Mimicking angiogenic microenvironment of alveolar soft-part sarcoma in a microfluidic coculture vasculature chip.

Alveolar soft-part sarcoma (ASPS) is a slow-growing soft tissue sarcoma with high mortality rates that affects adolescents and young adults. ASPS resists conventional chemotherapy; thus, decades of research have elucidated pathogenic mechanisms driving the disease, particularly its angiogenic capacities. Integrated blood vessels that are rich in pericytes (PCs) and metastatic potential are distinctive of ASPS. To mimic ASPS angiogenic microenvironment, a microfluidic coculture vasculature chip has been developed as a three-dimensional (3D) spheroid composed of mouse ASPS, a layer of PCs, and endothelial cells (ECs). This ASPS-on-a-chip provided functional and morphological similarity as the in vivo mouse model to elucidate the cellular crosstalk within the tumor vasculature before metastasis. We successfully reproduce ASPS spheroid and leaky vessels representing the unique tumor vasculature to assess effective drug delivery into the core of a solid tumor. Furthermore, this ASPS angiogenesis model enabled us to investigate the role of proteins in the intracellular trafficking of bioactive signals from ASPS to PCs and ECs during angiogenesis, including Rab27a and Sytl2. The results can help to develop drugs targeting the crosstalk between ASPS and the adjacent cells in the tumoral microenvironment.

Pericyte-Specific Secretome Profiling in Hypoxia Using TurboID in a Multicellular in Vitro Spheroid Model.

Cellular communication within the brain is imperative for maintaining homeostasis and mounting effective responses to pathological triggers like hypoxia. However, a comprehensive understanding of the precise composition and dynamic release of secreted molecules has remained elusive, confined primarily to investigations using isolated monocultures. To overcome these limitations, we utilized the potential of TurboID, a non-toxic biotin ligation enzyme, to capture and enrich secreted proteins specifically originating from human brain pericytes in spheroid cocultures with human endothelial cells and astrocytes. This approach allowed us to characterize the pericyte secretome within a more physiologically relevant multicellular setting encompassing the constituents of the blood-brain barrier. Through a combination of mass spectrometry and multiplex immunoassays, we identified a wide spectrum of different secreted proteins by pericytes. Our findings demonstrate that the pericytes secretome is profoundly shaped by their intercellular communication with other blood-brain barrier-residing cells. Moreover, we identified substantial differences in the secretory profiles between hypoxic and normoxic pericytes. Mass spectrometry analysis showed that hypoxic pericytes in coculture increase their release of signals related to protein secretion, mTOR signaling, and the complement system, while hypoxic pericytes in monocultures showed an upregulation in proliferative pathways including G2M checkpoints, E2F-, and Myc-targets. In addition, hypoxic pericytes show an upregulation of proangiogenic proteins such as VEGFA but display downregulation of canonical proinflammatory cytokines such as CXCL1, MCP-1, and CXCL6. Understanding the specific composition of secreted proteins in the multicellular brain microvasculature is crucial for advancing our knowledge of brain homeostasis and the mechanisms underlying pathology. This study has implications for the identification of targeted therapeutic strategies aimed at modulating microvascular signaling in brain pathologies associated with hypoxia.

Galectin-3-Binding Protein Inhibits Extracellular Heparan 6-O-Endosulfatase Sulf-2.

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented, and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). Despite its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the conditioned media of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed the importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration-dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited the invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP may regulate the physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

Mitochondrial transfer from cancer-associated fibroblasts increases migration in aggressive breast cancer.

Cancer-associated fibroblasts (CAFs) have distinct roles within the tumor microenvironment, which can impact the mode and efficacy of tumor cell migration. CAFs are known to increase invasion of less-aggressive breast cancer cells through matrix remodeling and leader-follower dynamics. Here, we demonstrate that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes (TNTs), which allow for the exchange of cargo between cell types. CAF mitochondria are an integral cargo component and are sufficient to increase the 3D migration of cancer cells. This cargo transfer results in an increase in mitochondrial ATP production in cancer cells, whereas it has a negligible impact on glycolytic ATP production. Manually increasing mitochondrial oxidative phosphorylation (OXPHOS) by providing extra substrates for OXPHOS fails to enhance cancer cell migration unless glycolysis is maintained at a constant level. Together, these data indicate that tumor-stromal cell crosstalk via TNTs and the associated metabolic symbiosis is a finely controlled mechanism by which tumor cells co-opt their microenvironment to promote cancer progression and may become a potential therapeutic target.

