Formation Of Multicellular Spheroids Research Articles

Abstract B095: Tenascin C promotes ovarian cancer stem cells interactions with the niche

Abstract Background: Effective treatment of ovarian cancer (OC) is greatly impacted by advanced, late-stage diagnosis which usually presents with intra-peritoneal metastases, chemoresistance and high recurrence. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids disseminated in the peritoneal fluid. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components that stiffen the tumor stroma and promote the aberrant activation of oncogenic processes, including survival, invasion, and stemness. Tenascin C (TNC) is one of the molecules secreted in the ECM and correlates with migration and metastatic progression in a variety of carcinomas. However, the functional role of TNC in spheroid formation and in the maintenance of the platinum resistant ovarian cancer stem phenotype has not been investigated yet. We tested the hypothesis that the increased expression of TNC and its secretion in the ECM compartment promotes survival of OCSCs, supports OC spheroid formation and tumor progression. Methods: We compared TNC expression in OC cells grown as spheroids and monolayers and in chemoresistant vs sensitive OC cells by realtime-PCR and Western Blot (WB). TNC blockade by siRNA knockdown (KD) was evaluated in combination with carboplatin on spheroid assay, colony formation, and by qPCR of stemness related gene expression. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the TNC promoter. Results: OC cells grown as spheroids showed a significant increase in TNC expression compared to monolayers. Our results also demonstrated an increase in TNC expression levels in platinum resistant OC cell lines when compared to the platinum sensitive counterpart. A comparison of normal adjacent ovarian epithelium and primary OC tumors on a multitissue array revealed that TNC expression coincided with a pro-tumorigenic phenotype (n=88, P<0.001), correlating with poor outcome. TNC-KD in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. Mechanistically, b-catenin-KD decreased TNC expression levels and β-catenin coimmunoprecipitated with the TNC promoter, suggesting that TNC is a direct β-catenin target. Additionally, OC spheroids treated with the Wnt ligand WNT-3A showed increased TNC expression compared to control untreated cells, as demonstrated by WB. Importantly, the enhanced expression of TNC was followed by its secretion in the ECM compartment, as observed via immunofluorescence imaging, implying a direct role of TNC in the ECM-rearrangement which is responsible for CSC maintenance and spheroid formation. Conclusions: TNC is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that strategies aimed at interfering with TNC expression and/or secretion along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Amber Rogers, Maya Shebeshi, Morganna Gordon, Rula Atwani, Virginie Lazar, Salvatore Condello. Tenascin C promotes ovarian cancer stem cells interactions with the niche [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B095.

Loss of phosphatase and tensin homolog (PTEN) increases Lysyl oxidase-like 2 (LOXL2) expression enhancing the growth of fallopian tube epithelial cells as three-dimensional spheroids

BackgroundHigh-grade serous ovarian cancer (HGSOC) accounts for 70–80% of all ovarian cancer-related deaths. Multiple studies have suggested that the fallopian tube epithelium (FTE) serves as the cell of origin of HGSOC. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss is sufficient to induce numerous tumorigenic changes in FTE, including increased migration, formation of multicellular tumor spheroids (MTSs), and ovarian colonization. In murine oviductal epithelial (MOE) cells (the equivalent of human FTE) loss of PTEN results in the upregulation of transcripts associated with the extracellular matrix, with a specific focus on the elevation of lysyl oxidase-like 2 (LOXL2). Although LOXL2 is known to drive transformation and invasion in solid tumors and is associated with a poor prognosis in ovarian cancer, its specific role in the tumorigenesis of ovarian cancer originating from FTE remains unclear. Therefore, we aim to investigate whether LOXL2 mediates tumorigenesis from the fallopian tube epithelium. MethodsIn this study, we utilized clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CAS9) technology to delete LOXL2 in PTEN-deficient MOE cells to understand its role in mediating the oncogenic effects of PTEN loss. In addition, CRISPR-CAS9 was used to delete LOXL2 in OVCAR8 ovarian cancer cells. We monitored the changes in tumorigenic properties, such as migration, invasion, and growth of three-dimensional (3D) spheroids, to assess whether the loss of LOXL2 resulted in any changes. ResultsWe found that a reduction in LOXL2 expression did not significantly change the migration or invasive capabilities of PTEN-depleted MOE or human ovarian cancer cells. However, we found that a reduction in LOXL2 expression resulted in a significant reduction in 3D MTS formation and survival in both lines. ConclusionsThese results reveal for the first time that PTEN loss in FTE cells increases LOXL2 expression through downregulation of Pax2, and LOXL2 deletion blocks 3D spheroid formation.

