestive and descriptive rather than conclusive evidence of nucleophagy. Importantly, our results also link nuclear remodeling to early cell cycle arrest and BrdU suppression, raising the possibility that nucleophagy may contribute to the observed proliferative blockade in lithium-treated cancer cells. Future studies using immunofluorescence, immunogold TEM, or autophagy-deficient models will be essential to confirm the molecular mechanisms underlying this response. Moreover, while the use of a 3D spheroid model provides a physiologically relevant platform to investigate subcellular stress responses, the clinical and translational relevance of these findings remains to be determined.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eThree-Dimensional (Spheroid) Cell Culture and LiCl Treatment\u003c/h2\u003e\u003cp\u003eA multicellular 3D spheroid model was established using a liquid-overlay technique. Ishikawa endometrial adenocarcinoma cells (ATCC) were cultured in DMEM/F12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, USA), 1% L-glutamine, and 1% penicillin\u0026ndash;streptomycin at 37\u0026deg;C in a humidified 5% CO₂ incubator. For spheroid formation, semi-confluent monolayers were trypsinized, and cells with 100% viability were seeded at a density of 3 \u0026times; 10⁵ cells/mL in ultra-low attachment 6-well plates (Corning, USA) pre-coated with 3% Noble agar (Difco, USA), and cultured for 48 h. Mature spheroids (120\u0026ndash;300 \u0026micro;m in diameter) were selected and treated with 50 mM lithium chloride (LiCl; Sigma-Aldrich, USA) for 24, 48, 72, or 96 hours. Control groups received vehicle treatment (distilled water). [\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBromodeoxyuridine (BrdU) Labeling and Immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eTo assess DNA synthesis, BrdU (5-bromo-2\u0026prime;-deoxyuridine; 20 \u0026micro;M; Roche, Germany) was added to the culture medium 4 hours before fixation. Spheroids were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 \u0026micro;m thickness. Sections were deparaffinized, and antigen retrieval was performed in citrate buffer (pH 6.0). DNA denaturation was carried out using 2N HCl at room temperature for 30 minutes, followed by neutralization in 0.1 M sodium borate (pH 8.5). Sections were incubated with mouse monoclonal anti-BrdU antibody (1:200; Roche) and HRP-conjugated secondary antibody (Dako, USA). Visualization was performed using 3,3'-diaminobenzidine (DAB). BrdU-positive nuclei were counted manually in five randomly selected fields by two blinded histologists. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCell Cycle Analysis\u003c/h3\u003e\n\u003cp\u003eFor flow cytometric cell cycle analysis, spheroids were dissociated into single-cell suspensions using Accutase (Sigma-Aldrich, USA) at 37\u0026deg;C for 15 minutes. Cells were fixed in 70% ethanol at \u0026minus;\u0026thinsp;20\u0026deg;C overnight. Following fixation, cells were treated with RNase A (100 \u0026micro;g/mL) and stained with propidium iodide (PI; 50 \u0026micro;g/mL) in the dark at room temperature for 30 minutes. DNA content was analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, USA), and cell cycle phases (G₀/G₁, S, and G₂/M) were quantified using ModFit LT software (Verity Software House, USA). All samples were analyzed in triplicate.\u003c/p\u003e\n\u003ch3\u003eAnnexin V-FITC/PI Apoptosis Assay\u003c/h3\u003e\n\u003cp\u003eApoptotic and necrotic cell populations were assessed using the Annexin V-FITC/PI apoptosis detection kit (BD Pharmingen, USA), following the manufacturer\u0026rsquo;s protocol. Briefly, 1 \u0026times; 10⁵ cells were washed with PBS and resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂). Cells were stained with 5 \u0026micro;L Annexin V-FITC and 5 \u0026micro;L PI in 100 \u0026micro;L of binding buffer for 15 minutes at room temperature in the dark. Samples were acquired using a BD FACSCalibur cytometer, and 10,000 events per sample were analyzed with CellQuest and FlowJo software to determine viable, early apoptotic, late apoptotic, and necrotic populations.\u003c/p\u003e\n\u003ch3\u003eTransmission Electron Microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eTo assess ultrastructural changes, spheroids were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4\u0026deg;C overnight. Post-fixation was carried out with 1% osmium tetroxide in the same buffer for 1 hour at 4\u0026deg;C. Following en bloc staining with 1% uranyl acetate at 4\u0026deg;C for 1 hour, samples were dehydrated in a graded acetone series and embedded in Epon 812 resin. Ultrathin sections (70\u0026ndash;90 nm) were cut using a Leica ultramicrotome and collected on copper grids. Sections were stained with 5% uranyl acetate and Reynold\u0026rsquo;s lead citrate, then examined under a JEOL JEM-1400 Plus transmission electron microscope (JEOL Ltd., Japan) operating at 80 kV. Images were captured at various magnifications.