JTC801 regresses uveal melanoma progression through novel methuosis-like cell death via lysosomal dysfunction

JTC801 regresses uveal melanoma progression through novel methuosis-like cell death via lysosomal dysfunction | 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 Article JTC801 regresses uveal melanoma progression through novel methuosis-like cell death via lysosomal dysfunction Qiuyan Liu, Mingyan Huang, Xinpei Ji, Ha Zhu, Wenjun Chang, Hao Shen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5718647/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Uveal melanoma (UM) is the most frequent primary intraocular malignancy in adults with high metastasis and mortality rate, whose effective therapeutic strategy is still in urgent need. Specifically, apoptosis-resistance is a great challenge for advanced UM patients, therefore novel therapeutic options targeting otherwise death modality, which may potentially enhance treatment effect, need to be further identified. Here, by a kinase inhibitor library of 113 approved drugs screening, JTC801, a selective antagonist of nociceptin receptor (NOP), exhibits a specifically strong tumor-killing ability in a lower dosage. JTC801 induces UM cell methuosis-like death characterized by cytoplasmic vacuolization, markedly regresses tumor progression and metastasis, prolongs the survival in multiple UM tumor models without apparent adverse effects. Mechanistically, JTC801-caused nutrient-deficient stress by mitochondrial damage which triggers macropinocytosis and cytoplasmic vacuolization in UM cells. Concomitantly, JTC801 is trapped into the macropinosomes that fuse with lysosomes, further causing lysosomal over-acidification, de-glycosylation of lysosomal associated membrane protein 1(LAMP1), inhibiting cathepsinsmaturation, and exacerbating lysosomal membrane permeabilization (LMP), eventually inducing UM cell methuosis-like death. Collectively, our findings identify JTC801 as a potential valuable antitumor drug especially for apoptosis-resistant advanced UM patients, and provide insight into the distinct tumor cytotoxicity role of JTC801 in UM treatment. Biological sciences/Cancer Health sciences/Medical research/Drug development JTC801 uveal melanoma methuosis cytoplasmic vacuolization lysosome dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Methuosis-like death contributes to JTC801-mediated antitumor efficacy in UM cells, that illumines the potential usage of JTC801 for apoptosis-resistant tumor therapy. JTC801-caused nutritional deficiency facilitates the accumulation of cytoplasmic vacuolization via micropinocytosis in UM cells. JTC801 can be engulfed into the macropinosomes which further fuse with lysosomes. JTC801-mediated lysosomal dysfunction results in UM cell methuosis-like death. Introduction As the most frequent primary intraocular tumor in adults, uveal melanoma (UM) has distinct biological and clinical phenotype compared with their more common cutaneous counterparts. Although the therapeutic regiments for primary UM including radiotherapy, laser therapy, local resection or enucleation achieve local control in more than 90% of patients, more than 50% patients develop liver metastases ultimately and mostly die within one year. Recently, targeted therapies such as tyrosine kinases inhibitors, anti-VEGF antibody (bevacizumab) or immunotherapies especially immune checkpoints inhibitors have been conducted in advanced UM patients [ 1 , 2 ], however, these therapeutic strategies have not shown definitive effectiveness in advanced UM patients. Tebentafusp, as the first-in-class immune-mobilizing monoclonal T cell receptor against cancer (ImmTAC) approved by the FDA in January 2022, has been explored to extend the overall survival (OS) in unresectable or metastatic UM patients [ 3 ]. However, only the patients with gp100 and HLA-A2:01 dual positive expression can benefit from this treatment, and the toxicities such as cytokine release syndrome (CRS), off-target or secondary resistance limit the therapeutic efficacy and clinical application [ 4 ], thus highlighting the urgent need for novel treatment options to reach more UM patients. Cell death is a complex and interconnected process that plays a crucial role in maintaining tissue homeostasis and preventing disease, and great efforts have been dedicated to targeting cell death process to hinder tumor progression. To find viable alternatives to conventional apoptosis-based tumor therapies that exhibit drug resistance, other types of programmed cell death (PCD) have recently attracted significant attention. Drugs inducing tumor cell pyroptosis [ 5 ], necroptosis [ 6 ] and ferroptosis [ 7 ], have been widely studied and shown promising antitumor effects. Besides, more PCD pathways have been identified as effective tumor therapeutic targets such as mitoptosis [ 8 ], paraptosis [ 9 ], alkaliptosis [ 10 ] and methuosis [ 11 ], each with distinct morphological features and relating to dysfunctional organelle and dysregulated metabolic process. However, the UM patients, most with aberrant activated kinase signaling pathway, are resistant to almost all existing kinase inhibitor treatment-induced apoptosis including PKC signaling inhibitors [ 12 ]. Therefore, identifying new valuable non-apoptotic death modality inducers for obstinate apoptosis-resistant UM, and uncovering the underlying mechanisms may help to provide more potential therapeutic strategies for the treatment of UM patients. JTC801 is a selective antagonist of nociceptin receptor (NOP) which belongs to the G-protein-coupled receptors (GPCRs) family [ 13 ], and well-known for reversing pain and anxiety symptoms [ 14 ]. Recently, the antitumor function of JTC801 raises extensively attention in multiple tumors. Accumulating evidences demonstrate that JTC801 efficiently inhibits tumor malignant phenotype in vitro and suppresses tumor progression and metastasis in vivo . Mechanistically, apoptosis [ 15 ], autophagy [ 16 ] or pH-dependent alkaliptosis [ 17 ] involve in JTC801-mediated antitumor efficacy have been demonstrated in difference tumor models. However, the role of JTC801 in regulating other tumor cell death modality has not been completely understood. It is worth investigating whether there is a unique role of JTC801 in regulating cell death in UM cells. Here, we screened a kinase inhibitor library of 113 approved drugs to identify the cytotoxic activity in human UM cell lines. Among these inhibitors, JTC801 exhibits a specifically strong tumor-killing ability in a lower dosage in vitro . Notably, JTC801 induces methuosis-like death with a predominant phenotype of cytoplasmic vacuolization in UM cells, profoundly regresses tumor progression and metastasis, prolongs the survival in multiple UM tumor models without apparent adverse effects. Mechanistically, JTC801 caused nutrition transporters inhibition and mitochondrial damage leading to nutritional deficiency-triggered macropinocytosis. Simultaneously, JTC801 is trapped into the macropinosomes that fuse with lysosomes, leading to lysosomal dysfunction results in methuosis-like death in UM cells. Our findings identify JTC801 as a potential promising drug for clinical application in advanced UM patients especially those resistant to apoptosis, and provide insight into the unique tumor cytotoxicity role of JTC801 in UM cells. Results JTC801 inhibits proliferation and induces cell death in UM cells Given that most UM patients exhibit aberrant activation of kinase signaling pathways [ 12 ], we aimed to identify novel antitumor agents for UM by screening a kinase inhibitor library of 113 approved drugs (ZK012, EFEBIO library service) under both normoxic and hypoxic conditions in human MUM2B cells. As shown in Fig. 1 A, 13 drugs exhibited higher tumor-killing ability, and the top 6 drugs were selected to evaluate their antitumor efficacy at different dosage and time point. Among these agents, JTC801 exhibited the best cytotoxic effect even at 3 µM dosage, and showed a dose- and time-dependent tumor-killing capacity in MUM2B and OMM2.5 UM cells, as well as in mouse melanoma B16F10 cells (Fig. 1 B-C and Fig. S1A-C ). Moreover, EdU incorporation and PI staining assay confirmed that JTC801 markedly decreased cell proliferation and induced G2/S-phase cell cycle arrest (Fig. S1D , E ). JTC801 also significantly impaired the migration and invasion abilities of MUM2B cells (Fig. S1F , G ). Furthermore, colony formation assay indicated elevated UM cell death after JTC801 treatment (Fig. 1 D, E), and annexin V/PI staining resulted in enhanced the ratio of cell death after JTC801 treatment in UM cells (Fig. 1 F, G). These results demonstrated that JTC801 could inhibit malignant phenotype and induce cell death in UM cells at lower dosage in vitro . Then, as a potential drug for tumor therapy, we are more interested in the cell death modality and underlying mechanisms triggered by JTC801 in UM cells. We performed RNA-sequencing (RNA-seq) in MUM2B cells with or without JTC801 treatment. Gene set enrichment analysis (GSEA) results showed that genes related to apoptosis, ferroptosis and mitophagy were enriched in JTC801-treated group (Fig. S2A ). So, we detected the expression of specifical markers of the above cell death forms by western blotting. Consistent with sequencing results, JTC801 treatment decreased the expression of BCL2 like 1 (BCL-XL) and glutathione peroxidase 4 (GPX4), while increased the expression of cleaved-caspase 3 and microtubule associated protein 1 light chain 3 beta (LC3B) in MUM2B cells (Fig. S2B ). Moreover, comet assay showed that MUM2B cells exhibited significant longer nuclear comet tails (tail length 13.4 µm v.s. 102.4 µm) after JTC801 treatment (Fig. S2C ), and the nuclear accumulation of γ-H2AX proteins was observed in JTC801-treated MUM2B cells (Fig. S2D ). Then, the specifical inhibitors including pan-caspase inhibitor (Z-VAD-FMK), caspase3 inhibitor (Z-DEVD-FMK), necroptosis inhibitor (Necrostatin-1) and ferroptosis inhibitor (Ferrostatin-1) were used to determine the key contributor of JTC801-triggered UM cell death. However, only the pan-caspase inhibitor (Z-VAD-FMK) could partially rescue cell viability induced by JTC801(Fig. S2E ). In addition, pre-treatment with Mdivi-1 or 3MA, the inhibitor of mitophagy and autophagy respectively, also failed to rescue JTC801-triggered cell death in MUM2B cells (Fig. S2F , G ). Thus, these results suggested that other novel cell death modality rather than apoptosis may play a key contributor in JTC801-induced UM cell death. JTC801 induces cytoplasmic vacuolization similar to methuosis in UM cells To figure out the cell death modality regulated by JTC801, we conducted live-cell imaging of JTC801-treated MUM2B cells. Massive cytoplasmic vacuoles were observed in JTC801-treated MUM2B cells, and the vacuoles were accumulated and coalesced to form progressively larger vacuoles in a time-dependent manner resulting in cell death eventually (Fig. 2 A and Video 1 ). Transmission electron microscopy (TEM) revealed that these cytoplasmic vacuoles were bound by a single membrane rather than the typical double membrane of autophagosomes (Fig. 2 B). These morphological characteristics is similar to methuosis, a non-apoptotic cell death modality, which is characterized by massive cytoplasmic single-membranous vacuoles accumulation derived from macropinocytosis or endosomal-lysosomal trafficking disruption without cellular shrinkage, chromatin condensation or nuclear fragmentation [ 18 ]. Since methuosis-related macropinosomes are decorated with the markers for late endosome and lysosomes RAB7 and LAMP1 [ 18 , 19 ], we further performed immunofluorescence (IF) assay to detect the expression and location of these two molecules in MUM2B cells. As expected, the cytoplasmic vacuoles incorporated RAB7 and LAMP1 were observed by confocal microscopy in JTC801-treated MUM2B cells (Fig. 2 C, D). Thus, these data indicated that JTC801 induced cytoplasmic vacuolization-associated cell death similar to methuosis in UM cells. Cytoplasmic vacuoles triggered by JTC801 origins from macropinocytosis and fuse with lysosomes To clarify the origin and composition of the cytoplasmic vacuoles, MUM2B cells were labeled by several organelle markers including MitoTracker (mitochondria), ER-Tracker (endoplasmic reticulum), LysoTracker (lysosome), and LysoSensor Green DND-189 (lysosomal acidification) after JTC801 treatment. As shown in Fig. 3 A-D, the dyes of LysoTracker and LysoSensor Green DND-189 were accumulated and colocalized with the cytoplasmic vacuoles, while the dyes of MitoTracker and ER-Tracker were excluded from the cytoplasmic vacuoles in JTC801-treated MUM2B cells. Moreover, enlarged lysosomes were observed in JTC801-treated MUM2B cells (Fig. 3 E). Therefore, these data suggested that JTC801-triggered cytoplasmic vacuoles might be related to the lysosomal compartments. As the cytoplasmic vacuoles in methuosis usually derived from micropinocytosis [ 18 ], then, living MUM2B cells were observed under a fluorescence microscope after adding dyes of bulk fluid-phase tracer Lucifer Yellow and LysoTracker to detect the origination of JTC801-induced cytoplasmic vacuoles. As shown in Fig. 3 F, Lucifer Yellow and LysoTracker were co-colocalization in cytoplasmic vacuoles forming yellow puncta in JTC801-treated cells. Moreover, dextran uptake assay confirmed that JTC801-triggered cytoplasmic vacuoles originated from macropinocytosis by flow cytometry analysis (Fig. 3 G). Next, to determine whether JTC801 can be trapped into macropinosomes via macropinocytosis concurrently, ultra performance liquid chromatography and mass spectrometry (UPLC-MS) experiment was performed. The analysis data showed that JTC801 was detected in lysosomal extraction lysates (Fig. 3 H). Taken together, these results indicated that JTC801 can be engulfed via macropinocytosis and trigger cytoplasmic vacuoles accumulation to form macropinosomes that fuse with lysosomes, a process distinct from methuosis. Thus, we termed JTC801-induced UM cell death as methuosis-like death. Lysosomal dysfunction contributes to JTC801-induced methuosis-like death in um cells Our above-mentioned results indicated that JTC801-triggered UM cell death matched the major characteristics of methuosis, except for the macropinosomes fused with lysosomes. Then, why JTC801-induced macropinosomes can fuse with lysosomes, but exhibit inefficiency in cellular