Multicellular Tumor Spheroid Model Research Articles

Optimal Control for a Parabolic–Hyperbolic Free Boundary Problem Modeling the Growth of Tumor with Drug Application

In this paper, we study two optimal control problems for a free boundary problem, which models tumor growth with drug application. This free boundary problem is a multicellular tumor spheroid model and includes five time-dependent partial differential equations. The tumor considered in this model consists of three kinds of cells: proliferative cells, quiescent cells and dead cells. Three different first-order hyperbolic equations are given, which describe the evolution of cells, and other two second-order parabolic equations describe the diffusions of nutrient (e.g., oxygen and glucose) and drug concentrations. Existence and uniqueness of optimal controls are also proved. We use tangent-normal cone techniques to obtain necessary conditions. Then, we employ the Ekeland variational principle to show that there exists unique optimal control for each optimal control problem.

A three-dimensional spheroidal cancer model based on PEG-fibrinogen hydrogel microspheres.

Three-dimensional (3D) invitro cancer models offer an attractive approach towards the investigation of tumorigenic phenomena and other cancer studies by providing dimensional context and higher degree of physiological relevance than that offered by conventional two-dimensional (2D) models. The multicellular tumor spheroid model, formed by cell aggregation, is considered to be the "gold standard" for 3D cancer models, due to its ease and simplicity of use. Although better than 2D models, tumor spheroids are unable to replicate key features of the native tumor microenvironment, particularly due to a lack of surrounding extracellular matrix components and heterogeneity in shape, size and aggregate forming tendencies. In order to address this issue, we have developed a 3D "tumor microsphere" model, formed by a dual-photoinitiator, aqueous-oil emulsion technique, for the encapsulation of cancer cells within PEG-fibrinogen hydrogel microspheres and for subsequent long-term 3D culture. In comparison to self-aggregated tumor spheroids, the tumor microspheres displayed a higher degree of size and shape homogeneity throughout long-term culture. In sharp contrast to cells in tumor spheroids, cells within tumor microspheres demonstrated significant loss in apico-basal polarity and cellular architecture, cellular and nuclear atypia, increased disorganization, elevated nuclear cytoplasmic ratio and nuclear volume density and reduction in cell-cell junction length, all of which are hallmarks of malignant transformation and tumorigenic progression. Additionally, the tumor microsphere model was extended for the 3D encapsulation and maintenance of a wide range of other cancer cell (metastatic and non-metastatic) types. Taken together, our results reinforce the importance of incorporating a biomimetic matrix in the cellular microenvironment of 3D tumor models and the influential effects of the matrix on the tumorigenic morphology of 3D cultured cells. The tumor microsphere system established in this study has the potential to be used in future investigations of 3D cancer cell-cell and cell-ECM interactions and in drug-testing applications.

Sensitization of multidrug-resistant malignant cells by liposomes co-encapsulating doxorubicin and chloroquine through autophagic inhibition

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters play a key role in the development of multidrug resistance (MDR) in cancer cells. P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) are important proteins in this superfamily which are widely expressed on the membranes of multidrug resistance (MDR) cancer cells. Besides, upregulation of cellular autophagic responses is considered a contributing factor for MDR in cancer cells. We designed a liposome system co-encapsulating a chemotherapeutic drug (doxorubicin hydrochloride, DOX) and a typical autophagy inhibitior (chloroquine phosphate, CQ) at a weight ratio of 1:2 and investigated its drug resistance reversal mechanism. MTT assay showed that the IC50 of DOX/CQ co-encapsulated liposome in DOX-resistant human breast cancer cells (MCF7/ADR) was 4.7 ± 0.2 μM, 5.7-fold less than that of free DOX (26.9 ± 1.9 μM), whereas it was 19.5-fold in doxorubicin-resistant human acute myelocytic leukemia cancer cells (HL60/ADR) (DOX/CQ co-encapsulated liposome 1.2 ± 0.1 μM, free DOX 23.4 ± 2.8 μM). The cellular uptake of DOX increased upon addition of free CQ, indicating that CQ may interact with P-gp and MRP1; however, the expressions of P-gp and MRP1 remained unchanged. In contrast, the expression of the autophagy-related protein LC3-II increased remarkably. Therefore, the mechanism of MDR reversal may be closely related to autophagic inhibition. Evaluation of anti-tumor activity was achieved in an MCF-7/ADR multicellular tumor spheroid model and transgenic zebrafish model. DOX/CQ co-encapsulated liposome exerted a better anti-tumor effect in both models than that of liposomal DOX or DOX alone. These findings suggest that encapsulating CQ with DOX in liposomes significantly improves the sensitivity of DOX in DOX-resistant cancer cells.

