3D Spheroid Formation Research Articles

Lysyl hydroxylase PLOD1 enhances actin network to promote confined migration of hepatocellular carcinoma cells via binding with Septin2.

e16111 Background: Hepatocellular carcinoma (HCC) is one of the most lethal cancers due to metastasis. Liver cirrhosis increases tumor stiffness and forms confine space for HCC cells. How HCC cells overcome the confined space to evade is largely unknown. Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase (PLOD) family members, mainly responsible for lysyl hydroxylation, play important roles in tumor progression. The role of PLOD1 in confine migration remains unclear. Methods: LC-MS was applied to determine the differentially expressed proteins between HCC tissues and corresponding adjacent tissues. Microfluidic confine migration device with 6μm high channels was designed to mimic the in vivo confined space. Real-time microscope was used to monitor cell migration. The high throughput 3D invasion assays using Matrigel and Collagen I mixture added to adherent cells was applied to evaluate 3D invasion with high content screening instrument. 3D spheroid formation assays developed by different substitution percentage GelMA created various stiffness environment. IP was performed to pull down the PLOD1-binding proteins. IF staining, Western blot, FRAP, and others were used to evaluate the target expression and cellular phenotype. Results: LC-MS analysis showed that PLOD1 is highly expressed in HCC tissues. High PLOD1 expression level was associated with advanced tumor stage and unfavorable prognosis in 130 HCC patients. We found that confine migration was enhanced by PLOD1 overexpression. With the PLOD1 knockdown or inhibition, the migration speed significantly decreased. Transwell migration also showed PLOD1-overexpressed cell line can migrate faster. 3D invasion and spheroid formation assays also supports the previous findings on chip that PLOD1-overexpressed cells can invade further and generate more spheroids under high stiffness. IP-MS analysis showed that PLOD1 could bind to actin related proteins, including Septin2 (SEPT2). We found that PLOD1 can promote the formation of F-actin network, Septin network and actin polymerization. In addition, knockdown of SEPT2 was able to reverse the effect of PLOD1 on confine migration and inhibited the formation of F-actin network. Co-IP experiments showed that SEPT2 can bind to α-actin. We further found that PLOD1 could promote the binding between SEPT2 and other Septin members. Mechanically, we found that PLOD1 can increase the hydroxylation of SEPT2 protein stabilize it. Finally, orthotopic transplantation assay showed that PLOD1 promoted intrahepatic or extrahepatic metastasis. Conclusions: We demonstrated that PLOD1 increased SEPT2 stability by modifying its hydroxylation, thereby promoting the formation of Septin and F-actin networks, which in turn promotes the confined migration of HCC cells. The current work revealed novel molecular mechanism of PLOD1 on HCC confine migration and metastasis.

Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells.

Phenothiazines (PTZ) were developed as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they have been used for decades as antipsychotic drugs. In addition, they possess significant anti-cancer properties and several attempts for their repurposing were made. However, their incompletely understood polypharmacology is challenging. Here we examined the potential of the PTZ fluphenazine (Flu) and its mustard derivative (Flu-M) to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras- and MAPK-pathway, cell viability and features of cancer cell stemness. Consistently, we show that the combination of a CaM inhibitor and the PP2A activator DT-061 synergistically inhibited the 3D-spheroid formation of MDA-MB-231 (K-Ras-G13D), NCI-H358 (K-Ras-G12C) and A375 (B-raf-V600E) cancer cells, and increased apoptosis in MDA-MB-231. We reasoned that these activities remain combined in PTZ, which were the starting point for PP2A activator development, while several PTZ are known CaM inhibitors. We show that both Flu and Flu-M retained CaM inhibitory activity in vitro and in cells, with a higher potency of the mustard derivative in cells. In line with the CaM dependence of Ras plasma membrane organization, the mustard derivative potently reduced the functional membrane organization of oncogenic Ras, while DT-061 had a negligible effect. Like DT-061, both PTZ potently decreased c-MYC levels, a hallmark of PP2A activation. Benchmarking against the KRAS-G12C specific inhibitor AMG-510 in MIA PaCa-2 cells revealed a higher potency of Flu-M than combinations of DT-061 and a CaM inhibitor on MAPK-output and a strong effect on cell proliferation. While our study is limited, our results suggest that improved PTZ derivatives that retain both, their CaM inhibitory and PP2A activating properties, but have lost their neurological side-effects, may be interesting to pursue further as anti-cancer agents.

