In vitro 2D and 3D cancer models to evaluate compounds that modulate macrophage polarization.

In vitro cancer models that can identify novel immunomodulating compounds are essential. Using a 3D multicellular tumor spheroid (MCTS) model comprising cancer cells, fibroblasts, and macrophages, we tested tumor-associated macrophage (TAM)-inhibiting compounds (CCL2 Ab, CSF1R inhibitor, CSF1R Ab) and TAM-reprograming compounds (poly I:C, CD40 Ab, CD40 ligand) for their effects on monocyte infiltration and polarization in tumor spheroids. For characterization of macrophage polarization, we measured the expression of CD206, CD163, CD86, MHC II, CD40, and CD14 and measured 43 soluble factors in the 3D MCTS cultures. 2D macrophage models were evaluated for comparison. A CSF1R inhibitor prevented infiltration of monocytes into pancreatic cancer spheroids, and macrophages treated with the inhibitor showed decreased expression of M2 markers. Treatment with a CD40 ligand and poly I:C induced M1 macrophage polarization in our models. We propose that these models can be used to improve the drug screening process of anti-cancer immunotherapies targeting macrophages.

A Novel 3D Culture Scaffold to Shorten Development Time for Multicellular Tumor Spheroids.

Multicellular tumor spheroids and tumoroids are considered ideal in vitro models that reflect the features of the tumor microenvironment. Biomimetic components resembling the extracellular matrix form scaffolds to provide structure to 3-dimensional (3D) culture systems, supporting the growth of both spheroids and tumoroids. Although Matrigel has long been used to support 3D culture systems, batch variations, component complexity, and the use of components derived from tumors are complicating factors. To address these issues, we developed the ACD 3D culture system to provide better control and consistency. We evaluated spheroid and tumoroid formation using the ACD 3D culture system, including the assessment of cell viability and cancer marker expression. Under ACD 3D culture conditions, spheroids derived from cancer cell lines exhibited cancer stem cell characteristics, including a sphere-forming size and the expression of stem cell marker genes. The ACD 3D culture system was also able to support patient-derived primary cells and organoid cell cultures, displaying adequate cell growth, appropriate morphology, and resistance to oxaliplatin treatment. These spheroids could also be used for drug screening purposes. In conclusion, the ACD 3D culture system represents an efficient tool for basic cancer research and therapeutic development.

In Vitro Evaluation of Lipopolyplexes for Gene Transfection: Comparing 2D, 3D and Microdroplet-Enabled Cell Culture.

Complexes combining nucleic acids with lipids and polymers (lipopolyplexes) show great promise for gene therapy since they enable compositional, physical and functional versatility to be optimized for therapeutic efficiency. When developing lipopolyplexes for gene delivery, one of the first evaluations performed is an in vitro transfection efficiency experiment. Many different in vitro models can be used, and the effect of the model on the experiment outcome has not been thoroughly studied. The objective of this work was to compare the insights obtained from three different in vitro models, as well as the potential limitations associated with each of them. We have prepared a series of lipopolyplex formulations with three different cationic polymers (poly-l-lysine, bioreducible poly-l-lysine and polyethyleneimine), and assessed their in vitro biological performance in 2D monolayer cell culture, 3D spheroid culture and microdroplet-based single-cell culture. Lipopolyplexes from different polymers presented varying degrees of transfection efficiency in all models. The best-performing formulation in 2D culture was the polyethyleneimine lipopolyplex, while lipoplexes prepared with bioreducible poly-l-lysine were the only ones achieving any transfection in microdroplet-enabled cell culture. None of the prepared formulations achieved significant gene transfection in 3D culture. All of the prepared formulations were well tolerated by cells in 2D culture, while at least one formulation (poly-l-lysine polyplex) delayed 3D spheroid growth. These results highlight the need for selecting the appropriate in vitro model depending on the intended application.

In vitro lung cancer multicellular tumor spheroid formation using a microfluidic device.