Size and Surface Charge of Engineered Poly(amidoamine) Dendrimers Modulate Tumor Accumulation and Penetration: A Model Study Using Multicellular Tumor Spheroids.

An enormous effort has been put into designing nanoparticles (NPs) with controlled biodistributions, prolonged plasma circulation times, and/or enhanced tissue targeting. However, little is known about how to design NPs with precise distributions in the target tissues. In particular, understanding NP tumor penetration and accumulation characteristics is crucial to maximizing the therapeutic potential of drug molecules carried by the NPs. In this study, we employed poly(amidoamine) (PAMAM) dendrimers, given their well-controlled size (<10 nm) and surface charge, to understand how the physical properties of NPs govern their tumor accumulation and penetration behaviors. We demonstrate for the first time that the size and surface charge of PAMAM dendrimers control their distributions in both a 3D multicellular tumor spheroid (MCTS) model and a separate extracellular matrix (ECM) model, which mimics the tumor microenvironment. Smaller PAMAM dendrimers not only diffused more rapidly in the ECM model but also efficiently penetrated to the MCTS core compared to their larger counterparts. Furthermore, cationic, amine-terminated PAMAM dendrimers exhibited the greatest accumulation in MCTS compared to either charge-neutral or anionic dendrimers. Our findings indicate that the size and surface charge of PAMAM dendrimers may tailor their tumor accumulation and penetration behaviors. These results suggest that controlled tumor accumulation and distinct intratumoral distributions can be achieved by simply controlling the size and surface charge of dendrimers, which may also be applicable for other similarly sized NPs.

Developing multi-cellular tumor spheroid model (MCTS) in the chitosan/collagen/alginate (CCA) fibrous scaffold for anticancer drug screening

In this work, a 3D MCTS-CCA system was constructed by culturing multi-cellular tumor spheroid (MCTS) in the chitosan/collagen/alginate (CCA) fibrous scaffold for anticancer drug screening. The CCA scaffolds were fabricated by spray-spinning. The interactions between the components of the spray-spun fibers were evidenced by methods of Coomassie Blue stain, X-ray diffraction (XRD) and Fourier transform-infrared spectroscopy (FTIR). Co-culture indicated that MCF-7 cells showed a spatial growth pattern of multi-cellular tumor spheroid (MCTS) in the CCA fibrous scaffold with increased proliferation rate and drug-resistance to MMC, ADM and 5-Aza comparing with the 2D culture cells. Significant increases of total viable cells were found in 3D MCTS groups after drug administration by method of apoptotic analysis. Glucose–lactate analysis indicated that the metabolism of MCTS in CCA scaffold was closer to the tumor issue in vivo than the monolayer cells. In addition, MCTS showed the characteristic of epithelial mesenchymal transition (EMT) which is subverted by carcinoma cells to facilitate metastatic spread. These results demonstrated that MCTS in CCA scaffold possessed a more conservative phenotype of tumor than monolayer cells, and anticancer drug screening in 3D MCTS-CCA system might be superior to the 2D culture system.

Three-Dimensional Spheroid Cell Culture Model for Target Identification Utilizing High-Throughput RNAi Screens.

The intrinsic limitations of 2D monolayer cell culture models have prompted the development of 3D cell culture model systems for in vitro studies. Multicellular tumor spheroid (MCTS) models closely simulate the pathophysiological milieu of solid tumors and are providing new insights into tumor biology as well as differentiation, tissue organization, and homeostasis. They are straightforward to apply in high-throughput screens and there is a great need for the development of reliable and robust 3D spheroid-based assays for high-throughput RNAi screening for target identification and cell signaling studies highlighting their potential in cancer research and treatment. In this chapter we describe a stringent standard operating procedure for the use of MCTS for high-throughput RNAi screens.

