Antimetastatic activity of (arene)ruthenium(II) complex of 4-aryl-4H-naphthopyran.

Metastatic cancer remains a formidable challenge in anticancer therapy. Despite efforts to develop effective antimetastasis drugs over the past half-century, currently approved treatments fall short of expectations. This report highlights the promising antiproliferative activity of a ruthenium-based therapeutic agent, namely dichlorido(p-cymene)[2-amino-4-(pyridin-3-yl)-4H-benzo[h]-chromene-3-carbonitrile]ruthenium(II) (complex 1) against metastatic cell lines. Complex 1 shows significant efficacy in metastatic LoVo and Du-145 cell lines at nanomolar concentrations, being markedly more active than clinically used anticancer cisplatin. Studies on the MDA-MB-231 cell line, which displays invasive characteristics, demonstrated that 1 significantly reduces cell invasion. This efficacy was confirmed by its impact on matrix metalloproteinase production in MDA-MB-231 cells. Given that cell migration drives cancer invasion and metastasis, complex 1's effect on MDA-MB-231 cell migration was evaluated via wound healing assay and vimentin network analysis. Results indicated a strong reduction in migration. A re-adhesion assay further demonstrated that 1 significantly lowers the re-adhesion ability of MDA-MB-231 cells compared to cisplatin. To better simulate the human body environment, a 3D spheroid invasion assay was used. This method showed that 1 effectively inhibits tumor spheroids from infiltrating the surrounding extracellular matrix. This study underscores the potential of (arene)ruthenium(II) complexes with naphthopyran ligands as potent antimetastatic agents for chemotherapy.

MCP-1 exerts the inflammatory response via ILK activation during endometriosis pathogenesis.

AIMS: MCP-1 has been shown to be elevated in endometriosis. ILK functions in several cellular events and interacts with MCP-1-signaling. In the current study, we evaluated the role of MCP-1-ILK signaling in human endometriotic cell's (Hs832(C).TCs) potential for colonization, invasion, adhesion, etc. and differentiation of macrophage along with inflammation in an endometriosis mouse model. MATERIALS AND METHODS: A mouse model of endometriosis with elevated levels of MCP-1 was developed by injecting MCP-1. We examined the migration, adhesion, colonization and invasion of Hs832(C).TCs in response to MCP-1-ILK signaling. We also examined the differentiation of THP-1 cells to macrophage in response to MCP-1-ILK signaling. KEY FINDINGS: We observed that MCP-1 increased Ser246 phosphorylation of ILK in Hs832(C).TCs and enhanced the migration, adhesion, colonization, and invasion of Hs832(C).TCs. In the mouse model of endometriosis, we found elevated chemokines (CCL-11, CCL-22 and CXCL13) levels. An increased level of MCP-1 mediated ILK activation, leading to increased inflammatory reaction and infiltration of residential and circulatory macrophages, and monocyte differentiation, but suppressed the anti-inflammatory reaction. The inhibitor (CPD22) of ILK reversed the MCP-1-mediated action by restoring Hs832(C).TCs and THP-1 phenotype. ILK inhibition in a mouse model of endometriosis reduced the effects of MCP-1 mediated pro-inflammatory cytokines, but increased anti-inflammatory response along with T-regulatory and T-helper cell restoration. SIGNIFICANCE: Targeting ILK restores MCP-1 milieu in the peritoneal cavity and endometrial tissues, reduces the inflammatory response, improves the T-regulatory and T-helper cells in the endometriosis mouse model and decreases the migration, adhesion, colonization and invasion of endometriotic cells.

A NSC-34 cell line-derived spheroid model: Potential and challenges for in vitro evaluation of neurodegeneration.