Cell jamming in a collagen-based interface assay is tuned by collagen density and proteolysis.

Tumor cell invasion into heterogenous interstitial tissues consisting of network-, channel- or rift-like architectures involves both matrix metalloproteinase (MMP)-mediated tissue remodeling and cell shape adaptation to tissue geometry. Three-dimensional (3D) models composed of either porous or linearly aligned architectures have added to the understanding of how physical spacing principles affect migration efficacy; however, the relative contribution of each architecture to decision making in the presence of varying MMP availability is not known. Here, we developed an interface assay containing a cleft between two high-density collagen lattices, and we used this assay to probe tumor cell invasion efficacy, invasion mode and MMP dependence in concert. In silico modeling predicted facilitated cell migration into confining clefts independently of MMP activity, whereas migration into dense porous matrix was predicted to require matrix degradation. This prediction was verified experimentally, where inhibition of collagen degradation was found to strongly compromise migration into 3D collagen in a density-dependent manner, but interface-guided migration remained effective, occurring by cell jamming. The 3D interface assay reported here may serve as a suitable model to better understand the impact of in vivo-relevant interstitial tissue topologies on tumor invasion patterning and responses to molecular interventions.

Vascular smooth muscle cells exhibit elevated hypoxia-inducible Factor-1α expression in human blood vessel organoids, influencing osteogenic performance.

Considering the importance of alternative methodologies to animal experimentation, we propose an organoid-based biological model for in vitro blood vessel generation, achieved through co-culturing endothelial and vascular smooth muscle cells (VSMCs). Initially, the organoids underwent comprehensive characterization, revealing VSMCs (α-SMA + cells) at the periphery and endothelial cells (CD31+ cells) at the core. Additionally, ephrin B2 and ephrin B4, genes implicated in arterial and venous formation respectively, were used to validate the obtained organoid. Moreover, the data indicates exclusive HIF-1α expression in VSMCs, identified through various methodologies. Subsequently, we tested the hypothesis that the generated blood vessels have the capacity to modulate the osteogenic phenotype, demonstrating the ability of HIF-1α to promote osteogenic signals, primarily by influencing Runx2 expression. Overall, this study underscores that the methodology employed to create blood vessel organoids establishes an experimental framework capable of producing a 3D culture model of both venous and arterial endothelial tissues. This model effectively guides morphogenesis from mesenchymal stem cells through paracrine signaling, ultimately leading to an osteogenic acquisition phenotype, with the dynamic involvement of HIF-1α.

Probing molecular crowding in compressed tissues with Brillouin light scattering.

Volume regulation is key in maintaining important tissue functions, such as growth or healing. This is achieved by modulation of active contractility as well as water efflux that changes molecular crowding within individual cells. Local sensors have been developed to monitor stresses or forces in model tissues, but these approaches do not capture the contribution of liquid flows to volume regulation. Here, we use a tool based on Brillouin light scattering (BLS) that uses the interaction of a laser light with inherent picosecond timescale density fluctuations in the sample. To investigate volume variations, we induced osmotic perturbations with a polysaccharide osmolyte, Dextran (Dx), and compress cells locally within multicellular spheroids (MCSs). During osmotic compressions, we observe an increase in the BLS frequency shift that reflects local variations in the compressibility. To elucidate these data, we propose a model based on a mixing law that describes the increase of molecular crowding upon reduction of the intracellular fluids. Comparison with the data suggests a nonlinear increase of the compressibility due to the dense crowding that induces hydrodynamic interactions between the cellular polymers.

14-3-3τ drives estrogen receptor loss via ERα36 induction and GATA3 inhibition in breast cancer.

About one-fourth of recurrent estrogen receptor-positive (ER+) breast cancers lose ER expression, leading to endocrine therapy failure. However, the mechanisms underlying ER loss remain to be fully explored. We now show that 14-3-3τ, up-regulated in ∼60% of breast cancer, drives the conversion of ER+ to ER- and epithelial-to-mesenchymal transition (EMT). We identify ERα36, an isoform of ERα66, as a downstream effector of 14-3-3τ. Overexpression of 14-3-3τ induces ERα36 in xenografts and tumor spheroids. The regulation is further supported by a positive correlation between ERα36 and 14-3-3τ expression in human breast cancers. ERα36 can antagonize ERα66 and inhibit ERα66 expression. Isoform-specific depletion of ERα36 blocks the ER conversion and EMT induced by 14-3-3τ overexpression in tumor spheroids, thus establishing ERα36 as a key mediator in 14-3-3τ-driven ER loss and EMT. ERα36 promoter is repressed by GATA3, which can be phosphorylated by AKT at consensus binding sites for 14-3-3. Upon AKT activation, 14-3-3τ binds phosphorylated GATA3 and facilitates the degradation of GATA3 causing GATA3 to lose transcriptional control over its target genes ERα66 and ERα36. We also demonstrate a role for the collaboration between 14-3-3τ and AKT in ERα36 induction and endocrine therapy resistance by three-dimensional spheroid and tamoxifen treatment models in MCF7 and T47D ER+ breast cancer cells. Thus, the 14-3-3τ-ERα36 regulation provides a previously unrecognized mechanism for ER loss and endocrine therapy failure.

