Search Results (38)

Chromosomal instability (CIN) arising from mitotic errors is a hallmark of cancer progression, yet how specific spindle assembly factors are co-opted to support aggressive tumor phenotypes remains incompletely understood. Hepatoma Upregulated Protein (HURP/DLGAP5), a Ran-regulated microtubule-associated protein essential for kinetochore fiber stabilization and chromosome congression, is frequently overexpressed in aggressive cancers. Here, we investigated HURP’s role across a breast cancer metastatic gradient—immortalized MCF10A, the low-metastatic luminal T47D, and the highly metastatic triple-negative MDA-MB-231 cell lines—integrating quantitative spindle analysis, kinetochore tension measurements, spindle checkpoint profiling, migration dynamics, and three-dimensional spheroid modeling. We show that total HURP protein levels increase with metastatic potential, yet spindle-bound HURP is paradoxically reduced in MDA-MB-231 cells, indicating cytoplasmic mislocalization despite increased total protein levels. HURP silencing induced cell-line-specific defects: moderate disorganization and misorientation in MCF10A and T47D cells, but catastrophic spindle collapse, apoptosis, and G2/M arrest in MDA-MB-231 cells. Mechanistically, HURP depletion disrupted the spindle-associated levels and distributions of TPX2, Aurora-A, and NuMA in a subtype-dependent manner, implicating HURP as a context-dependent stabilizer of this mitotic regulatory axis. HURP loss reduced interkinetochore tension in all cell lines, but only MCF10A and T47D cells mounted a proportional BubR1-dependent checkpoint response; MDA-MB-231 cells showed reduced checkpoint signaling, consistent with constitutive spindle assembly checkpoint (SAC) attenuation in triple-negative breast cancer. Beyond mitosis, HURP depletion impaired collective migration and converted MDA-MB-231 cells from super-diffusive, amoeboid-like motility to sub-diffusive behavior, while minimally affecting the less aggressive cell lines. HURP-depleted MDA-MB-231 spheroids were significantly larger, less compact, and less spherical than controls, linking spindle regulation to tissue-level architectural coherence. These findings establish HURP as a multifunctional regulator coordinating mitotic fidelity, migration plasticity, and tumor architecture in breast cancer, with a selective dependency in highly metastatic cells, positioning it as a promising therapeutic target for aggressive breast cancers. Full article

Rosmarinic acid (RA), a naturally occurring polyphenolic compound, has demonstrated promising anticancer activity; however, its combinatorial potential with conventional chemotherapeutic agents remains incompletely characterized. This study investigated the cytotoxic, pro-apoptotic, oxidative stress-associated, and cytokine-associated effects of RA alone and in combination with doxorubicin (DOX) in 4T1 murine breast cancer cells and HaCaT human keratinocyte cells as a non-cancerous control model. Cellular viability, apoptosis, cell cycle progression, oxidative stress, mitochondrial function, cytokine responses, and apoptosis-associated molecular alterations were evaluated using complementary cellular and molecular approaches. In addition, three-dimensional (3D) tumor spheroid experiments were performed to assess treatment responses under physiologically relevant tumor-like conditions. Results demonstrated that RA synergistically enhanced DOX-induced cytotoxicity in 4T1 cells while exhibiting comparatively lower toxicity toward HaCaT cells. Combination treatment significantly increased apoptotic cell death, mitochondrial depolarization, intracellular reactive oxygen species (ROS) accumulation, apoptotic DNA fragmentation, and G₂/M-phase accumulation. N-acetylcysteine (NAC)-mediated rescue experiments partially reversed ROS elevation and treatment-associated cytotoxicity in both monolayer and 3D spheroid models. Furthermore, the RA+DOX combination markedly disrupted spheroid integrity and reduced spheroid viability compared with monotherapies. Collectively, these findings indicate that RA enhances the anticancer activity of DOX and support further investigation of this combination strategy in breast cancer models. Full article