Effect of LOXL2 on metastasis through remodeling of the cell surface matrix in non-small cell lung cancer cells

Lung cancer is the prominent cause of cancer-associated death primarily because of distant metastatic disease. The metastatic potential of non-small cell lung cancer (NSCLC) is associated with tumor cell aggregation. However, the systemic mechanotransduction mechanism by which tumor cells dynamically aggregate and disseminate is poorly understood, especially in NSCLC. In this study, we examine whether the cell surface matrix plays an important role in metastasis. We used poly-2-hydroxyethyl methacrylate-based 3D spheroid formation methods to mimic in vivo metastatic lesions. Supra-structural analysis of human NSCLC A549 cells stained with ruthenium red for transmission electron microscopy (TEM) showed that glycocalyx surrounding the cell surface in 2D culture decreases in 3D culture. Comprehensive gene expression analysis revealed that the genes associated with cell adhesion were distinctly enriched in A549 cell spheroids. Of these, downregulation of the tumor metastatic microenvironment facilitator LOXL2, a copper-dependent enzyme catalyzing posttranslational oxidative deamination of peptidyl lysine, was of special interest. Knockdown of LOXL2 thickened the cell surface matrix in 2D culture and impaired compact aggregate formation in 3D culture. Moreover, A549 cell spheroids with endogenous overexpression of LOXL2 increased their dissemination on basement extracellular matrix Matrigel. Overall, these data imply that cell detachment-downregulated LOXL2 contributes to cell surface matrix remodeling, leading to collective dissemination of free-floating aggregates.

Prolonged exposure to simulated microgravity promotes stemness impairing morphological, metabolic and migratory profile of pancreatic cancer cells: a comprehensive proteomic, lipidomic and transcriptomic analysis

BackgroundThe impact of the absence of gravity on cancer cells is of great interest, especially today that space is more accessible than ever. Despite advances, few and contradictory data are available mainly due to different setup, experimental design and time point analyzed.MethodsExploiting a Random Positioning Machine, we dissected the effects of long-term exposure to simulated microgravity (SMG) on pancreatic cancer cells performing proteomic, lipidomic and transcriptomic analysis at 1, 7 and 9 days.ResultsOur results indicated that SMG affects cellular morphology through a time-dependent activation of Actin-based motility via Rho and Cdc42 pathways leading to actin rearrangement, formation of 3D spheroids and enhancement of epithelial-to-mesenchymal transition. Bioinformatic analysis reveals that SMG may activates ERK5/NF-κB/IL-8 axis that triggers the expansion of cancer stem cells with an increased migratory capability. These cells, to remediate energy stress and apoptosis activation, undergo a metabolic reprogramming orchestrated by HIF-1α and PI3K/Akt pathways that upregulate glycolysis and impair β-oxidation, suggesting a de novo synthesis of triglycerides for the membrane lipid bilayer formation.ConclusionsSMG revolutionizes tumor cell behavior and metabolism leading to the acquisition of an aggressive and metastatic stem cell-like phenotype. These results dissect the time-dependent cellular alterations induced by SMG and pave the base for altered gravity conditions as new anti-cancer technology.

Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma

Synovial sarcoma is typical aggressive malignant without satisfactory treatment outcome in adult series. Cyclin-dependent kinases (CDKs) in transcription have been considered promising molecular targets in cancer. Among these, CDK7 has been shown to play important roles in the pathogenesis of malignancies. However, the modulation mechanism of CDK7-regulated transcription in synovial sarcoma is unknown. In the present study, we aim to determine the expression and function of CDK7 in the transcription cycle of RNA polymerase II (RNAP II), and evaluate its prognostic and therapeutic significance in synovial sarcoma. Results showed that overexpression of CDK7 correlates with higher clinical stage and grade, and worse outcomes in clinic. High CDK7 expression was confirmed in all tested human synovial sarcoma cell lines and CDK7 was largely localized to the cell nucleus. Downregulation through siRNA or inhibition with the CDK7-targeting agent BS-181 exhibited dose-dependent cytotoxicity and prevented cell colony formation. Western blots demonstrated that inhibition of CDK7 paused transcription by a reduction of RNAP II phosphorylation. Blocking CDK7-dependent transcriptional addiction was accompanied by promotion of apoptosis. Furthermore, the CDK7-specific inhibitor reduced 3D spheroid formation and migration of synovial sarcoma. Collectively, our findings highlight the role of CDK7-dependent transcriptional addiction in human synovial sarcoma. CDK7-specific cytotoxic agents are therefore promising novel treatment options for synovial sarcoma.