The purpose of this study was to demonstrate self-organizing in vitro multicellular tumor spheroid (MCTS) formation in a microfluidic system and to observe the behavior of MCTSs under controlled microenvironment. The employed microfluidic system was designed for simple and effective formation of MCTSs by generating nutrient and oxygen gradients. The MCTSs were composed of cancer cells, vascular endothelial cells, and type I collagen matrix to mimic the in vivo tumor microenvironment (TME). Cell culture medium was perfused to the microfluidic device loaded with MCTSs by a passive fluidic pump at a constant flow rate. The dose response to an MMPs inhibitor was investigated to demonstrate the effects of biochemical substances. The result of long-term stability of MCTSs revealed that continuous perfusion of cell culture medium is one of the major factors for the successful MCTS formation. A continuous flow of cell culture medium in the in vitro TME greatly affected both the proliferation of cancer cells in the micro-wells and the sustainability of the endothelial cell-layer integrity in the lumen of microfluidic channels. Addition of MMP inhibitor to the cell culture medium improved the stability of the collagen matrix by preventing the detachment and shrinkage of the collagen matrix surrounding the MCTSs. In summary, the present constant flow assisted microfluidic system is highly advantageous for long-term observation of the MCTS generation, tumorous tissue formation process and drug responses. MCTS formation in a microfluidic system may serve as a potent tool for studying drug screening, tumorigenesis and metastasis.

miRNA expression profile in multicellular breast cancer spheroids.

Multicellular Tumor Spheroids develop a heterogeneous micromilieu and different cell populations, thereby constituting a cancer model with intermediate characteristics between in vitro bi-dimensional cultures and in vivo tumors. Multicellular Tumor Spheroids also acquire tumor aggressiveness features due to transcription modulation of coding and non-coding RNA. Utilizing microarray analyses, we evaluated the microRNAs expression profile in MCF-7 breast cancer cells cultured as Multicellular Tumor Spheroids. The expression data was used to predict associated cellular and molecular functions using different software tools. The biological importance of two dysregulated miRNAs (miR-221-3p and miR-187) was studied by functional assays. Finally, the clinical relevance of these dysregulated miRNAs was explored using previously reported data. Thirty-three dysregulated microRNAs were found in MCF-7 Multicellular Tumor Spheroids. miRNA expression changes were closely linked with growth, proliferation, and cell development. miRNA-221-3p and miR-187 were implicated in the acquisition of migration/invasion capacities, sensitivity to the deprivation of growth factors, cell cycle phase regulation, and cell death. A panel of 5 miRNAs, including miR-187, showed a good predictive value in discriminating between low and high-risk groups of breast cancer.

Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer.

The development of three-dimensional (3D) multicellular tumor spheroid models for cancer drug discovery research has increased in the recent years. The use of 3D tumor spheroid models may be more representative of the complex in vivo tumor microenvironments in comparison to two-dimensional (2D) assays. Currently, viability of 3D multicellular tumor spheroids has been commonly measured on standard plate-readers using metabolic reagents such as CellTiter-Glo® for end point analysis. Alternatively, high content image cytometers have been used to measure drug effects on spheroid size and viability. Previously, we have demonstrated a novel end point drug screening method for 3D multicellular tumor spheroids using the Celigo Image Cytometer. To better characterize the cancer drug effects, it is important to also measure the kinetic cytotoxic and apoptotic effects on 3D multicellular tumor spheroids. In this work, we demonstrate the use of PI and caspase 3/7 stains to measure viability and apoptosis for 3D multicellular tumor spheroids in real-time. The method was first validated by staining different types of tumor spheroids with PI and caspase 3/7 and monitoring the fluorescent intensities for 16 and 21 days. Next, PI-stained and nonstained control tumor spheroids were digested into single cell suspension to directly measure viability in a 2D assay to determine the potential toxicity of PI. Finally, extensive data analysis was performed on correlating the time-dependent PI and caspase 3/7 fluorescent intensities to the spheroid size and necrotic core formation to determine an optimal starting time point for cancer drug testing. The ability to measure real-time viability and apoptosis is highly important for developing a proper 3D model for screening tumor spheroids, which can allow researchers to determine time-dependent drug effects that usually are not captured by end point assays. This would improve the current tumor spheroid analysis method to potentially better identify more qualified cancer drug candidates for drug discovery research. © 2017 International Society for Advancement of Cytometry.