High throughput generating stable spheroids with tip-refill wafer.

Three-dimensional (3D) cell cultures have garnered significant attention in biomedical research due to their ability to mimic the in vivo cellular environment more accurately. The formation of 3D cell spheroids using hanging drops has emerged as a cost-effective and crucial method for generating uniformly-sized spheroids. This study aimed to validate the potential of a tip-refill wafer (TrW), a disposable laboratory item used to hold pipette tips, in facilitating 3D cell culture. The TrW allows for easy generation of hanging drops by pipetting the solution into the holes of the wafer. The mechanical stability of the hanging drops is ensured by the surface wettability and thickness of the TrW. Hanging drops containing 60-µL of solution remained securely attached to the TrW even when subjected to orbital shaking at 210rpm. The exceptional resistance to mechanical shaking enabled the use of inertial focusing to facilitate spheroid formation. This was demonstrated through live/dead cell staining, quantitative polymerase chain reaction (qPCR) analysis, and cytoskeleton staining, which revealed that horizontal orbiting at 60rpm for 15 min promoted cell aggregation and ultimately led to the formation of 3D spheroids. The spheroid harvest rate is 96.1%±3.5% across three TrWs, each containing 60 hanging drops. In addition to generating mono-culture 3D spheroids, the TrW-based hanging drop platform also enables the formation of multicellular spheroids, and on-demand pairing and fusion of spheroids. The TrW is a disposable item that does not require any fabrication or surface modification procedures, further enhancing its application potential in conventional biological laboratories.

Principles of Hanging Drop Method (Spheroid Formation) in Cell Culture.

A type of three-dimensional (3D) cell culture models which is simple and easy is hanging drop method. The hanging drop method emerges as a pivotal technique with diverse applications in cancer research and cell biology. This method facilitates the formation of multicellular spheroids, providing a unique environment for studying cell behavior dynamics. The hanging drop method's theoretical underpinning relies on gravity-enforced self-assembly, allowing for cost-effective, reproducible 3D cell cultures with controlled spheroid sizes. The advantages of this approach include its efficiency in producing cellular heterogeneity, particularly in non-adherent 3D cultures, and its ability to create hypoxic spheroids, making it a suitable model for studying cancer. Moreover, the hanging drop method has proven valuable in investigating various aspects such as tissue structure, signaling pathways, immune activation of cancer cells, and notably, cell proliferation. Researchers have utilized the hanging drop method to explore the dynamics of cell proliferation, studying the effects of mesenchymal stem cells (MSC) secretome on cancer cells. The method's application involves co-culturing different cell lines, assessing spheroid formations, and quantifying their sizes over time. These studies have unveiled intricate cell behavior dynamics, demonstrating how the MSC secretome influences cancer cell growth and viability within a three-dimensional co-culture paradigm.

Polyglycerol-Based Hydrogel as Versatile Support Matrix for 3D Multicellular Tumor Spheroid Formation.