Three-dimensional (3D) spheroid models aim to bridge the gap between traditional two-dimensional (2D) cultures and the complex in vivo tissue environment. These models, created by self-clustering cells to mimic a 3D environment with surrounding extracellular framework, provide a valuable research tool. The NSC-34 cell line, generated by fusing mouse spinal cord motor neurons and neuroblastoma cells, is essential for studying neurodegenerative diseases like amyotrophic lateral sclerosis (ALS), where abnormal protein accumulation, such as TAR-DNA-binding protein 43 (TDP-43), occurs in affected nerve cells. However, NSC-34 behavior in a 3D context remains underexplored, and this study represents the first attempt to create a 3D model to determine its suitability for studying pathology. We generated NSC-34 spheroids using a nonadhesive hydrogel-based template and characterized them for 6 days. Light microscopy revealed that NSC-34 cells in 3D maintained high viability, a distinct round shape, and forming stable membrane connections. Scanning electron microscopy identified multiple tunnel-like structures, while ultrastructural analysis highlighted nuclear bending and mitochondria alterations. Using inducible GFP-TDP-43-expressing NSC-34 spheroids, we explored whether 3D structure affected TDP-43 expression, localization, and aggregation. Spheroids displayed nuclear GFP-TDP-43 expression, albeit at a reduced level compared with 2D cultures and generated both TDP-35 fragments and TDP-43 aggregates. This study sheds light on the distinctive behavior of NSC-34 in 3D culture, suggesting caution in the use of the 3D model for ALS or TDP-43 pathologies. Yet, it underscores the spheroids' potential for investigating fundamental cellular mechanisms, cell adaptation in a 3D context, future bioreactor applications, and drug penetration studies. RESEARCH HIGHLIGHTS: 3D spheroid generation: NSC-34 spheroids, developed using a hydrogel-based template, showed high viability and distinct shapes for 6 days. Structural features: advanced microscopy identified tunnel-like structures and nuclear and mitochondrial changes in the spheroids. Protein dynamics: the study observed how 3D structures impact TDP-43 behavior, with altered expression but similar aggregation patterns to 2D cultures. Research implications: this study reveals the unique behavior of NSC-34 in 3D culture, suggests a careful approach to use this model for ALS or TDP-43 pathologies, and highlights its potential in cellular mechanism research and drug testing applications.

Identification of New Chemoresistance-Associated Genes in Triple-Negative Breast Cancer by Single-Cell Transcriptomic Analysis.

Triple-negative breast cancer (TNBC) is a particularly aggressive mammary neoplasia with a high fatality rate, mainly because of the development of resistance to administered chemotherapy, the standard treatment for this disease. In this study, we employ both bulk RNA-sequencing and single-cell RNA-sequencing (scRNA-seq) to investigate the transcriptional landscape of TNBC cells cultured in two-dimensional monolayers or three-dimensional spheroids, before and after developing resistance to the chemotherapeutic agents paclitaxel and doxorubicin. Our findings reveal significant transcriptional heterogeneity within the TNBC cell populations, with the scRNA-seq identifying rare subsets of cells that express resistance-associated genes not detected by the bulk RNA-seq. Furthermore, we observe a partial shift towards a highly mesenchymal phenotype in chemoresistant cells, suggesting the epithelial-to-mesenchymal transition (EMT) as a prevalent mechanism of resistance in subgroups of these cells. These insights highlight potential therapeutic targets, such as the PDGF signaling pathway mediating EMT, which could be exploited in this setting. Our study underscores the importance of single-cell approaches in understanding tumor heterogeneity and developing more effective, personalized treatment strategies to overcome chemoresistance in TNBC.

Mouse vascularized adipose spheroids: an organotypic model for thermogenic adipocytes.