Keratin 6A Is Expressed at the Invasive Front and Enhances the Progression of Colorectal Cancer.

Keratins (KRTs) are intermediate filament proteins in epithelial cells, and they are important for cytoskeletal organization. KRT6A, classified as a type II KRT, is normally expressed in stratified squamous epithelium and squamous cell carcinomas. Little is known about the expression and role of KRT6A in adenocarcinomas. We investigated the clinicopathologic and molecular biological significance of KRT6A in colorectal adenocarcinoma. Immunostaining of colorectal adenocarcinoma cases treated at our institution demonstrated that KRT6A showed significantly stronger expression at the invasive front than that at the tumor center (P < .0001). The high KRT6A-expression cases (n = 47) tended to have a high budding grade associated with significantly worse prognoses. A multivariate analysis revealed that the KRT6A expression status was an independent prognostic factor for overall survival (P = .0004), disease-specific survival (P = .0097), and progression-free survival (P = .0033). The correlation between KRT6A and patient prognoses was also validated in an external cohort from a published data set. To determine the function of KRT6A in vitro, KRT6A was overexpressed in 3 colon cancer cell lines: DLD-1, SW620, and HCT 116. KRT6A overexpression increased migration and invasion in DLD-1 but did not in SW620 and HCT116. In 3-dimensional sphere-forming culture, KRT6A expression enhanced the irregular protrusion around the spheroid in DLD-1. Our findings in this study indicated that KRT6A expression is a valuable prognostic marker of colorectal cancer and KRT6A may be involved the molecular mechanism in the progression of invasive areas of colorectal cancer.

NKCC1 inhibition reduces periaxonal swelling, increases white matter sparing, and improves neurological recovery after contusive SCI.

Ultrastructural studies of contusive spinal cord injury (SCI) in mammals have shown that the most prominent acute changes in white matter are periaxonal swelling and separation of myelin away from their axon, axonal swelling, and axonal spheroid formation. However, the underlying cellular and molecular mechanisms that cause periaxonal swelling and the functional consequences are poorly understood. We hypothesized that periaxonal swelling and loss of connectivity between the axo-myelinic interface impedes neurological recovery by disrupting conduction velocity, and glial to axonal trophic support resulting in axonal swelling and spheroid formation. Utilizing in vivo longitudinal imaging of Thy1YFP+ axons and myelin labeled with Nile red, we reveal that periaxonal swelling significantly increases acutely following a contusive SCI (T13, 30 kdyn, IH Impactor) versus baseline recordings (laminectomy only) and often precedes axonal spheroid formation. In addition, using longitudinal imaging to determine the fate of myelinated fibers acutely after SCI, we show that ∼73% of myelinated fibers present with periaxonal swelling at 1 h post SCI and âˆ¼ 51% of those fibers transition to axonal spheroids by 4 h post SCI. Next, we assessed whether cation-chloride cotransporters present within the internode contributed to periaxonal swelling and whether their modulation would increase white matter sparing and improve neurological recovery following a moderate contusive SCI (T9, 50 kdyn). Mechanistically, activation of the cation-chloride cotransporter KCC2 did not improve neurological recovery and acute axonal survival, but did improve chronic tissue sparing. In distinction, the NKKC1 antagonist bumetanide improved neurological recovery, tissue sparing, and axonal survival, in part through preventing periaxonal swelling and disruption of the axo-myelinic interface. Collectively, these data reveal a novel neuroprotective target to prevent periaxonal swelling and improve neurological recovery after SCI.

Simple 3D spheroid cell culture model for studies of prion infection.

Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 µm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, ß-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.

Olaparib enhances sensitization of BRCA-proficient breast cancer cells to x-rays and protons.