The autocrine loop of ALK receptor and ALKAL2 ligand is an actionable target in consensus molecular subtype 1 colon cancer

BackgroundIn the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs).MethodsIn this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use.ResultsALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo.ConclusionsCollectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

Investigation of anti-cancer effects of new pyrazino[1,2-a]benzimidazole derivatives on human glioblastoma cells through 2D in vitro model and 3D-printed microfluidic device

AimsRecent studies show targeted therapy of new pyrazino[1,2-a]benzimidazole derivatives with COX-II inhibitory effects on different cancer cells. This study aimed to investigate 2D cell culture and 3D spheroid formation of glioblastoma multiforme (GBM) cells using a microfluidic device after exposure to these compounds. Main methodsAfter isolating astrocytes from human GBM samples, IC50 of 2,6-dimethyl pyrazino[1,2-a]benzimidazole (L1) and 3,4,5-trimethoxy pyrazino[1,2-a]benzimidazole (L2) were determined as 13 μM and 85 μM, respectively. Then, in all experiments, cells were exposed to subtoxic concentrations of L1 (6.5 μM) and L2 (42.5 μM), which were ½IC50. In the following, in two phases, cell cycle, migration, and gene expression through 2D cell culture and tumor spheroid formation ability using a 3D-printed microfluidic chip were assessed. Key findingsThe obtained results showed that both compounds have positive effects in reducing G2/M cell population and GBM cell migration. Furthermore, real-time gene expression data showed that L1 and L2 significantly impact the upregulation of P21 and P53 and down-regulation of cyclin D1, MMP2, and MMP9. On the other hand, GBM spheroids exposed to L1 and L2 become smaller with fewer live cells. SignificanceOur data on human isolated astrocyte cells in 2D and 3D cell culture conditions showed that L1 and L2 compounds could reduce GBM cells' invasion by controlling gene expressions associated with migration and proliferation. Moreover, designing microfluidic platform and related cell culture protocols facilitates the broad screening of 3D multicellular tumor spheroids derived from GBM tumor biopsies and provides effective drug development for brain gliomas.

Low-Temperature Plasma-Activated Medium Inhibited Proliferation and Progression of Lung Cancer by Targeting the PI3K/Akt and MAPK Pathways.

Low-temperature plasma, an engineered technology to generate various reactive species, is actively studied in cancer treatment in recent years, yet mainly by using a traditional 2D cell culture system. In this study, we explored the effect of the plasma-activated medium (PAM) on lung cancer cells in vitro and in vivo by using a 3D cell culture model. The results showed that PAM markedly inhibited 3D spheroid formation and downregulated stemness-related gene expression. We found that reactive oxygen species (ROS) penetrated throughout the whole spheroids and induced cell death surrounding and in the core of the tumor spheroid. Besides, PAM treatment suppressed migration and invasion of lung cancer cells and downregulated epithelial-mesenchymal transition- (EMT-) related gene expression. In the mouse xenograft model, the tumor volume was significantly smaller in the PAM-treated group compared with the control group. By using transcriptome sequencing, we found that PI3K/Akt and MAPK pathways were involved in the inhibition effects of PAM on lung cancer cells. Therefore, our results indicated that PAM exhibits potential anticancer effects on lung cancer and provides insight into further exploration of PAM-induced cell death and translational preclinical use.