Adipose tissues, particularly beige and brown adipose tissue, play crucial roles in energy metabolism. Brown adipose tissues' thermogenic capacity and the appearance of beige cells within white adipose tissue have spurred interest in their metabolic impact and therapeutic potential. Brown and beige fat cells, activated by environmental factors like cold exposure or by pharmacology, share metabolic mechanisms that drive non-shivering thermogenesis. Understanding these two cell types requires advanced, yet broadly applicable in vitro models that reflect the complex microenvironment and vasculature of adipose tissues. Here we present mouse vascularized adipose spheroids of the stromal vascular microenvironment from inguinal white adipose tissue, a tissue with 'beiging' capacity in mice and humans. We show that adding a scaffold improves vascular sprouting, enhances spheroid growth, and upregulates adipogenic markers, thus reflecting increased adipocyte maturity. Transcriptional profiling via RNA sequencing revealed distinct metabolic pathways upregulated in our vascularized adipose spheroids, with increased expression of genes involved in glucose metabolism, lipid metabolism, and thermogenesis. Functional assessment demonstrated increased oxygen consumption in vascularized adipose spheroids compared to classical 2D cultures, which was enhanced by ß-adrenergic receptor stimulation correlating with elevated ß-adrenergic receptor expression. Moreover, stimulation with the naturally occurring adipokine, FGF21, induced Ucp1 mRNA expression in the vascularized adipose spheroids. In conclusion, vascularized inguinal white adipose tissue spheroids provide a physiologically relevant platform to study how the stromal vascular microenvironment shapes adipocyte responses and influence activated thermogenesis in beige adipocytes.

Exploring potential molecular targets and therapeutic efficacy of beauvericin in triple-negative breast cancer cells.

Triple negative breast cancer (TNBC) presents a significant global health concern due to its aggressive nature, high mortality rate and limited treatment options, highlighting the urgent need for targeted therapies. Beauvericin, a bioactive fungal secondary metabolite, possess significant anticancer potential, although its molecular targets in cancer cells remain unexplored. This study has investigated possible molecular targets of beauvericin and its therapeutic insights in TNBC cells. In silico studies using molecular docking and MD simulation predicted the molecular targets of beauvericin. The identified targets included MRP-1 (ABCC1), HDAC-1, HDAC-2, LCK and SYK with average binding energy of -90.1, -44.3, -72.1, -105 and -60.8 KJ/mol, respectively, implying its multifaceted roles in reversing drug resistance, inhibiting epigenetic modulators and oncogenic tyrosine kinases. Beauvericin has significantly reduced the viability of MDA-MB-231 and MDA-MB-468 cells, with IC50 concentrations of 4.4 and 3.9 µM, while concurrently elevating the intracellular ROS by 9.0 and 7.9 folds, respectively. Subsequent reduction of mitochondrial transmembrane potential in TNBC cells, has confirmed the induction of oxidative stress, leading to apoptotic cell death, as observed by flow cytometric analyses. Beauvericin has also arrested cell cycle at G1-phase and impaired the spheroid formation and clonal expansion abilities of TNBC cells. The viability of spheroids was reduced upon beauvericin treatment, exhibiting IC50 concentrations of 10.3 and 6.2 µM in MDA-MB-468 and MDA-MB-231 cells, respectively. In conclusion, beauvericin has demonstrated promising therapeutic potential against TNBC cells through possible inhibition of MRP-1 (ABCC1), HDAC-1, HDAC-2, LCK and SYK.

Three-dimensional environment sensitizes pancreatic cancer cells to the anti-proliferative effect of budesonide by reprogramming energy metabolism.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer with an aggressive metastatic phenotype and very poor clinical prognosis. Interestingly, a lower occurrence of PDAC has been described in individuals with severe and long-standing asthma. Here we explored the potential link between PDAC and the glucocorticoid (GC) budesonide, a first-line therapy to treat asthma. METHODS: We tested the effect of budesonide and the classical GCs on the morphology, proliferation, migration and invasiveness of patient-derived PDAC cells and pancreatic cancer cell lines, using 2D and 3D cultures in vitro. Furthermore, a xenograft model was used to investigate the effect of budesonide on PDAC tumor growth in vivo. Finally, we combined genome-wide transcriptome analysis with genetic and pharmacological approaches to explore the mechanisms underlying budesonide activities in the different environmental conditions. RESULTS: We found that in 2D culture settings, high micromolar concentrations of budesonide reduced the mesenchymal invasive/migrating features of PDAC cells, without affecting proliferation or survival. This activity was specific and independent of the Glucocorticoid Receptor (GR). Conversely, in a more physiological 3D environment, low nanomolar concentrations of budesonide strongly reduced PDAC cell proliferation in a GR-dependent manner. Accordingly, we found that budesonide reduced PDAC tumor growth in vivo. Mechanistically, we demonstrated that the 3D environment drives the cells towards a general metabolic reprogramming involving protein, lipid, and energy metabolism (e.g., increased glycolysis dependency). This metabolic change sensitizes PDAC cells to the anti-proliferative effect of budesonide, which instead induces opposite changes (e.g., increased mitochondrial oxidative phosphorylation). Finally, we provide evidence that budesonide inhibits PDAC growth, at least in part, through the tumor suppressor CDKN1C/p57Kip2. CONCLUSIONS: Collectively, our study reveals that the microenvironment influences the susceptibility of PDAC cells to GCs and provides unprecedented evidence for the anti-proliferative activity of budesonide on PDAC cells in 3D conditions, in vitro and in vivo. Our findings may explain, at least in part, the reason for the lower occurrence of pancreatic cancer in asthmatic patients and suggest a potential suitability of budesonide for clinical trials as a therapeutic approach to fight pancreatic cancer.