PURPOSE: This study aimed to compare the radiosensitizing effect of the PARP inhibitor, Olaparib, between proton and X-rays irradiations in BRCA-proficient breast cancer (BC) cells. METHODS: Two BRCA-proficient BC cell lines, MDA-MB-231 and T47D BC, were used. Cell proliferation was assessed using the CCK-8 assay, and radiosensitivity was determined through the clonogenic survival assay. Flow cytometry was employed to analyze cell cycle distribution and apoptosis. The kinetics of DNA damage repair were evaluated using γH2AX immunofluorescence imaging and the comet assay. Tumor spheroid assays were conducted to test radiosensitivity in a three-dimensional culture condition. RESULTS: Olaparib sensitized both MDA-MB-231 and T47D cells to proton and X-ray irradiation in the clonogenic assay. MDA-MB-231 cells exhibited a higher dose enhancement factor for Olaparib than T47D cells. Olaparib increased radiation-induced G2/M cell cycle arrest and apoptosis specifically in MDA-MB-231 cells. γH2AX immunostaining and the comet assay showed Olaparib augmented radiation-induced DNA damage and apoptosis. The enhancement effect of Olaparib was more pronounced in proton irradiation than in X-ray irradiation, particularly in MDA-MB-231 cells than T47D cells. Both radiation and Olaparib dose-dependently inhibited spheroid growth in both cell lines. The synergy scores demonstrated that Olaparib interacted more strongly with protons than X-rays. The addition of an ATR inhibitor further enhanced Olaparib-induced proton radiosensitization in MDA-MB-231 cells. CONCLUSION: This study found that Olaparib enhanced radiation efficacy in BRCA-proficient breast cancer cells, with a more pronounced effect observed with proton irradiation compared to X-ray irradiation. Combining Olaparib with an ATR inhibitor increased the radiosensitizing effect of protons.

Simultaneous Separating, Splitting, Collecting, and Dispensing by Droplet Pinch-Off for Droplet Cell Culture.

Simultaneous separating, splitting, collecting, and dispensing a cell suspension droplet has been demonstrated by aspiration and subsequent droplet pinch-off for use in microfluidic droplet cell culture systems. This method is applied to cell manipulations including aliquots and concentrations of microalgal and mammalian cell suspensions. Especially, medium exchange of spheroid droplets is successfully demonstrated by collecting more than 99% of all culture medium without damaging the spheroids, demonstrating its potential for a 3D cell culture system. Through dimensional analysis and systematic parametric studies, it is found that initial mother droplet size together with aspiration flow rate determines three droplet pinch-off regimes. By observing contact angle changes during aspiration, the difference in the large and the small droplet pinch-off can be quantitatively explained using force balance. It is found that the capillary number plays a significant role in droplet pinch-off, but the Bond number and the Ohnesorge number have minor effects. Since the dispensed droplet size is mainly determined by the capillary number, the dispensed droplet size can be controlled simply by adjusting the aspiration flow rate. It is hoped that this method can contribute to various fields using droplets, such as droplet cell culture and digital microfluidics, beyond the generation of small droplets.

Size-Dependent Penetration of Nanoparticles in Tumor Spheroids: A Multidimensional and Quantitative Study of Transcellular and Paracellular Pathways.

Tumor penetration of nanoparticles is crucial in nanomedicine, but the mechanisms of tumor penetration are poorly understood. This work presents a multidimensional, quantitative approach to investigate the tissue penetration behavior of nanoparticles, with focuses on the particle size effect on penetration pathways, in an MDA-MB-231 tumor spheroid model using a combination of spectrometry, microscopy, and synchrotron beamline techniques. Quasi-spherical gold nanoparticles of different sizes are synthesized and incubated with 2D and 3D MDA-MB-231 cells and spheroids with or without an energy-dependent cell uptake inhibitor. The distribution and penetration pathways of nanoparticles in spheroids are visualized and quantified by inductively coupled plasma mass spectrometry, two-photon microscopy, and synchrotron X-ray fluorescence microscopy. The results reveal that 15 nm nanoparticles penetrate spheroids mainly through an energy-independent transcellular pathway, while 60 nm nanoparticles penetrate primarily through an energy-dependent transcellular pathway. Meanwhile, 22 nm nanoparticles penetrate through both transcellular and paracellular pathways and they demonstrate the greatest penetration ability in comparison to other two sizes. The multidimensional analytical methodology developed through this work offers a generalizable approach to quantitatively study the tissue penetration of nanoparticles, and the results provide important insights into the designs of nanoparticles with high accumulation at a target site.