Background/Objectives: Gallic acid (GA) is a naturally occurring polyphenol with reported antioxidant and anticancer properties. This study investigated whether GA enhances carboplatin (CARB)-associated anticancer activity in HeLa cervical cancer cells through mechanisms related to oxidative stress, mitochondrial dysfunction, apoptosis, and cell cycle dysregulation, while comparatively evaluating cytotoxicity in HaCaT cells. Methods: The effects of GA and CARB, individually and in combination, were evaluated using cell viability assays, apoptosis and cell cycle analyses, intracellular reactive oxygen species (ROS) measurements, N-acetylcysteine (NAC)-mediated rescue experiments, mitochondrial membrane potential assessment, reverse transcription–quantitative polymerase chain reaction (RT-qPCR), immunocytochemistry, and three-dimensional (3D) tumor spheroid models. Bioinformatic analyses were performed to explore pathways associated with the observed molecular responses. Results: The GA + CARB combination demonstrated enhanced cytotoxicity and apoptotic activity in HeLa cells compared with either monotherapy, while exhibiting comparatively lower toxicity in HaCaT cells. Combination treatment increased intracellular ROS levels, whereas NAC pretreatment partially reversed ROS accumulation and cytotoxicity, supporting a contributory role of oxidative stress in treatment-associated responses. The combination also induced mitochondrial membrane depolarization, increased G2/M arrest and SubG1 accumulation, and modulated apoptosis- and cell cycle-related gene expression. In 3D spheroid models, GA + CARB reduced spheroid growth and viability and disrupted spheroid integrity more effectively than single-agent treatments. Bioinformatic analyses identified interconnected pathways associated with oxidative stress, apoptosis, and cell cycle regulation. Conclusions: GA may enhance CARB-associated anticancer activity through mechanisms linked to oxidative stress, mitochondrial dysfunction, apoptosis, and cell cycle dysregulation. The incorporation of ROS/NAC rescue experiments and 3D spheroid validation further supports the biological relevance of the observed effects. Nevertheless, these findings remain preliminary and require confirmation in advanced in vivo and translational cervical cancer models. Full article

Background/Objectives: The toxic side effects and resistance-associated limitations of conventional chemotherapeutic agents necessitate the development of more effective and selective combination strategies incorporating naturally derived compounds. In this study, the cytotoxic, apoptotic, oxidative stress-associated, and immunomodulatory effects of thymoquinone (TQ), a bioactive compound derived from Nigella sativa, and docetaxel (Dos), a taxane-based chemotherapeutic agent, were investigated alone and in combination in OVCAR3 ovarian cancer cells using integrated two-dimensional (2D) and three-dimensional (3D) experimental models. Materials and Methods: Cell viability was evaluated following treatment with TQ (10–500 µM), Dos (1–500 nM), and the TQ + Dos combination, and synergistic interactions were assessed by IC₅₀⁻ and combination index-based analyses. Apoptosis and cell cycle distribution were analyzed by flow cytometry. Cytokine levels were determined using ELISA, whereas apoptosis- and cell cycle-associated gene expression profiles were evaluated by RT-qPCR. Active caspase-3 expression was assessed by immunocytochemistry. Intracellular reactive oxygen species (ROS) accumulation was examined using DCFH-DA-based fluorescence imaging and antioxidant rescue experiments using N-acetyl-L-cysteine (NAC). In addition, the antitumor activity of the combination was further evaluated in OVCAR3-derived 3D tumor spheroid models using spheroid morphology, ATP-based viability, and live/dead fluorescence imaging analyses. Results: The TQ + Dos combination demonstrated enhanced cytotoxic and apoptotic activity in OVCAR3 cells compared with single-agent treatments and induced marked G2/M cell cycle arrest. Combination treatment increased pro-apoptotic gene expression and was associated with reduced expression of anti-apoptotic markers and modulated inflammatory cytokine profiles. Fluorescence-based analyses demonstrated marked intracellular ROS accumulation following TQ + Dos treatment, whereas NAC pretreatment partially attenuated oxidative stress and restored viability, suggesting partial involvement of ROS-associated mechanisms in treatment-induced cytotoxicity. Importantly, the combination maintained stronger cytotoxic and growth-inhibitory effects than either monotherapy in 3D ovarian cancer spheroids, where combination treatment induced pronounced spheroid shrinkage, viability loss, and structural disruption. Relatively lower toxicity observed in HaCaT cells suggested partial selectivity toward cancer cells. Conclusions: Collectively, these in vitro findings suggest that the TQ + Dos combination produces greater cytotoxic, apoptotic, and growth-inhibitory effects than either agent alone in ovarian cancer models and is associated with alterations in apoptosis-, cell cycle-, and oxidative stress-related responses. The observation of these effects in 3D spheroid models supports further investigation of this combination in more advanced preclinical systems. Full article

