Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis¹. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread^2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization ^4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces^6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used ⁸. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.Download video file.^{(60M, mov)}     

Morphogenetic and neuronal characterization of human neuroblastoma multicellular spheroids cultured under undifferentiated and all-trans-retinoic acid-differentiated conditions

In this study, we aimed to compare the morphogenetic and neuronal characteristics between monolayer cells and spheroids. For this purpose, we established spheroid formation by growing SH-SY5Y cells on the hydrophobic surfaces of thermally-collapsed elastin-like polypeptide. After 4 days of culture, the relative proliferation of the cells within spheroids was approximately 92% of the values for monolayer cultures. As measured by quantitative assays for mRNA and protein expressions, the production of synaptophysin and neuronspecific enolase (NSE) as well as the contents of cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins are much higher in spheroids than in monolayer cells. Under the all-trans-retinoic acid (RA)-induced differentiation condition, spheroids extended neurites and further up-regulated the expression of synaptophysin, NSE, CAMs, and ECM proteins. Our data indicate that RA-differentiated SH-SY5Y neurospheroids are functionally matured neuronal architectures. [BMB Reports 2013; 46(5): 276-281]     

Establishment of an in vitro organoid model of dermal papilla of human hair follicle

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three‐dimensional (3D) DP spheroids (DPS) with or without a silk–gelatin (SG) microenvironment, which showed enhanced DP‐specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil‐treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell–cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial–mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.     

Monitoring of integrin-mediated adhesion of human ovarian cancer cells to model protein surfaces by quartz crystal resonators: evaluation in the impedance analysis mode

The quartz crystal microbalance (QCM) was used to monitor specific, integrin-mediated adhesion of human ovarian cancer cells to distinct extracellular matrix (ECM) proteins immobilized on gold-coated quartz crystal resonators. The QCM was operated in the impedance analysis mode, where frequency shift as well as bandwidth are accessible on a broad range of overtones. The increase in bandwidth caused by covering the quartz resonator with cells was reproducible and largely independent of overtone order, whereas the frequency shift displayed some variability. Thus the bandwidth proved to be the more robust parameter for sensing cell adhesive events. The bandwidth increased in proportion to the number of seeded cells to the quartz crystal as long as the number was below 150,000 cells/ml. Comparing the resonance parameters on different harmonics, one finds that viscoelastic modeling with homogeneous layer systems cannot reproduce the results: lateral heterogeneity has to be taken into account. The differences in adhesive strength of human ovarian cancer cells towards selected ECM proteins monitored by QCM was in good agreement with data obtained by conventional cell adhesion assays. Strong cell adhesion was observed to the ECM proteins vitronectin (VN) and fibronectin (FN), while only weak attachment occurred on laminin. In order to prove specific, integrin alphavbeta3-mediated cell adhesion to its ligands FN and VN, the cyclic integrin alphavbeta3-directed peptide c(RGDfV) was used as competitor and significantly reversed cell adhesion. Since integrin interaction with ECM proteins is dependent on the presence of bivalent cations, cell detachment was also seen after treatment of cell monolayers with the chelator ethylene-dinitro-tetra-acetic acid (EDTA). The QCM technique is a reliable method to monitor cell adsorption to ECM-pretreated surfaces in real time. It may be an alternative tool for screening specific and selective antagonists of integrin/ECM interaction.     

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.     

Establishment and characterization of an in vitro 3D ovarian cancer model for drug screening assays