Hydrogel-based artificial scaffolds are essential for advancing cell culture models from 2D to 3D, enabling a more realistic representation of physiological conditions. These hydrogels can be customized through crosslinking to mimic the extracellular matrix. While the impact of extracellular matrix scaffolds on cell behavior is widely acknowledged, mechanosensing has become a crucial factor in regulating various cellular functions. cancer cells' malignant properties depend on mechanical cues from their microenvironment, including factors like stiffness, shear stress, and pressure. Developing hydrogels capable of modulating stiffness holds great promise for better understanding cell behavior under distinct mechanical stress stimuli. In this study, we aim to 3D culture various cancer cell lines, including MCF-7, HT-29, HeLa, A549, BT-474, and SK-BR-3. We utilize a non-degradable hydrogel formed from alpha acrylate-functionalized dendritic polyglycerol (dPG) and thiol-functionalized 4-arm polyethylene glycol (PEG) via the thiol-Michael click reaction. Due to its high multivalent hydroxy groups and bioinert ether backbone, dPG polymer was an excellent alternative as a crosslinking hub and is highly compatible with living microorganisms. The rheological viscoelasticity of the hydrogels is tailored to achieve a mechanical stiffness of approximately 1 kPa, suitable for cell growth. Cancer cells are in situ encapsulated within these 3D network hydrogels and cultured with cell media. The grown tumor spheroids were characterized by fluorescence and confocal microscopies. The average grown size of all tumoroid types was ca. 150 µm after 25 days of incubation. Besides, the stability of a swollen gel remains constant after 2 months at physiological conditions, highlighting the nondegradable potential. The successful formation of multicellular tumor spheroids (MCTSs) for all cancer cell types demonstrates the versatility of our hydrogel platform in 3D cell growth.

Cellulose nanofibril matrix drives the dynamic formation of spheroids.

Multicellular spheroids, which mimic the natural organ counterparts, allow the prospect of drug screening and regenerative medicine. However, their application is hampered by low processing efficiency or limited scale. This study introduces an efficient method to drive rapid multicellular spheroid formation by a cellulose nanofibril matrix. This matrix enables the facilitated growth of spheroids (within 48 h) through multiple cell assembly into size-controllable aggregates with well-organized physiological microstructure. The efficiency, dimension, and conformation of the as-formed spheroids depend on the concentration of extracellular nanofibrils, the number of assembled cells, and the heterogeneity of cell types. The above strategy allows the robust formation mechanism of compacted tumoroids and hepatocyte spheroids.

Effects of simulated zero gravity on adhesion, cell structure, proliferation, and growth behavior, in glioblastoma multiforme

All life on Earth has evolved under the influence of continuous gravity, and methods have been developed to balance this influence with the biological evolution of organisms at the cellular and system levels. However, when exposed to zero gravity in space, the balance between cell structure and external forces is destroyed, resulting in changes at the cellular level (e.g., cell morphology, adhesion, viability, apoptosis, etc.), and understanding the molecular mechanism of cell response to zero gravity will help to cope with diseases that rely on mechanical response. Therefore, biological research in space and zero gravity is a unique step in developing the best anti-cancer treatments, which is a great challenge to humanity. In this study, multicellular glioma cancer cells from a brain tumor in a 72-year-old Iraqi patient were subjected to simulated zero gravity for 24 h, and the results showed that most of the cells lost their adhesion, which is considered to be the first step toward cell apoptosis. In addition to the formation of multicellular spheroids, the results also showed that the inhibition rate for cell death was 32% in comparison to the control cells. Moreover, the cells showed a clear change in their cellular morphology and growth behavior. These results give new hope for fighting cancer distinctively, and such a treatment method has no side effects in comparison to traditional chemical and radiological ones.