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll experiments were conducted in biological triplicates (n\u0026thinsp;=\u0026thinsp;3). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, USA). One-way ANOVA with Dunnett\u0026rsquo;s, Sidak\u0026rsquo;s or Tukey\u0026rsquo;s post hoc tests was used to compare treated and control groups. Two-way ANOVA was applied to evaluate interaction effects (e.g., treatment and time). A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003e1. LiCl induces G1/G0 arrest in a time-dependent manner\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the impact of lithium chloride (LiCl) on cell cycle regulation in 3D Ishikawa spheroids, the distribution of cells in G1\u0026thinsp;+\u0026thinsp;G0, S, and G2\u0026thinsp;+\u0026thinsp;M phases was quantified at four time points (24, 48, 72, and 96 hours). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, cells treated with 50 mM LiCl showed time-dependent alterations in cell cycle progression. Two-way ANOVA analysis demonstrated a significant effect of treatment condition (column factor: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas the cell cycle phase itself (row factor) did not independently contribute to the variance (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999). Notably, LiCl exposure resulted in a pronounced accumulation of cells in the G1\u0026thinsp;+\u0026thinsp;G0 phase, particularly at 72 and 96 hours, accompanied by a concurrent decline in S-phase populations. These findings indicate that LiCl induces G1-phase arrest in a time-dependent manner and suppresses DNA synthesis activity. Tukey\u0026rsquo;s post hoc comparisons confirmed that the increase in G1/G0 population and the reduction in S-phase cells under 96h LiCl treatment were statistically significant (p\u0026thinsp;=\u0026thinsp;0.0007).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e2. BrdU incorporation indicates decreased S-phase entry upon LiCl treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBrdU labeling analysis demonstrated a consistent and significant reduction in proliferative capacity upon LiCl treatment across all time points examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Quantification of BrdU-positive cells revealed that LiCl (50 mM) significantly decreased S-phase cell percentage at 24h (59.95% \u0026plusmn; 1.52 vs. 49.97% \u0026plusmn; 1.68), 48h (55.14% \u0026plusmn; 2.47 vs. 37.14% \u0026plusmn; 1.61), 72h (41.57% \u0026plusmn; 1.00 vs. 30.73% \u0026plusmn; 1.00), and 96h (50.24% \u0026plusmn; 1.55 vs. 39.00% \u0026plusmn; 1.59) compared to untreated controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for all). Two-way ANOVA confirmed a significant effect of both time and treatment, with no significant interaction, indicating an overall suppressive effect of LiCl on proliferation independent of exposure duration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3. LiCl treatment reduces viable cell population without triggering classical apoptotic pathways\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFlow cytometric Annexin V-FITC/PI analysis demonstrated a progressive reduction in the viable (LL) cell fraction over time following LiCl treatment. Quantitative comparison revealed that the decrease in viability was statistically significant at all time points compared to 0h controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Dunnett\u0026rsquo;s test), whereas no significant increase was detected in early apoptotic (LR), late apoptotic/necrotic (UR), or necrotic (UL) populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e4. High-dose LiCl (50 mM) induces pronounced nuclear envelope remodeling consistent with nucleophagy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTransmission electron microscopy (TEM) analysis of Ishikawa spheroids treated with 50 mM lithium chloride for 72 hours revealed striking ultrastructural changes, especially involving the nuclear compartment (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Cells exhibited marked nuclear envelope elongation (NEE) and cytoplasmic lytic areas (LC) in the absence of classical apoptotic morphology. In addition, double-membraned (DM) vesicles were observed in the cytoplasm, some surrounding damaged mitochondria (M), suggestive of mitophagic activity as reported in lithium-related stress contexts.