The acquired drug chemoresistance represents the main challenge of the ovarian cancer treatment. In addition, the absence of an adequate in vitro model able to reproduce the native tumor environment can contribute to the poor success rate of pre-clinical studies of new compounds. Three-dimensional (3D) culture models have been recently used for drug screening purposes due to their ability to reproduce the main characteristics of in vivo solid tumors. Here we describe the establishment and characterization of 3D ovarian cancer spheroids using different adenocarcinoma tumor cell lines (SKOV-3 and OVCAR-3 cells) in two different 3D scaffold-free methods: forced-floating in ultra-low attachment (ULA) plates and hanging drop (HD). Spheroids were evaluated in both 3D cultures in order to establish the best condition to perform the drug response analysis with Paclitaxel, a common drug used to treat ovarian cancer. SKOV-3 and OVCAR-3 spheroids with the desired characteristics (roundness close to 1.0 and diameter in the 200–500 μm range) were obtained using both methods after addition of the methylcellulose (MC) in the culture medium (0.25% and 0.5%, w/v). We also observed the presence of microvilli on the surface of the spheroids, higher presence of apoptotic cells and higher drug resistance, when compared with 2D cultures. The 3D cultures obtained seem to provide more reliable results in terms of drug response than those provided by 2D monolayer culture. The forced floating method was considered more suitable and straightforward to generate ovarian cancer spheroids for drug screening/cytotoxicity assays.     

Fabrication of oxygen-carrying microparticles functionalized with liver ECM-proteins to improve phenotypic three-dimensional in vitro liver assembly,function, and responses

Oxygen and extracellular matrix (ECM)-derived biopolymers play vital roles in regulating many cellular functions in both the healthy and diseased liver. This study highlights the significance of synergistically tuning the internal microenvironment of three-dimensional (3D) cell aggregates composed of hepatocyte-like cells from the HepG2 human hepatocellular carcinoma cell line and hepatic stellate cells (HSCs) from the LX-2 cell line to enhance oxygen availability and phenotypic ECM ligand presentation for promoting the native metabolic functions of the human liver. First, fluorinated (PFC) chitosan microparticles (MPs) were generated with a microfluidic chip, then their oxygen transport properties were studied using a custom ruthenium-based oxygen sensing approach. Next, to allow for integrin engagements the surfaces of these MPs were functionalized using liver ECM proteins including fibronectin, laminin-111, laminin-511, and laminin-521, then they were used to assemble composite spheriods along with HepG2 cells and HSCs. After in vitro culture, liver-specific functions and cell adhesion patterns were compared between groups and cells showed enhanced liver phenotypic responses to laminin-511 and 521 as evidenced via enhanced E-cadherin and vinculin expression, as well as albumin and urea secretion. Furthermore, hepatocytes and HSCs exhibited more pronounced phenotypic arrangements when cocultured with laminin-511 and 521 modified MPs providing clear evidence that specific ECM proteins have distinctive roles in the phenotypic regulation of liver cells in engineering 3D spheroids. This study advances efforts to create more physiologically relevant organ models allowing for well-defined conditions and phenotypic cell signaling which together improve the relevance of 3D spheroid and organoid models.     

Optimization of the formation of embedded multicellular spheroids of MCF-7 cells: How to reliably produce a biomimetic 3D model

To obtain a multicellular MCF-7 spheroid model to mimic the three-dimensional (3D) of tumors, the microwell liquid overlay (A) and hanging-drop/agar (B) methods were first compared for their technical parameters. Then a method for embedding spheroids within collagen was optimized. For method A, centrifugation assisted cells form irregular aggregates but not spheroids. For method B, an extended sedimentation period of over 24 h for cell suspensions and increased viscosity of the culture medium using methylcellulose were necessary to harvest a dense and regular cell spheroid. When the number was less than 5000 cells/drop, embedded spheroids showed no tight cores and higher viability than the unembedded. However, above 5000 cells/drop, cellular viability of embedded spheroids was not significantly different from unembedded spheroids and cells invading through the collagen were in a sun-burst pattern with tight cores. Propidium Iodide staining indicated that spheroids had necrotic cores. The doxorubicin cytotoxicity demonstrated that spheroids were less susceptible to DOX than their monolayer cells. A reliable and reproducible method for embedding spheroids using the hanging-drop/agarose method within collagen is described herein. The cell culture model can be used to guide experimental manipulation of 3D cell cultures and to evaluate anticancer drug efficacy.     

The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells

Background aimsThe 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques.MethodsWe evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically.ResultsThe liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible.ConclusionsOur results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.     

3D in vitro co-culture models based on normal cells and tumor spheroids formed by cyclic RGD-peptide induced cell self-assembly

Objectives

To design novel 3D in vitro co-culture models based on the RGD-peptide-induced cell self-assembly technique.