Abstract 5658: MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies

Abstract The generation of bispecific molecules, in which two target-specific moieties are engineered into a single entity, offers an attractive approach to optimize biological activity which may improve clinical outcomes. Among various formats of bispecific molecules, the heterodimeric Fc currently is a favorable scaffold for the design of bispecific antibodies because of its structural similarity to the natural antibody. However, development of molecules based on this concept is challenged by the presence of potential homodimer contamination, reduced yield, and stability loss relative to the natural Fc. Here, we report the development of the MG-Ig platform by grafting a single chain fragment variable (scFv) onto the hinge region of a full-size immunoglobulin with a different specificity to produce a trivalent IgG-like bispecific antibody that retains natural homodimer Fc. Whereas the MG-Ig platform is evolved from the Dock-and-Lock (DNL) technology which exploits the interaction between the anchoring domain (AD) of A kinase anchor proteins and the dimerization and docking domain (DDD) of regulatory subunit of cAMP-dependent protein kinase, the main distinctions include 1) the AD-fused scFv monomer is judiciously designed to graft onto the DDD dimer inserted into the hinge region of IgG, forming a bispecific antibody with three (1+2) binding arms and 2) all modular subunits are expressed in a single host cell and self-assemble to generate the bioactive conjugate in situ. To validate the method, bispecific T-cell engagers, designated 3scFv × hL0125C (CD3 × Trop2) and 3scFv × hACD20C (CD3 × CD20), which redirect T cells to Trop2 and CD20, respectively, have been produced with high yield and purity. Flow cytometry analysis demonstrated the bispecific binding of 3scFv × hL0125C to both CD3 on Jurkat and Trop2 on ME-180 with unaltered affinities. These T-cell redirecting bispecific antibodies mediated dose-dependent killing of target cancer cells when co-incubated with human PBMCs. Based on the 60-h viability assays using MTS, the IC50 of 3scFv × hL0125C was about 6 pM for MDA-MB-468, 13 pM for MDA-MB-231, 20 pM for BT-474, and 18 pM for HCT116. In 3D culture of HCT116 cells, the formation of multicellular tumor spheroids was impeded by 3scFv × hL0125C at 19.5 pM or higher, which is consistent with the cytotoxicity assays for tumor cells grown in monolayer. In addition, 3scFv × hL0125C was stable in human plasma for 7 days at 37°C, retaining bispecific integrity and binding activity. These data are promising and merit further development of MG-Ig bispecific antibodies for preclinical and clinical studies. Citation Format: Donglin Liu, Li Zeng, Berenice E. Liu, Chien-Hsing Chang. MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5658.

Generation of multicellular tumor spheroids via 3D cell culture utilizing a hydrogel comprising chitosan and allylthiourea

The chitosan–allyl thiourea hydrogel demonstrates remarkable cell viability, enabling the formation of multicellular tumor spheroids. Moreover, it exhibits the ability to self-heal within a mere hour at room temperature.

Protective effect of TPP-Niacin on microgravity-induced oxidative stress and mitochondrial dysfunction of retinal epithelial cells

Adverse effects of spaceflight on the human body are attritubuted to microgravity and space radiation. One of the most sensitive organs affected by them is the eye, particularly the retina. The conditions that astronauts suffer, such as visual acuity, is collectively called a spaceflight-associated neuro-ocular syndrome (SANS); however, the underlying molecular mechanism of the microgravity-induced ocular pathogenesis is not clearly understood. The current study explored how microgravity affects the retina function in ARPE19 cells in vitro under time-averaged simulated microgravity (μG) generated by clinostat. We found multicellular spheroid (MCS) formation and a significantly decreased cell migration potency under μG conditions compared to 1G in ARPE19 cells. We also observed that μG increases intracellular reactive oxygen species (ROS) and causes mitochondrial dysfunction in ARPE19 cells. Subsequently, we showed that μG activates autophagic pathways and ciliogenesis. Furthermore, we demonstrated that mitophagy activation is triggered via the mTOR-ULK1-BNIP3 signaling axis. Finally, we validated the effectiveness of TPP-Niacin in mitigating μG-induced oxidative stress and mitochondrial dysfunction in vitro, which provides the first experimental evidence for TPP-Niacin as a potential therapeutic agent to ameliorate the cellular phenotypes caused by μG in ARPE19 cells. Further investigations are, however, required to determine its physiological functions and biological efficacies in primary human retinal cells, in vivo models, and target identification.