Temozolomide resistance mechanisms: unveiling the role of translesion DNA polymerase kappa in glioblastoma spheroids in vitro.

Temozolomide (TMZ) is the leading therapeutic agent for combating Glioblastoma Multiforme (GBM). Nonetheless, the persistence of chemotherapy-resistant GBM cells remains an ongoing challenge, attributed to various factors, including the translesion synthesis (TLS) mechanism. TLS enables tumor cells to endure genomic damage by utilizing specialized DNA polymerases to bypass DNA lesions. Specifically, TLS polymerase Kappa (Polκ) has been implicated in facilitating DNA damage tolerance against TMZ-induced damage, contributing to a worse prognosis in GBM patients. To better understand the roles of Polκ in TMZ resistance, we conducted a comprehensive assessment of the cytotoxic, antiproliferative, antimetastatic, and genotoxic effects of TMZ on GBM (U251MG) wild-type (WTE) and TLS Polκ knockout (KO) cells, cultivated as three-dimensional (3D) tumor spheroids in vitro. Initial results revealed that TMZ: (i) induces reductions in GBM spheroid diameter (10-200 µM); (ii) demonstrates significant cytotoxicity (25-200 µM); (iii) exerts antiproliferative effects (≤25 µM) and promotes cell cycle arrest (G2/M phase) in Polκ KO spheroids when compared with WTE counterparts. Furthermore, Polκ KO spheroids exhibit elevated levels of cell death (Caspase 3/7) and display greater genotoxicity (53BP1) than WTE following TMZ exposure. Concerning antimetastatic effects, TMZ impedes invadopodia (3D invasion) more effectively in Polκ KO than in WTE spheroids. Collectively, the results suggest that TLS Polκ plays a vital role in the survival, cell death, genotoxicity, and metastatic potential of GBM spheroids in vitro when subjected to TMZ treatment. While the precise mechanisms underpinning this resistance remain elusive, TLS Polκ emerges as a potential therapeutic target for GBM patients.

Development and in-depth characterization of BRAFi-resistant melanoma cell lines in vitro and in vivo.

Regardless of the clinical response and improved patient survival observed following treatment with BRAFi like Vemurafenib (Vem), rapid development of resistance still remains as a major obstacle in melanoma therapy. In this context, we developed and characterized two acquired Vem-resistant melanoma cell lines, A375V and SK-MEL-28V, and an intrinsically Vem-resistant cell line, RPMI-7951. Altered morphology and growth rate of the resistant cell lines displayed spindle-shaped cells with filopodia formation and enhanced proliferation rate as compared to parental cells. Further in vitro characterization in 2D models confirmed the emergence of a resistant phenotype in melanoma cells. To mimic the in vivo tumor microenvironment, spheroids were developed for both parental and resistant cell lines to recognize materialization of invadopodia structures demonstrating elevated invasiveness and proliferation of resistant cells-based spheroids, especially A375V. Importantly, we validated A375V cell line in vivo to prove its tumorigenicity and drug resistance in tumor xenograft model. Taken together, our established clinically relevant Vem-resistant tumor model could be beneficial to elucidate drug resistance mechanisms, screen and identify novel anticancer therapies to overcome BRAFi resistance in melanoma.