Results

Multicellular spheroids from M-3 murine melanoma cells and L-929 murine fibroblasts were obtained directly from monolayer culture by addition of culture medium containing cyclic RGD-peptide. To reach reproducible architecture of co-culture spheroids, two novel 3D in vitro models with well pronounced core–shell structure from tumor spheroids and single mouse fibroblasts were developed based on this approach. The first was a combination of a RGD-peptide platform with the liquid overlay technique with further co-cultivation for 1–2 days. The second allowed co-culture spheroids to generate within polyelectrolyte microcapsules by cultivation for 2 weeks. M-3 cells (a core) and L-929 fibroblasts (a shell) were easily distinguished by confocal microscopy due to cell staining with DiO and DiI dyes, respectively.

Conclusions

The 3D co-culture spheroids are proposed as a tool in tumor biology to study cell–cell interactions as well as for testing novel anticancer drugs and drug delivery vehicles.

     

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     

β1 Integrins restrict the growth of foci and spheroids

Extracellular matrices (ECM) have important roles for tissue architecture, both as structural and signaling components. Members of the integrin family are the main regulators of ECM assembly and transmitters of signals from the ECM to cells. In this study, we have analyzed the role of integrin subunit β1 in two-dimensional (2D) and three-dimensional (3D) cell cultures using integrin β1 null cells (MEFβ1^?/? and GD25) and their β1 integrin-expressing counterparts. GD25 and GD25β1 cells proliferated with similar kinetics in sub-confluent 2D cultures, whereas GD25 cells attained higher cell numbers in confluent culture and formed foci with fivefold higher frequency than GD25β1 cells. Fibronectin fibrils were abundantly deposited throughout the GD25β1 colonies but strictly limited to the central multilayered area (focus) of GD25 colonies. During 3D growth as spheroids, GD25 continuously increased in size for >21?days while the growth of GD25β1 spheroids ceased after 14?days. Similarly, MEFβ1^?/? cells formed foci and grew as spheroids, while the β1 integrin-expressing MEF did not. Expression levels of the cell cycle markers Ki67, PCNA, and histone H3-pSer10 were similar between GD25β1 and GD25 spheroids. Apoptotic cells accumulated earlier in GD25 spheroids; however, cell death increased with spheroid volumes in both spheroid types. In both cell systems, the presence of β1 integrins resulted in higher levels of active myosin light chain and inactive myosin light chain phosphatase, and a more compact spheroid structure. In conclusion, our results reveal that regulation of 3D growth in spheroids and foci is dependent on the β1 subfamily of integrins, and suggest that myosin-based spheroid contraction in combination with cell death limits the growth of β1-expressing spheroids.     

Quartz crystal microbalance biosensor study of endothelial cells and their extracellular matrix following cell removal: Evidence for transient cellular stress and viscoelastic changes during detachment and the elastic behavior of the pure matrix

A quartz crystal microbalance (QCM) cell biosensor utilizing living endothelial cells (ECs) or human breast cancer cells (MCF-7) adhering to the gold QCM surface was used to study the relative contributions of the cells and their underlying extracellular matrix (ECM) to the measured QCM Deltaf and DeltaR shifts. The ECM represents a natural biomaterial that is synthesized by the cells to enable their attachment to surfaces. We followed the detachment of the ECs or MCF-7 cells from their ECM using a nonproteolytic method and were able to apportion the total frequency, Deltaf, decrease of the biosensor into contributions from cell attachment and from the intact underlying ECM. We also demonstrated that the Deltaf shift remaining after EC removal corresponds to ECM as determined by light microscopic visualization of the stained protein. During the process of cell detachment, we observed a novel transient increase in viscoelastic behavior expressed as a transient increase in the motional resistance, DeltaR, parameter. Then we showed via a simulation experiment using ECs stained with fluorescent rhodamine-labeled phalloidin, an actin stain, that the transient viscoelastic increase correlated with cellular stress exhibited by the cells during removal with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'- tetraacetic acid. Prior to cells lifting from their ECM, the attached ECs rearrange their actin microfilaments first into peripheral stress fibers and second into internal aggregates, to maintain cell-cell connectivity, retain their spread morphology, and attempt to adhere more tightly to their underlying ECM. The decrease in DeltaR following its transient rise corresponds to cells finally losing their attachment focal points and lifting from the ECM. We also characterized the normalized f shifts, -Delta(Deltaf)(ECM)/attached cell and -Delta(Deltaf)(cells)/attached cell, as a function of varying the number of adherent cells. Finally, we demonstrate that the underlying native ECM biomaterial, from which all cells have been removed, does not exhibit any significant level of energy dissipation, in contrast to the cells when they are attached to the ECM.     