Although sorafenib (SOR) is effective for advanced hepatocellular carcinoma (HCC), significant metabolic heterogeneity limits its therapeutic effect. In this study, we employed high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to profile the spatial lipidomic alterations in 3D HepG2 spheroids following SOR treatment. Interestingly, sphingophospholipid and glycerophospholipid metabolism played crucial roles. In an orthotopic HCC mouse model, immunohistochemical and immunofluorescence staining confirmed that SOR induced immunological and inflammatory changes. Moreover, transcriptomic and Q-PCR analyses showed increased expression of Stat1, Zbp1, Parp14, Irf1, and Tifa along with decreased Eif4e2 in the SOR treatment group compared to the tumor control group. Bio-layer interferometry and molecular docking data also indicated that ZBP1 possessed favorable binding affinities with SOR. Overall, our findings demonstrated that SOR dramatically disrupted sphingolipid metabolism in tumor cell spheroids and, in an orthotopic model, activated the NOD-like receptor signaling pathway, accompanied by altered secretion of inflammatory factors and macrophage polarization. These results suggest that SOR exerts dual effects on tumor cell lipid metabolism and the tumor immune microenvironment. These findings provide a conceptual basis for future exploration of lipid-modulating therapeutic strategies in HCC. Full article

Background/Objectives: Retinoblastoma treatment remains limited by therapeutic resistance and toxicity. While melphalan is a key chemotherapeutic agent, its efficacy is constrained by adverse effects. Curcumin has emerged as a potential adjunct owing to its capacity to regulate oxidative stress and oncogenic signaling pathways, including STAT3. This study aimed to assess the synergistic tumor-inhibitory effects of melphalan–curcumin combined treatment and to investigate the roles of ROS, apoptosis, and STAT3-associated signaling, including validation in a three-dimensional (3D) tumor spheroid model. Materials and Methods: Human retinoblastoma (WERI-Rb-1) and normal keratinocyte (HaCaT) cells were exposed to melphalan, curcumin and the combined treatment regimen. Cell viability was analyzed by MTT assay, and drug interactions were analyzed using the Chou–Talalay method. Migration was evaluated by scratch assay. Intracellular ROS levels were quantified using the DCFH-DA assay and confirmed by flow cytometry. Apoptosis was quantified by Annexin V/PI staining, and caspase activity was assessed colorimetrically and by immunocytochemistry. Cytokine levels were determined by ELISA, and gene expression profiling of STAT3 and apoptosis-associated genes were performed using qRT-PCR. Three-dimensional tumor spheroids were established to evaluate treatment responses in a physiologically relevant model. The contribution of ROS was further investigated using N-acetyl-L-cysteine (NAC) pretreatment. Results: The combination of melphalan and curcumin notably reduced WERI-Rb-1 cell viability in a synergistic manner (CI < 1) while exhibiting lower cytotoxicity in HaCaT cells, indicating selective antitumor activity. Co-treatment markedly inhibited cell migration and increased intracellular ROS levels. Cells pretreated with NAC significantly reduced ROS levels accumulation and moderately restored cellular viability, supporting a contributory role of oxidative stress. The combination treatment induced pronounced apoptosis, with increased early and late apoptotic cell populations, enhanced caspase-7 and caspase-9 activity, and elevated caspase-9 protein expression. These effects were associated with upregulation of pro-apoptotic genes (BAX, CASP3, CASP7, CASP9), downregulation of anti-apoptotic genes (BCL2, SURVIVIN), and reduction in STAT3 mRNA expression. In addition, the combination reduced pro-inflammatory cytokine levels. Importantly, these effects were recapitulated in 3D tumor spheroids, where the combination treatment reduced spheroid size and viability and induced structural disruption. NAC-mediated rescue experiments in 3D models further supported the notion that ROS contributes to, but is not solely responsible for, the observed effects. Conclusions: Overall, these results suggest that melphalan and curcumin exert synergistic and selective antitumor effects in retinoblastoma cells, associated with changes consistent with ROS-related effects, mitochondrial apoptotic processes, and STAT3-related transcriptional alterations rather than definitive pathway activation. The validation of these effects in a 3D tumor spheroid model provides additional support for the potential clinical significance of this combined treatment; however, additional protein-level and functional validation is required. Full article

