Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue.     

Use of a laser diffraction particle sizer for the measurement of mean diameter of multicellular tumor spheroids

Summary Increasing use is being made of tumor cell lines cultured as cell aggregates (generally referred to as multicellular spheroids) in in vitro radiosensitivity and/or chemosensitivity tests. Conventional procedures for the determination of mean spheroid diameters for the construction of growth delay curves employ a microscope-image analyzer. However, this approach can prove excessively time consuming when large numbers of samples have to be, measured. We have, therefore, been exploring the use of a Laser Diffraction Particle Sizer, the Malvern 2600 long bench model, for the measurement of mean spheroid diameter and size distribution. We report here a direct comparison between measurements carried out by the instrument and under the microscope. Also a comparison of growth curves for six cell lines constructed from measurements by the microscope and by the instrument. A number of factors that might affect the accuracy of spheroid diameter measurement by the instrument have been investigated: The effect of stirring to maintain the spheroids in suspension during measurement. Sampling error due to removal of a series of spheroid samples from culture flasks for measurement. Optimum number of scans to be carried out by the instrument to reach a constant value for mean diameter, and minimum SE of the mean. This research was supported by the Yorkshire Cancer Research Campaign, Horrogate HG1 5LQ, United Kingdom, and by the Hospital Research Fund, Cookridge Hospitatl, Leeds LS16 6QB, United Kingdom.     

Penetration of anti-melanoma immunotoxin into multicellular tumor spheroids and cell kill effects

Summary In order to gain a better understanding of the interaction between immunotoxins and tumor cells at the level of three-dimensional tumor mass, we evaluated the cell kill effects of monoclonal antimelanoma-antibody/ricin-A-chain immunotoxin (ITN) on melanoma cells in multicellular tumor spheroids (MTS) as well as the penetration of ITN into MTS. For Minor melanoma cells in monolayer the ITN exerted cytotoxic effects after as little as 1 h of exposure. Increasing exposure time resulted in progressive increases in cytotoxic activity. In contrast, the cell kill effects of ITN were markedly delayed and reduced when Minor cells were in MTS. The ITN cytotoxic effects on the melanoma MTS were more than 100 fold less than those in monolayer. Patterns of ITN-induced cytotoxicities for Minor and for another melanoma cell line, DND-1A, were comparable. The native ricin A was more active against PC-10 squamous lung cancer cells than Minor cells, whereas the ITN was more cytotoxic against Minor cells than PC-10 cells, thus exhibiting selectivity. An autoradiographic study revealed time-dependent penetration of radiolabeled ITN from the surface of Minor MTS into the core. Incubation for 1 h resulted in the penetration of ITN into only the two or three outer layers of the Minor MTS, and low grain counts. Prolonged exposure resulted in inhomogeneous penetration of ITN into almost the entire melanoma MTS. Penetration of ITN into PC-10 MTS was extremely poor. The reduced cytotoxicity of ITN on melanoma cells in MTS as compared to cells grown in monolayer appears to correlate with its inhomogeneous distribution in the MTS. The delayed cytotoxicity of ITN is also consistent with its slow penetration into the core of the MTS.     

PDMS well platform for culturing millimeter‐size tumor spheroids

Multicellular tumor spheroids are widely used as in vitro models for testing of anticancer drugs. The advantage of this approach is that it can predict the outcome of a drug treatment on human cancer cells in their natural three‐dimensional environment without putting actual patients at risk. Several methods were utilized in the past to grow submillimeter‐size tumor spheroids. However, these small models are not very useful for preclinical studies of tumor ablation where the goal is the complete destruction of tumors that can reach several centimeters in diameter in the human body. Here, we propose a PDMS well method for large tumor spheroid culture. Our experiments with HepG2 hepatic cancer cells show that three‐dimensional aggregates of tumor cells with a volume as large as 44 mm³ can be grown in cylindrical PDMS wells after the initial culture of tumor cells by the hanging drop method. This is a 350 times more than the maximum volume of tumor spheroids formed inside hanging drops (0.125 mm³). © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1265–1269, 2013     

Active and inactive metabolic pathways in tumor spheroids: Determination by GC–MS

