Imaging Of Spheroids Research Articles

Abstract 363: Establishment of multicellular tumor spheroids-based assay for screening of novel therapeutics

Abstract 3D cell culture system is highlighted as new method to screen for new cancer therapies. The multicellular tumor spheroid cultures are closely similar to pathophysiological gradients of in vivo tumors and subsequently an ideal screening model for new drugs. Hepatocellular carcinomas (HCC) is resistant to conventional chemotherapeutic agents and remains an unmet medical need. Hence, we focused in developing tumor spheroids-based screening assay to identify new drugs to treat HCC. Tumor spheroid cultures were formed in hydrogel coated multi-well plates within 3 days followed by 3 days treatment with reference compounds (Doxorubicin, Etoposide, Methotrexate, Taxol, etc.). Bright images of tumor spheroids were visualized by Operetta imaging system in the presence and absence of reference compounds. Cell viability of spheroids were measured by resazurin assay. In the results, assay was developed and validated using multicellular tumor spheroids (MCTS) cultures composed of Huh7 (HCC cells), WI38 (fibroblasts), LX2 (hepatic stellate cells) and HUVEC (endothelial cells) to recapitulate HCC tumor microenvironments. We also addressed the effect of sorafenib on the growth of spheroids composed of Huh7 alone or MCTS. Results from this study indicate that sorafenib was less efficacious (IC50 = 8.0μM) in the MCTS compared to its effect on Huh7 spheroid (IC50 = 3.8 μM). In summary, we have established and validated a highly reproducible MCTS-based assay. Based on this results, MCTS based-HTS system can offer a new model for drug screening and significantly improve the efficiency of identifying new drugs for liver cancer therapy. Citation Format: Yeonhwa Song, Haengran Seo. Establishment of multicellular tumor spheroids-based assay for screening of novel therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 363.

Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening.

Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due to issues with running complex assays on large three-dimensional (3D) structures.To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned 96-well plates with automated production of nine uniform spheroids per well. Spheroids achieve diameters of up to 600 µm, and reproducibility was experimentally validated (interwell and interplate CV(diameter) <5%). Biphoton imaging confirmed that micropatterned spheroids exhibit characteristic cell heterogeneity with distinct microregions. Furthermore, central necrosis appears at a consistent spheroid size, suggesting standardized growth.Using three reference compounds (fluorouracil, irinotecan, and staurosporine), we validated HT-29 micropatterned spheroids on an HCS platform, benchmarking against hanging-drop spheroids. Spheroid formation and imaging in a single plate accelerate assay workflow, and fixed positioning prevents structures from overlapping or sticking to the well wall, augmenting image processing reliability. Furthermore, multiple spheroids per well increase the statistical confidence sufficiently to discriminate compound mechanisms of action and generate EC50 values for endpoints of cell death, architectural change, and size within a single-pass read. Higher quality data and a more efficient HCS work chain should encourage integration of micropatterned spheroid models within fundamental research and drug discovery applications.

Abstract 314: A rapid 3D tumor spheroid analysis method using the Celigo imaging cytometry

Abstract The current 2D methods for cancer drug discovery have had some difficulty in identifying potential drug candidates that can be used for clinical testing. To overcome this challenge, there has been an increase in research of 3D tissue culture that facilitated the development of new in-vitro tumor model assays. Traditional 2D and 3D analysis method relied heavily on visual observation using microscopy. However, the method is time-consuming and has high variations. Automated plate-based imaging cytometer can be employed to rapidly analyze and characterize 3D tumor spheroids, which can be used to generate both quantitative and qualitative results. In this work, we demonstrate a novel 3D tumor spheroid analysis method using the Celigo imaging cytometer for spheroid counting, size analysis, tumor migration and invasion, tumor viability, and dose response of drug induced/inhibited tumor growth. The plate-based imaging cytometer utilizes bright-field and three fluorescence channels (Blue, Green, and Red) for multi-channel analysis. By utilizing the F theta lens technology, uniform bright-field image is captured for more accurate counting of the entire well. In addition, Celigo analysis software is used to report numerous parameters allowing detailed spheroid characterization. In addition to direct spheroid counting in the well, the use of specific fluorescent dyes and probes allow the user to define viable and hypoxic areas within spheroids and monitor migration and invasion on or into supporting cells and/or tissues. The results showed that Celigo imaging cytometer can accurately count and measure spheroid sizes in response to drug induction. Furthermore, tumor migration and invasion were clearly observed and quantified in the captured images. By utilizing the 3D spheroid imaging cytometry method, researchers can quickly characterize and quantify tumor spheroids, which can improve the efficiency of identification of potential cancer drug candidates. Citation Format: Leo Li-Ying Chan, Scott Cribbes, Maria Vinci, Sarah Kessel, Lisa Patterson, Sue Eccles. A rapid 3D tumor spheroid analysis method using the Celigo imaging cytometry. [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 314. doi:10.1158/1538-7445.AM2015-314

High-content analysis of tumour cell invasion in three-dimensional spheroid assays.