Knock-out of 5-lipoxygenase in overexpressing tumor cells—consequences on gene expression and cellular function

5-Lipoxygenase (5-LO), the central enzyme in the biosynthesis of leukotrienes, is frequently expressed in human solid malignancies even though the enzyme is not present in the corresponding healthy tissues. There is little knowledge on the consequences of this expression for the tumor cells regarding gene expression and cellular function. We established a knockout (KO) of 5-LO in different cancer cell lines (HCT-116, HT-29, U-2 OS) and studied the consequences on global gene expression using next generation sequencing. Furthermore, cell viability, proliferation, migration and multicellular tumor spheroid (MCTS) formation were studied in these cells. Our results show that 5-LO influences the gene expression and cancer cell function in a cell type-dependent manner. The enzyme affected genes involved in cell adhesion, extracellular matrix formation, G protein signaling and cytoskeleton organization. Furthermore, absence of 5-LO elevated TGFβ2 expression in HCT-116 cells while MCP-1, fractalkine and platelet-derived growth factor expression was attenuated in U-2 OS cells suggesting that tumor cell-derived 5-LO shapes the tumor microenvironment. In line with the gene expression data, KO of 5-LO had an impact on cell proliferation, motility and MCTS formation. Interestingly, pharmacological inhibition of 5-LO only partly mimicked the KO suggesting that also noncanonical functions are involved.

Formation of Lymphoma Hybrid Spheroids and Drug Testing in Real Time with the Use of Fluorescence Optical Tweezers.

Interactions between stromal and lymphoma cells in the bone marrow are closely related to drug resistance and therapy failure. Physiologically relevant pre-clinical three-dimensional (3D) models recapitulating lymphoma microenvironmental complexity do not currently exist. In this study, we proposed a scheme for optically controlled hybrid lymphoma spheroid formation with the use of optical tweezers (OT). Following the preparation of stromal spheroids using agarose hydrogel, two aggressive non-Hodgkin lymphoma B-cell lines, Ri-1 (DLBCL) and Raji (Burkitt lymphoma), were used to conduct multi-cellular spheroid formation driven by in-house-developed fluorescence optical tweezers. Importantly, the newly formed hybrid spheroid preserved the 3D architecture for the next 24 h. Our model was successfully used for the evaluation of the influence of the anticancer agents doxorubicin (DOX), ibrutinib (IBR), and AMD3100 (plerixafor) on the adhesive properties of lymphoma cells. Importantly, our study revealed that a co-treatment of DOX and IBR with AMD3100 affects the adhesion of B-NHL lymphoma cells.

Abstract 906: Targeting CD166 to overcome platinum resistance in ovarian cancer

Abstract Background: Ovarian cancer (OC) is the most lethal gynecological cancer, characterized by chemoresistance and fatal tumor recurrence after primary treatment. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids by OC cells disseminated in the peritoneal fluid. Within a spheroid, OC cells undergo an epithelial-to-mesenchymal transition (EMT), which involves changes in the expression of cell adhesion molecules (CAMs) and acquisition of cancer stem cell (CSC) characteristics, therefore protecting OC cells during chemotherapy. Cluster of differentiation 166 (CD166) is one of the CAMs involved in tumor progression and its modulation impacts cell-cell adhesion, leading to impaired ability of tumor cells to metastasize. However, limited number of studies are available on the functional role of CD166 in the initiation, progression, and chemoresistance of OC. Our goal was to investigate the contribution of CD166 in supporting chemoresistance and the tumorigenic OCSC phenotype, thus proposing this molecule as a potential new therapeutic target in OC. Methods: We compared CD166 expression in primary OC cells grown as spheroids and monolayers and in chemoresistant versus sensitive OC cell lines by q-PCR and Western Blot (WB). CD166 blockade by shRNA knockdown (KD) or anti-CD166 inhibitory antibody (clone AZN-L50) was evaluated on spheroid assay and colony formation. Combinatorial carboplatin and AZN-L50 treatment was assessed on spheroid assays and by WB of pro-apoptotic proteins. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the CD166 promoter. Results: OC cells grown as spheroids showed a significant increase in CD166 expression compared to monolayers. Our results also demonstrated an increase in CD166 expression levels in chemoresistant OC cell lines when compared to the chemosensitive counterpart. A comparison of normal adjacent ovarian epithelium, localized, and metastatic OC tissues on a multitissue array revealed that CD166 expression coincided with an advanced promigratory phenotype (n=88, P<0.001), correlating with poor outcome. CD166 blockade by either shRNA-mediated KD or AZN-L50 treatment in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. The combination of carboplatin and CD166 blockade increased cleaved-PARP and -caspase-3 levels compared to single agent alone, indicating sustained DNA damage. Mechanistically, β-catenin-KD decreased CD166 expression levels and β-catenin coimmunoprecipitated with the CD166 promoter, suggesting that CD166 is a direct β-catenin target. Conclusions: CD166 is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that inhibition of CD166 along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Rula Atwani, Mayuri Prasad, George E Sandusky, Salvatore Condello. Targeting CD166 to overcome platinum resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 906.