Apoptotic Induction Mechanism of Artonin E in 3D Ovarian Cancer Cell Lines

Artonin E is a naturally isolated compound from Artocarpus elasticus and was evaluated for their anticancer property in the present study. This study aimed to develop a 3D ovarian cell culture model and to examine the cytotoxicity of artonin E in 2D and 3D cell culture models. The 3D cell culture was performed using BD™ Puramatrix™ Peptide Hydrogel. Alamar blue assay, selectivity index analysis, morphological apoptotic double staining and immunofluorescence study were conducted to study the antiproliferative and apoptotic induction mechanism of artonin E in 3D SKOV-3 spheroid culture. SKOV-3 cells encapsulated in BD™ Puramatrix™ Peptide Hydrogel clearly demonstrated 3D-spheroid formation. The Alamar blue assay result showed that artonin E inhibited the growth of SKOV-3 cells in 2D and 3D culture with the IC50 values at 72 h treatment of 6.0 ± 0.8 µg/mL and 25.0 ± 0.8 µg/mL, respectively. This compound was found to be less toxic to the normal human ovarian cell lines, T1074, with IC50 values at 72 h of 28.0 ± 0.8 µg/mL in 2D culture and 85.0 ± 0.5 µg/mL in 3D culture, respectively. The selectivity index analysis more than 2 indicated that artonin E was selective against cancer cells compared to normal cells. Artonin E treatment caused apoptotic morphological changes in 3D SKOV-3 spheroids. In a 3D immunofluorescence study, elevated levels of cleaved caspase-3, cleaved caspase-9, apoptotic proteins bax, and decreased levels of antiapoptotic proteins bcl-2, Hsp70, and survivin were observed. In conclusion, artonin E increases chemoresistance and induces cell death in a 3D SKOV-3 spheroids culture via a pro- and anti-apoptotic protein pathway. These findings demonstrate that 3D spheroid culture is an effective platform for testing artonin E therapeutic candidates in an in vivo mimic microenvironment.

Somatostatin Primes Endothelial Cells for Agonist-Induced Hyperpermeability and Angiogenesis In Vitro

Somatostatin is an inhibitory peptide, which regulates the release of several hormones, and affects neurotransmission and cell proliferation via its five Gi protein-coupled receptors (SST1-5). Although its endocrine regulatory and anti-tumour effects have been thoroughly studied, little is known about its effect on the vascular system. The aim of the present study was to analyse the effects and potential mechanisms of somatostatin on endothelial barrier function. Cultured human umbilical vein endothelial cells (HUVECs) express mainly SST1 and SST5 receptors. Somatostatin did not affect the basal HUVEC permeability, but primed HUVEC monolayers for thrombin-induced hyperpermeability. Western blot data demonstrated that somatostatin activated the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (MAPK) pathways by phosphorylation. The HUVEC barrier destabilizing effects were abrogated by pre-treating HUVECs with mitogen-activated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK), but not the Akt inhibitor. Moreover, somatostatin pre-treatment amplified vascular endothelial growth factor (VEGF)-induced angiogenesis (3D spheroid formation) in HUVECs. In conclusion, the data demonstrate that HUVECs under quiescence conditions express SST1 and SST5 receptors. Moreover, somatostatin primes HUVECs for thrombin-induced hyperpermeability mainly via the activation of MEK/ERK signalling and promotes HUVEC proliferation and angiogenesis in vitro.

Transplantation of 3D adipose-derived stem cell/hepatocyte spheroids alleviates chronic hepatic damage in a rat model of thioacetamide-induced liver cirrhosis

Cirrhosis refers to irreversible liver damage where healthy tissue is replaced by scar tissue, resulting in impaired liver function. There is no cure and current treatments only prevent further liver damage; thus, novel therapeutic options are urgently needed. Here, we report a new approach that enables the formation of self-assembled 3D spheroids of adipose-derived stem cells (ADSCs) and murine hepatocytes (AML12) via reconstituted collagen fibers. Compared with the spheroids formed in the commercially available EZSHERE dish, the collagen fiber-based ADSC/hepatocyte spheroids offer a notable benefit in structure formation and paracrine factor secretion. To test the regenerative capability of the collagen fiber-based 3D ADSC/hepatocyte spheroids, a rat model of thioacetamide (TAA)-induced liver cirrhosis was employed. The transplantation of the collagen fiber-based 3D ADSC/hepatocyte spheroids show an improvement in liver function and ameliorates pathological liver cirrhosis in TAA-treated rats. In summary, our data show collagen fiber-based self-assembled 3D ADSC/hepatocyte spheroids to possess the excellent regenerative capacity in response to TAA-induced liver injury, promising an alternative therapeutic strategy for liver cirrhosis.