Targeting infiltrating tumour cells is an attractive way of combating cancer invasion and metastasis. Here we describe a novel and reproducible method for high content analysis of invading cells using multicellular tumour spheroid assays in a high grade glioma model. Live cell imaging of spheroids generated from glioma cell lines, U87 and U251, gave insight into migration dynamics and cell morphology in response to anti-migratory drugs. Immunofluorescence imaging confirmed cytoskeletal rearrangements in the treated cells indicating a direct effect on cell morphology. Effect on migration was determined by a Migration Index (MI) from brightfield images which confirmed anti-migratory activity of the drugs. A marked effect on the core with treatment suggestive of disordered proliferation was also observed. A newly developed technique to prepare the spheroids and migratory cells for immunohistochemistry allowed an assessment of response to drug treatment with a selection of markers. A difference in protein expression was noted between cells maintained within the core and migratory cells indicative of the presence of cell subpopulations within the spheroid core. We conclude that this high content analysis allows researchers to perform screening of anti-tumour invasion compounds and study their effects on cellular dynamics, particularly in relation to protein expression, for the first time.

Live spheroid formation recorded with light sheet-based fluorescence microscopy.

We provide a detailed protocol for a three-dimensional long-term live imaging of cellular spheroids with light sheet-based fluorescence microscopy. The protocol allows the recording of all phases of spheroid formation in three dimensions, including cell proliferation, aggregation, and compaction. We employ the human hepatic cell line HepaRG transfected with the fusion protein H2B-GFP, i.e., a fluorescing histone. The protocol allows monitoring the effect of drugs or toxicants.

SU-E-T-565: RAdiation Resistance of Cancer CElls Using GEANT4 DNA: RACE

Purpose: The objective of the RACE project is to develop a comparison between Monte Carlo simulation using the Geant4-DNA toolkit and measurements of radiation damage on 3D melanoma and chondrosarcoma culture cells coupled with gadolinium nanoparticles. We currently expose the status of the developments regarding simulations. Methods: Monte Carlo studies are driven using the Geant4 toolkit and the Geant4-DNA extension. In order to model the geometry of a cell population, the opensource CPOP++ program is being developed for the geometrical representation of 3D cell populations including a specific cell mesh coupled with a multi-agent system. Each cell includes cytoplasm and nucleus. The correct modeling of the cell population has been validated with confocal microscopy images of spheroids. The Geant4 Livermore physics models are used to simulate the interactions of a 250 keV X-ray beam and the production of secondaries from gadolinium nanoparticles supposed to be fixed on the cell membranes. Geant4-DNA processes are used to simulate the interactions of charged particles with the cells. An atomistic description of the DNA molecule, from PDB (Protein Data Bank) files, is provided by the so-called PDB4DNA Geant4 user application we developed to score energy depositions in DNA base pairs and sugar-phosphate groups. Results: At the microscopic level, our simulations enable assessing microscopic energy distribution in each cell compartment of a realistic 3D cell population. Dose enhancement factors due to the presence of gadolinium nanoparticles can be estimated. At the nanometer scale, direct damages on nuclear DNA are also estimated. Conclusion: We successfully simulated the impact of direct radiations on a realistic 3D cell population model compatible with microdosimetry calculations using the Geant4-DNA toolkit. Upcoming validation and the future integration of the radiochemistry module of Geant4-DNA will propose to correlate clusters of ionizations with in vitro experiments. All those developments will be released publicly. This work was supported by grants from Plan Cancer 2009-2013 French national initiative managed by INSERM (Institut National de la Sante et de la Recherche Medicale)

Fluorometric assessment of acetaminophen-induced toxicity in rat hepatocyte spheroids seeded on micro-space cell culture plates