BIGH3: A Negative Regulator of Human Osteosarcoma Large Multicellular Spheroids

Numerous studies have demonstrated a relationship between the extracellular matrix protein BIGH3 and variations in the malignant properties of different cancer cell types, including osteosarcoma cells. BIGH3 protein can suppress and promote tumor growth, even on the same cancer cell type, indicating that contextual cues regulate BIGH3-mediated divergent outcomes. We employed a multicellular tumor spheroid model to study the effects of BIGH3 with respect to physical and molecular features of three-dimensional tumor growth. The results demonstrated that exogenous recombinant BIGH3 blocked the development of multicellular large tumor spheroids so that only small spheroids formed. The effect was dependent on the BIGH3 concentration in the growth medium and the time of incubation of BIGH3 with the osteosarcoma cells in the spheroid model. TGF-β1 signaling induced multicellular tumor spheroids to synthesize a greater quantity of BIGH3 relative to non-treated spheroids. The TGF-β1-mediated increase in BIGH3 protein antagonized the development of multicellular large spheroids. Anti-BIGH3 antibody, and an inhibitor of TGF-β1 signaling, blocked the antagonistic effect induced through TGF-β1 stimulation and BIGH3 protein expression, resulting in the formation of multicellular large spheroids. Immunohistochemistry detected BIGH3 at cell bodies within the spheroid stroma, suggesting osteosarcoma cell-surface proteins bind BIGH3. Flow cytometry demonstrates that osteosarcoma cells interact with soluble BIGH3, and solid-phase cell adhesion assays show that osteosarcoma adhesion to BIGH3 substratum is mediated by integrin α4β1. However, anti-α4 antibody did not attenuate the BIGH3-mediated antagonism toward formation of multicellular large spheroids. We conclude that TGFβ1 and BIGH3 suppress the development of large osteosarcoma tumors.

Noncovalent Ruthenium(II) Complexes-Single-Walled Carbon Nanotube Composites for Bimodal Photothermal and Photodynamic Therapy with Near-Infrared Irradiation.

To enhance the efficacy and optimize the treatment of cancers, the integration of multimodal treatment strategies leading to synergistic effects is a promising approach. The coassembly of multifunctional agents for systematic therapies has received considerable interest in cancer treatment. Herein, Ru(II) complex-functionalized single-walled carbon nanotubes (Ru@SWCNTs) are developed as nanotemplates for bimodal photothermal and two-photon photodynamic therapy (PTT-TPPDT). SWCNTs have the ability to load a great amount of Ru(II) complexes (Ru1 or Ru2) via noncovalent π-π interactions. The loaded Ru(II) complexes are efficiently released by the photothermal effect of irradiation from an 808 nm diode laser (0.25 W/cm(2)). The released Ru(II) complexes produce singlet oxygen species ((1)O2) upon two-photon laser irradiation (808 nm, 0.25 W/cm(2)) and can be used as a two-photon photodynamic therapy (TPPDT) agent. Based on the combination of photothermal therapy and two-photon photodynamic therapy, Ru@SWCNTs have greater anticancer efficacies than either PDT using Ru(II) complexes or PTT using SWCNTs in two-dimensional (2D) cancer cell and three-dimensional (3D) multicellular tumor spheroid (MCTS) models. Furthermore, in vivo tumor ablation is achieved with excellent treatment efficacy under a diode laser (808 nm) irradiation at the power density of 0.25 W/cm(2) for 5 min. This study examines an efficacious bimodal PTT and TPPDT nanoplat form for the development of cancer therapeutics.