degradation finally leading to cell death? We analyzed the KEGG pathway of cellular processes using differentially expressed genes from RNA sequence in UM cells treated with or without JTC801. The enrichment of lysosome pathway was observed in JTC801-treated cells (Fig. S3A ). And the abundant undegraded debris within the macropinosomes was observed by TEM in JTC801-treated UM cells (Fig. 4 A), indicating compromised lysosomal function. It’s well known that the most predominant lysosomal hydrolases, cathepsins in lysosomes are initially synthesized as inactive zymogens and must be converted into their mature forms to exhibit proteolytic activity [ 20 ]. As shown in Fig. 4 B, in JTC801-treated MUM2B cells, the precursor forms of both cathepsin B (CTSB) and CTSD were increased, however, their mature forms were decreased. And the impaired CTSB activity was also detected by MagicRed assay, indicating a loss of lysosomal hydrolytic capacity after JTC801 treatment (Fig. 4 C). In addition, the optimal pH of lysosomes is essential to ensure the cellular degradation process [ 21 ]. We then investigated whether JTC801 could interrupt the lysosomal acidification. As examined by LysoSensor Green DND-189 dye staining, the fluorescence of which increases in acidic environments, higher intensity of the LysoSensor fluorescence was observed in JTC801-treated MUM2B cells compared to the control cells (Fig. 4 D). Furthermore, LysoSensor Yellow/Blue dextran probe was performed to qualitatively measure the hydrogen ion concentration (pH) of lysosomes. As shown in Fig. 4 E, JTC801 treatment decreased the lysosomal pH in MUM2B cells compared to the control cells (2.99 v.s. 4.4). Similarly, PHrodo is a live-cell probe indicative of cytoplasmic pH, which has a low fluorescence intensity at neutral pH, with the fluorescence increasing as pH drops. The results showed that JTC801-treated MUM2B cells exhibited a brighter fluorescence compared to the control cells, suggesting a decreased cytoplasmic pH after JTC801 treatment (Fig. S3B ). Additionally, bafilomycin A (BafA1), a vacuolar-type H + ATPase (V-ATPase) inhibitor known to increase lysosomal pH [ 22 ], can significantly restore the viability of JTC801-treated MUM2B cells (Fig. 4 F). These results revealed that JTC801 facilitated lysosomal over-acidification and restrained the maturation of cathepsins in UM cells. The glycosylation of lysosomal membrane protein is essential for remaining membrane integrity by preventing rupture of lysosomal membrane induced by lumen proteolytic enzymes [ 23 , 24 ]. When lysosomal membrane permeabilization (LMP) occurred, membrane integrity is disrupted, allowing cathepsins to leak into the cytoplasm, resulting in cytoplasmic acidification and irreversible cell death [ 25 , 26 ]. Thereby, we next detected the glycosylation level of lysosomal membrane protein LAMP1. As shown in Fig. 4 G, JTC801 profoundly impaired LAMP1 glycosylation in a time-dependent manner in MUM2B cells. Then, acridine orange (AO) uptake assay was performed to detect LMP, whose FITC fluorescence indicating lysosomal membrane leakage while PE fluorescence indicating intact lysosome. The results showed that increased fluorescence intensity of FITC, accompanied by decreased of PE were detected by flow cytometry, and the ratio of FITC/PE was significantly increased after JTC801 treatment (Fig. 4 H, I). Accordantly, the red particles obviously descended, and green particles enhanced in JTC801-treated cells captured by fluorescence microscope (Fig. S3C ), suggesting loss of lysosomal membrane integrity. Taken together, these results revealed that JTC801 triggered UM cell methuosis-like death via lysosomal dysfunction including lysosomal over-acidification, LAMP1 de-glycosylation, cathepsins maturation inhibition and LMP. JTC801-caused nutritional deficiency by mitochondrial damage triggers macropinocytosis As a clathrin- and caveolin-independent endocytosis of extracellular fluid, macropinocytosis plays a vital role in nutrient uptake to meet the nutrition requirements in tumor cells under a nutrient-deficient microenvironment [ 27 , 28 ]. Then, we first explored whether JTC801 could affect the expression of nutritional transporters. The results showed in Fig. 5 A and B , JTC801 treatment significantly decreased the expression of glucose transporter solute carrier family 2 member 1 (SLC2A1) and amino acid transporter SLC7A5 in MUM2B cells compared to the control cells. However, the expression including SLC2A3, SLC1A5, carnitine palmitoyl transferase 2 (CPT2) and fatty acid synthase (FANS) did not show significant changes after JTC801 treatment (Fig. S4A ). Thus, these data suggested that JTC801 impaired glucose and amino acid collection and induced nutritional deficiency stress in UM cells. Then, whether JTC801-induced nutritional deficiency could affect mitochondrial function? GSEA analysis showed a significant downregulation of the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) in JTC801-treated MUM2B cells compared to the control cells (Fig. S4B , C ). And TEM images showed a significant swelling and cristae disorder of mitochondria in JTC801-treated MUM2B cells, while the control cells showed intact cristae and elongated mitochondria morphology (Fig. 5 C), indicating impaired mitochondrial function after JTC801 treatment. Then, a fluorometric analysis was conducted using the fluorescent dye JC-1 to assess mitochondrial damage. Compared to the control cells, JTC801 treatment significantly increased the green fluorescence of the JC-1 monomers, indicating damaged mitochondrial membrane potential (Fig. 5 D). Consistent with this, the decreased adenosine triphosphate (ATP) generation was observed as early as 2 hours in JTC801-treated MUM2B cells following JTC801 treatment (Fig. 4 E). Additionally, JTC801 treatment also increased reactive oxygen species (ROS) production and lipid peroxidation accumulation (Fig. 4 F, G). Collectively, these results indicated that JTC801 caused nutrient-deficient stress through mitochondrial damage triggering macropinocytosis in UM cells. JTC801 regresses tumor progression and metastasis in vivo Then, we want to know whether JTC801 can exhibit antitumor efficacy in vivo ? We firstly investigated the tumor-killing activity of JTC801 in a xenograft mouse model. Tumor-bearing mice was conducted by subcutaneous implantation of MUM2B cells in nude mice for one week, followed by JTC801 intratumoral delivery every other day for two consecutive weeks (Fig. 6 A). Reduced tumor growth and extended survival were observed in JTC801-treated tumor-bearing mice compared with control mice (Fig. 6 B, C). Similar experiments were established in immunocompetent C57BL/6 mice using luciferase-labeled B16F10 cells. Administration of JTC801 significantly suppressed tumor growth and improved the survival of tumor-bearing mice (Fig. 6 D, E). Moreover, JTC801 profoundly suppressed tumor progression, metastasis (75% v.s. 25%) and prolonged survival in an orthotopic mice model established by intravitreal injection of luciferase-labeled MUM2B cells (Fig. 6 F-I). In addition, tumor tissues were isolated and subjected to hematoxylin-eosin (HE) staining analysis after final treatment with JTC801. The images showed the formation of large vacuoles in JTC801-treated tumor tissues compared to their control counterparts (Fig. S5A ). Besides, daily oral administration of JTC801 in a xenograft mouse model and homograft mouse model performed (Fig. S5B ). Compared with the control group, the MUM2B tumor growth was obviously inhibited in the JTC801-treated group (Fig. S5C ). Similarly, mice treatment with JTC801 were associated with longer survival in B16F10-tumor bearing mice models (Fig. S5D ). Most importantly, there were no obvious adverse effects on the heart, liver, spleen, lung, kidney and stomach in both C57BL/6 and BALB/c mice following peritoneal injection of JTC801 for 3 weeks (Fig. S5E ). Taken together, JTC801 exhibited valuable antitumor efficacy via distinct tumor cytotoxicity, suggesting that JTC801 may be a promising drug with potential clinical application, especially for apoptosis-resistant advanced UM patients. Discussion Current cancer chemotherapeutic regimens primarily rely on the activation of apoptotic cell death pathway. However, after a period of clinical application, most antitumor compounds show reduced efficacy or various side effects, and majority of patients developed resistance toward those chemotherapeutic drugs [ 29 ]. Thus, resisting apoptotic cell death has been a common challenge and needs to be resolved urgently. Discovery of therapeutic regimens that induce non-apoptotic forms of cell death should be a promising strategy for breaking apoptotic resistance and increasing the antitumor curative effect. As a relatively new non-apoptotic form of cell death, methuosis can be triggered in both apoptosis-sensitive and apoptosis-resistant tumor cells, offering highly beneficial for those patients resistant to conventional chemotherapy [ 8 , 30 ]. Recently, several small molecule methuosis inducers such as indol-based chalcones [ 31 ], Jaspine B [ 32 ], Tubeimoside-1 (TBM1) [ 33 ], casein kinase II (CK2) inhibitor silmitasertib (CX-4945) [ 34 ] and phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) inhibitors [ 35 ] have been illustrated to exhibit effectively antitumor activity in multiple tumor models, specifically in apoptosis-resistant tumors. Therefore, methuosis becomes a potential promising cell death form which is expected to be effective for overcoming therapy-resistant tumors. In this study, we identified that methuosis-like cell death mainly contributed to JTC801-induced cell death in UM cells at a lower dose, and significantly regressed tumor progression and prolonged the survival of tumor-bearing mice. Notably, no obvious adverse effects were observed in JTC801-treated mice models. To our knowledge, our findings firstly identify JTC801 as a methuosis-like cell death inducer. These findings illumined the potential clinical application of JTC801, especially in apoptosis-resistant advanced UM patients. Methuosis is characterized by the accumulation of massive vacuoles within the cytoplasm derived from micropinocytosis [ 36 ]. Macropinocytosis plays a pivotal role in nutrient uptake which mediates non-selective internalization of extracellular fluid to meet the nutrition requirements in tumor cells under a nutrient-deficient microenvironment, promoting tumor cells survive and increasing the resistance to antitumor drugs [ 27 , 28 ]. However, hyperactive macropinocytosis or impaired lysosomal fusion with macropinosomes result in cell membrane rupture and final methuosis. Several small molecular methuosis inducers such as TBM1 [ 37 ], Bacoside A [ 38 ] and Ursolic acid derivatives (C17) [ 39 ] have been shown to exhibit antitumor activity in multiple solid tumors via stimulating hyperactive macropinocytosis, resulting in cell membrane rupture and final methuosis. DMBP [ 40 ], MIPP/MOMIPP [ 41 ] and Vacquinol-1 [ 42 ] restrain lysosomal fusion with macropinosomes to facilitate tumor cell methuosis, resulting in suppressing tumor progression and metastasis. Our results revealed that JTC801-caused nutrient-deficient stress through limiting nutritional collection and impairing mitochondrial function that triggers macropinocytosis in UM cells, concurrently, JTC801 is trapped into the macropinosomes. However, JTC801 did not affect the fusion of macropinosomes with lysosomes, a typical characteristic of methuosis, instead, it triggered methuosis-like death in UM cells, indicating JTC801 treatment may lead to lysosomal inefficiency. As a major regulatory platform for cellular processes and signaling hub, lysosome controls cell death and survival [ 21 ], the lysosomal optimal pH and the integrality of lysosomal membrane are essential for efficient cellular degradation, which is crucial for maintaining cellular homeostasis [ 43 ]. Both over-acidification and de-acidification of lysosomes can cause lysosomal functional defects [ 22 , 44 , 45 ] and different therapeutic intervention has been established via lysosomal acidification-modulating agents [ 46 ]. Moreover, the glycosylation of lysosomal membrane protein is important for lysosomal membrane integrity by preventing the lysosomal membrane rupture destroyed by lumen proteolytic enzymes [ 47 ]. TMEM175, a ubiquitously expressed lysosomal membrane protein, as a proton-activated proton channel for regulating the lysosomal proton efflux to maintain a steady-state pH [ 48 ]. Deficiency of TMEM175 can lead to lysosomal over-acidification, resulting in impaired the lysosomal degradation [ 49 ]. And prosapogenin A (PA), a bioactive compound prevalent in traditional Chinese herbs, has been demonstrated to trigger lysosomal over-acidification, which exacerbates LMP and subsequent lysosomal damage, exhibiting significant antitumor efficacy in anaplastic thyroid cancer [ 22 ]. Additionally, the methuosis inducer SGI-1027 has been documented that exerts synergistic antitumor activity with the mTOR inhibitor everolimus via apoptosis and GSDME-dependent pyroptosis by triggering LMP in renal cancer [ 11 ]. Consistent with these findings, our results revealed that the JTC801 is trapped into macropinosomes that fused with lysosomes, further caused lysosomal over-acidification, LAMP1 de-glycosylation, cathepsins maturation inhibition and LMP, leading to loss of lysosomal degradation capacity and membrane integrality, ultimately resulting in UM cell methuosis-like death via lysosomal dysfunction. In addition, Prof. Tang’s group first termed that JTC801 induces CA9 downregulation by ACSS2mediated acetylation and activation of NF-κB which specifically contributes to alkaliptosis in PDAC cells[ 17 , 50 ]. Recently, the group further revealed that JTC801 directly binds to ATP6V0D1 contributing to alkaliptosis via blocking STAT3-mediated lysosomal pH homeostasis, independent of the NF-κB-CA9 pathway [ 51 ]. However, we did not detect intracellular alkalization, CA9 downregulation and STAT3 phosphorylation inhibition after JTC801 treatment in UM cells (data not shown). We speculate that the discrepancy is likely due to the different cell lines, dosage or time points used in different studies. The mechanisms of JTC801-mediated cell death are diverse with heterogeneity and plasticity. Additionally, our RNA sequencing revealed elevated expression of SLC15A4 [ 52 ] and CLCN7 [ 53 ], the lysosome-resident, proton-coupled amino-acid transporter and H + /Cl – exchange transporter, respectively, which loss caused impaired lysosomal acidification, and a decreased expression of SLC16A3, a transporter mainly mediating intracellular lactic acid efflux [ 54 ] in JTC801-treated MUM2B cells (data not shown), suggested that these molecules might be involved in JTC801-mediated lysosomal over-acidification. Therefore, the precise effector molecules and signal pathways in JTC801-mediated cell death is worth further investigation. In sum, our results demonstrated that JTC801, acting as a methuosis-like cell death inducer, exhibited valuable antitumor efficacy via specific tumor cytotoxicity, presenting a new promising therapeutic option for apoptosis-resistant advanced UM treatment. Material and methods Mice and cell lines 6–8 weeks male athymic nude mice were purchased from Gempharmatech Co., Ltd. (Nanjing, China) and kept in specific pathogen-free conditions. 