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn more advanced sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), the nuclear envelope remodeling became more extensive and spatially distinct, occasionally appearing to surround electron-dense material. These features, together with the presence of autophagic vacuole\u0026ndash;like structures, support the occurrence of nucleophagic degradation under high-dose lithium stress. Unlike mitochondrial autophagy, nuclear envelope elongation and selective nuclear compartment alterations observed here represent a less-characterized but morphologically distinct process\u0026mdash;consistent with nucleophagy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003e1. Lithium-Induced G1 Arrest and Antiproliferative Effects\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur data demonstrate that high-dose lithium chloride (50 mM) induces robust G1/G0 cell cycle arrest in Ishikawa endometrial cancer spheroids, accompanied by a marked reduction in BrdU incorporation over time. These findings are consistent with earlier studies showing that lithium can modulate key cell cycle regulators and suppress DNA synthesis, potentially through inhibition of inositol monophosphatase and related signaling pathways [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The observed G1 enrichment suggests that LiCl may disrupt proliferative signaling, possibly via mitochondrial or metabolic checkpoints. Notably, this cell cycle blockade was temporally associated with early ultrastructural signs of nuclear stress observed by transmission electron microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), including nuclear envelope elongation and cytoplasmic lytic changes. While these features are compatible with the initiation of nucleophagy-like remodeling, it should be emphasized that our interpretations are based exclusively on TEM morphology without molecular confirmation. Thus, a potential link between G1 arrest and nucleophagic remodeling remains suggestive and requires further validation.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2. Non-Apoptotic Cell Death Under Lithium Exposure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnnexin V-FITC/PI analysis revealed a clear reduction in cell viability following LiCl treatment; however, this was not accompanied by a significant increase in apoptotic or necrotic populations. These findings suggest the involvement of non-apoptotic, regulated cell death pathways, possibly including autophagy-related mechanisms. Such results are in line with emerging concepts of non-canonical cell death, including nucleophagy, a form of selective autophagy targeting the nucleus. The absence of classical apoptotic features further supports previous reports in lithium-treated models, where cell death may occur via alternative, caspase-independent mechanisms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. It is important to note, however, that our data are descriptive and based solely on ultrastructural and cytometric analyses; thus, the precise molecular identity of the cell death pathway remains to be confirmed. Accordingly, we pursued further ultrastructural evaluation by TEM to explore whether LiCl triggers nuclear-selective degradation compatible with nucleophagy-like remodeling.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3. Ultrastructural Evidence for Lithium-Induced Nucleophagy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTransmission electron microscopy revealed pronounced structural changes in LiCl-treated spheroids, most notably involving the nuclear envelope. Cells exhibited nuclear envelope elongation (NEE), double-membraned vesicles (DM), and cytoplasmic lytic regions (LC), all observed in the absence of classical apoptotic morphology. These features are morphologically consistent with nucleophagy-like processes, as described in previous ultrastructural studies in yeast and mammalian models [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, nuclear envelope invaginations and sequestration of electron-dense nuclear material were frequently observed, suggesting the involvement of nuclear autophagic remodeling. While double-membraned vesicles compatible with mitophagy-like structures were also detected\u0026mdash;as previously reported under LiCl-induced stress [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], the predominant finding was the spatially distinct reorganization of nuclear architecture. Although mitophagy-related changes have been observed in our prior lithium-treated spheroid experiments, the present results underscore a more pronounced, nucleus-directed autophagic response. It is important to emphasize that these interpretations are based solely on TEM morphology, and molecular markers of nucleophagy or mitophagy were not analyzed. Thus, our findings should be regarded as descriptive and hypothesis-generating, highlighting the potential for LiCl to promote nuclear-selective autophagic remodeling in 3D endometrial cancer spheroids.\u003c/p\u003e\u003cp\u003e\u003cb\u003e4. Time and Dose Dependent Nuclear Stress Responses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur preliminary experiments with 10 mM LiCl revealed early ultrastructural indications of nuclear stress, such as mild nuclear envelope elongation (NEE), cytoplasmic autophagic vacuoles (AV), and alterations in Golgi and ER membranes (see Supplementary Figs.