Active metabolic pathways in three‐dimensional cancer‐cell cultures are potential chemotherapeutic targets that would be effective throughout tumors. Chaotic vasculature creates cellular regions in tumors with distinct metabolic behavior that are only present in aggregate cell masses. To quantify cancer cell metabolism, transformed mouse fibroblasts were grown as spheroids and fed isotopically labeled culture medium. Metabolite uptake and production rates were measured as functions of time. Gas chromatography–mass spectrometry was used to quantify the extent of labeling on amino acids present in cytoplasmic extracts. The labeling pattern identified several active and inactive metabolic pathways: Glutaminolysis was found to be active, and malic enzyme and gluconeogenesis were inactive. Transformed cells in spheroids were also found to actively synthesize serine, cysteine, alanine, aspartate, glutamate, and proline; and not synthesize glutamine. The activities of these pathways suggest that cancer cells consume glutamine for biosynthesis and not to provide cellular energy. Determining active metabolic pathways indicates how cells direct carbon flow and may lead to the discovery of novel molecular targets for anticancer therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Use of multicellular tumor spheroids to dissect endothelial cell-tumor cell interactions: a role for T-cadherin in tumor angiogenesis

This study addresses establishment of an "in vitro" melanoma angiogenesis model using multicellular tumor spheroids (MCTS) of differentiated (HBL) or undifferentiated (NA8) melanoma cell lines. DNA microarray assay and qRT-PCR indicated upregulation of pro-angiogenic factors IL-8, VEGF, Ephrin A1 and ANGPTL4 in NA8-MCTSs (vs. monolayers) whereas these were absent in MCTS and monolayer cultures of HBL. Upon co-culture with endothelial cell line HMEC-1 NA8-MCTS attract, whereas HBL-MCTS repulse, HMEC-1. Overexpression of T-cadherin in HMEC-1 leads to their increased invasion and network formation within NA8-MCTS. Given an appropriate angiogenic tumor microenvironment, T-cadherin upregulation on endothelial cells may potentiate intratumoral angiogenesis.     

Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy

Magnetic iron oxide (IO) nanoparticles with a long blood retention time, biodegradability and low toxicity have emerged as one of the primary nanomaterials for biomedical applications in vitro and in vivo. IO nanoparticles have a large surface area and can be engineered to provide a large number of functional groups for cross-linking to tumor-targeting ligands such as monoclonal antibodies, peptides, or small molecules for diagnostic imaging or delivery of therapeutic agents. IO nanoparticles possess unique paramagnetic properties, which generate significant susceptibility effects resulting in strong T2 and T*2 contrast, as well as T1 effects at very low concentrations for magnetic resonance imaging (MRI), which is widely used for clinical oncology imaging. We review recent advances in the development of targeted IO nanoparticles for tumor imaging and therapy.     

Three-dimensional multicellular cell culture for anti-melanoma drug screening: focus on tumor microenvironment

AbstractThe development of new treatments for malignant melanoma, which has the worst prognosis among skin neoplasms, remains a challenge. The tumor microenvironment aids tumor cells to grow and resist to chemotherapeutic treatment. One way to mimic and study the tumor microenvironment is by using three-dimensional (3D) co-culture models (spheroids). In this study, a melanoma heterospheroid model composed of cancer cells, fibroblasts, and macrophages was produced by liquid-overlay technique using the agarose gel. The size, growth, viability, morphology, cancer stem-like cells population and inflammatory profile of tumor heterospheroids and monospheroids were analyzed to evaluate the influence of stromal cells on these parameters. Furthermore, dacarbazine cytotoxicity was evaluated using spheroids and two-dimensional (2D) melanoma model. After finishing the experiments, it was observed the M2 macrophages induced an anti-inflammatory microenvironment in heterospheroids; fibroblasts cells support the formation of the extracellular matrix, and a higher percentage of melanoma CD271 was observed in this model. Additionally, melanoma spheroids responded differently to the dacarbazine than the 2D melanoma culture as a result of their cellular heterogeneity and 3D structure. The 3D model was shown to be a fast and reliable tool for drug screening, which can mimic the in vivo tumor microenvironment regarding interactions and complexity.Graphic abstract      

基于碳酸酐酶IX的肿瘤成像和治疗研究进展

碳酸酐酶IX (carbonic anhydrase IX, CAIX)是一种在乏氧肿瘤细胞表面特异性过表达的跨膜蛋白,具有调节肿瘤细胞内外酸碱度的功能,与肿瘤增殖、侵袭和转移息息相关。因此,CAIX是一个很有潜力的肿瘤成像和治疗靶点。本文详细阐述了基于CAIX的肿瘤成像、治疗和诊疗一体化的研究进展,并对CAIX作为抗肿瘤靶点的应用前景进行了展望。     

Formation of porcine hepatocyte spherical multicellular aggregates (spheroids) and analysis of drug metabolic functions

