Cancer cells frequently reside in a glucose-deprived microenvironment due to rapid tumor proliferation and insufficient angiogenesis. However, the mechanisms by which colorectal cancer cells (CRC) adapt to glucose starvation to sustain proliferation remain unclear. Succinylation, a novel post-translational modification, has been implicated in regulating tumor cell proliferation and survival under nutrient stress. Our study reveals that fumarate hydratase (FH), a key enzyme in the tricarboxylic acid (TCA) cycle, is downregulated in CRC and acts as a tumor suppressor. Under glucose starvation mimicked in vitro, FH protein expression is reduced, leading to abnormal accumulation of its upstream metabolites fumarate and succinate, which correlates with advanced clinical stage and poor prognosis in CRC patients. Mechanistically, accumulated fumarate specifically binds to and stabilizes the NRF2 protein, upregulating the expression of GPX4 and FTH1 to inhibit ferroptosis, thereby sustaining CRC cell proliferation. Meanwhile, glucose starvation induces CPT1A-mediated succinylation of FH at residues K66/K80, reducing FH protein stability and promoting its degradation via the autophagy-lysosome pathway. Our findings reveal the critical role of FH and its succinylation in CRC cell adaptation to glucose starvation, inhibiting ferroptosis, and maintaining cancer cell proliferation, providing novel potential targets and a theoretical basis for the clinical treatment of CRC.
Gastric cancer (GC) is one of the leading causes of global cancer mortality, underscoring an urgent need for novel therapeutic targets. Dysregulation of ubiquitination and deubiquitination plays a critical role in tumorigenesis. The deubiquitinating enzyme FAM105A, belonging to the OTULIN subfamily, has not yet been characterized in cancer. Preliminary bioinformatics analysis indicated that FAM105A is upregulated in gastric adenocarcinoma and positively correlates with the expression of CTGF, a downstream target of the Hippo/YAP signaling pathway, which is frequently dysregulated in GC. This study aimed to elucidate the role and mechanism of FAM105A in GC progression. Bioinformatics databases were used to analyze the expression of FAM105A and its clinical significance, which was further validated in clinical samples via immunohistochemistry (IHC) and Western blot (WB). In vitro and in vivo functional assays were conducted using stable FAM105A-knockdown and FAM105A-overexpressing GC cell lines. The interaction between FAM105A and YAP was investigated using co-immunoprecipitation (Co-IP), immunofluorescence, ubiquitination assays, and proximity ligation assay (PLA). The functional dependency on the Hippo pathway was assessed using the inhibitor XMU-MP-1. Domain mapping was performed by constructing truncated mutants of both FAM105A and YAP. FAM105A was significantly overexpressed in GC tissues, and its high expression correlated with advanced TNM stage and poor prognosis. Functionally, FAM105A promoted GC cell proliferation, migration, and inhibited apoptosis in vitro. In vivo evaluation using xenograft models demonstrated that FAM105A-overexpressing xenografts exhibited significantly larger tumor volumes compared to controls, while FAM105A-knockdown tumors showed reduced growth. FAM105A knockdown significantly decreased final tumor weight, whereas overexpression increased tumor mass. Immunohistochemical analysis revealed that knockdown tumors exhibited consistently reduced Ki-67 immunopositivity, while overexpression samples showed enhanced proliferation signatures. Mechanistically, Co-IP experiments further confirmed that FAM105A binds to YAP via its OTU domain, whereas full-length YAP, but not its truncation variants, efficiently interacts with FAM105A. FAM105A deubiquitinates and stabilizes YAP protein, promotes its nuclear translocation, and thereby activates the Hippo/YAP signaling pathway. The oncogenic effects of FAM105A overexpression were effectively reversed by the Hippo pathway inhibitor XMU-MP-1. This study provides comprehensive in vitro and in vivo evidence demonstrating that the deubiquitinating enzyme FAM105A functions as an oncogene in GC by stabilizing YAP and activating the Hippo/YAP pathway, thereby promoting tumor growth. FAM105A represents a promising prognostic biomarker and a potential therapeutic target for gastric cancer.