Melanocytes in regenerative medicine applications and disease modeling.

Melanocytes are dendritic cells localized in skin, eyes, hair follicles, ears, heart and central nervous system. They are characterized by the presence of melanosomes enriched in melanin which are responsible for skin, eye and hair pigmentation. They also have different functions in photoprotection, immunity and sound perception. Melanocyte dysfunction can cause pigmentary disorders, hearing and vision impairments or increased cancer susceptibility. This review focuses on the role of melanocytes in homeostasis and disease, before discussing their potential in regenerative medicine applications, such as for disease modeling, drug testing or therapy development using stem cell technologies, tissue engineering and extracellular vesicles.

Spatiotemporal analysis of multi-scale cell structure in spheroid culture reveals hypertrophic chondrocyte differentiation.

3D cell culture has emerged as a promising approach to replicate the complex behaviors of cells within living organisms. This study aims to analyze spatiotemporal behavior of the morphological characteristics of cell structure at multiscale in 3D scaffold-free spheroids using chondrogenic progenitor ATDC5 cells. Over a 14-day culture period, it exhibited cell hypertrophy in the spheroids regarding cellular and nuclear size as well as changes in morphology. Moreover, biological analysis indicated a signification up-regulation of normal chondrocyte as well as hypertrophic chondrocyte markers, suggesting early hypertrophic chondrocyte differentiation. Cell nuclei underwent changes in volume, sphericity, and distribution in spheroid over time, indicating alterations in chromatin organization. The ratio of chromatin condensation volume to cell nuclear volume decreased as the cell nuclei enlarged, potentially signifying changes in chromatin state during hypertrophic chondrocyte differentiation. Our image analysis techniques in this present study enabled detailed morphological measurement of cell structure at multi-scale, which can be applied to various 3D culture models for in-depth investigation.

Mechanical compression regulates tumor spheroid invasion into a 3D collagen matrix.

Uncontrolled growth of tumor cells in confined spaces leads to the accumulation of compressive stress within the tumor. Although the effects of tension within 3D extracellular matrices (ECMs) on tumor growth and invasion are well established, the role of compression in tumor mechanics and invasion is largely unexplored. In this study, we modified a Transwell assay such that it provides constant compressive loads to spheroids embedded within a collagen matrix. We used microscopic imaging to follow the single cell dynamics of the cells within the spheroids, as well as invasion into the 3D ECMs. Our experimental results showed that malignant breast tumor (MDA-MB-231) and non-tumorigenic epithelial (MCF10A) spheroids responded differently to a constant compression. Cells within the malignant spheroids became more motile within the spheroids and invaded more into the ECM under compression; whereas cells within non-tumorigenic MCF10A spheroids became less motile within the spheroids and did not display apparent detachment from the spheroids under compression. These findings suggest that compression may play differential roles in healthy and pathogenic epithelial tissues and highlight the importance of tumor mechanics and invasion.

Uncovering the Heterogeneity of Cardiac Lin-KIT+ Cells: A scRNA-seq Study on the Identification of Subpopulations.

The reparative potential of cardiac Lin-KIT+ (KIT) cells is influenced by their population, but identifying their markers is challenging due to changes in phenotype during in vitro culture. Resolving this issue requires uncovering cell heterogeneity and discovering new subpopulations. Single-cell RNA sequencing (scRNA-seq) can identify KIT cell subpopulations, their markers, and signaling pathways. We used 10× genomic scRNA-seq to analyze cardiac-derived cells from adult mice and found 3 primary KIT cell populations: KIT1, characterized by high-KIT expression (KITHI), represents a population of cardiac endothelial cells; KIT2, which has low-KIT expression (KITLO), expresses transcription factors such as KLF4, MYC, and GATA6, as well as genes involved in the regulation of angiogenic cytokines; KIT3, with moderate KIT expression (KITMOD), expresses the cardiac transcription factor MEF2C and mesenchymal cell markers such as ENG. Cell-cell communication network analysis predicted the presence of the 3 KIT clusters as signal senders and receivers, including VEGF, CXCL, and BMP signaling. Metabolic analysis showed that KIT1 has the low activity of glycolysis and oxidative phosphorylation (OXPHOS), KIT2 has high glycolytic activity, and KIT3 has high OXPHOS and fatty acid degradation activity, indicating distinct metabolic adaptations of the 3 KIT populations. Through the systemic infusion of KIT1 cells in a mouse model of myocardial infarction, we observed their involvement in promoting the formation of new micro-vessels. In addition, in vitro spheroid culture experiments demonstrated the cardiac differentiation capacity of KIT2 cells.