Porcine hepatocytes are used in the hybrid artificial liver support system that we are developing because of their high level of liver functions in vitro and because human hepatocytes can not be used in Japan for ethical reasons. Spherical multicellular aggregates or spheroids have been found to be effective in vitro for long-term maintenance of liver functions. Therefore, we formed spherical multicellular aggregates (spheroids) of primary porcine hepatocytes using a polyurethane foam (PUF) as a culture substratum and analyzed their drug metabolic functions in vitro. Primary porcine hepatocytes inoculated into the pores of a flat PUF plate (25 × 25 × 1 mm), spontaneously formed spheroids within the range of 100 to 150 μm in diameter 24 to 36 h after inoculation. The formed spheroids were attached to the bottom surface of the PUF pores, and their morphology and viability were maintained for more than 12 days. The P-450 activity in the spheroids of porcine hepatocytes was demonstrated by detecting production of monoethylglycinexylidide from lidocaine. In addition, the conjugation enzyme activity was demonstrated by detecting glucuronidation and sulfation of acetaminophen. These activities were maintained for 12 days at a level twice as high as in the monolayer culture. This result shows that the porcine hepatocyte spheroids formed by using PUF can maintain the drug metabolic functions important in a hybrid artificial liver device. Consequently, culturing porcine hepatocyte spheroids using PUF seems to be promising for development of a hybrid artificial liver. This revised version was published online in August 2006 with corrections to the Cover Date.     

Bringing 3D tumor models to the clinic – predictive value for personalized medicine

Current decision‐guiding algorithms in cancer drug treatment are based on decades of research and numerous clinical trials. For the majority of patients, this data is successfully applied for a systemic disease management. For a number of patients however, treatment stratification according to clinically based risk criteria will not be sufficient. The most effective treatment options are ideally identified prior to the start of clinical drug therapy. This review will discuss the implementation of three‐dimensional (3D) cell culture models as a preclinical testing paradigm for the efficacy of clinical cancer treatment. Patient tumor‐derived cells in 3D cultures duplicate the individual tumor microenvironment with a minimum of confounding factors. Clinical implementation of such personalized tumor models requires a high quality of methodological and clinical validation comparable to other biomarkers. A non‐systematic literature search demonstrated the small number of prospective studies that have been conducted in this area of research. This may explain the current reluctance of many physicians and insurance providers in implementing this type of assay into the clinical diagnostic routine despite potential benefit for patients. Achieving valid and reproducible results with a high level of evidence is central in improving the acceptance of preclinical 3D tumor models.     

Conductive carbon tape used for support and mounting of both whole animal and fragile heat-treated tissue sections for MALDI MS imaging and quantitation

Analysis of whole animal tissue sections by MALDI MS imaging (MSI) requires effective sample collection and transfer methods to allow the highest quality of in situ analysis of small or hard to dissect tissues. We report on the use of double-sided adhesive conductive carbon tape during whole adult rat tissue sectioning of carboxymethyl cellulose (CMC) embedded animals, with samples mounted onto large format conductive glass and conductive plastic MALDI targets, enabling MSI analysis to be performed on both TOF and FT-ICR MALDI mass spectrometers. We show that mounting does not unduly affect small molecule MSI detection by analyzing tiotropium abundance and distribution in rat lung tissues, with direct on-tissue quantitation achieved. Significantly, we use the adhesive tape to provide support to embedded delicate heat-stabilized tissues, enabling sectioning and mounting to be performed that maintained tissue integrity on samples that had previously been impossible to adequately prepare section for MSI analysis. The mapping of larger peptidomic molecules was not hindered by tape mounting samples and we demonstrate this by mapping the distribution of PEP-19 in both native and heat-stabilized rat brains. Furthermore, we show that without heat stabilization PEP-19 degradation fragments can detected and identified directly by MALDI MSI analysis.     

Modeling adaptive drug resistance of colorectal cancer and therapeutic interventions with tumor spheroids

Drug resistance is a major barrier against successful treatments of cancer patients. Various intrinsic mechanisms and adaptive responses of tumor cells to cancer drugs often lead to failure of treatments and tumor relapse. Understanding mechanisms of cancer drug resistance is critical to develop effective treatments with sustained anti-tumor effects. Three-dimensional cultures of cancer cells known as spheroids present a biologically relevant model of avascular tumors and have been increasingly incorporated in tumor biology and cancer drug discovery studies. In this review, we discuss several recent studies from our group that utilized colorectal tumor spheroids to investigate responses of cancer cells to cytotoxic and molecularly targeted drugs and uncover mechanisms of drug resistance. We highlight our findings from both short-term, one-time treatments and long-term, cyclic treatments of tumor spheroids and discuss mechanisms of adaptation of cancer cells to the treatments. Guided by mechanisms of resistance, we demonstrate the feasibility of designing specific drug combinations to effectively block growth and resistance of cancer cells in spheroid cultures. Finally, we conclude with our perspectives on the utility of three-dimensional tumor models and their shortcomings and advantages for phenotypic and mechanistic studies of cancer drug resistance.     