Interaction of Glia Cells with Glioblastoma and Melanoma Cells under the Influence of Phytocannabinoids.

Brain tumor heterogeneity and progression are subject to complex interactions between tumor cells and their microenvironment. Glioblastoma and brain metastasis can contain 30–40% of tumor-associated macrophages, microglia, and astrocytes, affecting migration, proliferation, and apoptosis. Here, we analyzed interactions between glial cells and LN229 glioblastoma or A375 melanoma cells in the context of motility and cell–cell interactions in a 3D model. Furthermore, the effects of phytocannabinoids, cannabidiol (CBD), tetrahydrocannabidiol (THC), or their co-application were analyzed. Co-culture of tumor cells with glial cells had little effect on 3D spheroid formation, while treatment with cannabinoids led to significantly larger spheroids. The addition of astrocytes blocked cannabinoid-induced effects. None of the interventions affected cell death. Furthermore, glial cell-conditioned media led to a significant slowdown in collective, but not single-cell migration speed. Taken together, glial cells in glioblastoma and brain metastasis micromilieu impact the tumor spheroid formation, cell spreading, and motility. Since the size of spheroid remained unaffected in glial cell tumor co-cultures, phytocannabinoids increased the size of spheroids without any effects on migration. This aspect might be of relevance since phytocannabinoids are frequently used in tumor therapy for side effects.

3D Models of Cellular Spheroids As a Universal Tool for Studying the Cytotoxic Properties of Anticancer Compounds In Vitro.

The aim of this work is to develop a 3D cell culture model based on cell spheroids for predicting the functional activity of various compounds in vivo. Agarose gel molds were made using 3D printing. The solidified agarose gel is a matrix consisting of nine low-adhesive U-shaped microwells of 2.3 × 3.3 mm for 3D cell spheroid formation and growth. This matrix is placed into a single well of a 12-well plate. The effectiveness of the cell culture method was demonstrated using human ovarian carcinoma SKOVip-kat cells stably expressing the red fluorescent protein Katushka in the cytoplasm and overexpressing the membrane-associated tumor marker HER2. The SKOVip-kat cell spheroids were visualized by fluorescence microscopy. The cell concentration required for the formation of same-shape and same-size spheroids with tight intercellular contacts was optimized. To verify the developed model, the cytotoxicity of the targeted immunotoxin anti-HER2 consisting of the anti-HER2 scaffold DARP 9_29 and a fragment of the Pseudomonas aeroginosa exotoxin, DARP-LoPE, was studied in 2D and 3D SKOVip-kat cell cultures. The existence of a difference in the cytotoxic properties of DARP-LoPE between the 2D and 3D cultures has been demonstrated: the IC50 value in the 3D culture is an order of magnitude higher than that in the monolayer culture. The present work describes a universal tool for 3D cultivation of mammalian cells based on reusable agarose gel molds that allows for reproducible formation of multicellular spheroids with tight contacts for molecular and cell biology studies.

Caffeine Induces G0/G1 Cell Cycle Arrest and Inhibits Migration through Integrin αv, β3, and FAK/Akt/c-Myc Signaling Pathway.

Lung cancer is recognized as a major cause of mortality worldwide owing to its metastatic activity. Given the lack of solid information regarding the possible effects of caffeine, one of the most consumed natural psychoactive substances, on molecular signaling pathways implicated in the aggressive behavior of lung cancer, our study aimed to evaluate the effect and mechanism of caffeine on metastasis-related mechanisms. The results revealed that caffeine treatment at concentrations of 0–500 µM caused no direct cytotoxic effects on NCI-H23 cells. Treatment of cells with caffeine showed good potential to inhibit cell proliferation at 48 h and induced significant cell cycle arrest at the G0/G1 phase. Concerning metastasis, caffeine was shown to reduce filopodia formation, inhibit migration and invasion capability, and reduce the ability of cancer cells to survive and grow in an anchorage-independent manner. Moreover, caffeine could attenuate the formation of 3D tumor spheroids in cancer stem cell (CSC)-enriched populations. With regard to mechanisms, we found that caffeine significantly altered the integrin pattern of the treated cells and caused the downregulation of metastasis-associated integrins, namely, integrins αv and β3. Subsequently, the downstream signals, including protein signaling and transcription factors, namely, phosphorylated focal adhesion kinase (p-FAK), phosphorylated protein kinase B (p-Akt), cell division cycle 42 (Cdc42), and c-Myc, were significantly decreased in caffeine-exposed cells. Taken together, our novel data on caffeine-inhibiting mechanism in relation to metastasis in lung cancer could provide insights into the impact of caffeine intake on human diseases and conditions.