\u0026nbsp;1 and 2). These features are morphologically compatible with the initial stages of nucleophagy-like remodeling, as described in previous reports, although no molecular markers were evaluated in this study. Importantly, these nuclear changes were observed even under sublethal lithium exposure, indicating that the initiation of nuclear remodeling is not limited to overt cytotoxicity. With 50 mM LiCl and 72-hour exposure, nuclear stress progressed to pronounced envelope elongation, invagination, and autophagic sequestration of electron-dense nuclear material\u0026mdash;hallmarks that are consistent with advanced nucleophagic processes described in the literature.\u003c/p\u003e\u003cp\u003eThis time- and dose-dependent evolution highlights the dynamic progression of lithium-induced nuclear remodeling and reinforces the view that the nucleus can become a primary target of autophagic activity under pharmacological stress. Notably, these ultrastructural alterations were observed in parallel with G1/G0 arrest and BrdU suppression, suggesting a possible association between nuclear envelope remodeling and early proliferative blockade. However, since all observations are based exclusively on transmission electron microscopy, our interpretations remain descriptive and should be validated with molecular assays in future studies. This conclusion is further supported by statistical analysis, demonstrating a significant increase in the G1/G0 fraction and a reduction in S-phase cells in LiCl-treated spheroids (p\u0026thinsp;=\u0026thinsp;0.0007, two-way ANOVA with Tukey\u0026rsquo;s post hoc test).\u003c/p\u003e\u003cp\u003e\u003cb\u003e5. Mechanistic Considerations and Future Directions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhile direct molecular confirmation of LC3 or Lamin B involvement was not performed in the present study, the observed double-membraned vesicles and nuclear envelope elongation are morphologically consistent with LC3-associated nucleophagy-like processes described in prior reports [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Previous studies have indicated that LC3 can translocate into the nucleus and interact with Lamin B, thereby mediating the selective autophagic degradation of nuclear components.\u003c/p\u003e\u003cp\u003eThe double membranes identified by our TEM analysis may reflect isolation membranes originating from the endoplasmic reticulum (ER) or nuclear envelope extensions, potentially regulated by the Atg8/LC3 lipidation system [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, these interpretations are based exclusively on ultrastructural criteria and should be regarded as descriptive rather than definitive.\u003c/p\u003e\u003cp\u003eTo conclusively validate these structures as nucleophagosomes, future studies should employ immunogold TEM, LC3/Lamin B co-localization by immunofluorescence, and autophagy-deficient (e.g., Atg5/Atg7 knockout) spheroid models. These approaches will also clarify the contribution of upstream regulators, such as mTOR inhibition or ER stress signaling, in orchestrating nuclear autophagic responses under lithium exposure [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our findings reveal that high-dose lithium chloride induces nuclear envelope remodeling and autophagy-related features in 3D endometrial cancer spheroids, independent of classical apoptosis. Transmission electron microscopy uncovered hallmark features morphologically consistent with nucleophagy-like remodeling, such as nuclear envelope elongation and double-membraned vesicles, in a time- and dose-dependent manner. It is important to emphasize that all interpretations are based exclusively on ultrastructural findings (TEM), without direct molecular validation; therefore, these results should be regarded as descriptive rather than definitive. While LC3/Lamin B interactions were not directly assessed, the observed double-membraned structures and nuclear envelope changes are compatible with LC3-associated nucleophagy pathways reported in previous literature. Future studies incorporating nuclear-specific autophagy markers, immunofluorescence, genetic loss-of-function models, and physiologically relevant LiCl concentrations, ideally \u003cem\u003ein vivo\u003c/em\u003e, are warranted to confirm the mechanistic basis and translational impact of these observations.\u003c/p\u003e\u003cp\u003eImportantly, the use of a 3D spheroid model provides a physiologically relevant platform to investigate the spatial and temporal dynamics of nuclear remodeling under pharmacological stress, more accurately recapitulating \u003cem\u003ein vivo\u003c/em\u003e tumor architecture than monolayer cultures. However, it should be noted that the highest LiCl concentration tested (50 mM) exceeds physiological and therapeutic ranges, which may limit the direct clinical relevance of these findings. Nevertheless, these results expand our understanding of lithium\u0026rsquo;s intracellular effects beyond mitochondrial targets, positioning the nucleus as a potential site of autophagy-related regulation in cancer cells.