Quercetin, a naturally occurring flavonoid, exhibits antiproliferative and pro-apoptotic effects across various cancer models. Topotecan, a topoisomerase I inhibitor, is used in the treatment of retinoblastoma; however, its clinical utility is limited by dose-dependent toxicity. This study aimed to investigate whether quercetin is associated with enhanced topotecan-induced cytotoxicity in retinoblastoma and to explore the underlying mechanisms under both two-dimensional (2D) and three-dimensional (3D) conditions. Cell viability was assessed using the MTT assay, and drug interactions were evaluated using the combination index (CI) based on the Chou–Talalay method. Apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry. Reactive oxygen species (ROS) levels and mitochondrial membrane potential were evaluated using fluorometric methods, and N-acetyl-L-cysteine (NAC) was used for functional modulation of oxidative stress. Three-dimensional tumor spheroid models were used to assess treatment effects under conditions that partially recapitulate tumor architecture. Gene expression levels of apoptosis-related markers and PI3K/Akt/mTOR pathway components were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The combination of quercetin and topotecan was associated with synergistic cytotoxic effects in Y79 cells (CI < 1), accompanied by increased ROS levels, mitochondrial membrane depolarization, and elevated apoptotic cell death. NAC co-treatment partially attenuated ROS levels and restored cell viability. In 3D spheroid models, combination treatment induced structural disruption, reduced viability, and increased cell death, effects that were partially reversed by NAC. Gene expression analysis revealed upregulation of pro-apoptotic genes and downregulation of survival-related genes, along with increased PTEN expression. Quercetin is associated with enhanced topotecan-induced cytotoxicity in retinoblastoma cells under both 2D and 3D conditions. These effects were associated with ROS-associated cellular stress, mitochondrial dysfunction, and modulation of apoptotic and survival-related pathways. The partial rescue by NAC supports a contributory, but not exclusive, role of oxidative stress. These findings should be interpreted within a preclinical context and suggest that quercetin may represent a potential adjunct strategy warranting further validation in translational and in vivo models. Full article