6–8 weeks male C57BL/6 and BALB/c were purchased from Joint Ventures Sipper BK Experimental Animal (Shanghai, China). All mice experimental manipulations were approved by the Scientific Investigation Board of Navy Medical University (Shanghai, China) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Human UM cell lines including MUM2B, OMM2.5 and MP41 cells were kind gifts from Prof. Jiaxu Hong (Department of Ophthalmology and Vision Science, Shanghai Eye Ear Nose and Throat Hospital, Fudan University, Shanghai, China). Mouse melanoma B16F10 cells were purchased from the American Type Culture Collection (ATCC). MUM2B and B16F10 were transfected with lentivirus encoding luciferase (Genechem, GV633) as previously described [ 55 ], then termed as MUM2B-luc cells or B16F10-luc cells. All cell lines were cultured in RPMI 1640 medium (11875-093, Gibco) supplemented with 10% fetal bovine serum (S1580-500, Biowest) at 37℃ in a humidified incubator containing 5% CO 2 . Cell viability assay Cell viability assay was measured by the Cell Counting Kit 8 assay (CCK8) Kit (HY-K0301, MCE). Cells (5 × 10 3 cells per well) were cultivated in 96-well plates and JTC801 (HY-13274, MCE) was added to plates. At the appropriated time point, each well was replaced with 100 µl of fresh medium containing 10 µl of CCK8 solutions and incubated for 1 h at 37℃. The results were assayed with a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, USA). Colony formation assay MUM2B cells and OMM2.5 cells were treated with or without JTC801 for 8 h. Then, cells were harvested and seeded (1000 counts per well) into a new 6-well plate. 14 days later, visible colonies were fixed and then stained with 0.1% crystal violet for 30 min at room temperature (RT). Images were acquired using a scanner (OLYMPUS, Tokyo, Japan). Flow cytometry For cell apoptosis, MUM2B cells and OMM2.5 cells were treated with or without JTC801 for 24 h. Cells were harvested and stained with Annexin V-FITC and Propidium Iodide (556547, BD biosciences) for 15 min in the dark at RT. The Annexin V-positive cells were regarded as apoptotic cells. For dextran uptake analysis, MUM2B cells (1 × 10 5 ) were seeded into 12-well plate and incubated overnight. The cells were then incubated with dextran-Alexa Fluor 488 (D1821, Invitrogen) at presence or absence of JTC801 for 2 h. Mitochondrial membrane potential was assessed using JC-1 dye (T3168, Invitrogen). MUM2B cells (3 × 10 5 ) were seeded into 6-well plate and treated with or without JTC801 for 16 h. Then, cells were stained with JC-1 dye at 37°C in the dark for 30 minutes. Red fluorescence from JC-1-aggregate (Ex/Em = 525/590 nm) was observed in normal cells, while green fluorescence from JC-1 monomer (Ex/Em = 490/530 nm) was generated as membrane potential decreased. ROS generation was detected using a ROS assay kit (S0033S, Beyotime) according to the manufacturer’s instructions. MUM2B cells (3 × 10 5 ) were seeded into 6-well plate and treated with or without JTC801 for 24 h. Then, DCFH-DA dilution (1:1000) was added into each well and incubated at 37 ℃ for 30 min in the dark. Lipid peroxidation was measured by BODIPY 581/591 C11 (D3861, Invitrogen). MUM2B cells (3 × 10 5 ) were seeded into 6-well plate and treated with or without JTC801 for 24 h. Then the cells were washed with PBS and incubated with BODIPY dilution (1:1000) for 30 min at 37°C in the dark. After washing, cells were determined by flow cytometry (Fortessa X-20, Becton Dickinson, San Jose, CA, USA) as described previously [ 55 ]. The data were analyzed using FlowJo 10 software (TreeStar, USA). RNAsequencing Total RNA was extracted from the samples using TRIzol reagent (127401, Invitrogen) according to the manufacturer’s protocol. RNA quality control, library construction and sequencing were performed by BGI Genomics Corporation (Shenzhen, China), data analysis was performed online ( https://biosys.bgi.com ). The differentially expressed genes were identified using edgeR. The set of potential target genes was screened using the following filter criteria: q-value (corrected P value) 2. Gene set enrichment analysis (GSEA) was used to screen significantly enriched signaling pathways with default parameters and Gene Ontology (GO) framework was used to identify the specific biological processes. Transmission electron microscopy (TEM) The ultrastructure of MUM2B cells were observed by TEM. MUM2B cells treated with or without JTC801 were placed in 3% glutaraldehyde at 4℃ for 24 h. Electron microscope photography was performed and imaged by Servicebio Technology Company (Wuhan, China) using Hitachi HT7700 TEM (Hitachi, Japan). Time-lapse imaging Time-lapse imaging of MUM2B cells treated with or without JTC801 was performed using an IncuCyte Zoom Live-content imaging system (IncuCyte S3, Sartorius, Goettingen, Germany). Immunofluorescence (IF) IF was performed as we previously described [ 56 ]. MUM2B cells (5 × 10 4 ) were seeded into coverslips placed in a 24-well plate at presence or absence of JTC801. The cells were washed twice with ice-cold PBS and fixed with 4% formaldehyde for 30 min at RT. After washing, the cells were permeabilized with 0.1% TritonX-100 in PBS for 15 min followed by blocking with 5% BSA for 1 h. Subsequently, the cells were incubated with the primary antibodies against LAMP1 (9091S, CST) or RAB7 (9367S, CST) or γ-H2AX (ab22551, Abcam) at the dilution of 1:200 overnight at 4℃, and then incubated with Alexa Fluor 488-conjugated secondary antibodies (A21210, Invitrogen) at the dilution of 1:100. Nuclear were labeled with DAPI stain (36308ES20, Yeasen). Fluorescence images were performed using a fluorescence microscope (Zeiss, Oberkochen, Germany or OLYMPUS, Tokyo, Japan). For live cell imaging, MUM2B cells (5 × 10 4 ) were seeded into a 24-well plate at presence or absence of JTC801. The cells were washed twice with ice-cold PBS and stained with LysoTracker (C1046, Beyotime), MitoTracker Red (M7512, Invitrogen), ER-Tracker (E34251, Invitrogen), LysoSensor Green DND-189 (L7535, Invitrogen), Magic Red (ab270772, Abcam) or pHrodo Green (P35373, Invitrogen), according to the manufacturer’s instructions. Additionally, cells were cultured in medium containing Lucifer Yellow (abs47048137, Absin) and LysoTracker (C1046, Beyotime) for another 4 h. The cells were washed twice with fresh culture medium and observed using an IncuCyte S3 imaging system (Sartorius, Goettingen, Germany) or a fluorescence microscope (OLYMPUS, Tokyo, Japan). Mass spectrometry (MS) analysis MUM2B cells (2 × 10 6 ) were seeded into 6 cm dish and treated with or without JTC801 for 16 h. The lysosome lysates were acquired using lysosome extraction kit (EX1230, Solarbio) according to the manufacturer’s instructions and were analyzed by Agilent UPLC-QTOF/MS (Agilent, Santa Clara, California, USA). Standard as a positive control, medium as a negative control. Cathepsin B (CTSB) activity CTSB activity was measured using fluorometric Cathepsin B Activity Assay (ab270772, Abcam) according to the manufacturer’s instructions. MUM2B cells (5 × 10 4 ) were seeded into a 24-well plate at presence or absence of JTC801 for 16 h followed by incubating with Magic Red staining solution for 30 mins at 37°C while protected from light. After rinsing twice in fresh culture medium, the cells were observed by the IncuCyte S3 imaging system (Sartorius, Goettingen, Germany). RNA isolation and reverse transcription-quantitative real-time PCR (RT-qPCR) RNA isolation and RT-qPCR were performed as we previously describe [ 55 ]. Briefly, Total RNA from MUM2B cells was extracted using TRIzol reagent (127401, Invitrogen) and the complementary DNA (cDNA) was synthesized using ReverTra Ace™ qPCR RT Kit (FSQ-101, TOYOBO) according to the manufacturer’s instructions. RT-qPCR was conducted using gene-specific primers with SYBR® Green Real time PCR Master Mix (QPK-201, TOYOBO) on a LightCycler480II (Roche, Basel, Switzerland). All the samples were normalized to human GAPDH according to the ΔΔ Ct method. All primers used in this study were synthesized by Sangen Biotech (Shanghai, China). The sequences of the relevant primers were as follows: GAPDH (F: 5′-CTGGGCTACACTGAGCACC-3′, R: 5′-AAGTGGTCGTTGAGGGCAATG-3′), SLC7A5 (F: 5′-CCGTGAACTGCTACAGCGT-3′, R: 5′-CTTCCCGATCTGGACGAAGC-3′), SLC2A1 (F: 5′-GTATCGTCAACACGGCCTTC-3′, R: 5′-ATGGCCACGATGCTCAGATAG-3′), FASN (F: 5′-GCAAGCTGAAGGACCTGTCT-3′, R: 5′-TCCTCGGAGTGAATCTGGGT-3′), SLC2A3 (F: 5′-GCTGGGCATCGTTGTTGGA-3′, R: 5′-GCACTTTGTAGGATAGCAGGAAG-3′), SLC1A5 (F: 5′-AACTCGACAGGATATTGAGGGG-3′, R: 5′-CACCCTGGTTCCGGTGATATT-3′), CPT2 (F: 5′-ATGATGGTTGAGTGCTCCAAG-3′, R: 5′-TTGCAGCCTATCCAGTTGTCA-3′). Western blotting (WB) WB was performed as we previously described [ 55 ]. Briefly, whole cell lysates were prepared using cell lysis buffer (9803, CST) containing proteinase inhibitor cocktail (539138, Calbiochem). Lysosome proteins and non-lysosomal cytoplasmic proteins were acquired using lysosome extraction kit (EX1230, Solarbio) according to the manufacturer’s instructions. Protein concentrations were determined via the BCA protein assay (23225, Thermo Fisher Scientific). Then, proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose blotting membranes (10600002, GE Healthcare). After blocking with TBST solution containing 5% non-fat dry milk, the membranes were incubated with the primary antibodies overnight at 4°C, including anti-GAPDH (1:1000, Abs830030, Absin, Shanghai, China), anti-BCL-XL (1:1000, 2764, Cell Signaling Technology [CST], Danvers, MA), anti-GPX4 (1:1000, 52455S, CST), anti- LC3B (1:1000, 3868S, CST), anti-cleaved-Caspase 3 (1:1000, 9661S, CST), anti- LAMP1 (1:1000, 9091S, CST), anti-SLC7A5 (1:1000, 32683, CST), anti-SLC2A1 (1:1000, 12939S, CST), anti-CTSB (1:1000, PA1296, Abmart, Shanghai, China), anti-CTSD (1:1000, T55339, Abmart, Shanghai, China), followed by incubation with the secondary antibodies, including anti-rabbit (1:2000, 7074, CST) and anti-mouse (1:2000, 7076, CST). Immunoblots were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (34095, Thermo Fisher Scientiffc) and detected by chemiluminescent western blotting scanner (Gene Company, HongKong, China). All loaded samples were normalized by GAPDH staining. Lysosomal membrane permeabilization (LMP) Acridine orange (AO) (ab270772, Abcam) was performed to assess the LMP. MUM2B cells were treated with or without JTC801 for 16 h, and then stained with AO (1 µM) for 30 min at 37°C. After washed 3 times with PBS, cells were visualized using a fluorescence microscope (OLYMPUS, Tokyo, Japan), or detected by a flow cytometer (Fortessa X-20, Becton Dickinson, San Jose, CA, USA). Intracellular ATP detection Intracellular ATP levels were determined with an enhanced ATP assay kit (S0026, Beyotime) according to the manufacturer’s instructions. Briefly, MUM2B cells (1 × 10 5 ) were seeded into 96-well plate and treated with or without JTC801 for indicated time. Then, Cells were lysed in 100 µL buffer, followed by centrifugation at 12,000 × g for 15 min to collect the cell supernatant. An aliquot of ATP detection working solution was added to a 96-well culture plate and incubated at RT for 5 min. Then, the cell lysate supernatant was added to the wells and the luminescence was measured immediately by a microplate reader (Synergy™ H1, BioTek). Lysosomal pH measurements The LysoSensor Yellow/Blue dextran (L22460, Invitrogen) ratiometric lysosomal pH probe was used for quantifying the lysosomal pH. Briefly, 50 µg/ml LysoSensor was loaded into the cells overnight. Then, cells were chased without the dye in the medium for 2 h. The cells incubated with various pH standard solutions (BB-489113, Bestbio) containing 10 µM nigericin/monensin for 5 min to generate the calibration curve. Thereafter, the fluorescent microplate reader (Synergy™ H1, BioTek) was used to monitor fluorescence at the 365 nm excitation and 450/510 nm emission wavelengths, respectively. The decreased 510/450 ratio suggested higher lysosomal pH (less acidic). The calibration curve drawn based on pH standard solutions served as the standard curve for determining the experimental pH. Murine tumor models and antitumor therapy For subcutaneous tumor model, MUM2B cells (5 × 10 6 ), B16F10 (3 × 10 5 ) or B16F10-luc were inoculated subcutaneously in the right flank of nude mouse or C57BL/6 mouse, respectively. For orthotopic MUM2B tumor model, MUM2B (5 × 10 6 ) cells were administered intraocularly choroid under surgical microscope (OPMI pico, ZEISS, Jena, Germany). 