The regulation of organ size is a fundamental question in developmental biology, and insect wings provide a powerful model for elucidating the genetic mechanisms underlying morphogenesis. Although the conserved Hippo signaling pathway plays a central role in controlling tissue growth, its precise regulatory network during wing development remains incompletely understood. Here, we identify the zinc finger transcription factor OVOL as a critical mediator of Hippo signaling in insect wing development. We indicate that OVOL is essential for normal wing formation in both Locusta migratoria and Drosophila melanogaster, regulating cell proliferation and trichome patterning. Through transcriptomic analysis and functional validation, we further identify the small GTPase Rac1 as a key downstream effector of OVOL that promotes proliferative growth. Moreover, we find that OVOL expression is directly activated by the Yorkie/Scalloped (Yki/Sd) complex, the core transcriptional effector of the Hippo pathway, without forming a feedback loop. This regulation is mediated through a specific Sd-binding motif (GATAA) within the OVOL promoter. Importantly, Yki/Sd-induced Rac1 expression is dependent on OVOL. Collectively, our findings establish the Yorkie/Sd-OVOL-Rac1 pathway that governs insect wing development by promoting cell proliferation, providing mechanistic insights into organ size regulation in animals.
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Objective: To investigate the effects of miR-1910-3p on proliferation, invasion, epithelial-mesenchymal transition (EMT) and in vivo tumor growth of lung adenocarcinoma (LUAD) cells. Methods: Bioinformatics analysis was performed using The Cancer Genome Atlas (TCGA) database and the Starbase database to compare the expression of miR-1910-3p between LUAD tissues and adjacent normal tissues. In vitro experiments were conducted using human LUAD cell lines (H1299, A549) and normal lung epithelial cells (BEAS-2B). The expression level of miR-1910-3p was verified by real-time quantitative polymerase chain reaction (RT-qPCR). H1299 and A549 cells were transfected with liposomes to establish miR-1910-3p overexpression (mimic group), knockdown (inhibitor group) and negative control (NC group) cell models. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay and plate colony formation assay. Cell invasion was evaluated by Transwell invasion assay. The protein expression changes of EMT markers (E-cadherin, N-cadherin, Vimentin, Snail) were quantitatively analyzed by Western blot. For in vivo experiments, a subcutaneous xenograft model was established in nude mice, and tumor growth was monitored on days 7, 14, 21 and 28. At the end of the experiment, the nude mice were euthanized and the tumors were harvested for analysis. Results: Bioinformatics analysis showed that the expression level of miR-1910-3p was significantly higher in LUAD tissues than in adjacent normal tissues (P＜0.01). RT-qPCR results confirmed that compared with BEAS-2B cells, the relative expression levels of miR-1910-3p were significantly upregulated in A549 and H1299 LUAD cell lines (both P＜0.05). CCK-8 assay results showed that compared with the mimic NC group, overexpression of miR-1910-3p significantly enhanced the proliferative activity of A549 cells [mimic NC group (111.00±7.69)% vs. miR-1910-3p mimic group (119.70±7.54)%, P＜0.01] and H1299 cells [mimic NC group (113.40±15.21)% vs. miR-1910-3p mimic group (118.3±18.82)%, P＜0.01]; conversely, compared with the inhibitor NC group, knockdown of miR-1910-3p significantly inhibited the proliferative activity of A549 cells [inhibitor NC group (115.90±11.39)% vs. miR-1910-3p inhibitor group (111.90±8.83)%, P＜0.05] and H1299 cells [inhibitor NC group (113.20±15.34)% vs. miR-1910-3p inhibitor group (109.60±12.53)%, P＜0.05]. Plate colony