Metastatic bladder cancer cells distinctively sense and respond to physical cues of collagen fibril-mimetic nanotopography

Tumor metastasis is characterized by enhanced invasiveness and migration of tumor cells through the extracellular matrix (ECM), resulting in extravasation into the blood and lymph and colonization at secondary sites. The ECM provides a physical scaffold consisting of components such as collagen fibrils, which have distinct dimensions at the nanoscale. In addition to the interaction of peptide moieties with tumor cell integrin clusters, the ECM provides a physical guide for tumor cell migration. Using nanolithography we set out to mimic the physical dimensions of collagen fibrils using lined nanotopographical silicon surfaces and to explore whether metastatic tumor cells are uniquely able to respond to these physical dimensions. Etched silicon surfaces containing nanoscale lined patterns with varying trench and ridge sizes (65–500 nm) were evaluated for their ability to distinguish between a non-metastatic (253J) and a highly metastatic (253J-BV) derivative bladder cancer cell line. Enhanced alignment was distinctively observed for the metastatic cell lines on feature sizes that mimic the dimensions of collagen fibrils (65–100 nm lines, 1:1–1:1.5 pitch). Further, these sub-100 nm lines acted as guides for migration of metastatic cancer cells. Interestingly, even at this subcellular scale, metastatic cell migration was abrogated when cells were forced to move perpendicular to these lines. Compared to flat surfaces, 65 nm lines enhanced the formation of actin stress fibers and filopodia of metastatic cells. This was accompanied by increased formation of focal contacts, visualized by immunofluorescent staining of phospho-focal adhesion kinase along the protruding lamellipodia. Simple lined nanotopography appears to be an informative platform for studying the physical cues of the ECM in a pseudo-3D format and likely mimics physical aspects of collagen fibrils. Metastatic cancer cells appear distinctively well adapted to sense these features using filopodia protrusions to enhance their alignment and migration.     

Hydrogel‐based three‐dimensional cell culture for organ‐on‐a‐chip applications

Recent studies have reported that three‐dimensionally cultured cells have more physiologically relevant functions than two‐dimensionally cultured cells. Cells are three‐dimensionally surrounded by the extracellular matrix (ECM) in complex in vivo microenvironments and interact with the ECM and neighboring cells. Therefore, replicating the ECM environment is key to the successful cell culture models. Various natural and synthetic hydrogels have been used to mimic ECM environments based on their physical, chemical, and biological characteristics, such as biocompatibility, biodegradability, and biochemical functional groups. Because of these characteristics, hydrogels have been combined with microtechnologies and used in organ‐on‐a‐chip applications to more closely recapitulate the in vivo microenvironment. Therefore, appropriate hydrogels should be selected depending on the cell types and applications. The porosity of the selected hydrogel should be controlled to facilitate the movement of nutrients and oxygen. In this review, we describe various types of hydrogels, external stimulation‐based gelation of hydrogels, and control of their porosity. Then, we introduce applications of hydrogels for organ‐on‐a‐chip. Last, we also discuss the challenges of hydrogel‐based three‐dimensional cell culture techniques and propose future directions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:580–589, 2017     