7 days after tumor inoculation, JTC801 was administered intratumoral or orally at indicated dosage every two days for 2 weeks. Tumor volume was calculated by a vernier caliper using the formula a×b 2 /2 (a, the maximal diameter of tumor; b, the minimal diameter of tumor) every 2 days, and mice were sacrificed when tumors volume reached 2000 mm 3 . The tumor growth or metastatic colonies were assessed weekly by using luciferase-based bioluminescence imaging platform of IVIS Lumina III (PerkinElmer Inc., Hopkinton, MA, USA) following our previously described [ 55 ]. The survival of tumor-bearing mice was monitored daily starting from the death of the first mouse, and the survival rate was calculated based on the lifespan from the day of tumor inoculation. For toxicological analysis, C57BL/6 and BALB/c mice were given peritoneal administration of JTC801 (10 mg/kg) every other day for 3 weeks. Mice were sacrificed by CO2 asphyxia, and the tissues including heart, liver, spleen, lung, kidney and stomach were collected for HE analysis. Statistical analysis GraphPad Prism 8 software (GraphPad, CA, USA) was used for statistical analyses. A two-tailed Student’s t-test was conducted to compare two independent groups, and ANOVA was used to compare multiple groups. For mice survival analysis, a Log-rank (Mantel-Cox) test was used to determine statistically significant differences between groups. P < 0.05 was considered statistically significant. Declarations Data Availability Statement All data supporting this study are presented in this published article and in its Supplementary information files. Ethics declarations Ethics approval for animal work was provided by the Institutional Animal Care and Use Committee of Naval Medical University. Acknowledgments: Mingyan Huang, Xinpei Ji and Ha Zhu contributed equally to this work. MYH, XPJ and HZ performed the experiments and analyzed the data. QYL and MYH wrote the original manuscript. QYL, MYH and HZ modified the writing. QYL designed, supervised and supported the study. All authors read and approved the final manuscript. We thank Ms Yingying Liu for her excellent technical assistance. This work was supported by grants from the National Natural Science Foundation of China (31770966, 32200743), the National Key Research Program of China (2014CB542102). Conflicts of Interest: The authors declare no competing financial interests. References Bai, H., J.J. Bosch, and L.M. Heindl. 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Additional Declarations There is no duality of interest Supplementary Files SupportingInformation.docx Supporting Information 1 Originalfulllengthwesternblots.docx Supporting information 2 Video1.mp4 Video 1 Sourcedatafortumorgrowth.xlsx Dataset 1 Graphicalabstract.docx SupplFig1.tif SupplFig2.tif SupplFig3.tif SupplFig4.tif SupplFig5.tif Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5718647","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":396791761,"identity":"8fde807f-a6a7-4b27-8cf1-866ef3d381bb","order_by":0,"name":"Qiuyan Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYBACPmYQWSEB4T0gRgsbWMsZqJYEorSACMY2BlK0sPMY3uadZxGtOyP34IMEBpt8eQeCDuMxtubdJpG77UZeskECQ5rlxgOEtZhJQ7TkmEkkMBw2MGwgSssc0rU0IGmRJ6ADqIWt2HLOMaCWM2+MDRIM0gwMCGnh5z+88cabmrrcbcdzDB98qLAxkCfkMBCQ4oEzgVYYHCBCi+QPZB5RtoyCUTAKRsGIAgDI6jYbHCnUEQAAAABJRU5ErkJggg==","orcid":"","institution":"Naval Medical University","correspondingAuthor":true,"prefix":"","firstName":"Qiuyan","middleName":"","lastName":"Liu","suffix":""},{"id":396791762,"identity":"2548b0cc-b90f-4486-9f5a-132844c1d037","order_by":1,"name":"Mingyan Huang","email":"","orcid":"","institution":"Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingyan","middleName":"","lastName":"Huang","suffix":""},{"id":396791763,"identity":"6da19941-5221-4c8a-84d8-10ea40895293","order_by":2,"name":"Xinpei Ji","email":"","orcid":"","institution":"Department of Ophthalmology, Shanghai East Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xinpei","middleName":"","lastName":"Ji","suffix":""},{"id":396791764,"identity":"3087b893-03ce-4006-8b3d-a03604ce73cd","order_by":3,"name":"Ha Zhu","email":"","orcid":"","institution":"Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ha","middleName":"","lastName":"Zhu","suffix":""},{"id":396791765,"identity":"a6e9481d-7fb2-48b9-9bce-7312ff87abd1","order_by":4,"name":"Wenjun Chang","email":"","orcid":"","institution":"The Second Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenjun","middleName":"","lastName":"Chang","suffix":""},{"id":396791766,"identity":"dd343cb5-b94b-497f-9e9e-89579e7f2995","order_by":5,"name":"Hao Shen","email":"","orcid":"","institution":"Navy Military Medical University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Shen","suffix":""},{"id":396791767,"identity":"9f9f9d68-c495-4739-87b9-ab99c7b9c3db","order_by":6,"name":"Yizhi Yu","email":"","orcid":"https://orcid.org/0000-0002-3793-1154","institution":"Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yizhi","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-12-27 03:05:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5718647/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5718647/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75405901,"identity":"35dd2def-4095-4e41-a559-dc9251eb0846","added_by":"auto","created_at":"2025-02-04 08:49:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1282962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJTC801 inhibits the proliferation and induces cell death in UM cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) MUM2B cells were treated with kinase inhibitors (10 μM) from kinase inhibitor library (ZK012) for 24 h under normoxic (20% O\u003csub\u003e2\u003c/sub\u003e) or hypoxic (2% O\u003csub\u003e2\u003c/sub\u003e) condition. Cell viability was detected by CCK8 assay and the inhibition rate of cell growth was calculated accordingly. Each square represents a kinase inhibitor.\u003c/p\u003e\n\u003cp\u003e(B-C) Cell viability of MUM2B cells and OMM2.5 cells were detected by CCK8 assay following JTC801 treatment for 24 h at indicated dosage (B), or with 3 μM JTC801 treatment at indicated time (C).\u003c/p\u003e\n\u003cp\u003e(D-E) The cytotoxic effect of JTC801 (3 μM) in MUM2B cells (D) and OMM2.5 cells (E) were detected by clone formation assay.\u003c/p\u003e\n\u003cp\u003e(F-G) Cell death was analyzed by flow cytometry using annexin V/PI staining\u0026nbsp;in MUM2B cells (F) or OMM2.5 cells (G) after treatment with JTC801 (3 μM) at indicated time.\u003c/p\u003e\n\u003cp\u003eData are presented as the mean ± SD from three independent experiments. The \u003cem\u003eP\u003c/em\u003e values were calculated by unpaired two-tailed Student’s t test (B and D-G) or one-way ANOVA (C). ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5718647/v1/f7f0c5157122228df99af514.png"},{"id":75408001,"identity":"0c8c2e56-dff0-49ef-a6df-9dd5ed88bd41","added_by":"auto","created_at":"2025-02-04 08:57:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2561174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJTC801 triggers\u003c/strong\u003e \u003cstrong\u003ecytoplasmic vacuoles accumulation in UM cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of cytoplasmic vacuole formation over time in JTC801- treated (3 µM) MUM2B cells observed by IncuCyte real-time imaging system. Bottom panels show enlarged images. Arrowheads indicate cytoplasmic vacuoles. Bar = 50 μm for enlarged images.\u003c/p\u003e\n\u003cp\u003e(B) Representative TEM images of MUM2B cells treated with or without JTC801 (3 μM) for 24 h. Right panels show enlarged images. Arrowheads indicate the single membrane. Bar = 5 μm for original images (left) and bar = 1 μm for enlarged images (right).\u003c/p\u003e\n\u003cp\u003e(C-D) MUM2B cells were stained with RAB7 (C) and LAMP1 (D) in the absence or presence of JTC801 (3 μM) for 2 h or 16 h, respectively. Representative images were obtained by confocal laser scanning microscopy (CLSM). Nucleus were stained with 4',6-diamidino-2-phenylindole (DAPI). Arrowheads indicate cytoplasmic vacuoles. BF, bright field; bar = 10 μm.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5718647/v1/ffb348247e5b25e15ebc9840.png"},{"id":75409726,"identity":"2a51bbf1-abbb-4fdf-8286-f03a9717ae81","added_by":"auto","created_at":"2025-02-04 09:05:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2196431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJTC801 induces cytoplasmic vacuoles via macropinocytosis and fuse with lysosomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Representative fluorescence images of MUM2B cells stained with MitoTracker (A), ER-Tracker (B) and LysoTracker (C) after treated with or without JTC801 (3 μM) for 16 h. Arrowheads indicate cytoplasmic vacuoles. Bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e(D) Representative fluorescence images of MUM2B cells treated with JTC801 (3 μM) for 16 h followed by staining with MitoTracker (red) and LysoSensor (green). Arrowheads indicate cytoplasmic vacuoles. Bar = 20 μm.\u003c/p\u003e\n\u003cp\u003e(E) The diameter of LAMP1-positive vacuoles in MUM2B cells treated with or without JTC801 (3 μM) for 16 h was calculated. Data were presented as the mean ± SD from three independent experiments. The \u003cem\u003eP\u003c/em\u003e values were calculated by unpaired two-tailed Student’s t test. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e(F) Representative images of MUM2B cells captured by fluorescence microscope stained with LuciferYellow and LysoTracker dye after incubated with the JTC801 (3 μM) for 4 h. Arrowheads point to yellow indicate co-localization. Bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e(G) The mean fluorescence intensity of dextran-Alexa Fluor 488 in MUM2B cells treated with or without JTC801(3 μM) for 2 h measured by flow cytometry. Data are presented as the mean ± SD from three independent experiments. The \u003cem\u003eP\u003c/em\u003e value was calculated by unpaired two-tailed Student’s t test. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e(H) The isotopic peaks of the JTC801 feature ([C25H25N3O2] + H\u003csup\u003e+\u003c/sup\u003e, 7.7 min, 412.2 ms) in MUM2B lysosomal lysates analyzed by UPLC-MS with JTC801 (3 μM) treatment for 16 h. Standard served as a positive control, medium served as a negative control, derived from MUM2B lysosomal lysates without JTC801 (3 μM) treatment. The molecular structure of JTC801 ([C25H25N3O2]·HCl) was shown in right panel.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5718647/v1/fddf0ad026b154d06d4ee339.png"},{"id":75408016,"identity":"dc8cde26-33dc-4394-8776-e4ae413a47e2","added_by":"auto","created_at":"2025-02-04 08:57:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1822738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJTC801 leads to lysosomal dysfunctions in UM cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative TEM images of MUM2B cells treated with JTC801(3 μM) for 24 h. Bottom panel shows enlarged image. Arrowheads indicate undigested fragments. Bar = 5 μm for original images (top) and bar = 1 μm for enlarged images (bottom).\u003c/p\u003e\n\u003cp\u003e(B) MUM2B cells were treated with or without JTC801 (3 µM) for the indicated time. Cellular proteins were collected and the levels of CTSB and CTSD protein were analyzed by western blotting. GAPDH served as an internal reference.\u003c/p\u003e\n\u003cp\u003e(C) Representative immunofluorescence images of MUM2B cells stained with MagicRed following treatment with or without JTC801 (3 μM) for 16 h (top), and semi-quantitative analysis of the average fluorescence intensity was performed using ImageJ software (bottom). Bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e(D) Representative images of MUM2B cellsstained with LysoSensor following treatment with or without JTC801 (3 μM) for 16 h (left), and semi-quantitative analysis of the average fluorescence intensity was performed using ImageJ (right). Bar = 50 μm for enlarged images.\u003c/p\u003e\n\u003cp\u003e(E) Lysosomal pH was determined using Lysosensor Yellow/Blue dextran. Each dot represents a lysosomal pH value in an independent experiment (n=6).\u003c/p\u003e\n\u003cp\u003e(F) Cell viability of MUM2B cells was assessed using the CCK8 assay after treatment with JTC801 (3μM) in combination with BafA1 (100 nM, pre-treated for 2 h) for 24 h.\u003c/p\u003e\n\u003cp\u003e(G) MUM2B cells were treated with or without JTC801 (3 µM) for the indicated time. Cellular proteins were collected and the levels of LAMP1 protein were analyzed by western blotting. GAPDH served as an internal reference.\u003c/p\u003e\n\u003cp\u003e(H-I) Representative flow cytometry images of MUM2B cells stained with acridine orange (AO) after treatment with or without JTC801 (3μM) for 16 h (H). The average fluorescence intensity of FITC and PE, and the ratio of FITC to PE were quantified by flow cytometry (I).\u003c/p\u003e\n\u003cp\u003eData are presented as the mean ± SDfrom three independent experiments. The \u003cem\u003eP \u003c/em\u003evalues were calculated by unpaired two-tailed Student’s t test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5718647/v1/098e5b718012a3479c81503b.png"},{"id":75405926,"identity":"2d273038-852d-4968-8b05-089277ec7d94","added_by":"auto","created_at":"2025-02-04 08:49:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":964949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJTC801 augments nutritional deficiency by compromising glucose and amino acid transport and mitochondrial damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) MUM2B cells were treated with or without JTC801 (3 μM) for 12 h. Expression of SLC2A1 and SLC7A5 mRNA were determined by RT-qPCR.\u003c/p\u003e\n\u003cp\u003e(B) The protein expression of SLC2A1 and SLC7A5 in MUM2B cells following treatment with JTC801 (3 μM) at indicated time analyzedby western blotting.\u003c/p\u003e\n\u003cp\u003e(C) MUM2B cells were treated with or without JTC801 (3 µM) for 16 h. After fixation, the cells were observed by TEM. Right images represent higher magnifications. Red arrows indicate damaged mitochondria. Bar = 5 μm for left images and 1 μm for right images.\u003c/p\u003e\n\u003cp\u003e(D) Representative flow cytometry images of MUM2B cells stained with JC-1 after treatment JTC801 (3 μM) for 16 h (left). The average fluorescence intensity of FITC(indicates damaged mitochondria) was quantified by flow cytometry (right).\u003c/p\u003e\n\u003cp\u003e(E) The level of intracellular ATP in MUM2B cells following treatment with or without JTC801 (3 μM) for indicated time detected via a Synergy H1 microplate reader.\u003c/p\u003e\n\u003cp\u003e(F) Representative flow cytometry images of MUM2B cells stained with DCFH-DA after treatment with or without JTC801 (3 μM) for 24 h (left). The average fluorescence intensity of FITC(indicates ROS production) was quantified by flow cytometry (right).\u003c/p\u003e\n\u003cp\u003e(G) Representative flow cytometry images of MUM2B cells stained with BODIPY 581/591 C11 after treatment with JTC801 at indicated concentration for 24 h (left). The average fluorescence intensity of FITC (indicates lipid peroxidation) was quantified by flow cytometry (right).\u003c/p\u003e\n\u003cp\u003eData are presented as the mean ± SDfrom three independent experiments. The \u003cem\u003eP\u003c/em\u003evalues were calculated by unpaired two-tailed Student’s t test. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5718647/v1/994e39f2a1dd56488d710a77.png"},{"id":75405915,"identity":"7e68a0a3-1f19-4f49-9141-1b38b8b99b26","added_by":"auto","created_at":"2025-02-04 08:49:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1311883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJTC801 exhibits antitumor efficacy \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of the evaluation of antitumor activities of intratumoral JTC801 delivery \u003cem\u003ein vivo\u003c/em\u003e. s.c.= subcutaneous injection.\u003c/p\u003e\n\u003cp\u003e(B-C) Nude mice were injected subcutaneously with MUM2B cells and then given intratumoral treatment with JTC801 for 2 weeks as shown in (A). Tumor volumes were calculated at indicated time (B) and the survival of tumor-bearing mice was monitored every day (C).\u003c/p\u003e\n\u003cp\u003e(D-E) C57BL/6 mice were injected subcutaneously with luciferase-labeled B16F10 cells and then intratumoral treatment with JTC801 for 2 weeks as shown in (A). The tumor burden was monitored by measuring luminescence using IVIS imaging (D left), and total fluorescence intensity was used to quantify tumor growth (D right). The survival curve of B16F10 tumor-bearing mice was recorded in two groups with or without JTC801 (20 mg/kg) treatment (E). n=5 for each group.\u003c/p\u003e\n\u003cp\u003e(F-I) Nude mice were injected intraocularly with luciferase-labeled MUM2B cells and then intratumoral treatment with JTC801 every two days for 2 weeks. The tumor burden was monitored by measuring luminescence using IVIS imaging, the representative IVIS images showing tumor growth at indicated time (F). Total fluorescence intensity of each mouse at the indicted time calculated by IVIS (G). Metastasis (H) and survival (I) of MUM2B tumor-bearing mice with or without JTC801 (10 mg/kg) treatment were recorded.\u003c/p\u003e\n\u003cp\u003eData are shown as mean ± SEM, The \u003cem\u003eP\u003c/em\u003evalues were calculated by unpaired two-tailed Student’s t test (D), two-way ANOVA (B and G) or log-rank test (C, E and I); *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5718647/v1/58ddfe0cc8a9c7fb333aa2dc.png"},{"id":75411647,"identity":"782888fb-3d87-4e1c-94e9-79a645290bb4","added_by":"auto","created_at":"2025-02-04 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08:49:16","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":9316804,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFig5.tif","url":"https://assets-eu.researchsquare.com/files/rs-5718647/v1/9287bf08802409c7af6e93a9.tif"}],"financialInterests":"There is no duality of interest","formattedTitle":"JTC801 regresses uveal melanoma progression through novel methuosis-like cell death via lysosomal dysfunction","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eMethuosis-like death contributes to JTC801-mediated antitumor efficacy in UM cells, that illumines the potential usage of JTC801 for apoptosis-resistant tumor therapy.\u003c/li\u003e\n \u003cli\u003eJTC801-caused nutritional deficiency facilitates the accumulation of cytoplasmic vacuolization via micropinocytosis in UM cells.\u003c/li\u003e\n \u003cli\u003eJTC801 can be engulfed into the macropinosomes which further fuse with lysosomes.\u003c/li\u003e\n \u003cli\u003eJTC801-mediated lysosomal dysfunction results in UM cell methuosis-like death.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eAs the most frequent primary intraocular tumor in adults, uveal melanoma (UM) has distinct biological and clinical phenotype compared with their more common cutaneous counterparts. Although the therapeutic regiments for primary UM including radiotherapy, laser therapy, local resection or enucleation achieve local control in more than 90% of patients, more than 50% patients develop liver metastases ultimately and mostly die within one year. Recently, targeted therapies such as tyrosine kinases inhibitors, anti-VEGF antibody (bevacizumab) or immunotherapies especially immune checkpoints inhibitors have been conducted in advanced UM patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], however, these therapeutic strategies have not shown definitive effectiveness in advanced UM patients. Tebentafusp, as the first-in-class immune-mobilizing monoclonal T cell receptor against cancer (ImmTAC) approved by the FDA in January 2022, has been explored to extend the overall survival (OS) in unresectable or metastatic UM patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, only the patients with gp100 and HLA-A2:01 dual positive expression can benefit from this treatment, and the toxicities such as cytokine release syndrome (CRS), off-target or secondary resistance limit the therapeutic efficacy and clinical application [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], thus highlighting the urgent need for novel treatment options to reach more UM patients.\u003c/p\u003e \u003cp\u003eCell death is a complex and interconnected process that plays a crucial role in maintaining tissue homeostasis and preventing disease, and great efforts have been dedicated to targeting cell death process to hinder tumor progression. To find viable alternatives to conventional apoptosis-based tumor therapies that exhibit drug resistance, other types of programmed cell death (PCD) have recently attracted significant attention. Drugs inducing tumor cell pyroptosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], necroptosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and ferroptosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], have been widely studied and shown promising antitumor effects. Besides, more PCD pathways have been identified as effective tumor therapeutic targets such as mitoptosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], paraptosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], alkaliptosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and methuosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], each with distinct morphological features and relating to dysfunctional organelle and dysregulated metabolic process. However, the UM patients, most with aberrant activated kinase signaling pathway, are resistant to almost all existing kinase inhibitor treatment-induced apoptosis including PKC signaling inhibitors [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, identifying new valuable non-apoptotic death modality inducers for obstinate apoptosis-resistant UM, and uncovering the underlying mechanisms may help to provide more potential therapeutic strategies for the treatment of UM patients.\u003c/p\u003e \u003cp\u003eJTC801 is a selective antagonist of nociceptin receptor (NOP) which belongs to the G-protein-coupled receptors (GPCRs) family [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and well-known for reversing pain and anxiety symptoms [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Recently, the antitumor function of JTC801 raises extensively attention in multiple tumors. Accumulating evidences demonstrate that JTC801 efficiently inhibits tumor malignant phenotype \u003cem\u003ein vitro\u003c/em\u003e and suppresses tumor progression and metastasis \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, apoptosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], autophagy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] or pH-dependent alkaliptosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] involve in JTC801-mediated antitumor efficacy have been demonstrated in difference tumor models. However, the role of JTC801 in regulating other tumor cell death modality has not been completely understood. It is worth investigating whether there is a unique role of JTC801 in regulating cell death in UM cells.\u003c/p\u003e \u003cp\u003eHere, we screened a kinase inhibitor library of 113 approved drugs to identify the cytotoxic activity in human UM cell lines. Among these inhibitors, JTC801 exhibits a specifically strong tumor-killing ability in a lower dosage \u003cem\u003ein vitro\u003c/em\u003e. Notably, JTC801 induces methuosis-like death with a predominant phenotype of cytoplasmic vacuolization in UM cells, profoundly regresses tumor progression and metastasis, prolongs the survival in multiple UM tumor models without apparent adverse effects. Mechanistically, JTC801 caused nutrition transporters inhibition and mitochondrial damage leading to nutritional deficiency-triggered macropinocytosis. Simultaneously, JTC801 is trapped into the macropinosomes that fuse with lysosomes, leading to lysosomal dysfunction results in methuosis-like death in UM cells. Our findings identify JTC801 as a potential promising drug for clinical application in advanced UM patients especially those resistant to apoptosis, and provide insight into the unique tumor cytotoxicity role of JTC801 in UM cells.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eJTC801 inhibits proliferation and induces cell death in UM cells\u003c/h2\u003e \u003cp\u003eGiven that most UM patients exhibit aberrant activation of kinase signaling pathways [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], we aimed to identify novel antitumor agents for UM by screening a kinase inhibitor library of 113 approved drugs (ZK012, EFEBIO library service) under both normoxic and hypoxic conditions in human MUM2B cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, 13 drugs exhibited higher tumor-killing ability, and the top 6 drugs were selected to evaluate their antitumor efficacy at different dosage and time point. Among these agents, JTC801 exhibited the best cytotoxic effect even at 3 \u0026micro;M dosage, and showed a dose- and time-dependent tumor-killing capacity in MUM2B and OMM2.5 UM cells, as well as in mouse melanoma B16F10 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C and Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS1A-C\u003c/span\u003e). Moreover, EdU incorporation and PI staining assay confirmed that JTC801 markedly decreased cell proliferation and induced G2/S-phase cell cycle arrest (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS1D\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eE\u003c/span\u003e). JTC801 also significantly impaired the migration and invasion abilities of MUM2B cells (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS1F\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eG\u003c/span\u003e). Furthermore, colony formation assay indicated elevated UM cell death after JTC801 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E), and annexin V/PI staining resulted in enhanced the ratio of cell death after JTC801 treatment in UM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). These results demonstrated that JTC801 could inhibit malignant phenotype and induce cell death in UM cells at lower dosage \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThen, as a potential drug for tumor therapy, we are more interested in the cell death modality and underlying mechanisms triggered by JTC801 in UM cells. We performed RNA-sequencing (RNA-seq) in MUM2B cells with or without JTC801 treatment. Gene set enrichment analysis (GSEA) results showed that genes related to apoptosis, ferroptosis and mitophagy were enriched in JTC801-treated group (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS2A\u003c/span\u003e). So, we detected the expression of specifical markers of the above cell death forms by western blotting. Consistent with sequencing results, JTC801 treatment decreased the expression of BCL2 like 1 (BCL-XL) and glutathione peroxidase 4 (GPX4), while increased the expression of cleaved-caspase 3 and microtubule associated protein 1 light chain 3 beta (LC3B) in MUM2B cells (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS2B\u003c/span\u003e). Moreover, comet assay showed that MUM2B cells exhibited significant longer nuclear comet tails (tail length 13.4 \u0026micro;m v.s. 102.4 \u0026micro;m) after JTC801 treatment (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS2C\u003c/span\u003e), and the nuclear accumulation of γ-H2AX proteins was observed in JTC801-treated MUM2B cells (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS2D\u003c/span\u003e). Then, the specifical inhibitors including pan-caspase inhibitor (Z-VAD-FMK), caspase3 inhibitor (Z-DEVD-FMK), necroptosis inhibitor (Necrostatin-1) and ferroptosis inhibitor (Ferrostatin-1) were used to determine the key contributor of JTC801-triggered UM cell death. However, only the pan-caspase inhibitor (Z-VAD-FMK) could partially rescue cell viability induced by JTC801(Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS2E\u003c/span\u003e). In addition, pre-treatment with Mdivi-1 or 3MA, the inhibitor of mitophagy and autophagy respectively, also failed to rescue JTC801-triggered cell death in MUM2B cells (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS2F\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eG\u003c/span\u003e). Thus, these results suggested that other novel cell death modality rather than apoptosis may play a key contributor in JTC801-induced UM cell death.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eJTC801 induces cytoplasmic vacuolization similar to methuosis in UM cells\u003c/h3\u003e\n\u003cp\u003eTo figure out the cell death modality regulated by JTC801, we conducted live-cell imaging of JTC801-treated MUM2B cells. Massive cytoplasmic vacuoles were observed in JTC801-treated MUM2B cells, and the vacuoles were accumulated and coalesced to form progressively larger vacuoles in a time-dependent manner resulting in cell death eventually (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Video \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e). Transmission electron microscopy (TEM) revealed that these cytoplasmic vacuoles were bound by a single membrane rather than the typical double membrane of autophagosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These morphological characteristics is similar to methuosis, a non-apoptotic cell death modality, which is characterized by massive cytoplasmic single-membranous vacuoles accumulation derived from macropinocytosis or endosomal-lysosomal trafficking disruption without cellular shrinkage, chromatin condensation or nuclear fragmentation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Since methuosis-related macropinosomes are decorated with the markers for late endosome and lysosomes RAB7 and LAMP1 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], we further performed immunofluorescence (IF) assay to detect the expression and location of these two molecules in MUM2B cells. As expected, the cytoplasmic vacuoles incorporated RAB7 and LAMP1 were observed by confocal microscopy in JTC801-treated MUM2B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Thus, these data indicated that JTC801 induced cytoplasmic vacuolization-associated cell death similar to methuosis in UM cells.