Abstract 323: Spheroid-based high throughput screening for identification of molecules targeting different tumor microenvironment characteristics

Abstract In recent years, cell-based evaluation of drug-induced phenotypic changes has become an important strategy in high throughput drug screening (HTS). However, due to the lack of clinically relevant in vitro models, phenotype-based screening has not resulted in the identification of as many successful compounds as initially assumed. Thus, an application of the long-known multicellular tumor spheroid (MCTS) model for HTS has recently gained attention, as MCTS mimic the complex in vivo situation more closely than standard monolayer cell cultures. However, due to major difficulties with formation of uniform spheroids in a large scale, application of MCTS for HTS has been limited. We focused our efforts on the development of spheroid-based HTS systems in 384-well format for the identification of molecules targeting different tumor microenvironment characteristics. For spheroid formation we used two colorectal cancer cell lines, HCT116 and HT-29, both constitutively expressing green fluorescent protein (GFP). During the spheroid formation process, culture medium was either changed every 4 days or left unchanged. This approach resulted in the generation of two different spheroid models from the each cell line - one representing tumor regions with the sufficient access to nutrients; the other simulating parts with more harsh conditions. Spheroids formed with medium change were more proliferative and less dependent on oxygen. By measuring spheroid GFP fluorescence intensity we could non-invasively monitor drug action over time. First screens revealed a group of drugs particularly effective in quiescent spheroids compared with proliferating spheroids and monolayer cell cultures. All of the compounds inhibited mitochondrial oxidative phosphorylation. The screen confirmed mitochondrial respiration as a promising target for treatment of dormant cells in nutrient-deprived tumor regions and identified molecules with previously unrecognized potential for further clinical advancement. Another screening system was developed to identify drugs that directly influence the immune components of tumor microenvironment. Spheroids are formed by co-culturing cancer cells with human primary monocytes. Over the culture period, monocytes differentiate into tumor-associated macrophages. First pilot experiments showed promising results and more extensive screens are planned. For characterization of drug effects at the transcriptome level, a spheroid-based gene expression profiling method is under development. The method will enable high throughput analysis of transcripts from spheroids treated with chemical libraries. In conclusion, we here present a novel approach to HTS, which by utilizing diverse MCTS models has a potential to identify molecules directly targeting tumor-specific microenvironment components. Citation Format: Wojciech Senkowski, Xiaonan Zhang, Peter Nygren, Maria Hägg Olofsson, Mats Gustafsson, Stig Linder, Rolf Larsson, Mårten Fryknäs. Spheroid-based high throughput screening for identification of molecules targeting different tumor microenvironment characteristics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 323. doi:10.1158/1538-7445.AM2015-323

Abstract LB-077: Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer

Abstract The Wnt signaling pathway, which controls TCF/Lef/β-catenin (TCF) transcription, is well accepted as a dominate player in CRC tumor initiation and progression. More recently, this pathway has been implicated as a major driving force inducing epithelial-mesenchymal transition (EMT), acquiring a drug resistant tumor initiating cell (TIC) phenotype that may promote metastasis. As a result, this pathway has been seriously pursued for novel drug development that has yielded no real translational success due to the lack of “druggable” targets. Thus, the major challenge moving forward is to identify a druggable target within the Wnt/TCF pathway that may result in effective clinical translation. Using a chemical biology approach targeting EMT we have identified topoisomerase IIα (TopoIIα) as a master regulator EMT by participating as a required component of TCF-transcription in CRC. Specifically, we show that inhibiting TopoIIα using small molecule ATP-competitive inhibitors as well as shRNA results in the blockade of TCF-transcription using the TOP/FOP flash reporter system. Inhibition of TopoIIα-dependent TCF-transcription results in significant reversion of EMT characterized by Western blot analysis in a panel of CRC cell lines. Time course studies reveal that inhibiting TopoIIα results in downregulation of TCF-transcription followed by the induction of apoptosis using 3D multicellular tumor spheroid models. We show that TopoIIα participates in protein-protein complexation with β-catenin using co-immunoprecipitation studies, which is not prevented by small molecule inhibitors. However, chromatin immunoprecipitation (ChIP) studies reveal that ATP-competitive TopoIIα inhibitors can significantly block TCF-transcription by preventing TCF-complex-DNA binding. In addition, inhibiting TopoIIα-dependent TCF-transcription correlates with the inhibition of TIC function characterized by loss of colony formation using the clonogenic assay. Likewise, inhibiting TopoIIα-dependent TCF-transcription inhibits the invasive potential in a panel of CRC tumor cell lines. Interestingly, the clinically used TopoIIα drug, etoposide, had no effect on TCF-transcription, EMT, or invasion. In conclusion, we hypothesize that TopoIIα participates as a component of the TCF-complex via the C-terminus and is required for binding chromatin DNA and initiating gene transcription. TopoIIα participation in TCF-transcription may convey drug resistance due to altered poison binding sites. However, our data indicates that TopoIIα-dependent TCF-transcription requires ATP. Hence, central to our hypothesis is that N-terminal ATP sites are conserved providing an Achilles heel to drug resistance. Therefore, N-terminal ATP binding sites provide optimal druggable target sites that may be utilized for translational drug development preventing TCF-transcription, EMT and potentially metastasis in CRC patients. Citation Format: Daniel V. LaBarbera, Qiong Zhou, Adedoyin D. Abraham, Linfeng Li, Wells A. Messersmith. Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-077. doi:10.1158/1538-7445.AM2015-LB-077

Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model.

Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.

Radiation-induced bystander effect in non-irradiated glioblastoma spheroid cells.

Radiation-induced bystander effects (RIBEs) are detected in cells that are not irradiated but receive signals from treated cells. The present study explored these bystander effects in a U87MG multicellular tumour spheroid model. A medium transfer technique was employed to induce the bystander effect, and colony formation assay was used to evaluate the effect. Relative changes in expression of BAX, BCL2, JNK and ERK genes were analysed using RT-PCR to investigate the RIBE mechanism. A significant decrease in plating efficiency was observed for both bystander and irradiated cells. The survival fraction was calculated for bystander cells to be 69.48% and for irradiated cells to be 34.68%. There was no change in pro-apoptotic BAX relative expression, but anti-apoptotic BCL2 showed downregulation in both irradiated and bystander cells. Pro-apoptotic JNK in bystander samples and ERK in irradiated samples were upregulated. The clonogenic survival data suggests that there was a classic RIBE in U87MG spheroids exposed to 4 Gy of X-rays, using a medium transfer technique. Changes in the expression of pro- and anti-apoptotic genes indicate involvement of both intrinsic apoptotic and MAPK pathways in inducing these effects.

Modulation of extracellular matrix in cancer is associated with enhanced tumor cell targeting by bacteriophage vectors.

BackgroundGene therapy has been an attractive paradigm for cancer treatment. However, cancer gene therapy has been challenged by the inherent limitation of vectors that are able to deliver therapeutic genes to tumors specifically and efficiently following systemic administration. Bacteriophage (phage) are viruses that have shown promise for targeted systemic gene delivery. Yet, they are considered poor vectors for gene transfer. Recently, we generated a tumor-targeted phage named adeno-associated virus/phage (AAVP), which is a filamentous phage particle whose genome contains the adeno-associated virus genome. Its effectiveness in delivering therapeutic genes to tumors specifically both in vitro and in vivo has been shown in numerous studies. Despite being a clinically useful vector, a multitude of barriers impede gene transduction to tumor cells. We hypothesized that one such factor is the tumor extracellular matrix (ECM).MethodsWe used a number of tumor cell lines from different species and histological types in 2D monolayers or 3D multicellular tumor spheroid (MCTS) models. To assess whether the ECM is a barrier to tumor cell targeting by AAVP, we depleted the ECM using collagenase, hyaluronidase, or combination of both. We employed multiple techniques to investigate and quantify the effect of ECM depletion on ECM composition (including collagen type I, hyaluronic acid, fibronectin and laminin), and how AAVP adsorption, internalisation, gene expression and therapeutic efficacy are subsequently affected. Data were analyzed using a student’s t test when comparing two groups or one-way ANOVA and post hoc Tukey tests when using more than two groups.ResultsWe demonstrate that collagenase and hyaluronidase-mediated degradation of tumor ECM affects the composition of collagen, hyaluronic acid and fibronectin. Consequently, AAVP diffusion, internalisation, gene expression and tumor cell killing were enhanced after enzymatic treatment. Our data suggest that enhancement of gene transfer by the AAVP is solely attributed to ECM depletion. We provide substantial evidence that ECM modulation is relevant in clinically applicable settings by using 3D MCTS, which simulates in vivo environments more accurately.ConclusionOur findings suggest that ECM depletion is an effective strategy to enhance the efficiency of viral vector-guided gene therapy.