Self‐Organization Formation of Multicellular Spheroids Mediated by Mechanically Tunable Hydrogel Platform: Toward Revealing the Synergy of Chemo‐ and Noninvasive Photothermal Therapy against Colon Microtumor

Front Cover: In this artwork, a novel method for inducing multicellular spheroids (MCSs) formation is proposed based on hydrogel with tunable microenvironmental properties (the lower half of the cover design). The synergism of chemotherapeutic drugs (the bubbles around the MCSs) and noninvasive photothermal therapy based on graphene device (the upper half of the cover design) is furtherly verified in our MCSs model. This is reported by Ting-ting Yu, Ke Hu, and co-workers in article number 2100498.

Three-Dimensional Growth of Prostate Cancer Cells Exposed to Simulated Microgravity.

Prostate cancer metastasis has an enormous impact on the mortality of cancer patients. Factors involved in cancer progression and metastasis are known to be key players in microgravity (µg)-driven three-dimensional (3D) cancer spheroid formation. We investigated PC-3 prostate cancer cells for 30 min, 2, 4 and 24 h on the random positioning machine (RPM), a device simulating µg on Earth. After a 24 h RPM-exposure, the cells could be divided into two groups: one grew as 3D multicellular spheroids (MCS), the other one as adherent monolayer (AD). No signs of apoptosis were visible. Among others, we focused on cytokines involved in the events of metastasis and MCS formation. After 24 h of exposure, in the MCS group we measured an increase in ACTB, MSN, COL1A1, LAMA3, FN1, TIMP1, FLT1, EGFR1, IL1A, IL6, CXCL8, and HIF1A mRNA expression, and in the AD group an elevation of LAMA3, COL1A1, FN1, MMP9, VEGFA, IL6, and CXCL8 mRNAs compared to samples subjected to 1 g conditions. Significant downregulations in AD cells were detected in the mRNA levels of TUBB, KRT8, IL1B, IL7, PIK3CB, AKT1 and MTOR after 24 h. The release of collagen-1α1 and fibronectin protein in the supernatant was decreased, whereas the secretion of IL-6 was elevated in 24 h RPM samples. The secretion of IL-1α, IL-1β, IL-7, IL-2, IL-8, IL-17, TNF-α, laminin, MMP-2, TIMP-1, osteopontin and EGF was not significantly altered after 24 h compared to 1 g conditions. The release of soluble factors was significantly reduced after 2 h (IL-1α, IL-2, IL-7, IL-8, IL-17, TNF-α, collagen-1α1, MMP-2, osteopontin) and elevated after 4 h (IL-1β, IL-2, IL-6, IL-7, IL-8, TNF-α, laminin) in RPM samples. Taken together, simulated µg induced 3D growth of PC-3 cancer cells combined with a differential expression of the cytokines IL-1α, IL-1β, IL-6 and IL-8, supporting their involvement in growth and progression of prostate cancer cells.

3D bioprinted tumor model with extracellular matrix enhanced bioinks for nanoparticle evaluation

The traditional evaluation of nanoparticles (NPs) is mainly based on 2D cell culture and animal models. However, these models are difficult to accurately represent human tumor microenvironment (TME) and fail to systematically study the complex transportation of NPs, thus limiting the translation of nano-drug formulations to clinical studies. This study reports a tumor model fabricated via 3D bioprinting with adipose decellularized extracellular matrix (ECM) enhanced hybrid bioink. Compared with 2D cultured cells, the 3D printed tumor models with multicellular spheroids formation are closer to real tumor in protein, gene expression and tumorigenicity both in vitro and in vivo. Two characteristics of TME, ECM remodeling and epithelial-mesenchymal transition, are tracked simultaneously under 3D conditions. Furthermore, the cellular uptake efficiency of two different NPs is significantly lower in the printed 3D tumor model than the 2D individual cells, and higher drug resistance is observed in 3D group, which suggest the ECM barrier of tumor can significantly affect the permeability of NPs. These results suggest that this 3D printed tumor model is capable of mimicking the multiple TME, potentially providing a more accurate platform for the design and development of NPs before moving into animal and clinical trials.