Targeting cholesterol impairs cell invasion of all breast cancer types.

BACKGROUND: Breast cancer clinical outcome relies on its intrinsic molecular subtype and mortality is almost exclusively due to metastasis, whose mechanism remains unclear. We recently revealed the specific contribution of plasma membrane cholesterol to the invasion of malignant MCF10CAIa but not premalignant MCF10AT and normal MCF10A cell lines in 2D, through invadopodia formation and extracellular matrix (ECM) degradation. In the present study, we address the impact of breast cancer subtypes, mutations and aggressiveness on cholesterol implication in breast cancer cell invasion and 3D spheroid invasion and growth. METHODS: We used nine breast cancer cell lines grouped in four subtypes matching breast tumor classification. Four of these cell lines were also used to generate 3D spheroids. These cell lines were compared for cell invasion in 2D and 3D, spheroid growth in 3D, gelatin degradation, cortactin expression, activation and subcellular distribution as well as cell surface cholesterol distribution and lipid droplets. The effect of plasma membrane cholesterol depletion on all these parameters was determined in parallel and systematically compared with the impact of global matrix metalloproteinase (MMP) inhibition. RESULTS: The six invasive cell lines in 2D were sensitive to partial cholesterol depletion, independently of their subtype, aggressiveness or mutation. Nevertheless, the effect was stronger in the three cell lines able to degrade gelatin. 3D spheroid invasion was also reduced after cholesterol depletion in all breast cancer subtypes tested. Notably, targeting cholesterol was more powerful than MMP inhibition in reducing invasion in both 2D and 3D culture models. Moreover, cholesterol depletion in the six invasive cell lines impaired cortactin distribution in the perinuclear region where invadopodia localized. Breast cancer cell line aggressiveness relied on cholesterol-enriched domains at the ECM-free side and intracellular lipid droplets. Furthermore, the three gelatin-degrading cell lines were characterized by increased cholesterol-enriched submicrometric domains at their ECM-contact side. CONCLUSION: Together, our data suggest cell surface cholesterol combined with lipid droplet labeling as a breast cancer cell aggressiveness marker. They also open the way to test other cholesterol-targeting drugs in more complex models to further evaluate whether cholesterol could represent a strategy in breast cancer therapy.

Characterization of EpCAM in thyroid cancer biology by three-dimensional spheroids in vitro model.

BACKGROUND: Thyroid cancer (TC) is the most common endocrine malignancy. Nowadays, undifferentiated thyroid cancers (UTCs) are still lethal, mostly due to the insurgence of therapy resistance and disease relapse. These events are believed to be caused by a subpopulation of cancer cells with stem-like phenotype and specific tumor-initiating abilities, known as tumor-initiating cells (TICs). A comprehensive understanding of how to isolate and target these cells is necessary. Here we provide insights into the role that the protein Epithelial Cell Adhesion Molecule (EpCAM), a known TICs marker for other solid tumors, may have in TC biology, thus considering EpCAM a potential marker of thyroid TICs in UTCs. METHODS: The characterization of EpCAM was accomplished through Western Blot and Immunofluorescence on patient-derived tissue samples, adherent cell cultures, and 3D sphere cultures of poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) cell lines. The frequency of tumor cells with putative tumor-initiating ability within the 3D cultures was assessed through extreme limiting dilution analysis (ELDA). EpCAM proteolytic cleavages were studied through treatments with different cleavages' inhibitors. To evaluate the involvement of EpCAM in inducing drug resistance, Vemurafenib (PLX-4032) treatments were assessed through MTT assay. RESULTS: Variable EpCAM expression pattern was observed in TC tissue samples, with increased cleavage in the more UTC. We demonstrated that EpCAM is subjected to an intense cleavage process in ATC-derived 3D tumor spheres and that the 3D model faithfully mimics what was observed in patient's samples. We also proved that the integrity of the protein appears to be crucial for the generation of 3D spheres, and its expression and cleavage in a 3D system could contribute to drug resistance in thyroid TICs. CONCLUSIONS: Our data provide novel information on the role of EpCAM expression and cleavage in the biology of thyroid TICs, and our 3D model reflects the variability of EpCAM cleavage observed in tissue samples. EpCAM evaluation could play a role in clinical decisions regarding patient therapy since its expression and cleavage may have a fundamental role in the switch to a drug-resistant phenotype of UTC cells.