Transcriptomic profiling analysis of the effect of palmitic acid on 3D spheroids of ß-like cells derived from induced pluripotent stem cells.

Type 2 diabetes (T2D) is posing a serious public health concern with a considerable impact on human life and health expenditures worldwide. The disease develops when insulin plasma level is insufficient for coping insulin resistance, caused by the decline of pancreatic ß-cell function and mass. In ß-cells, the lipotoxicity exerted by saturated free fatty acids in particular palmitate (PA), which is chronically elevated in T2D, plays a major role in ß-cell dysfunction and mass. However, there is a lack of human relevant in vitro model to identify the underlying mechanism through which palmitate induces ß-cell failure. In this frame, we have previously developed a cutting-edge 3D spheroid model of ß-like cells derived from human induced pluripotent stem cells. In the present work, we investigated the signaling pathways modified by palmitate in ß-like cells derived spheroids. When compared to the 2D monolayer cultures, the transcriptome analysis (FDR set at  0.1) revealed that the 3D spheroids upregulated the pancreatic markers (such as GCG, IAPP genes), lipids metabolism and transporters (CD36, HMGSC2 genes), glucose transporter (SLC2A6). Then, the 3D spheroids are exposed to PA 0.5 mM for 72 h. The differential analysis demonstrated that 32 transcription factors and 135 target genes were mainly modulated (FDR set at  0.1) including the upregulation of lipid and carbohydrates metabolism (HMGSC2, LDHA, GLUT3), fibrin metabolism (FGG, FGB), apoptosis (CASP7). The pathway analysis using the 135 selected targets extracted the fibrin related biological process and wound healing in 3D PA treated conditions. An overall pathway gene set enrichment analysis, performed on the overall gene set (with pathway significance cutoff at 0.2), highlighted that PA perturbs the citrate cycle, FOXO signaling and Hippo signaling as observed in human islets studies. Additional RT-PCR confirmed induction of inflammatory (IGFBP1, IGFBP3) and cell growth (CCND1, Ki67) pathways by PA. All these changes were associated with unaffected glucose-stimulated insulin secretion (GSIS), suggesting that they precede the defect of insulin secretion and death induced by PA. Overall, we believe that our data demonstrate the potential of our spheroid 3D islet-like cells to investigate the pancreatic-like response to diabetogenic environment.

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing.

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Experimental and Computational Analysis of High-Intensity Focused Ultrasound Thermal Ablation in Breast Cancer Cells: Monolayers vs. Spheroids.

High-intensity focused ultrasound (HIFU) is a non-invasive therapeutic modality that uses precise acoustic energy to ablate cancerous tissues through coagulative necrosis. In this context, we investigate the efficacy of HIFU ablation in two distinct cellular configurations, namely 2D monolayers and 3D spheroids of epithelial breast cancer cell lines (MDA-MB 231 and MCF7). The primary objective is to compare the response of these two in vitro models to HIFU while measuring their ablation percentages and temperature elevation levels. HIFU was systematically applied to the cell cultures, varying ultrasound intensity and duty cycle during different sonication sessions. The results indicate that the degree of ablation is highly influenced by the duty cycle, with higher duty cycles resulting in greater ablation percentages, while sonication duration has a minimal impact. Numerical simulations validate experimental observations, highlighting a significant disparity in the response of 2D monolayers and 3D spheroids to HIFU treatment. Specifically, tumor spheroids require lower temperature elevations for effective ablation, and their ablation percentage significantly increases with elevated duty cycles. This study contributes to a comprehensive understanding of acoustic energy conversion within the biological system during HIFU treatment for 2D versus 3D ablation targets, holding potential implications for refining and personalizing breast cancer therapeutic strategies.

Polymeric nanofiber leveraged co-delivery of anti-stromal PAK1 inhibitor and paclitaxel enhances therapeutic effects in stroma-rich 3D spheroid models.