This study aimed to investigate the effects of resveratrol (RES) and carboplatin (CPT), alone and in combination, on cell viability, apoptosis, cell cycle progression, mitochondrial function, and oxidative stress in Y79 retinoblastoma (RB) cells. Particular emphasis was placed on evaluating the synergistic potential of the combination and elucidating the interconnected molecular mechanisms underlying its anticancer effects. Y79 cells were treated with RES, CPT, and their combinations. Cell viability and synergy were assessed using the MTT assay and combination index (CI) analysis. Apoptosis (annexin V/PI), cell cycle distribution (propidium iodide (PI) staining), intracellular ROS production (DCFH-DA), and mitochondrial membrane potential (JC-1) were evaluated by flow cytometry. ROS dependency was further examined using N-acetylcysteine (NAC) pretreatment. Expression levels of apoptosis- and cell cycle-related genes (BAX, BCL-2, CASP3, CASP9, CCNB1, and CDK1) were analyzed by RT-qPCR. Cytoskeletal alterations were assessed by immunocytochemistry. In addition, the antitumor effects of the combination were validated in a three-dimensional (3D) tumor spheroid model. RES and CPT reduced cell viability in a dose- and time-dependent manner and demonstrated synergistic effects (CI < 1) at selected concentrations. Combination treatment significantly increased apoptosis, induced G2/M phase arrest, enhanced ROS accumulation, and promoted mitochondrial depolarization compared with single-agent treatments. NAC pretreatment attenuated ROS generation and partially restored cell viability, supporting a contributory role of oxidative stress in combination-induced cytotoxicity. At the transcriptional level, the RES + CPT combination significantly increased the BAX/BCL-2 ratio and upregulated CASP3 and CASP9 expression, while downregulating CCNB1 and CDK1, consistent with mitochondrial apoptotic activation and G2/M arrest. Immunocytochemical analysis revealed pronounced cytoskeletal disruption and apoptotic morphology in the combination group. Importantly, in the 3D spheroid model, co-treatment markedly reduced spheroid size and viability and enhanced cell death compared with monotherapies. The combination of RES and CPT exerts a synergistic anticancer effect in Y79 RB cells through coordinated mechanisms involving ROS accumulation, mitochondrial dysfunction, caspase activation, and G2/M phase arrest. The attenuation of cytotoxicity by NAC and the validation of efficacy in a 3D tumor spheroid model strengthen the mechanistic relevance of these findings. These results support further preclinical investigation of this combination strategy in in vivo models and normal retinal cell systems. Full article

Background/Objectives: Retinoblastoma represents the most common intraocular malignancy in childhood; however, the clinical applicability of mitomycin C (MMC) is restricted by dose-dependent ocular toxicity. Consequently, the development of pharmacological strategies that sensitize tumor cells to MMC while allowing dose reduction remains an unmet therapeutic objective. In this context, quercetin, a bioactive flavonoid with pleiotropic anticancer properties, has emerged as a potential chemosensitizing agent. Methods: Human retinoblastoma cell lines Y79 and WERI-Rb1 were exposed to MMC and quercetin, administered either individually or in fixed-ratio combinations. Cytotoxic responses were quantified through dose–response modeling and IC₅₀ determination following 24 and 48 h of treatment. Drug–drug interactions were quantitatively characterized using the Chou–Talalay combination index (CI) approach and isobologram analysis. Cell cycle distribution was assessed by propidium iodide (PI)-based flow cytometric analysis to evaluate treatment-associated alterations in cell cycle progression. Apoptotic cell death was assessed by Annexin V-FITC/PI flow cytometry, while transcriptional modulation of genes associated with apoptosis, cell cycle regulation, and oxidative stress (BAX, BCL-2, TP53, CASP3, CDKN1A, and HMOX1) was evaluated by qRT-PCR. Modulation of tumor-supportive signaling was examined by measuring VEGF and IL-6 secretion. Translational relevance was further investigated using a three-dimensional (3D) tumor spheroid model, and the functional contribution of reactive oxygen species (ROS) was interrogated through N-acetyl-L-cysteine (NAC) rescue experiments. Results: Quercetin significantly enhanced the cytotoxic activity of MMC in both retinoblastoma cell lines, with CI values below 1 across IC₅₀–IC₉₀ effect levels, indicating a synergistic pharmacological interaction. PI–FACS analysis revealed that combined MMC and quercetin treatment induced a pronounced accumulation of cells in the G2/M phase, consistent with cell cycle arrest, with a more marked effect observed in Y79 cells compared with WERI-Rb1 cells. Combination treatment resulted in a pronounced increase in apoptotic cell populations compared with single-agent exposure and triggered a coordinated pro-apoptotic transcriptional response, characterized by increased expression of BAX, TP53, CASP3, CDKN1A, and HMOX1, alongside suppression of BCL-2 and a marked shift in the BAX/BCL-2 ratio. Concurrently, VEGF and IL-6 secretion were significantly reduced, reflecting attenuation of pro-angiogenic and pro-inflammatory signaling. Notably, synergistic cytotoxicity was maintained in 3D tumor spheroids, where combined treatment induced spheroid shrinkage, architectural disruption, and reduced viability. NAC pretreatment diminished ROS accumulation and partially restored cell viability, indicating that oxidative stress contributes to, but does not solely account for, the observed synergistic cytotoxic effect. Conclusions: Collectively, these findings indicate that quercetin appears to function as an effective chemosensitizing adjuvant to MMC in retinoblastoma models, through transcriptional changes consistent with p53-associated apoptotic signaling at the transcriptional level, G2/M cell cycle arrest, and partial involvement of ROS-related cellular stress responses, along with suppression of tumor-supportive signaling pathways. The preservation of synergistic activity in 3D tumor spheroids supports the potential preclinical relevance of this combination. However, these findings are based on transcriptional and phenotypic analyses and should be interpreted as hypothesis-generating, requiring further validation through protein-level and in vivo studies before translational application. Full article