\u003c/p\u003e\n\u003ch3\u003eCytoplasmic vacuoles triggered by JTC801 origins from macropinocytosis and fuse with lysosomes\u003c/h3\u003e\n\u003cp\u003eTo clarify the origin and composition of the cytoplasmic vacuoles, MUM2B cells were labeled by several organelle markers including MitoTracker (mitochondria), ER-Tracker (endoplasmic reticulum), LysoTracker (lysosome), and LysoSensor Green DND-189 (lysosomal acidification) after JTC801 treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D, the dyes of LysoTracker and LysoSensor Green DND-189 were accumulated and colocalized with the cytoplasmic vacuoles, while the dyes of MitoTracker and ER-Tracker were excluded from the cytoplasmic vacuoles in JTC801-treated MUM2B cells. Moreover, enlarged lysosomes were observed in JTC801-treated MUM2B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Therefore, these data suggested that JTC801-triggered cytoplasmic vacuoles might be related to the lysosomal compartments.\u003c/p\u003e \u003cp\u003eAs the cytoplasmic vacuoles in methuosis usually derived from micropinocytosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], then, living MUM2B cells were observed under a fluorescence microscope after adding dyes of bulk fluid-phase tracer Lucifer Yellow and LysoTracker to detect the origination of JTC801-induced cytoplasmic vacuoles. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, Lucifer Yellow and LysoTracker were co-colocalization in cytoplasmic vacuoles forming yellow puncta in JTC801-treated cells. Moreover, dextran uptake assay confirmed that JTC801-triggered cytoplasmic vacuoles originated from macropinocytosis by flow cytometry analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Next, to determine whether JTC801 can be trapped into macropinosomes via macropinocytosis concurrently, ultra performance liquid chromatography and mass spectrometry (UPLC-MS) experiment was performed. The analysis data showed that JTC801 was detected in lysosomal extraction lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Taken together, these results indicated that JTC801 can be engulfed via macropinocytosis and trigger cytoplasmic vacuoles accumulation to form macropinosomes that fuse with lysosomes, a process distinct from methuosis. Thus, we termed JTC801-induced UM cell death as methuosis-like death.\u003c/p\u003e\n\u003ch3\u003eLysosomal dysfunction contributes to JTC801-induced methuosis-like death in um cells\u003c/h3\u003e\n\u003cp\u003eOur above-mentioned results indicated that JTC801-triggered UM cell death matched the major characteristics of methuosis, except for the macropinosomes fused with lysosomes. Then, why JTC801-induced macropinosomes can fuse with lysosomes, but exhibit inefficiency in cellular degradation finally leading to cell death? We analyzed the KEGG pathway of cellular processes using differentially expressed genes from RNA sequence in UM cells treated with or without JTC801. The enrichment of lysosome pathway was observed in JTC801-treated cells (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS3A\u003c/span\u003e). And the abundant undegraded debris within the macropinosomes was observed by TEM in JTC801-treated UM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), indicating compromised lysosomal function. It\u0026rsquo;s well known that the most predominant lysosomal hydrolases, cathepsins in lysosomes are initially synthesized as inactive zymogens and must be converted into their mature forms to exhibit proteolytic activity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, in JTC801-treated MUM2B cells, the precursor forms of both cathepsin B (CTSB) and CTSD were increased, however, their mature forms were decreased. And the impaired CTSB activity was also detected by MagicRed assay, indicating a loss of lysosomal hydrolytic capacity after JTC801 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In addition, the optimal pH of lysosomes is essential to ensure the cellular degradation process [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We then investigated whether JTC801 could interrupt the lysosomal acidification. As examined by LysoSensor Green DND-189 dye staining, the fluorescence of which increases in acidic environments, higher intensity of the LysoSensor fluorescence was observed in JTC801-treated MUM2B cells compared to the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Furthermore, LysoSensor Yellow/Blue dextran probe was performed to qualitatively measure the hydrogen ion concentration (pH) of lysosomes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, JTC801 treatment decreased the lysosomal pH in MUM2B cells compared to the control cells (2.99 v.s. 4.4). Similarly, PHrodo is a live-cell probe indicative of cytoplasmic pH, which has a low fluorescence intensity at neutral pH, with the fluorescence increasing as pH drops. The results showed that JTC801-treated MUM2B cells exhibited a brighter fluorescence compared to the control cells, suggesting a decreased cytoplasmic pH after JTC801 treatment (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS3B\u003c/span\u003e). Additionally, bafilomycin A (BafA1), a vacuolar-type H\u003csup\u003e+\u003c/sup\u003e ATPase (V-ATPase) inhibitor known to increase lysosomal pH [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], can significantly restore the viability of JTC801-treated MUM2B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results revealed that JTC801 facilitated lysosomal over-acidification and restrained the maturation of cathepsins in UM cells.\u003c/p\u003e \u003cp\u003eThe glycosylation of lysosomal membrane protein is essential for remaining membrane integrity by preventing rupture of lysosomal membrane induced by lumen proteolytic enzymes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. When lysosomal membrane permeabilization (LMP) occurred, membrane integrity is disrupted, allowing cathepsins to leak into the cytoplasm, resulting in cytoplasmic acidification and irreversible cell death [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thereby, we next detected the glycosylation level of lysosomal membrane protein LAMP1. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, JTC801 profoundly impaired LAMP1 glycosylation in a time-dependent manner in MUM2B cells. Then, acridine orange (AO) uptake assay was performed to detect LMP, whose FITC fluorescence indicating lysosomal membrane leakage while PE fluorescence indicating intact lysosome. The results showed that increased fluorescence intensity of FITC, accompanied by decreased of PE were detected by flow cytometry, and the ratio of FITC/PE was significantly increased after JTC801 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I). Accordantly, the red particles obviously descended, and green particles enhanced in JTC801-treated cells captured by fluorescence microscope (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS3C\u003c/span\u003e), suggesting loss of lysosomal membrane integrity. Taken together, these results revealed that JTC801 triggered UM cell methuosis-like death via lysosomal dysfunction including lysosomal over-acidification, LAMP1 de-glycosylation, cathepsins maturation inhibition and LMP.\u003c/p\u003e\n\u003ch3\u003eJTC801-caused nutritional deficiency by mitochondrial damage triggers macropinocytosis\u003c/h3\u003e\n\u003cp\u003eAs a clathrin- and caveolin-independent endocytosis of extracellular fluid, macropinocytosis plays a vital role in nutrient uptake to meet the nutrition requirements in tumor cells under a nutrient-deficient microenvironment [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Then, we first explored whether JTC801 could affect the expression of nutritional transporters. The results showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eand B\u003c/span\u003e, JTC801 treatment significantly decreased the expression of glucose transporter solute carrier family 2 member 1 (SLC2A1) and amino acid transporter SLC7A5 in MUM2B cells compared to the control cells. However, the expression including SLC2A3, SLC1A5, carnitine palmitoyl transferase 2 (CPT2) and fatty acid synthase (FANS) did not show significant changes after JTC801 treatment (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS4A\u003c/span\u003e). Thus, these data suggested that JTC801 impaired glucose and amino acid collection and induced nutritional deficiency stress in UM cells.\u003c/p\u003e \u003cp\u003eThen, whether JTC801-induced nutritional deficiency could affect mitochondrial function? GSEA analysis showed a significant downregulation of the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) in JTC801-treated MUM2B cells compared to the control cells (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS4B\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003e). And TEM images showed a significant swelling and cristae disorder of mitochondria in JTC801-treated MUM2B cells, while the control cells showed intact cristae and elongated mitochondria morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), indicating impaired mitochondrial function after JTC801 treatment. Then, a fluorometric analysis was conducted using the fluorescent dye JC-1 to assess mitochondrial damage. Compared to the control cells, JTC801 treatment significantly increased the green fluorescence of the JC-1 monomers, indicating damaged mitochondrial membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Consistent with this, the decreased adenosine triphosphate (ATP) generation was observed as early as 2 hours in JTC801-treated MUM2B cells following JTC801 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Additionally, JTC801 treatment also increased reactive oxygen species (ROS) production and lipid peroxidation accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G). Collectively, these results indicated that JTC801 caused nutrient-deficient stress through mitochondrial damage triggering macropinocytosis in UM cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eJTC801 regresses tumor progression and metastasis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThen, we want to know whether JTC801 can exhibit antitumor efficacy \u003cem\u003ein vivo\u003c/em\u003e? We firstly investigated the tumor-killing activity of JTC801 in a xenograft mouse model. Tumor-bearing mice was conducted by subcutaneous implantation of MUM2B cells in nude mice for one week, followed by JTC801 intratumoral delivery every other day for two consecutive weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Reduced tumor growth and extended survival were observed in JTC801-treated tumor-bearing mice compared with control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). Similar experiments were established in immunocompetent C57BL/6 mice using luciferase-labeled B16F10 cells. Administration of JTC801 significantly suppressed tumor growth and improved the survival of tumor-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E). Moreover, JTC801 profoundly suppressed tumor progression, metastasis (75% v.s. 25%) and prolonged survival in an orthotopic mice model established by intravitreal injection of luciferase-labeled MUM2B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-I). In addition, tumor tissues were isolated and subjected to hematoxylin-eosin (HE) staining analysis after final treatment with JTC801. The images showed the formation of large vacuoles in JTC801-treated tumor tissues compared to their control counterparts (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS5A\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBesides, daily oral administration of JTC801 in a xenograft mouse model and homograft mouse model performed (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS5B\u003c/span\u003e). Compared with the control group, the MUM2B tumor growth was obviously inhibited in the JTC801-treated group (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS5C\u003c/span\u003e). Similarly, mice treatment with JTC801 were associated with longer survival in B16F10-tumor bearing mice models (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS5D\u003c/span\u003e). Most importantly, there were no obvious adverse effects on the heart, liver, spleen, lung, kidney and stomach in both C57BL/6 and BALB/c mice following peritoneal injection of JTC801 for 3 weeks (Fig. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS5E\u003c/span\u003e). Taken together, JTC801 exhibited valuable antitumor efficacy via distinct tumor cytotoxicity, suggesting that JTC801 may be a promising drug with potential clinical application, especially for apoptosis-resistant advanced UM patients.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrent cancer chemotherapeutic regimens primarily rely on the activation of apoptotic cell death pathway. However, after a period of clinical application, most antitumor compounds show reduced efficacy or various side effects, and majority of patients developed resistance toward those chemotherapeutic drugs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thus, resisting apoptotic cell death has been a common challenge and needs to be resolved urgently. Discovery of therapeutic regimens that induce non-apoptotic forms of cell death should be a promising strategy for breaking apoptotic resistance and increasing the antitumor curative effect.