The role of tumor stroma in solid tumors has been widely recognized in cancer progression, metastasis and chemoresistance. Cancer-associated fibroblasts (CAFs) play a crucial role in matrix remodeling and promoting cancer cell stemness and resistance via reciprocal crosstalk. Residual tumor tissue after surgical removal as well as unresectable tumors face therapeutic challenges to achieve curable outcome. In this study, we propose to develop a dual delivery approach by combining p21-activated kinase 1 (PAK1) inhibitor (FRAX597) to inhibit tumor stroma and chemotherapeutic agent paclitaxel (PTX) to kill cancer cells using electrospun nanofibers. First, the role of the PAK1 pathway was established in CAF differentiation, migration and contraction using relevant in vitro models. Second, polycaprolactone polymer-based nanofibers were fabricated using a uniaxial electrospinning technique to incorporate FRAX597 and/or PTX, which showed a uniform texture and a prolonged release of both drugs for 16 days. To test nanofibers, stroma-rich 3D heterospheroid models were set up which showed high resistance to PTX nanofibers compared to stroma-free homospheroids. Interestingly, nanofibers containing PTX and FRAX597 showed strong anti-tumor effects on heterospheroids by reducing the growth and viability by > 90 % compared to either of single drug-loaded nanofibers. These effects were reflected by reduced intra-spheroidal expression levels of collagen 1 and α-smooth muscle actin (α-SMA). Overall, this study provides a new therapeutic strategy to inhibit the tumor stroma using PAK1 inhibitor and thereby enhance the efficacy of chemotherapy using nanofibers as a local delivery system for unresectable or residual tumor. Use of 3D models to evaluate nanofibers highlights these models as advanced in vitro tools to study the effect of controlled release local drug delivery systems before animal studies.

Sexual Dimorphism's impact on adipogenesis: A three-dimensional in vitro model treated with 17ß-estradiol and testosterone.

Using a three-dimensional (3-D) in vitro culture model, we report the dose dependent effect of 17ß-estradiol and testosterone on the adipogenic differentiation and maturation of human adipose derived stem cells (hASCs) obtained from female and male patients. Considering sexual dimorphism, we expected male and female adipocytes to respond differently to the sex steroids. Both male and female hASC spheroids were exposed to 100 nM and 500 nM of 17ß-estradiol and testosterone either at the beginning of the adipogenic maturation (Phase I) to discourage intracellular triglyceride accumulation or exposed after adipogenic maturation (Phase II) to reduce the intracellular triglyceride accumulation. The results show that 17ß-estradiol leads to a dose dependent reduction in intracellular triglyceride accumulation in female hASC spheroids compared to the both untreated and testosterone-treated cells. Affirming our hypothesis, 17ß-estradiol prevented intracellular triglyceride accumulation during Phase I, while it stimulated lipolysis during Phase II. PPAR-γ and adiponectin gene expression also reduced upon 17ß-estradiol treatment in female cells. Interestingly, 17ß-estradiol and testosterone had only a modest effect on the male hASC spheroids. Collectively, our findings suggest that 17ß-estradiol can prevent fat accumulation in adipocytes during early and late stages of maturation in females.

Assessment of cellular senescence potential of PM2.5 using 3D human lung fibroblast spheroids in vitro model.

Background: Epidemiological studies demonstrate that particulate matter 2.5 (PM2.5) exposure closely related to chronic respiratory diseases. Cellular senescence plays an important role in many diseases. However, it is not fully clear whether PM2.5 exposure could induce cellular senescence in the human lung. In this study, we generated a three-dimensional (3D) spheroid model using isolated primary human lung fibroblasts (HLFs) to investigate the effects of PM2.5 on cellular senescence at the 3D level. Methods: 3D spheroids were exposed to 25-100 µg/ml of PM2.5 in order to evaluate the impact on cellular senescence. SA-ß-galactosidase activity, cell proliferation, and the expression of key genes and proteins were detected. Results: Exposure of the HLF spheroids to PM2.5 yielded a more sensitive cytotoxicity than 2D HLF cell culture. Importantly, PM2.5 exposure induced the rapid progression of cellular senescence in 3D HLF spheroids, with a dramatically increased SA-ß-Gal activity. In exploiting the mechanism underlying the effect of PM2.5 on senescence, we found a significant increase of DNA damage, upregulation of p21 protein levels, and suppression of cell proliferation in PM2.5-treated HLF spheroids. Moreover, PM2.5 exposure created a significant inflammatory response, which may be at least partially associated with the activation of TGF-ß1/Smad3 axis and HMGB1 pathway. Conclusions: Our results indicate that PM2.5 could induce DNA damage, inflammation, and cellular senescence in 3D HLF spheroids, which may provide a new evidence for PM2.5 toxicity based on a 3D model which has been shown to be more in vivo-like in their phenotype and physiology than 2D cultures.

Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors.

Tumor acidosis is associated with increased invasiveness and drug resistance. Here, we take an unbiased approach to identify vulnerabilities of acid-exposed cancer cells by combining pH-dependent flow cytometry cell sorting from 3D colorectal tumor spheroids and transcriptomic profiling. Besides metabolic rewiring, we identify an increase in tetraploid cell frequency and DNA damage response as consistent hallmarks of acid-exposed cancer cells, supported by the activation of ATM and ATR signaling pathways. We find that regardless of the cell replication error status, both ATM and ATR inhibitors exert preferential growth inhibitory effects on acid-exposed cancer cells. The efficacy of a combination of these drugs with 5-FU is further documented in 3D spheroids as well as in patient-derived colorectal tumor organoids. These data position tumor acidosis as a revelator of the therapeutic potential of DNA repair blockers and as an attractive clinical biomarker to predict the response to a combination with chemotherapy.

Assessing the gene silencing potential of AuNP-based approaches on conventional 2D cell culture versus 3D tumor spheroid.

Three-dimensional (3D) cell culture using tumor spheroids provides a crucial platform for replicating tissue microenvironments. However, effective gene modulation via nanoparticle-based transfection remains a challenge, often facing delivery hurdles. Gold nanoparticles (AuNPs) with their tailored synthesis and biocompatibility, have shown promising results in two-dimensional (2D) cultures, nevertheless, they still require a comprehensive evaluation before they can reach its full potential on 3D models. While 2D cultures offer simplicity and affordability, they lack physiological fidelity. In contrast, 3D spheroids better capture in vivo conditions, enabling the study of cell interactions and nutrient distribution. These models are essential for investigating cancer behavior, drug responses, and developmental processes. Nevertheless, transitioning from 2D to 3D models demands an understanding of altered internalization mechanisms and microenvironmental influences. This study assessed ASO-AuNP conjugates for silencing the c-MYC oncogene in 2D cultures and 3D tumor spheroids, revealing distinctions in gene silencing efficiency and highlighting the microenvironment's impact on AuNP-mediated gene modulation. Herein, we demonstrate that increasing the number of AuNPs per cell by 2.6 times, when transitioning from a 2D cell model to a 3D spheroid, allows to attain similar silencing efficiencies. Such insights advance the development of targeted gene therapies within intricate tissue-like contexts.

Biomolecular fingerprints of the effect of zoledronic acid on prostate cancer stem cells: Comparison of 2D and 3D cell culture models.

Revealing the potential of candidate drugs against different cancer types without disrupting normal cells depends on the drug mode of action. In the current study, the drug response of prostate cancer stem cells (PCSCs) to zoledronic acid (ZOL) grown in two-dimensional (2D) and three-dimensional (3D) culture systems was compared using Fourier transform-infrared (FT-IR) spectroscopy which is a vibrational spectroscopic technique, supporting by biochemical assays and imaging techniques. Based on our data, in 2D cell culture conditions, the ZOL treatment of PCSCs isolated according to both C133 and CD44 cell surface properties induced early/late apoptosis and suppressed migration ability. The CD133 gene expression and protein levels were altered, depending on culture systems. CD133 expression was significantly reduced in 2D cells upon ZOL treatment. FT-IR data revealed that the integrity, fluidity, and ordering/disordering states of the cell membrane and nucleic acid content were altered in both 2D and 3D cells after ZOL treatment. Regular protein structures decrease in 2D cells while glycogen and protein contents increase in 3D cells, indicating a more pronounced cytotoxic effect of ZOL for 2D cells. Untreated 3D PCSCs exhibited an even different spectral profile associated with IR signals of lipids, proteins, nucleic acids, and glycogen in comparison to untreated 2D cells. Our study revealed significant differences in the drug response and cellular constituents between 2D and 3D cells. Exploring molecular targets and/or drug-action mechanisms is significant in cancer treatment approaches; thus, FT-IR spectroscopy can be successfully applied as a novel drug-screening method in clinical research.