formation assay showed that compared with the mimic NC group, overexpression of miR-1910-3p significantly enhanced the colony formation ability of A549 cells [mimic NC group (18.96±1.92)% vs. miR-1910-3p mimic group (37.33±3.66)%, P＜0.01] and H1299 cells [mimic NC group (22.86±2.78)% vs. miR-1910-3p mimic (42.33±2.58)%, P＜0.01]; conversely, compared with the inhibitor NC group, knockdown of miR-1910-3p significantly inhibited the colony forming ability of A549 cells [inhibitor NC group (19.46±3.33)% vs. miR-1910-3p inhibitor group (10.79±2.86)%, P＜0.01] and H1299 cells [inhibitor NC group (22.63±1.27)% vs. miR-1910-3p inhibitor group (12.96±1.45)%, P＜0.01]. Western blot analysis showed that compared with the mimic NC group, overexpression of miR-1910-3p significantly upregulated the expression of N-cadherin, Vimentin and the transcription factor Snail, while downregulating the expression level of E-cadherin (all P＜0.05). Transwell invasion assay showed that compared with the mimic NC group, overexpression of miR-1910-3p significantly enhanced the invasion ability of A549 cells [mimic NC group (333.00±35.68) vs. miR-1910-3p mimic group (521.67±46.92), P＜0.01] and H1299 cells [mimic NC group (341.67 ±32.87) vs. miR-1910-3p mimic group (537.66±33.13), P＜0.01]; conversely, compared with the inhibitor NC group, knockdown of miR-1910-3p significantly inhibited the invasive ability of A549 cells [inhibitor NC group (363.67±49.24) vs. miR-1910-3p inhibitor group (211.33±27.79), P＜0.01] and H1299 cells [inhibitor NC group (351.67±24.11) vs. miR-1910-3p inhibitor group (154.33±9.29), P＜0.01]. Subcutaneous xenograft experiment in nude mice showed that compared with the mimic NC group, overexpression of miR-1910-3p significantly promoted tumor growth in vivo, manifested as an increased tumor weight and volume (both P＜0.01). In the mimic NC group, tumor weights on days 7, 14, 21 and 28 were (0.08±0.01) g, (0.18±0.03) g, (0.41±0.06) g and (0.73±0.06) g, respectively; in the the miR-1910-3p mimic group, tumor weights on days 7, 14, 21 and 28 were (0.07±0.01) g, (0.35±0.06) g, (0.72±0.08) g, and (0.96±0.09) g, respectively. In the mimic NC group, tumor volumes on days 7, 14, 21 and 28 were (132.00±1.00) mm3, (254.67±7.10) mm3, (530.67± 42.71) mm3 and (853.33±74.10) mm3; in the miR-1910-3p mimic group, tumor volumes on days 7, 14, 21 and 28 were (132.00±2.00) mm3, (425.33±29.94) mm3, (829.00±62.00) mm3, and (1 123.33±95.38) mm3, respectively. Conclusion: miR-1910-3p acts an oncogene in LUAD, promoting tumor cell proliferation, invasion and in vivo tumorigenesis by activating EMT process. 目的： 探讨miR-1910-3p对肺腺癌细胞增殖、侵袭、上皮-间质转化（EMT）和体内肿瘤生长的影响。 方法： 通过癌症基因组图谱数据库和Starbase数据库进行生物信息学分析，分析miR-1910-3p在肺腺癌组织与癌旁正常组织中的表达差异。体外实验采用人肺腺癌细胞系（H1299、A549）及正常肺上皮细胞（BEAS-2B），应用实时荧光定量聚合酶链反应（RT-qPCR）验证miR-1910-3p表达水平。H1299和A549细胞分别通过脂质体转染法构建miR-1910-3p过表达（mimic组）、沉默（inhibitor组）及阴性对照（NC组）细胞模型。采用细胞计数试剂盒8（CCK-8）、平板克隆形成实验评估细胞增殖能力，Transwell侵袭实验检测细胞侵袭能力，Western blot技术定量分析EMT标志物（E-cadherin、N-cadherin、Vimentin、Snail）的蛋白表达变化。体内实验建立裸鼠皮下移植瘤模型，分别于第7天、第14天、第21天及第28天监测肿瘤生长情况，实验终点处死裸鼠并取瘤分析。 结果： 生物信息学分析显示，与癌旁正常组织比较，miR-1910-3p在肺腺癌组织中表达水平升高（P<0.01）。RT-qPCR验证结果显示，与BEAS-2B细胞比较，miR-1910-3p在肺腺癌细胞系A549细胞和H1299细胞中的相对表达水平均上调（均P<0.05）。CCK-8检测结果显示，与mimic NC组比较，过表达miR-1910-3p能够促进A549细胞［mimic NC组（111.00±7.69）%，miR-1910-3p mimic组（119.70±7.54）%，P<0.01］和H1299细胞［mimic NC组（113.40±15.21）%，miR-1910-3p mimic组（118.3±18.82）%，P<0.01］的增殖能力；反之，与inhibitor NC组比较，敲低miR-1910-3p能够抑制A549细胞［inhibitor NC组（115.90±11.39）%，miR-1910-3p inhibitor组（111.90±8.83）%，P<0.05］和H1299细胞［inhibitor NC组（113.20±15.34）%，miR-1910-3p inhibitor组（109.60±12.53）%，P<0.05］的增殖能力。平板克隆实验表明，与mimic NC组比较，过表达miR-1910-3p能够促进A549细胞［mimic NC组（18.96±1.92）%，miR-1910-3p mimic组（37.33±3.66）%，P<0.01］和H1299细胞［mimic NC组（22.86±2.78）%，miR-1910-3p mimic组（42.33±2.58）%，P<0.01］的克隆形成能力；反之，与inhibitor NC组比较，敲低miR-1910-3p能够抑制A549细胞［inhibitor NC组（19.46±3.33）%，miR-1910-3p inhibitor组（10.79±2.86）%，P<0.01］和H1299细胞［inhibitor NC组（22.63±1.27）%，miR-1910-3p inhibitor组（12.96±1.45）%，P<0.05］的克隆形成能力。