Three-Dimensional Cell Culture Models of Hepatocellular Carcinoma - a Review.

Three-dimensional (3D) cell culture studies are becoming extremely common because of theircapability to mimic tumor architecture, such as cell-cell and cell-ECM interactions, more efficiently than 2Dmonolayer systems. These interactions have important roles in defining the tumor cell behaviors, such asproliferation, differentiation, and most importantly, tumor drug response. This review aims to provide an overview of the methods for 3D tumor spheroid formation to modelhuman tumors, specifically concentrated on studies using hepatocellular carcinoma (HCC) cells. We obtained information from previously published articles. In this review, there is discussion of thescaffold and non-scaffold-based approaches, including hanging drop, bioreactors and 3D bioprinting. The mimicking of the tumor microenvironment (TME) as tumor spheroids could provide avaluable platform for studying tumor biology. Multicellular tumor spheroids are self-assembled cultures of mixedcells (tumor and stromal cells) organized in a 3D arrangement. These spheroids closely mimic the main features ofhuman solid tumors, such as structural organization, central hypoxia, and overall oxygen and nutrient gradients.Hepatocellular carcinoma (HCC) is the most common liver malignancy, and most difficult to overcome because ofits drug resistance and tumor heterogeneity. In order to mimic this highly heterogeneous environment, 3D cellculture systems are needed.

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling.

DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of β-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.

Engineering injectable vascularized tissues from the bottom-up: Dynamics of in-gel extra-spheroid dermal tissue assembly

Modular tissue engineering approaches open up exciting perspectives for the biofabrication of vascularized tissues from the bottom-up, using micro-sized units such as spheroids as building blocks. While several techniques for 3D spheroid formation from multiple cell types have been reported, strategies to elicit the extra-spheroid assembly of complex vascularized tissues are still scarce. Here we describe an injectable approach to generate vascularized dermal tissue, as an example application, from spheroids combining fibroblasts and endothelial progenitors (OEC) in a xeno-free (XF) setting. Short-term cultured spheroids (1 day) were selected over mature spheroids (7 days), as they showed significantly higher angiogenic sprouting potential. Embedding spheroids in fibrin was crucial for triggering cell migration into the external milieu, while providing a 3D framework for in-gel extra-spheroid morphogenesis. Migrating fibroblasts proliferated and produced endogenous ECM forming a dense tissue, while OEC self-assembled into stable capillaries with lumen and basal lamina. Massive in vitro interconnection between sprouts from neighbouring spheroids rapidly settled an intricate vascular plexus. Upon injection into the chorioallantoic membrane of chick embryos, fibrin-entrapped pre-vascularized XF spheroids developed into a macrotissue with evident host vessel infiltration. After only 4 days, perfused chimeric capillaries with human cells were present in proximal areas, showing fast and functional inosculation between host and donor vessels. This method for generating dense vascularized tissue from injectable building blocks is clinically relevant and potentially useful for a range of applications.

A Substrate-Mimicking Basement Membrane Drives the Organization of Human Mesenchymal Stromal Cells and Endothelial Cells Into Perivascular Niche-Like Structures.