Engineering Mesoscopic 3D Tumor Models with a Self-Organizing Vascularized Matrix.

Advanced in vitro systems such as multicellular spheroids and lab-on-a-chip devices have been developed, but often fall short in reproducing the tissue scale and self-organization of human diseases. A bioprinted artificial tumor model is introduced with endothelial and stromal cells self-organizing into perfusable and functional vascular structures. This model uses 3D hydrogel matrices to embed multicellular tumor spheroids, allowing them to grow to mesoscopic scales and to interact with endothelial cells. It is shown that angiogenic multicellular tumor spheroids promote the growth of a vascular network, which in turn further enhances the growth of cocultivated tumor spheroids. The self-developed vascular structure infiltrates the tumor spheroids, forms functional connections with the bioprinted endothelium, and can be perfused by erythrocytes and polystyrene microspheres. Moreover, cancer cells migrate spontaneously from the tumor spheroid through the self-assembled vascular network into the fluid flow. Additionally, tumor type specific characteristics of desmoplasia, angiogenesis, and metastatic propensity are preserved between patient-derived samples and tumors derived from this same material growing in the bioreactors. Overall, this modular approach opens up new avenues for studying tumor pathophysiology and cellular interactions in vitro, providing a platform for advanced drug testing while reducing the need for in vivo experimentation.

Overexpression of SLC2A1, ALDOC, and PFKFB4 in the glycolysis pathway drives strong drug resistance in 3D HeLa tumor cell spheroids.

The 3D multicellular tumor spheroid (MTS) model exhibits enhanced fidelity in replicating the tumor microenvironment and demonstrates exceptional resistance to clinical drugs compared to the 2D monolayer model. In this study, we used multiomics (transcriptome, proteomics, and metabolomics) tools to explore the molecular mechanisms and metabolic differences of the two culture models. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways revealed that the differentially expressed genes between the two culture models were mainly enriched in cellular components and biological processes associated with extracellular matrix, extracellular structural organization, and mitochondrial function. An integrated analysis of three omics data revealed 11 possible drug resistance targets. Among these targets, seven genes, AKR1B1, ALDOC, GFPT2, GYS1, LAMB2, PFKFB4, and SLC2A1, exhibited significant upregulation. Conversely, four genes, COA7, DLD, IFNGR1, and QRSL1, were significantly downregulated. Clinical prognostic analysis using the TCGA survival database indicated that high-expression groups of SLC2A1, ALDOC, and PFKFB4 exhibited a significant negative correlation with patient survival. We further validated their involvement in chemotherapy drug resistance, indicating their potential significance in improving prognosis and chemotherapy outcomes. These results provide valuable insights into potential therapeutic targets that can potentially enhance treatment efficacy and patient outcomes.

Dual Nanozyme-Driven PtSn Bimetallic Nanoclusters for Metal-Enhanced Tumor Photothermal and Catalytic Therapy.

Specific generation of reactive oxygen species (ROS) within tumors in situ catalyzed by nanozymes is a promising strategy for cancer therapeutics. However, it remains a significant challenge to fabricate highly efficient nanozymes acting in the tumor microenvironment. Herein, we develop a bimetallic nanozyme (Pt50Sn50) with the photothermal enhancement of dual enzymatic activities for tumor catalytic therapy. The structures and activities of PtSn bimetallic nanoclusters (BNCs) with different Sn content are explored and evaluated systematically. Experimental comparisons show that the Pt50Sn50 BNCs exhibit the highest activities among all those investigated, including enzymatic activity and photothermal property, due to the generation of SnO2-x with oxygen vacancy (Ovac) sites on the surface of Pt50Sn50 BNCs. Specifically, the Pt50Sn50 BNCs exhibit photothermal-enhanced peroxidase-like and catalase-like activities, as well as a significantly enhanced anticancer efficacy in both multicellular tumor spheroids and in vivo experiments. Due to the high X-ray attenuation coefficient and excellent light absorption property, the Pt50Sn50 BNCs also show dual-mode imaging capacity of computed tomography and photoacoustic imaging, which could achieve in vivo real-time monitoring of the therapeutic process. Therefore, this work will advance the development of noble-metal nanozymes with optimal composition for efficient tumor catalytic therapy.