Integration of hyper-compliant microparticles into a 3D melanoma tumor model

Multicellular spheroids provide a physiologically relevant platform to study the microenvironment of tumors and therapeutic applications, such as microparticle-based drug delivery. The goal of this study was to investigate the incorporation/penetration of compliant polyacrylamide microparticles (MPs), into either cancer or normal human cell spheroids. Incorporation of collagen-1-coated MPs (stiffness: 0.1 and 9 kPa; diameter: 15–30 µm) into spheroids (diameter ∼100 µm) was tracked for up to 22 h. Results indicated that cells within melanoma spheroids were more influenced by MP mechanical properties than cells within normal cell spheroids. Melanoma spheroids had a greater propensity to incorporate and displace the more compliant MPs over time. Mature spheroids composed of either cell type were able to recognize and integrate MPs. While many tumor models exist to study drug delivery and efficacy, the study of uptake and incorporation of cell-sized MPs into established spheroids/tissues or tumors has been limited. The ability of hyper-compliant MPs to successfully penetrate 3D tumor models with natural extracellular matrix deposition provides a novel platform for potential delivery of drugs and other therapeutics into the core of tumors and micrometastases.     

Novel fluid shear‐based dissociation device for improved single cell dissociation of spheroids and cell aggregates

Biological industries commonly rely on bioreactor systems for the large‐scale production of cells. Cell aggregation, clumping, and spheroid morphology of certain suspension cells make their large‐scale culture challenging. Growing stem cells as spheroids is indispensable to retain their stemness, but large spheroids (>500 µm diameter) suffer from poor oxygen and nutrient diffusion, ultimately resulting in premature cell death in the centers of the spheroids. Despite this, most large‐scale bioprocesses do not have an efficient method for dissociating cells into single cells, but rely on costly enzymatic dissociation techniques. Therefore, we tested a proof‐of‐concept fluid shear‐based mechanical dissociator that was designed to dissociate stem cell spheroids and aggregates. Our prototype was able to dissociate cells while retaining high viability and low levels of apoptosis. The dissociator also did not impact long‐term cell growth or spheroid formation. Thus, the dissociator introduced here has the potential to replace traditional dissociation methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:293–298, 2018     

Synthesis and selective cytotoxicity of a hyaluronic acid-antitumor bioconjugate.

A cell-targeted prodrug was developed for the anti-cancer drug Taxol, using hyaluronic acid (HA) as the drug carrier. HA-Taxol bioconjugates were synthesized by linking the Taxol 2'-OH via a succinate ester to adipic dihydrazide-modified HA (HA-ADH). The coupling of Taxol-NHS ester and HA-ADH provided several HA bioconjugates with different levels of ADH modification and different Taxol loadings. A fluorescent BODIPY-HA was also synthesized to illustrate cell targeting and uptake of chemically modified HA using confocal microscopy. HA-Taxol conjugates showed selective toxicity toward the human cancer cell lines (breast, colon, and ovarian) that are known to overexpress HA receptors, while no toxicity was observed toward a mouse fibroblast cell line at the same concentrations used with the cancer cells. The drug carrier HA-ADH was completely nontoxic. The selective cytotoxicity is consistent with the results from confocal microscopy, which demonstrated that BODIPY-HA only entered the cancer cell lines.     

Differential sensitivity to short-chain ceramide analogues of human intestinal carcinoma cells grown in tumor spheroids versus monolayer culture

The cytotoxic activity of short-chain (C(2)) ceramide was evaluated in human intestinal carcinoma cells grown as multicellular tumor spheroids versus the same cells cultured as monolayers under closely comparable conditions. A decrease in cell number was seen in monolayer cultures of HT-29, Caco-2, and HRT-18 cells, with an EC(50) (concentration for half-maximal toxicity) of between 13 and 23 microM. However, when the same cells were grown in the multicellular spheroid format, C(2) was markedly less potent in reducing cell number, with an EC(50) of between 44 and 63 microM, representing a 1.9- to 4.9-fold decrease in its potency. The chemotherapeutic agents 5-fluorouracil and cisplatin were equally potent against spheroids and monolayer cultures, indicating that although drug access is a problem in conventionally grown tumor spheroids it is not a problem for spheroids grown under the conditions used in this study. Our results suggest that although ceramide is capable of inducing cell death in intestinal carcinoma cells grown in spheroid culture, its cellular toxicity is constrained by influences that are independent of drug access and may be the consequence of the altered cellular relationships. Carcinoma cell populations show an intrinsically decreased responsiveness to the effects of ceramide when they are grown in a three-dimensional culture format.