Western blot分析结果显示，与mimic NC组比较，过表达miR-1910-3p能够同时上调N-cadherin、Vimentin及Snail转录因子的表达，下调E-cadherin的表达水平（均P<0.05）。Transwewll检测结果显示，与mimic NC组比较，过表达miR-1910-3p能够促进A549细胞［mimic NC组（333.00±35.68）个，miR-1910-3p mimic组（521.67±46.92）个，P<0.01］和H1299细胞［mimic NC组（341.67±32.87）个，miR-1910-3p mimic组（537.66±33.13）个，P<0.01］的侵袭能力；反之，与inhibitor NC组比较，敲低miR-1910-3p能够抑制A549细胞［inhibitor NC组（363.67±49.24）个，miR-1910-3p inhibitor组（211.33±27.79）个，P<0.01］和H1299细胞［inhibitor NC组（351.67±24.11）个，miR-1910-3p inhibitor组（154.33±9.29）个，P<0.01］的侵袭能力。裸鼠皮下成瘤实验显示，与mimic NC组比较，过表达miR-1910-3p能够促进裸鼠体内肿瘤生长，肿瘤重量和体积增加（均P<0.01）。mimic NC组裸鼠第7天、第14天、第21天及第28天肿瘤重量分别为（0.08±0.01）g、（0.18±0.03）g、（0.41±0.06）g和（0.73±0.06）g；miR-1910-3p mimic组分别为（0.07±0.01）g、（0.35±0.06）g、（0.72±0.08）g和（0.96±0.09）g。mimic NC组裸鼠第7天、第14天、第21天及第28天肿瘤体积分别为（132.00±1.00）mm3、（254.67±7.10）mm3、（530.67±42.71）mm3和（853.33±74.10）mm3；miR-1910-3p mimic组分别为（132.00±2.00）mm3、（425.33±29.94）mm3、（829.00±62.00）mm3和（1 123.33±95.38）mm3。 结论： miR-1910-3p在肺腺癌中发挥促癌基因作用，其通过激活EMT进程促进肿瘤细胞增殖、侵袭及体内成瘤。.
Glioblastoma is the most lethal and frequent type of primary brain tumors, characterized by a high proliferative and infiltrative capacity. Here, we used live cell imaging to analyze the effect of cell density variations on the migratory capacity of established and primary glioblastoma cell lines. We found that proliferation events promoted local velocity of glioblastoma cells, up to three cell-length away from the proliferation event. Furthermore, two phenotypes were found when subjecting glioblastoma cells to a cell density gradient. While one phenotype was characterized by the active migration of cells, independent of proliferation, the other was mostly driven by cell proliferation. Lastly, the analysis of the effects of an overall increasing cell density demonstrated that cells showing signs of self-organization, forming liquid crystal-like structures are able to maintain a high migratory potential even for high cell densities. Notably, the emergence of small-scale liquid crystal-like order was associated with a better maintenance of cellular migration, even in cell populations that were largely in a state of migratory arrest. Thus, structure formation might help glioblastoma cells to move efficiently in states of high confinement, thereby maintaining infiltrative properties.
Understanding cellular dynamics represents a critical challenge in biomedical research. Optical microscopy remains a key technique for observing live-cell behaviors in vitro. This paper introduces an enhanced cell-tracking algorithm designed to address dynamic changes in cell populations, including mitosis, migration, and cell-cell interactions, even within complex co-culture models. The proposed method involves three main steps: 1) modeling the movements and interactions of different cell types in co-culture experiments via tailored open multi-agent systems; 2) identifying parameters via real data for a multi-agent, multi-culture framework; 3) embedding the model within an Extended Kalman Filter, to predict the dynamics of heterogeneous cell populations across video frames. To validate the approach, we used a novel dataset involving the interplay between tumor and normal cells, namely osteosarcoma and mesenchymal stromal cells, respectively. This dataset offers a challenging and clinically relevant framework to track cell proliferation and study how cancer cells evolve and interact with stromal cells within their surroundings. Performance metrics demonstrated the effectiveness of the algorithm over state-of-the-art methodologies, highlighting its ability to track heterogeneous cell types, capture their interactions, and generate the estimated cell lineage tree.