Metastasis accounts for most cancer-related deaths and hepatocellular carcinoma (HCC) is no exception. Midkine (MDK) is a multifunctional secreted protein elevated in HCC with a vague role in HCC. In this study, we used bioinformatics to verify MDK expression in HCC tumors, and next, we inhibited the MDK protein in invasive Hep3B cells using an MDK inhibitor (iMDK) both in vitro and in vivo. Our results showed that iMDK promoted cell migration and enhanced lamellipodia formation while at the same time downregulating the expression of cell–matrix adhesion genes. In order to also consider forces exerted by the surrounding matrix, we performed cell adhesion, transwell invasion, and 3D tumor spheroid invasion assays in two different stiffness conditions. Adhesion and invasion always exhibited opposite patterns, with adhesion being inhibited in soft matrix environments, accompanied by increased invasion, and a reverse effect in stiff environments. In vivo experiments where cells pre-treated with iMDK were implanted to zebrafish embryos showed overall reduced metastasis, verifying that MDK is a central mechanotransduction regulator that enables HCC cells to adapt their metastatic strategies to ECM stiffness. Thus, MDK inhibition effectively disrupts mechanosensitive coordination during metastasis, highlighting its potential as a therapeutic target. Full article

Algal carotenoids play a promising role in handling chronic diseases due to their diverse bioactive properties, including anti-inflammatory, antioxidant, and anticancer effects. This study assesses the activity of the antioxidant xanthophyll diatoxanthin (Dt), derived from marine diatoms, against triple-negative breast cancer (TNBC) cells using in vitro models, gene expression evaluation, and explores its role in potentiating the cytotoxic effect of chemotherapy. Dt exhibited selective activity against MDA-MB-231 and BT-549 TNBC cells at concentrations ≥12.5 ng/mL, with maximal effects observed at 25 ng/mL while sparing human umbilical vein endothelial cells (HUVECs) at these doses. When combined with doxorubicin (0.1–0.5 μM), Dt enhanced the anti-tumor efficacy in both TNBC cell lines, further reducing cell viability compared with doxorubicin alone (p < 0.05–0.001). Dt also exerted its activity in inhibiting migration and chemotaxis by approximately 30–50% compared with the controls (p < 0.01) and suppressing 3D-tumor spheroid growth at day 12 (up to >50% reduction, p < 0.001). Notably, secretome analysis revealed Dt-induced changes in inflammatory, oxidative and angiogenic mediators, highlighting its ability to modulate the TNBC microenvironment. Dt also downregulated key pro-survival, pro-angiogenic and pro-tumorigenic genes in both TNBC cell lines, supporting its role in disrupting oncogenic pathways. Angiogenesis-related genes were significantly reduced. Dt also decreased the expression of angiogenic mediators in HUVECs, supporting Dt’s role in inhibiting tumor vascularization. Results on gene expression regulation were also confirmed by RNA-Seq analysis. These findings pose Dt as a promising chemopreventing candidate in the challenging fight against TNBC, a well-known type of cancer that is aggressive and resistant to conventional therapies, targeting critical pathways for tumor survival, such as inflammation, angiogenesis, tumor cell growth, and cell migration. Given its selective activity against TNBC cells, ability to enhance chemotherapy efficacy, and modulation of the tumor microenvironment, Dt holds promise as a complementary drug for cancer prevention and interception. Future studies should focus on validating these effects in vivo and exploring Dt’s potential in combinatorial treatment strategies for cancer. Full article

Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in hematologic malignancies but has limited efficacy in solid tumors due to tumor microenvironment (TME) barriers that impede CAR T cell recognition, infiltration, and sustained function. Traditional 2D assays inadequately recapitulate these constraints, necessitating improved in vitro models. This study validated a 3D tumor spheroid platform using an agarose microwell system to generate uniform B7-H3-positive spheroids from multiple solid tumor cell lines, enabling the evaluation of CAR T cell activity. TME-relevant immune modulation under 3D conditions was analyzed by flow cytometry for B7-H3, MHC I/II, and antigen processing machinery (APM), followed by co-culture with B7-H3 CAR T cells to assess cytotoxicity, spheroid integrity, tumor viability, and CAR T cell activation, exhaustion, and cytokine production. Two human cancer-cell-line-derived spheroids, DU 145 (prostate cancer) and SUM159 (breast cancer), retained B7-H3 expression, while MC38 (mouse colon cancer)-derived spheroids served as a B7-H3 negative control. Under 3D culture conditions, DU 145 and SUM159 spheroids acquire TME-like immune evasion characteristics and specifically downregulated MHC-I and APM (TAP1, TAP2, LMP7) with concurrent upregulation of MHC-II and calreticulin. Co-culture showed effective spheroid infiltration, cytotoxicity, and structural disruption, with infiltrating CAR T cells displaying higher CD4⁺ fraction, activation, exhaustion, effector/terminal differentiation, and IFN-γ/TNF-α production. This 3D platform recapitulates critical TME constraints and provides a cost-effective, feasible preclinical tool to assess CAR T therapies beyond conventional 2D assays. Full article

Deep alterations in tumor cell gene profiles resulting in the loss of their specific functions are frequently the cause of resistance to traditional cancer treatments. Therefore, reprogramming the expression pattern of cancer cells toward a differentiated phenotype represents a promising therapeutic strategy. In this study, we investigated whether resveratrol (RSV) and its natural analogs—3,4′,5-trimethoxystilbene (3-MET-OX) and isorhapontigenin (ISOR-H-PG)—can modulate the SOX2/SOX17 balance and promote re-differentiation in anaplastic thyroid cancer (ATC) cells. Two human ATC cell lines (SW1736 and 8505c) and non-tumoral thyroid cells (Nthy-ori 3-1) were cultured in two-dimensional (2D) or three-dimensional (3D) systems and treated with polyphenols at sub-cytotoxic doses. In 2D cultures, cell viability and cell cycle analyses confirmed a cytostatic effect characterized by G1 arrest. In 3D cultures, polyphenol treatment caused morphological disruption of ATC spheroids and significantly modulated the gene expression profile. RSV and 3-MET-OX reduced stemness markers (SOX2, NANOG), increased the thyroid lineage transcription factor (SOX17), and enhanced differentiation genes (TTF-1, TPO, NIS). Overall, these results support our hypothesis that modulation of the SOX2/SOX17 ratio by polyphenols provides a mechanistic basis for re-differentiation, thereby improving therapeutic responsiveness in ATC. Full article