Roles of Tumor Microenvironment in Hepatocelluar Carcinoma

Hepatocellular carcinoma (HCC) is one of the most malignant human cancers, with a high mortality rate worldwide despite its early diagnosis in patients and improvement in therapeutic technology. Most cases of liver cancer show a strong resistance to anticancer therapy. Moreover, liver cancer patients generally have poor tolerance to chemotherapy due to liver dysfunction. In these situations, liver-targeting drugs with fewer side effects and a high efficacy are urgently needed during drug discovery for liver cancer. Researchers have aimed to derive target genes and drug candidates for HCC; however, the development of targeted drugs has not yet improved the outcome significantly.Recently, the role of the tumor microenvironment (TME) in HCC has been probed to combat this deadly disease. A deeper knowledge of the crosstalk between tumor cells and their TME is needed to fully understand tumor development, progression and chemo-resistance in HCC because this cancer develops from chronically damaged tissue that contains large amounts of inflammation and fibrosis.In this review, we summarize how distinct stromal cells of TME are involved in tumorigenesis and chemo-resistance in HCC and the significant challenge to recapitulate tumor complexity and heterogeneity enhancement.

A collagen-based multicellular tumor spheroid model for evaluation of the efficiency of nanoparticle drug delivery

Targeted drug delivery systems, especially those that use nanoparticles, have been the focus of research into cancer therapy during the last decade, to improve the bioavailability and delivery of anticancer drugs to specific tumor sites, thereby reducing the toxicity and side effects to normal tissues. However, the positive antitumor effects of these nanocarriers observed in conventional monolayer cultures frequently fail in vivo, due to the lack of physical and biological barriers resembling those seen in the actual body. Therefore, the collagen-based 3-D multicellular culture system, to screen new nanocarriers for drug delivery and to obtain more adequate and better prediction of therapeutic outcomes in preclinical experiments, was developed. This 3-D culture model was successfully established using optimized density of cells. Our result showed that 3-D cell colonies were successfully developed from 95-D, U87 and HCT116 cell lines respectively, after a seven-day culture in the collagen matrix. The coumarin-conjugated nanoparticles were able to penetrate the matrix gel to reach the tumor cells. The model is supposedly more accurate in reflecting/predicting the dynamics and therapeutic outcomes of candidates for drug transport in vivo, and/or investigation of tumor biology, thus speeding up the pace of discovery of novel drug delivery systems for cancer therapy.

Live Multicellular Tumor Spheroid Models For High-Content Imaging and Screening In Cancer Drug Discovery

The multi cellular tumor spheroid (MCTS) model has been used for decades with proven superiority over monolayer cell culture models at recapitulating in vivo tumor growth. Yet its use in high-throughput drug discovery has been limited, particularly with image based screening, due to practical and technical hurdles. Here we report a significant advance in utilizing live MCTS models for high-content image based drug discovery. Using a validated GFP reporter (CK5Pro-GFP) of luminal breast cancer stem cells (CSC), we developed an algorithm to quantify changes in CK5Pro-GFP expression levels for individual Z-stack planes (local) or as maximal projections of the summed Z-stacks (global) of MCTS. From these image sets, we can quantify the cross-sectional area of GFP positive cells, the fluorescence intensity of the GFP positive cells, and the percent of spheroid cross-sectional area that expresses CK5Pro-GFP.We demonstrate that acquiring data in this manner can be done in real time and is statistically robust (Z’=0.85) for use in primary high-content screening cancer drug discovery.