Myogenesis involves sequential stages of muscle satellite cell activation, myoblast proliferation, differentiation, and fusion into multinucleated myotubes. Teleost muscle exhibits indeterminate growth and is highly sensitive to environmental temperature, yet the underlying mechanisms by which temperature regulate proliferation and differentiation remain poorly understood. In this study, we established a primary skeletal muscle cell culture from the yellow drum (Nibea albiflora), an economically important marine fish, and integrated morphological observations with comparative transcriptomics analysis to characterize cellular and molecular responses at 28 °C and 20 °C during both proliferation and differentiation stages. Phenotypic analysis revealed that 28 °C significantly enhanced both myoblast proliferation and myogenic differentiation ability compared with 20 °C. Transcriptomic profiling revealed that at 28 °C, differentiation upregulated extracellular matrix(ECM) organization, calcium signaling, and sarcomere assembly, while proliferation enhanced focal adhesion, growth factor signaling, and lipid metabolism. At 20 °C, differentiation was characterized by glutathione metabolism, and ferroptosis, while proliferation involved cytokine-cytokine receptor interaction and negative regulation of signal transduction. Core myogenic regulatory factors (MRFs), particularly myogenin, were strongly upregulated at 28 °C during the differentiation stage, serving as an internal control. Based on these findings, we propose a testable model that elevated temperature coordinates Ca2+-dependent MRF activation with ECM-integrin signaling to drive sarcomere assembly and muscle growth. Key differentially expressed genes (DEGs) regulating myogenesis in N. albiflora were also identified. This study provides a mechanistic framework for temperature adaptation in teleost skeletal muscle and identifies candidate genes for functional validation and marker-assisted selection, as well as a rationale for temperature management strategies to improve aquaculture yield of N. albiflora.
Three-dimensional (3D) dynamic conditions are necessary for physiologically relevant mesenchymal stem cell (MSC) culture. Furthermore, bioreactor-based processes and hydrogel-encapsulation substantially improve niche standardization, expansion control, and process scalability. The integration of these technologies can overcome some challenges that prevent the clinical translation of MSCs. However, few studies have investigated the synergistic use of bioreactors and hydrogels, and none have explored the combination of high-throughput encapsulation platforms, such as microfluidics or millifluidics-generated microgels, with standard stirred-tank systems. In this study, we characterized the continuous culture of MSCs in a stirred-tank reactor, encapsulating the cells in gelatin methacryloyl (GelMA) microgels using a low-cost, user-friendly approach. The effects of seeding density, GelMA's degree of functionalization (DoF), bioreactor sampling protocol, and donor screening were assessed using cell metabolic activity, differentiation, proliferation, and extracellular vesicle (EV) secretion. Gentle dynamic culture enhanced MSCs' metabolic activity. However, cell proliferation was inhibited within the microgels, and no cell migration was observed on the hydrogel's surface. GelMA with a low DoF and high cell seeding density favored cell survival during culture, whereas pronounced donor-dependent differences were observed in cell proliferation and metabolism. The yield, size distribution, and protein content of MSC-EVs were affected by seeding density under dynamic 3D conditions. Furthermore, MSC differentiation led to readily measurable changes in the microgels. Our findings highlight the platform's potential for high throughput microtissue generation and efficient assessment of bioactive compounds.
Benign prostatic hyperplasia (BPH) is an age-related prostate disorder with incompletely defined mechanisms. We integrated laser capture microdissection RNA sequencing, public single-cell RNA sequencing, public spatial transcriptomics, human tissue validation, mouse-model analyses, and in vitro perturbation assays to characterize hypoxia-associated epithelial senescence in BPH. SA-β-gal-positive cells were predominantly epithelial. RNA sequencing of SA-β-gal-positive epithelial cells revealed enrichment of hypoxia/HIF-1, NF-κB, cell-cycle arrest, and senescence-associated programs, with increased HIF1A, senescence-marker, and SASP-associated gene expression. Single-cell and spatial analyses showed coordinated activation of hypoxia-response, NF-κB-related, and senescence-associated programs in luminal epithelial cells or luminal-dominant spots. In 52 human BPH specimens, epithelial HIF-1α staining was modestly but significantly associated with p21, p27, and Rb. In vitro, 1% O₂ exposure or HIF1A overexpression induced senescence-associated phenotypes in BPH-1 and RWPE-1 cells, whereas HIF-1α inhibition or HIF1A knockdown attenuated these effects. NF-κB inhibition or RELA knockdown partially reversed HIF-1α-associated senescence phenotypes. Conditioned media from hypoxia-treated epithelial cells promoted stromal-cell proliferation and SASP-associated cytokine secretion in a partly HIF1A-dependent manner. In a testosterone propionate-induced BPH-like mouse model, HIF-1α or NF-κB inhibition attenuated prostatic hyperplasia, epithelial remodeling, and senescence-marker expression. These findings link hypoxia-associated HIF-1α/NF-κB signaling to epithelial senescence and epithelial-stromal crosstalk in BPH.