Extracellular matrix-derived products (e.g. Matrigel) are widely used for in vitro cell cultures both as two-dimensional (2D) substrates and as three-dimensional (3D) encapsulation gels because of their ability to control cell phenotypes through biospecific cues. However, batch-to-batch variations, poor stability, cumbersome handling, and the relatively high costs strictly limit their use. Recently, a new substrate known as PhenoDrive-Y has been used as 2D coating of tissue culture plastic showing to direct the bone marrow mesenchymal stromal cells (MSCs) toward the formation of 3D spheroids. When organized into 3D spheroids, the MSCs expressed levels of pluripotency markers and of paracrine angiogenic activity higher than those of the MSCs adhering as fibroblast-like colonies on tissue culture plastic. The formation of the spheroids was attributed to the properties of this biomaterial that resemble the main features of the basement membrane by mimicking the mesh structure of collagen IV and by presenting the cells with orderly spaced laminin bioligands. In this study, PhenoDrive-Y was compared to Matrigel for its ability to drive the formation of perivascular stem cell niche-like structures in 2D co-culture conditions of human endothelial cells and adult bone marrow MSCs. Morphological analyses demonstrated that, when compared to Matrigel, PhenoDrive-Y led endothelial cells to sprout into a more consolidated tubular network and that the MSCs nestled as compact spheroids above the anastomotic areas of this network resemble more closely the histological features of the perivascular stem cell niche. A study of the expressions of relevant markers led to the identification of the pathways linking the PhenoDrive-Y biomimicking properties to the acquired histological features, demonstrating the enhanced levels of stemness, renewal potential, predisposition to migration, and paracrine activities of the MSCs.

A new cell-sized support for 3D cell cultures based on recombinant spider silk fibers.

It is now generally accepted that 2D cultures cannot accurately replicate the rich environment and complex tissue architecture that exists in vivo, and that classically cultured cells tend to lose their original function. Growth of spheroids as opposed to 2D cultures on plastic has now been hailed as an efficient method to produce quantities of high-quality cells for cancer research, drug discovery, neuroscience, and regenerative medicine. We have developed a new recombinant protein that mimics dragline spidersilk and that self-assembles into cell-sized coils. These have high thermal and shelf-life stability and can be readily sterilized and stored for an extended period of time. The fibers are flexible, elastic, and biocompatible and can serve as cell-sized scaffold for the formation of 3D cell spheroids. As a proof of concept, recombinant spidersilk was integrated as a scaffold in spheroids of three cell types: primary rat hepatocytes, human mesenchymal stem cells, and mouse L929 cells. The scaffolds significantly reduced spheroid shrinkage and unlike scaffold-free spheroids, spheroids did not disintegrate over the course of long-term culture. Cells in recombinant spidersilk spheroids showed increased viability, and the cell lines continued to proliferate for longer than control cultures without spidersilk. The spidersilk also supported biological functions. Recombinant spidersilk primary hepatocyte spheroids exhibited 2.7-fold higher levels of adenosine triphosphate (ATP) continued to express and secrete albumin and exhibited significantly higher basal and induced CYP3A activity for at least 6 weeks in culture, while control spheroids without fibers stopped producing albumin after 27 days and CPY3A activity was barely detectable after 44 days. These results indicate that recombinant spidersilk can serve as a useful tool for long-term cell culture of 3D cell spheroids and specifically that primary hepatocytes can remain active in culture for an extended period of time which could be of great use in toxicology testing.

Non-cell adhesive hexanoyl glycol chitosan hydrogels for stable and efficient formation of 3D cell spheroids with tunable size and density

Three-dimensional (3D) culture systems that provide a more physiologically similar environment than conventional two-dimensional (2D) cultures have been extensively developed. Previously we have provided a facile method for the formation of 3D spheroids using non-adhesive N-hexanoyl glycol chitosan (HGC) hydrogel-coated dishes, but with limitations such as low gel stability and weak mechanical properties. In this study, chemically crosslinked hydrogels were prepared by photocrosslinking of methacrylated HGCs (M-HGCs), and their spheroid-forming abilities were evaluated for long-term 3D cell cultures. The M-HGC hydrogels demonstrated not only enhanced gel stability, but also good spheroid-forming abilities. Furthermore, the M-HGC-coated dishes were effective in generating spheroids of larger size and higher cell density depending on the crosslinking density of the M-HGCs. These results indicate that our hydrogel-coated dish system could be widely applied as an effective technique to produce cell spheroids with customized sizes and densities that are essential for tissue engineering and drug screening.