Proteomic-miRNA Biomics Profile Reveals 2D Cultures of Human iPSC-Derived Neural Progenitor Cells More Sensitive than 3D Spheroid System Against the Experimental Exposure to Arsenic.

The iPSC-derived 3D models are considered to be a connective link between 2D culture and in vivo studies. However, the sensitivity of such 3D models is yet to be established. We assessed the sensitivity of the hiPSC-derived 3D spheroids against 2D cultures of neural progenitor cells. The sub-toxic dose of Sodium Arsenite (SA) was used to investigate the alterations in miRNA-proteins in both systems. Though SA exposure induced significant alterations in the proteins in both 2D and 3D systems, these proteins were uncommon except for 20 proteins. The number and magnitude of altered proteins were higher in the 2D system compared to 3D. The association of dysregulated miRNAs with the target proteins showed their involvement primarily in mitochondrial bioenergetics, oxidative and ER stress, transcription and translation mechanism, cytostructure, etc., in both culture systems. Further, the impact of dysregulated miRNAs and associated proteins on these functions and ultrastructural changes was compared in both culture systems. The ultrastructural studies revealed a similar pattern of mitochondrial damage, while the cellular bioenergetics studies confirm a significantly higher energy failure in the 2D system than to 3D. Such a higher magnitude of changes could be correlated with a higher amount of internalization of SA in 2D cultures than in 3D spheroids. Our findings demonstrate that a 2D culture system seems better responsive than a 3D spheroid system against SA exposure.

Identification of MCM4 and PRKDC as new regulators of osteosarcoma cell dormancy based on 3D cell cultures.

Dormancy is a potential way for tumors to develop drug resistance and escape treatment. However, the mechanisms involved in cancer dormancy remain poorly understood. This is mainly because there is no in vitro culture model making it possible to spontaneously induce dormancy. In this context, the present work proposes the use of three-dimensional (3D) spheroids developed from osteosarcoma cell lines as a relevant model for studying cancer dormancy. MNNG-HOS, SaOS-2, 143B, MG-63, U2OS and SJSA-1 cell lines were cultured in 3D using the Liquid Overlay Technique (LOT). Dormancy was studied by staining cancer cells with a lipophilic dye (DiD), and long-term DiD+ cells were considered as dormant cancer cells. The role of the extracellular matrix in inducing dormancy was investigated by embedding cells into methylcellulose or Geltrex™. Gene expression of DiD+ cells was assessed with a Nanostring™ approach and the role of the genes detected in dormancy was validated by a transient down-expression model using siRNA treatment. Proliferation was measured using fluorescence microscopy and the xCELLigence technology. We observed that MNNG-HOS, 143B and MG-G3 cell lines had a reduced proliferation rate in 3D compared to 2D. U2OS cells had an increased proliferation rate when they were cultured in Geltrex™ compared to other 3D culture methods. Using 3D cultures, a transcriptomic signature of dormancy was obtained and showed a decreased expression of 18 genes including ETV4, HELLS, ITGA6, MCM4, PRKDC, RAD21 and UBE2T. The treatment with siRNA targeting these genes showed that cancer cell proliferation was reduced when the expression of ETV4 and MCM4 were decreased, whereas proliferation was increased when the expression of RAD21 was decreased. 3D culture facilitates the maintenance of dormant cancer cells characterized by a reduced proliferation and less differential gene expression as compared to proliferative cells. Further studies of the genes involved has enabled us to envisage their role in regulating cell proliferation.