This protocol describes a methodological approach to investigate the expression and functional role of CUB domain-containing protein 1 in nasopharyngeal carcinoma and its potential involvement in epithelial-mesenchymal transition. Clinical tissue samples from patients with nasopharyngeal carcinoma and rhinitis were collected to analyze CDCP1 expression using real-time quantitative PCR and immunohistochemistry. In vitro experiments were performed using CNE2 and HK1 nasopharyngeal carcinoma cell lines. CDCP1 overexpression and knockdown were achieved by transfection with a CDCP1 plasmid or specific siRNA. Cell proliferation was assessed by MTT assay, apoptosis was evaluated by Caspase-3 activity measurement, and the expression of EMT-related markers and phosphorylation levels of ERK1/2 were detected by western blot and quantitative PCR. To validate pathway involvement, rescue experiments were conducted using the ERK1/2-specific inhibitor U0126. This protocol provides a systematic in vitro and ex vivo. framework for elucidating the molecular mechanisms by which CDCP1 may regulate tumor progression in nasopharyngeal carcinoma.
Extrahepatic cholangiocarcinoma (eCCA) is characterized by marked molecular heterogeneity and limited therapeutic options. MicroRNAs (miRNAs) are key post-transcriptional regulators of cancer-related pathways, but their contribution to tumor adaptation in physiologically relevant models remains poorly understood. Three-dimensional (3D) tumor spheroids better mimic in vivo conditions than conventional two-dimensional (2D) cultures. We compared miRNA expression profiles in two eCCA cell lines (Sk-ChA-1 and Mz-ChA-1) grown as monolayers (2D) or multicellular tumor spheroids (3D). MiRNA profiling was performed using NanoString technology. Predicted targets were analyzed by over-representation analysis, and selected miRNAs and genes were validated by RT-qPCR and ELISA-based assays. 3D growth induced extensive miRNA remodeling, with distinct (54 deregulated in Sk-ChA-1 and 29 in Mz-ChA-1 cells) and partially overlapping signatures (miR-1283, miR-577, and miR-2113). Among the shared miRNAs, predicted targets included DUSP10 and RBFOX1, while in spheroids, cell-specific multiple miRNAs converged on shared targets (TNRC6B, SMARCAD1, ATG14, HMGA2, and CLOCK) displaying inverse expression patterns. The transcriptional program impacted MAPK signaling, enhanced EMT, and activated stress-adaptive networks but attenuated proliferation in 3D Sk-ChA-1 cells, while Mz-ChA-1 cells retained a more epithelial and proliferative profile. In this context, we point out the involvement of miR-19b-3p using anti-miR transfection experiments. Our findings reveal a miRNA-driven regulatory landscape associated with 3D growth in eCCA, linking tumor architecture to signaling rewiring and cellular plasticity, and highlight potentially druggable candidate targets and pathways to investigate as candidates using inhibitors or gene therapy-based interventions.
Cancer treatment using immune checkpoint blockade (ICB) with anti-PD-1 and anti-CTLA-4 has been successful. However, primary and acquired resistance limits clinical benefit. To improve the effectiveness of ICB therapies, strategies that reorchestrate anti-tumor immunity through mechanism-based drug combinations are being actively explored. The alkylating chemotherapeutic agent cyclophosphamide (CTX) has direct tumoricidal and immunomodulatory properties, including the induction of homeostatic proliferation of T cells. Since ICB suppresses inhibitory signals in T cells, ICB might be able to augment CTX-induced homeostatic proliferation of antigen-specific T cells, thereby resetting the T cell receptor (TCR) repertoire in favor of tumor-specific T cells. Here, we showed that a single dose of CTX one day prior to starting αPD-1+αCTLA-4 treatment was sufficient to delay tumor progression in established melanoma and prolong survival in tumor-bearing mouse models. These effects extended to other lymphodepleting treatments, such as gemcitabine and radiation therapy. The anti-tumor immune response was mainly driven by the clonal expansion of activated/effector CD8+ tumor infiltrating lymphocytes. Furthermore, combined CTX and αPD-1+αCTLA-4 treatment demonstrated efficacy across additional preclinical tumor models, including colorectal cancer and triple negative breast cancer. Overall, these findings highlight that the combination of CTX and ICB represents a clinically relevant approach in the treatment of immunotherapy-refractory tumors.