\u003c/p\u003e\u003cp\u003eAs lithium salts continue to be explored for repurposing in oncology, characterizing such non-classical stress responses may uncover new therapeutic vulnerabilities, especially in tumors with aberrant nuclear architecture, and may inform the development of targeted autophagy modulators for endometrial and other nuclear-dysregulated cancers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.B. designed and supervised the study, conducted all experimental procedures including cell culture, immunohistochemistry, flow cytometry, and transmission electron microscopy, and contributed to data analysis. B.Y. performed data interpretation, prepared the figures, and wrote the initial draft of the manuscript. K.K. provided technical assistance and critical input during manuscript revision. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are included in the manuscript and its figures. Additional data are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011;147:728\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark Y-E, et al. Autophagic degradation of nuclear components in mammalian cells. Autophagy. 2009;5:795\u0026ndash;804.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDou Z, et al. 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Pamukkale Med J. 2024;17:560\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRashid H-O, Yadav RK, Kim H-R, Chae H-J. ER stress: Autophagy induction, inhibition and selection. Autophagy. 2015;11:1956\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMotoi Y, Shimada K, Ishiguro K, Hattori N. Lithium and autophagy. ACS Chem Neurosci. 2014;5:434\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nucleophagy, Lithium chloride, Endometrial cancer, 3D spheroid, Ultrastructure","lastPublishedDoi":"10.21203/rs.3.rs-7547146/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7547146/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNucleophagy, the selective autophagic degradation of nuclear components, remains poorly characterized in mammalian systems, especially in the context of cancer. Lithium chloride (LiCl) has been reported to modulate cellular stress responses and induce mitophagy-like processes; however, its effects on nuclear architecture have not been clearly defined at the ultrastructural level. Here, we used three-dimensional spheroids of Ishikawa endometrial cancer cells exposed to 10 mM or 50 mM LiCl for up to 96 hours. Cell cycle distribution, BrdU incorporation, and viability were evaluated using flow cytometry and immunohistochemistry, while ultrastructural changes were examined by transmission electron microscopy (TEM). High-dose LiCl (50 mM) induced marked G1/G0 arrest and a sustained decrease in BrdU-positive cells, while Annexin V-FITC/PI staining revealed reduced viability without a proportional increase in apoptotic or necrotic fractions. TEM analysis revealed nuclear envelope elongation, double-membraned vesicles, and cytoplasmic lysis, morphological features that are suggestive of nucleophagy-like remodeling. Notably, preliminary data with 10 mM LiCl revealed early nuclear envelope changes and autophagic vacuole formation, supporting a time- and dose-dependent nuclear response to lithium. It should be emphasized that all findings are descriptive and based solely on ultrastructural (TEM) analysis, without direct molecular validation; therefore, these results should be interpreted as suggestive rather than definitive evidence of nucleophagy. The use of a 3D spheroid model offers a physiologically relevant platform to investigate nuclear remodeling under pharmacological stress. Future studies incorporating molecular markers and loss-of-function approaches will be essential to confirm these observations and assess their clinical relevance in endometrial cancer.\u003c/p\u003e","manuscriptTitle":"Nucleophagy-Like Ultrastructural Remodeling in 3D Endometrial Carcinoma Spheroids Exposed to Lithium Chloride","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-12-15 16:19:42","doi":"10.21203/rs.3.rs-7547146/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2025-09-11 14:28:15","doi":"10.21203/rs.3.rs-7547146/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0943c336-6d2f-4884-932d-3b7f26e9f4cd","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-11T09:08:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 16:19:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-7547146","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7547146","identity":"rs-7547146","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}