\u003c/p\u003e \u003cp\u003eAs a relatively new non-apoptotic form of cell death, methuosis can be triggered in both apoptosis-sensitive and apoptosis-resistant tumor cells, offering highly beneficial for those patients resistant to conventional chemotherapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Recently, several small molecule methuosis inducers such as indol-based chalcones [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], Jaspine B [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], Tubeimoside-1 (TBM1) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], casein kinase II (CK2) inhibitor silmitasertib (CX-4945) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) inhibitors [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] have been illustrated to exhibit effectively antitumor activity in multiple tumor models, specifically in apoptosis-resistant tumors. Therefore, methuosis becomes a potential promising cell death form which is expected to be effective for overcoming therapy-resistant tumors. In this study, we identified that methuosis-like cell death mainly contributed to JTC801-induced cell death in UM cells at a lower dose, and significantly regressed tumor progression and prolonged the survival of tumor-bearing mice. Notably, no obvious adverse effects were observed in JTC801-treated mice models. To our knowledge, our findings firstly identify JTC801 as a methuosis-like cell death inducer. These findings illumined the potential clinical application of JTC801, especially in apoptosis-resistant advanced UM patients.\u003c/p\u003e \u003cp\u003eMethuosis is characterized by the accumulation of massive vacuoles within the cytoplasm derived from micropinocytosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Macropinocytosis plays a pivotal role in nutrient uptake which mediates non-selective internalization of extracellular fluid to meet the nutrition requirements in tumor cells under a nutrient-deficient microenvironment, promoting tumor cells survive and increasing the resistance to antitumor drugs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, hyperactive macropinocytosis or impaired lysosomal fusion with macropinosomes result in cell membrane rupture and final methuosis. Several small molecular methuosis inducers such as TBM1 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], Bacoside A [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and Ursolic acid derivatives (C17) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] have been shown to exhibit antitumor activity in multiple solid tumors via stimulating hyperactive macropinocytosis, resulting in cell membrane rupture and final methuosis. DMBP [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], MIPP/MOMIPP [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and Vacquinol-1 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] restrain lysosomal fusion with macropinosomes to facilitate tumor cell methuosis, resulting in suppressing tumor progression and metastasis. Our results revealed that JTC801-caused nutrient-deficient stress through limiting nutritional collection and impairing mitochondrial function that triggers macropinocytosis in UM cells, concurrently, JTC801 is trapped into the macropinosomes. However, JTC801 did not affect the fusion of macropinosomes with lysosomes, a typical characteristic of methuosis, instead, it triggered methuosis-like death in UM cells, indicating JTC801 treatment may lead to lysosomal inefficiency.\u003c/p\u003e \u003cp\u003eAs a major regulatory platform for cellular processes and signaling hub, lysosome controls cell death and survival [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the lysosomal optimal pH and the integrality of lysosomal membrane are essential for efficient cellular degradation, which is crucial for maintaining cellular homeostasis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Both over-acidification and de-acidification of lysosomes can cause lysosomal functional defects [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and different therapeutic intervention has been established via lysosomal acidification-modulating agents [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, the glycosylation of lysosomal membrane protein is important for lysosomal membrane integrity by preventing the lysosomal membrane rupture destroyed by lumen proteolytic enzymes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. TMEM175, a ubiquitously expressed lysosomal membrane protein, as a proton-activated proton channel for regulating the lysosomal proton efflux to maintain a steady-state pH [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Deficiency of TMEM175 can lead to lysosomal over-acidification, resulting in impaired the lysosomal degradation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. And prosapogenin A (PA), a bioactive compound prevalent in traditional Chinese herbs, has been demonstrated to trigger lysosomal over-acidification, which exacerbates LMP and subsequent lysosomal damage, exhibiting significant antitumor efficacy in anaplastic thyroid cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, the methuosis inducer SGI-1027 has been documented that exerts synergistic antitumor activity with the mTOR inhibitor everolimus via apoptosis and GSDME-dependent pyroptosis by triggering LMP in renal cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Consistent with these findings, our results revealed that the JTC801 is trapped into macropinosomes that fused with lysosomes, further caused lysosomal over-acidification, LAMP1 de-glycosylation, cathepsins maturation inhibition and LMP, leading to loss of lysosomal degradation capacity and membrane integrality, ultimately resulting in UM cell methuosis-like death via lysosomal dysfunction.\u003c/p\u003e \u003cp\u003eIn addition, Prof. Tang\u0026rsquo;s group first termed that JTC801 induces CA9 downregulation by ACSS2mediated acetylation and activation of NF-κB which specifically contributes to alkaliptosis in PDAC cells[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Recently, the group further revealed that JTC801 directly binds to ATP6V0D1 contributing to alkaliptosis via blocking STAT3-mediated lysosomal pH homeostasis, independent of the NF-κB-CA9 pathway [\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e51\u003c/span\u003e]. However, we did not detect intracellular alkalization, CA9 downregulation and STAT3 phosphorylation inhibition after JTC801 treatment in UM cells (data not shown). We speculate that the discrepancy is likely due to the different cell lines, dosage or time points used in different studies. The mechanisms of JTC801-mediated cell death are diverse with heterogeneity and plasticity. Additionally, our RNA sequencing revealed elevated expression of SLC15A4 [\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e52\u003c/span\u003e] and CLCN7 [\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e53\u003c/span\u003e], the lysosome-resident, proton-coupled amino-acid transporter and H\u003csup\u003e+\u003c/sup\u003e/Cl\u003csup\u003e\u0026ndash;\u003c/sup\u003e exchange transporter, respectively, which loss caused impaired lysosomal acidification, and a decreased expression of SLC16A3, a transporter mainly mediating intracellular lactic acid efflux [\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e54\u003c/span\u003e] in JTC801-treated MUM2B cells (data not shown), suggested that these molecules might be involved in JTC801-mediated lysosomal over-acidification. Therefore, the precise effector molecules and signal pathways in JTC801-mediated cell death is worth further investigation.\u003c/p\u003e \u003cp\u003eIn sum, our results demonstrated that JTC801, acting as a methuosis-like cell death inducer, exhibited valuable antitumor efficacy via specific tumor cytotoxicity, presenting a new promising therapeutic option for apoptosis-resistant advanced UM treatment.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eMice and cell lines\u003c/h2\u003e\n \u003cp\u003e6\u0026ndash;8 weeks male athymic nude mice were purchased from Gempharmatech Co., Ltd. (Nanjing, China) and kept in specific pathogen-free conditions. 6\u0026ndash;8 weeks male C57BL/6 and BALB/c were purchased from Joint Ventures Sipper BK Experimental Animal (Shanghai, China). All mice experimental manipulations were approved by the Scientific Investigation Board of Navy Medical University (Shanghai, China) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Human UM cell lines including MUM2B, OMM2.5 and MP41 cells were kind gifts from Prof. Jiaxu Hong (Department of Ophthalmology and Vision Science, Shanghai Eye Ear Nose and Throat Hospital, Fudan University, Shanghai, China). Mouse melanoma B16F10 cells were purchased from the American Type Culture Collection (ATCC). MUM2B and B16F10 were transfected with lentivirus encoding luciferase (Genechem, GV633) as previously described [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e], then termed as MUM2B-luc cells or B16F10-luc cells. All cell lines were cultured in RPMI 1640 medium (11875-093, Gibco) supplemented with 10% fetal bovine serum (S1580-500, Biowest) at 37℃ in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eCell viability assay\u003c/h2\u003e\n \u003cp\u003eCell viability assay was measured by the Cell Counting Kit 8 assay (CCK8) Kit (HY-K0301, MCE). Cells (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well) were cultivated in 96-well plates and JTC801 (HY-13274, MCE) was added to plates. At the appropriated time point, each well was replaced with 100 \u0026micro;l of fresh medium containing 10 \u0026micro;l of CCK8 solutions and incubated for 1 h at 37℃. The results were assayed with a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eColony formation assay\u003c/h2\u003e\n \u003cp\u003eMUM2B cells and OMM2.5 cells were treated with or without JTC801 for 8 h. Then, cells were harvested and seeded (1000 counts per well) into a new 6-well plate. 14 days later, visible colonies were fixed and then stained with 0.1% crystal violet for 30 min at room temperature (RT). Images were acquired using a scanner (OLYMPUS, Tokyo, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eFlow cytometry\u003c/h2\u003e\n \u003cp\u003eFor cell apoptosis, MUM2B cells and OMM2.5 cells were treated with or without JTC801 for 24 h. Cells were harvested and stained with Annexin V-FITC and Propidium Iodide (556547, BD biosciences) for 15 min in the dark at RT. The Annexin V-positive cells were regarded as apoptotic cells.\u003c/p\u003e\n \u003cp\u003eFor dextran uptake analysis, MUM2B cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were seeded into 12-well plate and incubated overnight. The cells were then incubated with dextran-Alexa Fluor 488 (D1821, Invitrogen) at presence or absence of JTC801 for 2 h.\u003c/p\u003e\n \u003cp\u003eMitochondrial membrane potential was assessed using JC-1 dye (T3168, Invitrogen). MUM2B cells (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were seeded into 6-well plate and treated with or without JTC801 for 16 h. Then, cells were stained with JC-1 dye at 37\u0026deg;C in the dark for 30 minutes. Red fluorescence from JC-1-aggregate (Ex/Em\u0026thinsp;=\u0026thinsp;525/590 nm) was observed in normal cells, while green fluorescence from JC-1 monomer (Ex/Em\u0026thinsp;=\u0026thinsp;490/530 nm) was generated as membrane potential decreased.\u003c/p\u003e\n \u003cp\u003eROS generation was detected using a ROS assay kit (S0033S, Beyotime) according to the manufacturer\u0026rsquo;s instructions. MUM2B cells (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were seeded into 6-well plate and treated with or without JTC801 for 24 h. Then, DCFH-DA dilution (1:1000) was added into each well and incubated at 37 ℃ for 30 min in the dark.\u003c/p\u003e\n \u003cp\u003eLipid peroxidation was measured by BODIPY 581/591 C11 (D3861, Invitrogen). MUM2B cells (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were seeded into 6-well plate and treated with or without JTC801 for 24 h. Then the cells were washed with PBS and incubated with BODIPY dilution (1:1000) for 30 min at 37\u0026deg;C in the dark.\u003c/p\u003e\n \u003cp\u003eAfter washing, cells were determined by flow cytometry (Fortessa X-20, Becton Dickinson, San Jose, CA, USA) as described previously [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. The data were analyzed using FlowJo 10 software (TreeStar, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eRNAsequencing\u003c/h2\u003e\n \u003cp\u003eTotal RNA was extracted from the samples using TRIzol reagent (127401, Invitrogen) according to the manufacturer\u0026rsquo;s protocol. RNA quality control, library construction and sequencing were performed by BGI Genomics Corporation (Shenzhen, China), data analysis was performed online (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biosys.bgi.com\u003c/span\u003e\u003c/span\u003e). The differentially expressed genes were identified using edgeR. The set of potential target genes was screened using the following filter criteria: q-value (corrected \u003cem\u003eP\u003c/em\u003e value)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold-change\u0026thinsp;\u0026gt;\u0026thinsp;2. Gene set enrichment analysis (GSEA) was used to screen significantly enriched signaling pathways with default parameters and Gene Ontology (GO) framework was used to identify the specific biological processes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e\n \u003cp\u003eThe ultrastructure of MUM2B cells were observed by TEM. MUM2B cells treated with or without JTC801 were placed in 3% glutaraldehyde at 4℃ for 24 h. Electron microscope photography was performed and imaged by Servicebio Technology Company (Wuhan, China) using Hitachi HT7700 TEM (Hitachi, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eTime-lapse imaging\u003c/h2\u003e\n \u003cp\u003eTime-lapse imaging of MUM2B cells treated with or without JTC801 was performed using an IncuCyte Zoom Live-content imaging system (IncuCyte S3, Sartorius, Goettingen, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunofluorescence (IF)\u003c/h2\u003e\n \u003cp\u003eIF was performed as we previously described [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]. MUM2B cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were seeded into coverslips placed in a 24-well plate at presence or absence of JTC801. The cells were washed twice with ice-cold PBS and fixed with 4% formaldehyde for 30 min at RT. After washing, the cells were permeabilized with 0.1% TritonX-100 in PBS for 15 min followed by blocking with 5% BSA for 1 h. Subsequently, the cells were incubated with the primary antibodies against LAMP1 (9091S, CST) or RAB7 (9367S, CST) or \u0026gamma;-H2AX (ab22551, Abcam) at the dilution of 1:200 overnight at 4℃, and then incubated with Alexa Fluor 488-conjugated secondary antibodies (A21210, Invitrogen) at the dilution of 1:100. Nuclear were labeled with DAPI stain (36308ES20, Yeasen). Fluorescence images were performed using a fluorescence microscope (Zeiss, Oberkochen, Germany or OLYMPUS, Tokyo, Japan).