Casein kinase 2 (CK2) is a key regulator of cancer cell survival and proliferation, making it an attractive therapeutic target. Quinalizarin is a potent CK2 inhibitor; however, its clinical application is limited by suboptimal delivery and bioavailability. This study aimed to develop and characterize a quinalizarin-gold nanoparticle nanocomplex (QGNPs) and evaluate its potential to enhance anticancer activity in non-small cell lung cancer (NSCLC). QGNPs were synthesized using sulfhydryl calcium acetate as a linking agent. The nanocomplex was characterized by UV-Vis spectroscopy, dynamic light scattering, zeta potential analysis, and transmission electron microscopy. Drug loading and release profiles were assessed using UV-Vis spectroscopy. In vitro cytotoxicity was evaluated in A549 lung cancer cells using MTT assay, and cellular uptake was examined by confocal laser scanning microscopy. QGNPs demonstrated successful conjugation, with an increase in particle size from ~ 15 to 85.18 ± 8.25 nm and a zeta potential shift to - 19.59 ± 5.62 mV. The nanocomplex exhibited pH-responsive drug release, with significantly higher quinalizarin release under acidic conditions (pH 5.0) compared to physiological pH (7.4). In vitro studies showed that QGNPs significantly enhanced cytotoxic activity compared to free quinalizarin, with lower IC₅₀ values. Confocal microscopy confirmed efficient intracellular uptake of QGNPs in A549 cells. QGNPs represent a promising nanocarrier system for improving the delivery and anticancer efficacy of quinalizarin in NSCLC. The enhanced cytotoxicity and pH-responsive release suggest potential for targeted cancer therapy. However, further studies are required to elucidate underlying mechanisms and to evaluate radiosensitization and in vivo therapeutic efficacy.
Toxoplasma gondii is a globally prevalent foodborne zoonotic pathogen that threatens animal production and human health. Consumption of undercooked meat like pork and lamb is a major route of human infection. In this study, we performed a genome-wide CRISPR knockout screen in the porcine cell line PK15 to identify host factors that are critical for T. gondii replication. The results showed that disrupting the ELFN2 (extracellular leucine rich repeat and fibronectin type III domain containing 2) gene in PK15 did not affect host cell growth, but significantly reduced the proliferation of Toxoplasma parasites. Loss of ELFN2 decreased macroautophagy/autophagy in PK15 cells and impaired lipid metabolism, resulting in reduced lipid availability for the parasites and consequent suppression of T. gondii proliferation. Exogenous lipid supplementation or pharmacological activation of autophagy could fully restore the replication of parasites in ΔELFN2 cells. The requirement of host ELFN2 for optimal parasite proliferation in vivo was validated by constructing elfn2-/- mice, which showed increased resistance to T. gondii infection and reduced parasite burden, highlighting the value of ELFN2 in breeding Toxoplasma-resistant animals. Notably, naturally occurring loss-of-function mutations in ELFN2 could be found in certain pig breeds, further indicating the feasibility of breeding T. gondii-resistant animals like pigs, to reduce the transmission of Toxoplasma.
This protocol describes a hydrostatic pressure-loading device that facilitates real-time microscopic observation of adherent cells during sustained hydrostatic pressure stimulation, and is compatible with 3.5 cm commercial cell culture dishes. The apparatus consists of an airtight culture chamber fabricated from an aluminum base, an optically transparent poly(methyl methacrylate) cover, gas inlet/outlet ports integrated into the cover, and a sealed observation window. By connecting to a regulated gas source, the device maintains a stable hydrostatic pressure (0-200 kPa, adjustable) while enabling continuous phase-contrast or fluorescence imaging. Using this pressure-loading device, pressure-induced dose-dependent effects on cell phenotype and behaviors, such as morphology, proliferation, and migration, can be recorded. Furthermore, fluorescent signals can also be recorded in real time. Here, pressure-triggered Ca2⁺ signaling heterogeneity and dynamics in breast cancer MDA-MB-231 cells and cervical cancer HeLa cells were observed and quantified by inverted fluorescence microscopy using time-lapse imaging. This platform integrates mechanical loading with live‑cell imaging to overcome limitations of conventional endpoint systems, providing a universal tool for mechanobiological studies.
RHEBL1 (RHEB2), a member of the Ras superfamily, has established roles in tumor-promoting signaling pathways including mTOR activation and NF-κB transcription; however, its specific role in oral squamous cell carcinoma (OSCC) and its regulation by the stem cell transcription factors Oct4 and Sox2 have not been previously characterized. This study aimed to elucidate the function and mechanism of RHEBL1 in OSCC development and analyze the regulatory influence of Oct4 and Sox2 on RHEBL1 expression. Immunohistochemistry and immunofluorescence assessed RHEBL1, Oct4, and Sox2 expression in normal and precancerous tissues. RHEBL1-overexpressing and knockout cell lines were created for in vitro assessment of proliferation and self-renewal; mice models were used to evaluate tumor formation in vivo. Bioinformatics analyses predicted Oct4 and Sox2 binding sites within the RHEBL1 promoter, validated by dual-luciferase reporter assays and ChIP-PCR. RHEBL1 showed high expression in OSCC and adjacent tissues, with linear arrangement of positive cells in the basal layer. High levels of RHEBL1, Oct4, and Sox2 were observed at the tumor invasion front and the basal layer of adjacent oral epithelia. RHEBL1 overexpression enhanced sphere formation and induced subcutaneous tumor-like lesions in immunodeficient mice, characterized histologically by invasive growth patterns and vascular structures, whereas control cells showed no such phenomenon. Furthermore, RHEBL1 knockout significantly reduced in vitro sphere formation and in vivo tumorigenicity. Oct4-Sox2 complexes bound two sites in the RHEBL1 promoter; mutations in these sites reduced transcriptional activation. Thus, this study demonstrated that Oct4 and Sox2 promote OSCC initiation and proliferation by regulating RHEBL1 expression. Clinical trial number: not applicable.