Tumor Spheroids as Model to Design Acoustically Mediated Drug Therapies: A Review.

Tumor spheroids as well as multicellular tumor spheroids (MCTSs) are promising 3D in vitro tumor models for drug screening, drug design, drug targeting, drug toxicity, and validation of drug delivery methods. These models partly reflect the tridimensional architecture of tumors, their heterogeneity and their microenvironment, which can alter the intratumoral biodistribution, pharmacokinetics, and pharmacodynamics of drugs. The present review first focuses on current spheroid formation methods and then on in vitro investigations exploiting spheroids and MCTS for designing and validating acoustically mediated drug therapies. We discuss the limitations of the current studies and future perspectives. Various spheroid formation methods enable the easy and reproducible generation of spheroids and MCTSs. The development and assessment of acoustically mediated drug therapies have been mainly demonstrated in spheroids made up of tumor cells only. Despite the promising results obtained with these spheroids, the successful evaluation of these therapies will need to be addressed in more relevant 3D vascular MCTS models using MCTS-on-chip platforms. These MTCSs will be generated from patient-derived cancer cells and nontumor cells, such as fibroblasts, adipocytes, and immune cells.

The interface stiffness and topographic feature dictate interfacial invasiveness of cancer spheroids.

During cancer metastasis, tumor cells likely navigate, in a collective manner, discrete tissue spaces comprising inherently heterogeneous extracellular matrix microstructures where interfaces may be frequently encountered. Studies have shown that cell migration modes can be determined by adaptation to mechanical/topographic cues from interfacial microenvironments. However, less attention has been paid to exploring the impact of interfacial mechnochemical attributes on invasive and metastatic behaviors of tumor aggregates. Here, we excogitated a collagen matrix-solid substrate interface platform to investigate the afore-stated interesting issue. Our data revealed that stiffer interfaces stimulated spheroid outgrowth by motivating detachment of single cells and boosting their motility and velocity. However, stronger interfacial adhesive strength between matrix and substrate led to the opposite outcomes. Besides, this interfacial parameter also affected the morphological switch between migration modes of the detached cells and their directionality. Mechanistically, myosin II-mediated cell contraction, compared to matrix metalloproteinases-driven collagen degradation, was shown to play a more crucial role in the invasive outgrowth of tumor spheroids in interfacial microenvironments. Thus, our findings highlight the importance of heterogeneous interfaces in addressing and combating cancer metastasis.

Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines.

In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for cancer drug discovery experience 20%-21% O2 levels (normoxic) which are 4-fold higher than O2 levels in normal tissues and ≥10-fold higher than in the hypoxic regions of solid tumors. The oxygen electrodes, exogenous bio-reductive markers, and increased expression of endogenous hypoxia-regulated proteins like HIF-1α generally used to mark hypoxic regions in solid tumors are impractical in large sample numbers and longitudinal studies. We used a novel homogeneous live-cell permeant HypoxiTRAK™ (HPTK) molecular probe compatible with high content imaging detection, analysis, and throughput to identify and quantify hypoxia levels in live HNC multicellular tumor spheroid (MCTS) cultures over time. Accumulation of fluorescence HPTK metabolite in live normoxic HNC MCTS cultures correlated with hypoxia detection by both pimonidazole and HIF-1α staining. In HNC MCTSs, hypoxic cytotoxicity ratios for the hypoxia activated prodrugs (HAP) evofosfamide and tirapazamine were much smaller than have been reported for uniformly hypoxic 2D monolayers in gas chambers, and many viable cells remained after HAP exposure. Cells in solid tumors and MCTSs experience three distinct O2 microenvironments dictated by their distances from blood vessels or MCTS surfaces, respectively; oxic, hypoxic, or intermediate levels of hypoxia. These studies support the application of more physiologically relevant in vitro 3D models that recapitulate the heterogeneous microenvironments of solid tumors for preclinical cancer drug discovery.