Assessment of the number of local cytotoxic T lymphocytes required for degradation of micrometer-size tumor spheroids

Adoptive immunotherapy with human cytotoxic T lymphocytes (CTL) is a promising cancer treatment. Previously we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor. Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to 6 days and then increased in number with apparently lethal activity against the tumor spheroid. In contrast to CTLs, the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor site, they can destroy micrometer-size tumors after considerable local proliferation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination

The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light‐dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm² and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm² and light doses of 140 J/cm², 170 J/cm² and 200 J/cm². Irradiation of KillerRed‐expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP‐like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed.

Representative fluorescence image of two KillerRed‐expressing spheroids before and immediately after CW irradiation.     

Targeting cell signaling pathways for drug discovery: an old lock needs a new key

In this age of targeted therapy, the failure of most current drug-discovery efforts to yield safe, effective, and inexpensive drugs has generated widespread concern. Successful drug development has been stymied by a general focus on target selection rather than clinical safety and efficacy. The very process of validating the targets themselves is inefficient and in many cases leads to drugs having poor efficacy and undesirable side effects. Indeed, some rationally designed drugs (e.g., inhibitors of receptor tyrosine kinases, tumor necrosis factor (TNF), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), bcr-abl, and proteasomes) are ineffective against cancers and other inflammatory conditions and produce serious side effects. Since any given cancer carries mutations in an estimated 300 genes, this raises an important question about how effective these targeted therapies can ever be against cancer. Thus, it has become necessary to rethink drug development strategies. This review analyzes the shortcomings of rationally designed target-specific drugs against cancer cell signaling pathways and evaluates the available options for future drug development.     

Cystine-knot peptides: emerging tools for cancer imaging and therapy

Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature’s repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.     

Optical fluorescence imaging with shortwave infrared light emitter nanomaterials for in vivo cell tracking in regenerative medicine

In vivo tracking and monitoring of adoptive cell transfer has a distinct importance in cell‐based therapy. There are many imaging modalities for in vivo monitoring of biodistribution, viability and effectiveness of transferred cells. Some of these procedures are not applicable in the human body because of low sensitivity and high possibility of tissue damages. Shortwave infrared region (SWIR) imaging is a relatively new technique by which deep biological tissues can be potentially visualized with high resolution at cellular level. Indeed, scanning of the electromagnetic spectrum (beyond 1000 nm) of SWIR has a great potential to increase sensitivity and resolution of in vivo imaging for various human tissues. In this review, molecular imaging modalities used for monitoring of biodistribution and fate of administered cells with focusing on the application of non‐invasive optical imaging at shortwave infrared region are discussed in detail.     

Nanosized multifunctional liposomes for tumor diagnosis and molecular imaging by SPECT/CT

Among currently used cancer imaging methods, nuclear medicine modalities provide metabolic information, whereas modalities in radiology provide anatomical information. However, different modalities, having different acquisition times in separate machines, decrease the specificity and accuracy of images. To solve this problem, hybrid imaging modalities were developed as a new era, especially in the cancer imaging field. With widespread usage of hybrid imaging modalities, specific contrast agents are essentially needed to use in both modalities, such as single-photon emission computed tomography/computed tomography (SPECT/CT). Liposomes are one of the most desirable drug delivery systems, depending on their suitable properties. The aim of this study was to develop a liposomal contrast agent for the diagnosis and molecular imaging of tumor by SPECT/CT. Liposomes were prepared nanosized, coated with polyethylene glycol to obtain long blood circulation, and modified with monoclonal antibody 2C5 for specific tumor targeting. Although DTPA-PE and DTPA-PLL-NGPE (polychelating amphilic polymers; PAPs) were loaded onto liposomes for stable radiolabeling for SPECT imaging, iopromide was encapsulated into liposomes for CT imaging. Liposomes [(DPPC:PEG₂₀₀₀-PE:Chol:DTPA-PE), (PL 90G:PEG₂₀₀₀-PE:Chol:DTPA-PE), (DPPC:PEG₂₀₀₀-PE:Chol:PAPs), (PL 90G:PEG₂₀₀₀-PE:Chol:PAPs), (60:0.9:39:0.1% mol ratio)] were characterized in terms of entrapment efficiency, particle size, physical stability, and release kinetics. Additionally, in vitro cell-binding studies were carried out on two tumor cell lines (MCF-7 and EL 4) by counting radioactivity. Tumor-specific antibody-modified liposomes were found to be effective multimodal contrast agents by designating almost 3–8 fold more uptake than nonmodified ones in different tumor cell lines. These results could be considered as an important step in the development of tumor-targeted SPECT/CT contrast agents for cancer imaging.