Editorial High Content Screening for Lead Identification and Optimization

Automated microscope-based High Content Screening (HCS or HCA) has gained tremendous momentum recently because of its ability to capture many subcellular features simultaneously in complex biology systems, and/or to monitor cellular processes that are otherwise intractable using conventional technologies. HCS can be utilized in early drug discovery and preclinical development to accelerate drug discovery. It has been widely used in target validation, lead generation, toxicity studies, and drug mechanism studies. HCS also has the potential to be used to support clinical trials, such as companion diagnostics. In this special issue on HCS, three HCS-based assays that are applicable for lead identification and optimization are highlighted. To many institutions, the initiation of primary screening is a serious commitment to the targets, pathways or disease hypothesis. Therefore, patho-physiological relevancy, cost, throughput, scalability, quality, etc. must be carefully weighted when designing an assay for primary screening. Unlike traditional HTS, which has one or two measurements, HCS enables one to measure many parameters or features of individual cells or organisms simultaneously. With currently available technologies, HCS-based HTS is still labor-intensive and could be very costly if multiple dyes and/or antibodies are used. Therefore, HCS-based HTS should be considered only if there are no adequate conventional screening technologies that could be used to obtain similar information. In this issue, Peppard and colleagues present an HCS-based HTS entitled “Identifying small molecules which inhibit autophagy: a phenotypic screen using image-based high-content cell analysis” [1] to identify small-molecule autophagy inhibitors by following the cytoplasmic redistribution of GFP tagged LC3, a component of the autophagosome, from diffuse to punctate dots in HeLa cells. Overexpression of tagged proteins may interrupt the interaction between the protein and its partners in the cellular network, thus leading to non-physiologically relevant phenotypes. Therefore, many use antibody stains to monitor the translocation and expression of target proteins, or changes of organelle and cell structures and functions. Antibodies in general are expensive and the use of multiple antibody stains in primary screening could be cost prohibitive, and the multiple wash steps maybe not HTS friendly. However, antibody staining-based HCS assays have played and will continue to play key roles in secondary and tertiary assays, as well as mechanism and toxicity studies. Sum and his colleagues describe the development of an antibody stain-based HCS for microtubule structure, entitled “Establishing a High-Content Analysis Method for Tubulin Polymerization to Evaluate Both the Stabilizing and Destabilizing Activities of Compounds” [2]. This assay enables the understanding of the mechanisms of action for tubulin-interacting compounds. Two-dimensional (2-D) monolayer culture cells are routinely employed in primary HTS and in lead generation and optimization. However, these 2-D model systems do not recapitulate the complexity of the 3-D organization and the pathophysiology in tissues and organs. Therefore, the practice of using 2-D cellular models for drug testing is being questioned for its inability to mimic physiological environments of diseased cells in the human body. An increased number of laboratories has started to establish 3-D cellular models for drug testing. Although much less complex than in vivo models or complete organisms, these 3-D cellular models are one-step closer to physiological conditions. In the manuscript of “Live Multicellular Tumor Spheroid Models For High-Content Imaging and Screening In Cancer Drug Discovery” [3], Reid and his colleagues report the development of a multicellular tumor spheroid (MCTS) model for drug screening and a simple yet predictive 3-D image analysis algorithm to quantify changes induced by drugs. In summary, due to its flexibility and information-rich nature, HCS has become the choice for many scientists to examine the temporal and spatial effects of compounds on cells, not only for the intended targets, but also for other cellular targets and pathways. This feature not only enables the identification of the best hits and leads from screening, but also facilitates multi-parameter optimization of leads.

Multicellular Tumor Spheroids as a Model for Assessing Delivery of Oligonucleotides in Three Dimensions

Oligonucleotides have shown promise in selectively manipulating gene expression in vitro, but that success has not translated to the clinic for cancer therapy. A potential reason for this is that cells behave differently in monolayer than in the three-dimensional tumor, resulting in limited penetration and distribution of oligonucleotides in the tumor. This may be especially true when oligonucleotides are associated with nanocarriers such as lipoplexes and polyplexes, commonly used delivery vehicles for oligonucleotides. The multicellular tumor spheroid (MCTS), a three-dimensional model that closely resembles small avascular tumors and micrometastases, has been utilized as an intermediate between monolayer culture and in vivo studies for the screening of small-molecule drugs. However, spheroids have been little used for the study of various oligonucleotide delivery formulations. Here, we have evaluated the uptake and efficacy of splice-switching antisense oligonucleotides using various delivery modalities in two- and three-dimensional culture models. We find that the size of the delivery agent dramatically influences penetration into the spheroid and thus the biological effect of the oligonucleotides. We hypothesize that the MCTS model will prove to be a useful tool in the future development of oligonucleotide delivery formulations.