Three-dimensional in vitro culture models in oncology research.

Cancer is a multifactorial disease that is responsible for 10 million deaths per year. The intra- and inter-heterogeneity of malignant tumors make it difficult to develop single targeted approaches. Similarly, their diversity requires various models to investigate the mechanisms involved in cancer initiation, progression, drug resistance and recurrence. Of the in vitro cell-based models, monolayer adherent (also known as 2D culture) cell cultures have been used for the longest time. However, it appears that they are often less appropriate than the three-dimensional (3D) cell culture approach for mimicking the biological behavior of tumor cells, in particular the mechanisms leading to therapeutic escape and drug resistance. Multicellular tumor spheroids are widely used to study cancers in 3D, and can be generated by a multiplicity of techniques, such as liquid-based and scaffold-based 3D cultures, microfluidics and bioprinting. Organoids are more complex 3D models than multicellular tumor spheroids because they are generated from stem cells isolated from patients and are considered as powerful tools to reproduce the disease development in vitro. The present review provides an overview of the various 3D culture models that have been set up to study cancer development and drug response. The advantages of 3D models compared to 2D cell cultures, the limitations, and the fields of application of these models and their techniques of production are also discussed.

TIMP-1-expressing breast tumor spheroids for the evaluation of drug penetration and efficacy.

Abundance of stromal cells and extracellular matrix (ECM) is observed in breast cancer, acting as a barrier for drug penetration and presenting a key issue for developing efficient therapeutics. In this study, we aimed to develop a three-dimensional (3D) multicellular tumor model comprising cancer and stromal cells that could effectively mimic the drug resistance properties of breast cancer. Three different types of spheroid models were designed by co-culturing breast cancer cells (MDA-MB-231) with three different types of stromal cells: human adipose-derived stromal cells (hASCs), human bone marrow stromal cells, or human dermal fibroblasts. Compared with other models, in the hASC co-culture model, tissue inhibitor of metalloproteinases-1 (TIMP-1) was highly expressed and the activity of matrix metalloproteinases was decreased, resulting in a higher ECM deposition on the spheroid surfaces. This spheroid model showed less drug penetration and treatment efficacy than the other models. TIMP-1 silencing in hASCs reduced ECM protein expression and increased drug penetration and vulnerability. A quantitative structure-activity relationship study using multiple linear regression drew linear relationships between the chemical properties of drugs and experimentally determined permeability values. Drugs that did not match the drug-likeness rules exhibited lower permeability in the 3D tumor model. Taken together, our findings indicate that this 3D multicellular tumor model may be used as a reliable platform for efficiently screening therapeutics agents for solid tumors.

Prediction and validation of avascular tumor growth pattern in different metabolic conditions using in silico and in vitro models.

Objectives: In recent years, scientists have taken many efforts for in vitro and in silico modeling of cancerous tumors. In fact, three-dimensional (3D) cultures of multicellular tumor spheroids (MCTSs) are good validators for computational results. The goal of this study is to simulate the 3D early growth of avascular tumors using MCTSs and to compare the in vitro models with the results and predictions of a specific computational modeling framework. Using these two types of models, the importance of metabolic condition on tumor growth behavior and necrosis could be predicted. Materials and methods: We took advantage of a previously developed computational model of tumor growth (constructed by integrating a generic metabolic network model of cancer cells with a multiscale agent-based framework). Among the computational predictions is the importance of glucose accessibility on tumor growth behavior. To study the effect of glucose concentration experimentally, MCTSs were grown in high and low glucose culture media. After that, tumor growth pattern was analyzed by MTT assay, cell counting and propidium iodide (PI) staining. Results: We obviously observed that the rate of necrosis increases and the rate of tumor growth and cell activity decreases as the glucose availability reduces, which is in line with the computational model prediction.