\u003c/p\u003e\n \u003cp\u003eFor live cell imaging, MUM2B cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were seeded into a 24-well plate at presence or absence of JTC801. The cells were washed twice with ice-cold PBS and stained with LysoTracker (C1046, Beyotime), MitoTracker Red (M7512, Invitrogen), ER-Tracker (E34251, Invitrogen), LysoSensor Green DND-189 (L7535, Invitrogen), Magic Red (ab270772, Abcam) or pHrodo Green (P35373, Invitrogen), according to the manufacturer\u0026rsquo;s instructions. Additionally, cells were cultured in medium containing Lucifer Yellow (abs47048137, Absin) and LysoTracker (C1046, Beyotime) for another 4 h. The cells were washed twice with fresh culture medium and observed using an IncuCyte S3 imaging system (Sartorius, Goettingen, Germany) or a fluorescence microscope (OLYMPUS, Tokyo, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eMass spectrometry (MS) analysis\u003c/h2\u003e\n \u003cp\u003eMUM2B cells (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) were seeded into 6 cm dish and treated with or without JTC801 for 16 h. The lysosome lysates were acquired using lysosome extraction kit (EX1230, Solarbio) according to the manufacturer\u0026rsquo;s instructions and were analyzed by Agilent UPLC-QTOF/MS (Agilent, Santa Clara, California, USA). Standard as a positive control, medium as a negative control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eCathepsin B (CTSB) activity\u003c/h2\u003e\n \u003cp\u003eCTSB activity was measured using fluorometric Cathepsin B Activity Assay (ab270772, Abcam) according to the manufacturer\u0026rsquo;s instructions. MUM2B cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were seeded into a 24-well plate at presence or absence of JTC801 for 16 h followed by incubating with Magic Red staining solution for 30 mins at 37\u0026deg;C while protected from light. After rinsing twice in fresh culture medium, the cells were observed by the IncuCyte S3 imaging system (Sartorius, Goettingen, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA isolation and reverse transcription-quantitative real-time PCR (RT-qPCR)\u003c/h2\u003e\n \u003cp\u003eRNA isolation and RT-qPCR were performed as we previously describe [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. Briefly, Total RNA from MUM2B cells was extracted using TRIzol reagent (127401, Invitrogen) and the complementary DNA (cDNA) was synthesized using ReverTra Ace\u0026trade; qPCR RT Kit (FSQ-101, TOYOBO) according to the manufacturer\u0026rsquo;s instructions. RT-qPCR was conducted using gene-specific primers with SYBR\u0026reg; Green Real time PCR Master Mix (QPK-201, TOYOBO) on a LightCycler480II (Roche, Basel, Switzerland). All the samples were normalized to human GAPDH according to the \u003csup\u003e\u0026Delta;\u0026Delta;\u003c/sup\u003eCt method. All primers used in this study were synthesized by Sangen Biotech (Shanghai, China). The sequences of the relevant primers were as follows: GAPDH (F: 5\u0026prime;-CTGGGCTACACTGAGCACC-3\u0026prime;, R: 5\u0026prime;-AAGTGGTCGTTGAGGGCAATG-3\u0026prime;), SLC7A5 (F: 5\u0026prime;-CCGTGAACTGCTACAGCGT-3\u0026prime;, R: 5\u0026prime;-CTTCCCGATCTGGACGAAGC-3\u0026prime;), SLC2A1 (F: 5\u0026prime;-GTATCGTCAACACGGCCTTC-3\u0026prime;, R: 5\u0026prime;-ATGGCCACGATGCTCAGATAG-3\u0026prime;), FASN (F: 5\u0026prime;-GCAAGCTGAAGGACCTGTCT-3\u0026prime;, R: 5\u0026prime;-TCCTCGGAGTGAATCTGGGT-3\u0026prime;), SLC2A3 (F: 5\u0026prime;-GCTGGGCATCGTTGTTGGA-3\u0026prime;, R: 5\u0026prime;-GCACTTTGTAGGATAGCAGGAAG-3\u0026prime;), SLC1A5 (F: 5\u0026prime;-AACTCGACAGGATATTGAGGGG-3\u0026prime;, R: 5\u0026prime;-CACCCTGGTTCCGGTGATATT-3\u0026prime;), CPT2 (F: 5\u0026prime;-ATGATGGTTGAGTGCTCCAAG-3\u0026prime;, R: 5\u0026prime;-TTGCAGCCTATCCAGTTGTCA-3\u0026prime;).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern blotting (WB)\u003c/h2\u003e\n \u003cp\u003eWB was performed as we previously described [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. Briefly, whole cell lysates were prepared using cell lysis buffer (9803, CST) containing proteinase inhibitor cocktail (539138, Calbiochem). Lysosome proteins and non-lysosomal cytoplasmic proteins were acquired using lysosome extraction kit (EX1230, Solarbio) according to the manufacturer\u0026rsquo;s instructions. Protein concentrations were determined via the BCA protein assay (23225, Thermo Fisher Scientific). Then, proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose blotting membranes (10600002, GE Healthcare). After blocking with TBST solution containing 5% non-fat dry milk, the membranes were incubated with the primary antibodies overnight at 4\u0026deg;C, including anti-GAPDH (1:1000, Abs830030, Absin, Shanghai, China), anti-BCL-XL (1:1000, 2764, Cell Signaling Technology [CST], Danvers, MA), anti-GPX4 (1:1000, 52455S, CST), anti- LC3B (1:1000, 3868S, CST), anti-cleaved-Caspase 3 (1:1000, 9661S, CST), anti- LAMP1 (1:1000, 9091S, CST), anti-SLC7A5 (1:1000, 32683, CST), anti-SLC2A1 (1:1000, 12939S, CST), anti-CTSB (1:1000, PA1296, Abmart, Shanghai, China), anti-CTSD (1:1000, T55339, Abmart, Shanghai, China), followed by incubation with the secondary antibodies, including anti-rabbit (1:2000, 7074, CST) and anti-mouse (1:2000, 7076, CST). Immunoblots were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (34095, Thermo Fisher Scientiffc) and detected by chemiluminescent western blotting scanner (Gene Company, HongKong, China). All loaded samples were normalized by GAPDH staining.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eLysosomal membrane permeabilization (LMP)\u003c/h2\u003e\n \u003cp\u003eAcridine orange (AO) (ab270772, Abcam) was performed to assess the LMP. MUM2B cells were treated with or without JTC801 for 16 h, and then stained with AO (1 \u0026micro;M) for 30 min at 37\u0026deg;C. After washed 3 times with PBS, cells were visualized using a fluorescence microscope (OLYMPUS, Tokyo, Japan), or detected by a flow cytometer (Fortessa X-20, Becton Dickinson, San Jose, CA, USA).\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eIntracellular ATP detection\u003c/h2\u003e\n \u003cp\u003eIntracellular ATP levels were determined with an enhanced ATP assay kit (S0026, Beyotime) according to the manufacturer\u0026rsquo;s instructions. Briefly, MUM2B cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were seeded into 96-well plate and treated with or without JTC801 for indicated time. Then, Cells were lysed in 100 \u0026micro;L buffer, followed by centrifugation at 12,000 \u0026times; g for 15 min to collect the cell supernatant. An aliquot of ATP detection working solution was added to a 96-well culture plate and incubated at RT for 5 min. Then, the cell lysate supernatant was added to the wells and the luminescence was measured immediately by a microplate reader (Synergy\u0026trade; H1, BioTek).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eLysosomal pH measurements\u003c/h2\u003e\n \u003cp\u003eThe LysoSensor Yellow/Blue dextran (L22460, Invitrogen) ratiometric lysosomal pH probe was used for quantifying the lysosomal pH. Briefly, 50 \u0026micro;g/ml LysoSensor was loaded into the cells overnight. Then, cells were chased without the dye in the medium for 2 h. The cells incubated with various pH standard solutions (BB-489113, Bestbio) containing 10 \u0026micro;M nigericin/monensin for 5 min to generate the calibration curve. Thereafter, the fluorescent microplate reader (Synergy\u0026trade; H1, BioTek) was used to monitor fluorescence at the 365 nm excitation and 450/510 nm emission wavelengths, respectively. The decreased 510/450 ratio suggested higher lysosomal pH (less acidic). The calibration curve drawn based on pH standard solutions served as the standard curve for determining the experimental pH.\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eMurine tumor models and antitumor therapy\u003c/h2\u003e\n \u003cp\u003eFor subcutaneous tumor model, MUM2B cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e), B16F10 (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) or B16F10-luc were inoculated subcutaneously in the right flank of nude mouse or C57BL/6 mouse, respectively. For orthotopic MUM2B tumor model, MUM2B (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) cells were administered intraocularly choroid under surgical microscope (OPMI pico, ZEISS, Jena, Germany). 7 days after tumor inoculation, JTC801 was administered intratumoral or orally at indicated dosage every two days for 2 weeks. Tumor volume was calculated by a vernier caliper using the formula a\u0026times;b\u003csup\u003e2\u003c/sup\u003e/2 (a, the maximal diameter of tumor; b, the minimal diameter of tumor) every 2 days, and mice were sacrificed when tumors volume reached 2000 mm\u003csup\u003e3\u003c/sup\u003e. The tumor growth or metastatic colonies were assessed weekly by using luciferase-based bioluminescence imaging platform of IVIS Lumina III (PerkinElmer Inc., Hopkinton, MA, USA) following our previously described [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. The survival of tumor-bearing mice was monitored daily starting from the death of the first mouse, and the survival rate was calculated based on the lifespan from the day of tumor inoculation. For toxicological analysis, C57BL/6 and BALB/c mice were given peritoneal administration of JTC801 (10 mg/kg) every other day for 3 weeks. Mice were sacrificed by CO2 asphyxia, and the tissues including heart, liver, spleen, lung, kidney and stomach were collected for HE analysis.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eGraphPad Prism 8 software (GraphPad, CA, USA) was used for statistical analyses. A two-tailed Student\u0026rsquo;s t-test was conducted to compare two independent groups, and ANOVA was used to compare multiple groups. For mice survival analysis, a Log-rank (Mantel-Cox) test was used to determine statistically significant differences between groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting this study are presented in this published article and in its Supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval for animal work was provided by the Institutional Animal Care and Use Committee of Naval Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eMingyan Huang, Xinpei Ji and Ha Zhu contributed equally to this work. MYH, XPJ and HZ performed the experiments and analyzed the data. QYL and MYH wrote the original manuscript. QYL, MYH and HZ modified the writing. QYL designed, supervised and supported the study. All authors read and approved the final manuscript. We thank Ms Yingying Liu for her excellent technical assistance. This work was supported by grants from the National Natural Science Foundation of China (31770966, 32200743), the National Key Research Program of China (2014CB542102).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBai, H., J.J. Bosch, and L.M. Heindl. Current management of uveal melanoma: A review. Clin Exp Ophthalmol. 2023;51:484-494.\u003c/li\u003e\n \u003cli\u003eCarvajal, R.D., J.J. Sacco, M.J. Jager, D.J. Eschelman, R. Olofsson Bagge, J.W. Harbour, et al. 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Cancer Lett. 2024;589:216824.\u003c/li\u003e\n \u003cli\u003eHuang, M., J. Luo, X. Ji, M. Hu, Y. Xue, and Q. Liu. Deficiency of tumor-expressed B7-H3 augments anti-tumor efficacy of anti-PD-L1 monotherapy rather than the combined chemoimmunotherapy in ovarian cancer. Pharmacol Res. 2022;186:106512.\u003c/li\u003e\n \u003cli\u003eZhu, H., M. Huang, J. Luo, X. Ji, and Q. Liu. Deficiency of GFRalpha1 promotes hepatocellular carcinoma progression but enhances oxaliplatin-mediated anti-tumor efficacy. Pharmacol Res. 2021;172:105815.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"JTC801, uveal melanoma, methuosis, cytoplasmic vacuolization, lysosome dysfunction ","lastPublishedDoi":"10.21203/rs.3.rs-5718647/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5718647/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUveal melanoma (UM) is the most frequent primary intraocular malignancy in adults with high metastasis and mortality rate, whose effective therapeutic strategy is still in urgent need. Specifically, apoptosis-resistance is a great challenge for advanced UM patients, therefore novel therapeutic options targeting otherwise death modality, which may potentially enhance treatment effect, need to be further identified. Here, by a kinase inhibitor library of 113 approved drugs screening, JTC801, a selective antagonist of nociceptin receptor (NOP), exhibits a specifically strong tumor-killing ability in a lower dosage. JTC801 induces UM cell methuosis-like death characterized by cytoplasmic vacuolization, markedly regresses tumor progression and metastasis, prolongs the survival in multiple UM tumor models without apparent adverse effects. Mechanistically, JTC801-caused nutrient-deficient stress by mitochondrial damage which triggers macropinocytosis and cytoplasmic vacuolization in UM cells. Concomitantly, JTC801 is trapped into the macropinosomes that fuse with lysosomes, further causing lysosomal over-acidification, de-glycosylation of lysosomal associated membrane protein 1(LAMP1), inhibiting cathepsinsmaturation, and exacerbating lysosomal membrane permeabilization (LMP), eventually inducing UM cell methuosis-like death. Collectively, our findings identify JTC801 as a potential valuable antitumor drug especially for apoptosis-resistant advanced UM patients, and provide insight into the distinct tumor cytotoxicity role of JTC801 in UM treatment.\u003c/p\u003e","manuscriptTitle":"JTC801 regresses uveal melanoma progression through novel methuosis-like cell death via lysosomal dysfunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-04 08:49:09","doi":"10.21203/rs.3.rs-5718647/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0e08a8d-a201-4fbf-ad42-9f1b09ea2d9a","owner":[],"postedDate":"February 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":42273155,"name":"Biological sciences/Cancer"},{"id":42273156,"name":"Health sciences/Medical research/Drug development"}],"tags":[],"updatedAt":"2025-02-04T08:49:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-04 08:49:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5718647","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5718647","identity":"rs-5718647","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}