Background/Objectives: Paclitaxel (PTX) faces clinical limitations in melanoma treatment due to poor solubility, P-glycoprotein (P-gp)-mediated efflux, and systemic toxicity. This study aimed to develop PTX-loaded mesoporous silica nanoparticles (PS), which would be co-administered with curcumin (CUR) and D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) to enhance intracellular accumulation and improve anti-tumor activity. CUR and TPGS were integrated with PS to inhibit P-gp-mediated PTX-efflux, to enhance the intracellular accumulation of PTX, and to improve anti-tumor activity in B16F10 cells. Methods: The physicochemical properties of PS were analyzed using standard characterization methods. The antitumor activity of PS co-administered with CUR and TPGS was evaluated using two-dimensional (2D) culture and three-dimensional (3D) spheroid assays, and also assessed in B16F10 tumor-bearing mice. The therapeutic mechanism of the PS combination was compared using apoptosis and microtubule disruption through flow cytometry and confocal microscopy. The pharmacokinetics and biodistribution of the PS combination were compared in B16F10 tumor-bearing mice. Results: PS formulations exhibited amorphous transformation with an approximate particle size of 200 nm. PS co-administered with CUR and TPGS reduced the IC₅₀ to 178.7 nM compared with 283.3 nM for free PTX in B16F10 melanoma cells and achieved significant tumor growth inhibition in B16F10 melanoma spheroid culture. The intracellular accumulation of PTX correlated with its therapeutic efficacy. Flow cytometry revealed a significant induction of both early and late apoptosis in cells treated with the PS + CUR + TPGS combination, while confocal imaging confirmed enhanced microtubule disruption. In B16F10 tumor-bearing mice, PS co-administered with CUR and TPGS demonstrated higher and selective distribution of PTX into tumor tissue without affecting systemic exposure of PTX in B16F10-xenografted mice. Conclusions: PS + CUR + TPGS combination enhanced PTX delivery by improving solubility and enhancing distribution to tumor tissue through P-gp inhibition, thereby increasing its therapeutic potential. The combination of CUR and TPGS offers synergistic apoptosis induction and microtubule disruption. Thus, the PS + CUR + TPGS combination represents a promising approach for treating drug-resistant melanomas. Full article

Organoid and spheroid technologies have rapidly become pivotal in thyroid cancer research, offering models that are more physiologically relevant than traditional two-dimensional culture. In the study of papillary and anaplastic thyroid carcinomas, two subtypes that differ both histologically and clinically, three-dimensional (3D) models offer unparalleled insights into tumor biology, therapeutic vulnerabilities, and resistance mechanisms. These models maintain essential tumor characteristics such as cellular diversity, spatial structure, and interactions with the microenvironment, making them extremely valuable for disease modeling and drug testing. This review emphasizes recent progress in the development and use of thyroid cancer organoids and spheroids, focusing on their role in replicating disease features, evaluating targeted therapies, and investigating epithelial–mesenchymal transition (EMT), cancer stem cell behavior, and treatment resistance. Patient-derived organoids have shown potential in capturing individualized drug responses, supporting precision oncology strategies for both differentiated and aggressive subtypes. Additionally, new platforms, such as thyroid organoid-on-a-chip systems, provide dynamic, high-fidelity models for functional studies and assessments of endocrine disruption. Despite ongoing challenges, such as standardization, limited inclusion of immune and stromal components, and culture reproducibility, advancements in microfluidics, biomaterials, and machine learning have enhanced the clinical and translational potential of these systems. Organoids and spheroids are expected to become essential in the future of thyroid cancer research, particularly in bridging the gap between laboratory discoveries and patient-focused therapies. Full article