This study aimed to define the functional role of the HIF-1α target gene NDUFA4L2 in clear cell renal cell carcinoma (ccRCC), specifically its regulation of mitochondrial function and the ferroptosis cell death pathway. Through TCGA data analysis and in vitro and in vivo models, we confirmed that HIF-1α induces NDUFA4L2 expression and mitochondrial localization. Using shRNA-mediated knockdown combined with rescue experiments employing the ferroptosis inhibitor ferrostatin-1 and the mitochondrial antioxidant mitoTEMPO, we demonstrated that silencing NDUFA4L2 triggered mitochondrial lipid peroxidation, altered mitochondrial ultrastructure, and suppressed proliferation via a mitochondria-associated ferroptotic mechanism. Mechanistically, NDUFA4L2 functioned parallel to Lactate Dehydrogenase B (LDHB); their genetic or pharmacological co-inhibition synergistically enhanced ferroptosis and suppressed cell viability in vitro and tumor growth in vivo, associated with elevated ferroptosis markers (PTGS2, 4-HNE). Furthermore, NDUFA4L2 knockdown sensitized tumors to radiotherapy by amplifying ferroptotic cell death. In conclusion, NDUFA4L2 is a critical suppressor of mitochondria-associated ferroptosis in ccRCC, acting cooperatively with LDHB to maintain redox homeostasis, and targeting the NDUFA4L2/LDHB axis represents a promising therapeutic strategy, particularly in combination with radiotherapy.
Extracellular matrix (ECM) stiffness critically regulates stem cell behavior. Previously, we demonstrated that pathological increases in matrix stiffness during aging disrupt stem Leydig cell (SLC) homeostasis, leading to a decline in testosterone. Building on this discovery, we here present a detailed protocol-originally developed in our laboratory-for fabricating polyacrylamide (PA) hydrogels with tunable stiffness to model the testicular microenvironment in vitro. This method enables reproducible casting of gels across a stiffness range of 1-100 kPa, covering physiological to pathological conditions. Key steps include precise mixing of acrylamide/bis-acrylamide, gel swelling equilibration, surface activation with Sulfo-SANPAH, and collagen coating to support SLC adhesion and culture. We provide optimized formulations for target stiffnesses and troubleshooting guidance for common issues such as incomplete polymerization and poor cell attachment. This system allows systematic investigation of how substrate stiffness modulates SLC proliferation, differentiation, and steroidogenic function under defined 2D conditions. Beyond reproductive biology, it also serves as a valuable platform for mechanobiological studies in other cell types and for screening therapeutics targeting stiffness-related dysfunction.
Gallbladder cancer (GBC) is a rare but aggressive biliary tract malignancy. This study explores the transcriptomic profile of GBC to identify differentially expressed genes (DEGs) and dysregulated pathways involved in its pathogenesis. RNA sequencing was performed on 13 GBC tumors and 6 matched controls. Functional enrichment analysis (FEA) as well as weighted gene co-expression network analysis (WGCNA) were used to identify dysregulated pathways, functionally relevant gene modules and hub genes. Key targets were validated in patient tissues and cell lines. A total of 1319 DEGs were identified (528 upregulated, 791 downregulated). Gene set enrichment analysis revealed activation of E2F targets and G2/M checkpoint, with downregulation of bile acid metabolism and estrogen response pathways. A tumor grade-correlated gene module was identified by WGCNA. FEA of the gene module highlighted pathways related to cell cycle and cell division. Co-expression analysis identified TPX2 as a central hub gene. Inhibitors of aurora kinase, TPX2 dependent enzyme, significantly reduced proliferation, migration, and invasion in GBC cells. Elevated Aurora kinases expression was also observed in GBC. This first transcriptomic analysis of GBC in South-East Asian Indians uncovers key drivers like TPX2 and Aurora kinases in disease progression. The study highlights cell cycle dysregulation and sex-linked signatures, offering insights for biomarker discovery and targeted therapies.