Pleiotropic effects of probenecid on three-dimensional cultures of prostate cancer cells.

AIMS: Chemoresistance remains a persistent challenge in advanced prostate cancer therapy. Probenecid reportedly inhibits multiple drug-efflux transporters; hence, it can be employed as a potential sensitizer for chemotherapy. In the present study, we evaluated the effects of probenecid on three-dimensional (3D)-cultures of prostate cancer cells. MAIN METHODS: Prostate cancer cell lines, 22Rv1 and PC-3 were cultured as multicellular tumor spheroids. The effects of probenecid were evaluated using the MTT assay for viability, microscopy for spheroid size, and soft agar colony formation assay for anchorage-independent growth. KEY FINDINGS: The 3D-cultured 22Rv1 cells were less sensitive to cisplatin and doxorubicin than two-dimensional (2D) cell culture. Co-administration of probenecid at a low (100 or 300 µM), but not high (500 µM), concentration increased the sensitivity to cisplatin or doxorubicin in 22Rv1 spheroids. Probenecid increased the expression of ABCG2, a multidrug resistance transporter, in a dose-dependent manner. Furthermore, treatment with probenecid alone reduced the growth of 22Rv1 spheroids. Conversely, probenecid inhibited spheroid compaction rather than growth inhibition in 3D-cultured PC-3 cells. Moreover, probenecid inhibited colony formation of 22Rv1 and PC-3 cells in soft agar, as well as downregulated focal adhesion kinase (FAK), a crucial factor in anchorage-independent growth. SIGNIFICANCE: In 3D-cultured prostate cancer cells, probenecid demonstrated pleiotropic effects such as chemosensitization, growth suppression, inhibition of spheroid compaction, and suppression of anchorage-independent growth. Elucidating the detailed mechanism underlying these probenecid actions could result in the identification of novel therapeutic targets toward the advanced prostate cancer.

Caffeic Acid, Quercetin and 5-Fluorocytidine-Functionalized Au-Fe3O4 Nanoheterodimers for X-ray-Triggered Drug Delivery in Breast Tumor Spheroids.

Au-Fe3O4 nanoheterodimers (NHD) were functionalized with the natural and synthetic anticancer drugs caffeic acid (CA), quercetin (Q) and 5-fluorocytidine (5FC). Their X-radiation dose-enhancing potential and chemotherapeutic efficacy for bimodal cancer therapy were investigated by designing multicellular tumor spheroids (MCTS) to in vitro avascular tumor models. MCTS were grown from the breast cancer cell lines MCF-7, MDA-MB-231, and MCF-10A. The MCF-7, MDA-MB-231 and MCF-10A MCTS were incubated with NHD-CA, NHD-Q, or NHD-5FC and then exposed to fractionated X-radiation comprising either a single 10 Gy dose, 2 daily single 5 Gy doses or 5 daily single 2 Gy doses. The NHD-CA, NHD-Q, and NHD-5FC affected the growth of X-ray irradiated and non-irradiated MCTS in a different manner. The impact of the NHDs on the glycolytic metabolism due to oxygen deprivation inside MCTS was assessed by measuring lactate secretion and glucose uptake by the MCTS. The NHD-CA and NHD-Q were found to act as X-radiation dose agents in MCF-7 MCTS and MDA-MB-231 MCTS and served as radioprotector in MCF-10A MCTS. X-ray triggered release of CA and Q inhibited lactate secretion and thereupon disturbed glycolytic reprogramming, whereas 5FC exerted their cytotoxic effects on both, healthy and tumor cells, after their release into the cytosol.

Multi-omics analysis defines 5-fluorouracil drug resistance in 3D HeLa carcinoma cell model.