Self-Organization Formation of Multicellular Spheroids Mediated by Mechanically Tunable Hydrogel Platform: Toward Revealing the Synergy of Chemo- and Noninvasive Photothermal Therapy against Colon Microtumor.

Three-dimensional (3D) tumor cell culture offers a more tissue-recapitulating model in cancer treatment evaluation. However, conventional models based on cell-substrate adhesion deprivation are still of insufficient real tumor mimic. In this work, a novel method is proposed for inducing multicellular spheroids (MCSs) formation based on hydrogel with tunable microenvironmental properties. Colon tumor cells DLD1 cultured on hydrogel substrate with proper physical stimulation form MCSs via self-organization. Chemotherapy based on clinical drug and far-infrared photothermal therapy is evaluated with DLD1 MCSs obtained by this method. The synergism of chemotherapy and noninvasive photothermal therapy based on graphene device is further verified in MCSs model and it is believed this method holds potential in in vitro anti-tumor strategies evaluation for precision medicine.

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.

Cancer Studies under Space Conditions: Finding Answers Abroad.

In this review article, we discuss the current state of knowledge in cancer research under real and simulated microgravity conditions and point out further research directions in this field. Outer space is an extremely hostile environment for human life, with radiation, microgravity, and vacuum posing significant hazards. Although the risk for cancer in astronauts is not clear, microgravity plays a thought-provoking role in the carcinogenesis of normal and cancer cells, causing such effects as multicellular spheroid formation, cytoskeleton rearrangement, alteration of gene expression and protein synthesis, and apoptosis. Furthermore, deleterious effects of radiation on cells seem to be accentuated under microgravity. Ground-based facilities have been used to study microgravity effects in addition to laborious experiments during parabolic flights or on space stations. Some potential ‘gravisensors’ have already been detected, and further identification of these mechanisms of mechanosensitivity could open up ways for therapeutic influence on cancer growth and apoptosis. These novel findings may help to find new effective cancer treatments and to provide health protection for humans on future long-term spaceflights and exploration of outer space.

Polyanion-induced, microfluidic engineering of fragmented collagen microfibers for reconstituting extracellular environments of 3D hepatocyte culture

Artificial biological scaffolds made of extracellular matrix (ECM) components, such as type I collagen, provide ideal physicochemical cues to various cell culture platforms. However, it remains a challenge to fabricate micrometer-sized ECM materials with precisely controlled morphologies that could reconstitute the 3-dimensional (3D) microenvironments surrounding cells. In the present study, we proposed a unique process to fabricate fragmented collagen microfibers using a microfluidic laminar-flow system. The continuous flow of an acidic collagen solution was neutralized to generate solid fibers, which were subsequently fragmented by applying a gentle shear stress in a polyanion-containing phosphate buffer. The morphology of the fiber fragment was controllable in a wide range by changing the type and/or concentration of the polyanion and by tuning the applied shear stress. The biological benefits of the fragmented fibers were investigated through the formation of multicellular spheroids composed of primary rat hepatocytes and microfibers on non-cell-adhesive micro-vessels. The microfibers enhanced the survival and functions of the hepatocytes and reproduced proper cell polarity, because the fibers facilitated the formation of cell−cell and cell−matrix interactions while modulating the close packing of cells. These results clearly indicated that the microengineered fragmented collagen fibers have great potential to reconstitute extracellular microenvironments for hepatocytes in 3D culture, which will be of significant benefit for cell-based drug testing and bottom-up tissue engineering.