Protein Transfection Study Using Multicellular Tumor Spheroids of Human Hepatoma Huh-7 Cells

Several protein transfection reagents are commercially available and are powerful tools for elucidating function of a protein in a cell. Here we described protein transfection studies of the commercially available reagents, Pro-DeliverIN, Xfect, and TuboFect, using Huh-7 multicellular tumor spheroid (MCTS) as a three-dimensional in vitro tumor model. A cellular uptake study using specific endocytosis inhibitors revealed that each reagent was internalized into Huh-7 MCTS by different mechanisms, which were the same as monolayer cultured Huh-7 cells. A certain amount of Pro-DeliverIN and Xfect was uptaken by Huh-7 cells through caveolae-mediated endocytosis, which may lead to transcytosis through the surface-first layered cells of MCTS. The results presented here will help in the choice and use of protein transfection reagents for evaluating anti-tumor therapeutic proteins against MCTS models.

Abstract A108: Induction of mitochondrial dysfunction as a strategy for targeting tumor cells in hypoxic microenvironment.

Abstract A fundamental problem in cancer drug discovery is to identify compounds that eliminate dormant malignant cells responsible for tumor relapse. Abnormal vascularization of solid tumors leads to the generation of tissue microenvironments that are chronically starved of oxygen and nutrients. Cells residing in such environments are slowly growing or quiescent and display altered phenotypic characteristics when compared to cells located in more vascularized regions. Such non-dividing cells are often resistant to mainstay standard chemotherapies that rely on DNA replication and cell division to elicit their anti-tumor effect. The altered phenotype of quiescent cells enables them to survive chemotherapeutic regimes and reseed nascent tumors following secession of chemotherapy. Indeed resistance to agents such as doxorubicin, cisplatin and vinblastine has been correlated with poor vasculature, tumor relapse and poor patient survival. Thus there is a need to alter the scope of cancer drug discovery and focus more on screening for agents that can exploit the altered phenotype of quiescent metabolically stressed cells to eliminate tumors. Cell-based screening for novel anticancer drugs is typically performed using monolayer cultures of tumor cells, however such monolayer cultures do not represent the characteristics of 3-D solid tumors, frequently leading to the failure of subsequent in vivo models. The multicellular tumor spheroid model is of intermediate complexity between in vivo tumors and in vitro monolayer cultures and is more suitable for drug screening and evaluation. Spheroids are known to be more resistant to drug effects compared to monolayer cultures. Resistance is due not only to pharmacokinetic obstacles limiting drug penetrance to inner layers, but also to multi-cellular interactions leading to altered expression of genes and proteins regulating drug response. An additional benefit of growing cells three- dimensionally is the opportunity to explore the core regions’ potential vulnerability related to hypoxia and nutrient defiency and to reflect the heterogeneous milieu in tumor microregions relative to the supply. Poorly vascularized and perfused tumor microareas in many aggressive cancers have limited access not only to oxygen but also to glucose. Core regions are also associated with acidic pHsince these tumor cells change their metabolism towards increased glycolysis, resulting in increased lactic acid production. We here employed spheroid cultures of HCT116 colon cancer cells to screen a diverse chemical library with the aim to find compounds with cytotoxic activity in core, hypoxic, regions. The screen identified a compound (VLX600) demonstrating anti-cancer activity with a large therapeutic window both in vitro and in vivo. Here we characterized the mechanism of action and potential of VLX600 as anti-cancer therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A108. Citation Format: Xiaonan Zhang, Mårten Fryknäs, Emma Hernlund, Walid Fayad, Angelo De Milito, Maria Hägg Olofsson, Stig Linder. Induction of mitochondrial dysfunction as a strategy for targeting tumor cells in hypoxic microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A108.