Cervical cancer is a serious health problem in women around the globe. However, the use of clinical drug is seriously dampened by the development of drug resistance. Efficient in vitro tumor model is essential to improve the efficiency of drug screening and the accuracy of clinical application. Multicellular tumor spheroids (MTSs) can in a way recapitulates tumor traits in vivo, thereby representing a powerful transitional model between 2D monolayer culture and xenograft. In this study, based on the liquid overlay method, a protocol for rapid generation of the MTSs with uniform size and high reproducibility in a high-throughput manner was established. As expected, the cytotoxicity results showed that there was enhanced 5-fluorouracil (5-FU) resistance of HeLa carcinoma cells in 3D MTSs than 2D monolayer culture with a resistance index of 5.72. In order to obtain a holistic view of the molecular mechanisms that drive 5-FU resistance in 3D HeLa carcinoma cells, a multi-omics study was applied to discover hidden biological regularities. It was observed that in the 3D MTSs mitochondrial function-related proteins and the metabolites of the tricarboxylic acid cycle (TCA cycle) were significantly decreased, and the cellular metabolism was shifted towards glycolysis. The differences in the protein synthesis, processing, and transportation between 2D monolayer cultures and 3D MTSs were significant, mainly in the heat shock protein family, with the up-regulation of protein folding function in endoplasmic reticulum (ER), which promoted the maintenance of ER homeostasis in the 3D MTSs. In addition, at the transcript and protein level, the expression of extracellular matrix (ECM) proteins (e.g., laminin and collagen) were up-regulated in the 3D MTSs, which enhanced the physical barrier of drug penetration. Summarizing, this study formulates a rapid, scalable and reproducible in vitro model of 3D MTS for drug screening purposes, and the findings establish a critical role of glycolytic metabolism, ER hemostasis and ECM proteins expression profiling in tumor chemoresistance of HeLa carcinoma cells towards 5-FU.

Overexpression of miR-21-5p in colorectal cancer cells promotes self-assembly of E-cadherin-dependent multicellular tumor spheroids.

Three-dimensional (3D) multicellular tumor spheroid (MCTS) cultures are increasingly popular as an in vitro tumor model for drug screening because they can mimic the complexity and heterogeneity of tumors compared to 2D monolayer cell cultures. The oncogenic microRNA, miR-21-5p (hereafter denoted as miR-21), is one of the most upregulated miRNAs in colorectal cancer (CRC). Herein, we established a stable miR-21-overexpressing clone in the DLD-1 human CRC cell line to investigate its impact on MCTS formation. We found that miR-21 overexpression enhanced cell-cell interactions/aggregations in both 2D monolayer and 3D suspension cultures. Cell aggregates in 3D suspension culture further formed MCTSs in miR-21-overexpressing cells. miR-21 overexpression was associated with the upregulation of proteins involved in E-cadherin-associated cell-cell adhesion. Furthermore, miR-21 induction of MCTSs could be reversed by the antibody-induced blockade of E-cadherin. Our results showed that miR-21 overexpression promoted MCTS formation through enhancing E-cadherin-dependent cell-cell interactions, which represents an advance in vitro model for investigating CRC biology.

Natural and Synthetic Biomaterials for Engineering Multicellular Tumor Spheroids.

The lack of in vitro models that represent the native tumor microenvironment is a significant challenge for cancer research. Two-dimensional (2D) monolayer culture has long been the standard for in vitro cell-based studies. However, differences between 2D culture and the in vivo environment have led to poor translation of cancer research from in vitro to in vivo models, slowing the progress of the field. Recent advances in three-dimensional (3D) culture have improved the ability of in vitro culture to replicate in vivo conditions. Although 3D cultures still cannot achieve the complexity of the in vivo environment, they can still better replicate the cell-cell and cell-matrix interactions of solid tumors. Multicellular tumor spheroids (MCTS) are three-dimensional (3D) clusters of cells with tumor-like features such as oxygen gradients and drug resistance, and represent an important translational tool for cancer research. Accordingly, natural and synthetic polymers, including collagen, hyaluronic acid, Matrigel®, polyethylene glycol (PEG), alginate and chitosan, have been used to form and study MCTS for improved clinical translatability. This review evaluates the current state of biomaterial-based MCTS formation, including advantages and disadvantages of the different biomaterials and their recent applications to the field of cancer research, with a focus on the past five years.