Hepatotoxicity induced by the metabolic activation of drugs is a major concern in drug discovery and development. Three-dimensional (3-D) cultures of hepatocyte spheroids may be superior to monolayer cultures for evaluating drug metabolism and toxicity because hepatocytes in spheroids maintain the expression of various metabolizing enzymes and transporters, such as cytochrome P450 (CYP). In this study, we examined the hepatotoxicity due to metabolic activation of acetaminophen (APAP) using fluorescent indicators of cell viability and intracellular levels of glutathione (GSH) in rat hepatocyte spheroids grown on micro-space cell culture plates. The mRNA expression levels of some drug-metabolizing enzymes were maintained during culture. Additionally, this culture system was compatible with microfluorometric imaging under confocal laser scanning microscopy. APAP induced a decrease in intracellular ATP at 10mM, which was blocked by the CYP inhibitor 1-aminobenzotriazole (ABT). APAP (10mM, 24h) decreased the levels of both intracellular ATP and GSH, and GSH-conjugated APAP (APAP-GSH) were formed. All three effects were blocked by ABT, confirming a contribution of APAP metabolic activation by CYP to spheroid toxicity. Fluorometric imaging of hepatocyte spheroids on micro-space cell culture plates may allow the screening of drug-induced hepatotoxicity during pharmaceutical development.

Small molecule phosphorescent probes for O2 imaging in 3D tissue models.

Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O2 sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present several new cell-penetrating phosphorescent conjugates of a Pt(ii)-tetrakis(pentafluorophenyl)porphine (PtPFPP) dye produced by click-modification with thiols, and perform their evaluation as O2 imaging probes for 3D tissue models. The hydrophilic glucose (Pt-Glc) and galactose (Pt-Gal) conjugates demonstrated minimal aggregation and self-quenching in aqueous media, and efficient in-depth staining of different cell types and multi-cellular aggregates at working concentrations ≤10 μM. The Pt-Glc probe was applied in high-resolution phosphorescence lifetime based O2 imaging (PLIM) in multi-cellular spheroids of cancer cells (PC12), primary neural cells (neurospheres) and slices of brain tissue, where it showed good analytical performance, minimal effects on cell viability and appropriate responses to O2 with phosphorescence lifetimes changing from 20 μs in air-saturated to 57 μs under deoxygenated conditions. In contrast, mono- and tetra-substituted oligoarginine conjugates of PtPFPP showed marked aggregation and unstable photophysical properties precluding their use as O2 sensing probes.

Quantifying the autophagy-triggering effects of drugs in cell spheroids with live fluorescence microscopy.

We present a 3D assay for the quantification of the autophagic flux in live cell spheroids by using the fluorescent reporter mRFP-GFP-LC3. The protocol describes the formation of the spheroids from the astrocytoma cell line U343, live long-term 3D fluorescence imaging of drug-treated spheroids, and the image processing workflow required to extract quantitative data on the autophagic flux.

Transfer, Imaging, and Analysis Plate for Facile Handling of 384 Hanging Drop 3D Tissue Spheroids

Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated high-fidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type-dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging.

Acoustic and photoacoustic imaging of spheroids

Acoustic and photoacoustic high frequency imaging (50–100 MHz) can be used to generate images of cell constructs and spheroids with good spatial resolution and contrast. Here we demonstrate how co-registered acoustic and photoacoustic imaging can be used for imaging spheroids. Spheroids are widely used in cancer research and biology since they emulate a three-dimensional environment such as that experienced in tumors. Spheroids were made by the hanging-drop method using the MCF-7 cancer cell line. To generate photoacoustic contrast, MCF-7 cells were incubated with optical absorbing nanoparticles (e.g., gold nanorods, 780 nm absorption) for 24 h and mixed with native MCF-7 cells prior to spheroid formation. The spheroids were between 0.5 mm and 1mm in diameter. Imaging was performed with the VisualSonics VEVO 770 (25–55 MHz) and a high-resolution SASAM acoustic/photoacoustic microscope for frequencies over 80 MHz (Kibero GmbH, Germany). The spheroid was imaged first using pulse echo ultrasound, then with photoacoustics immediately after. The necrotic core of the spheroid had a 20 dB increase in ultrasound backscatter compared the viable cells surrounding the core, and the ultrasound/photoacoustic images of the spheroid were co-registered showing the distribution of the optical absorbing agents.

Multicellular Tumor Spheroids as an in Vivo–Like Tumor Model for Three-Dimensional Imaging of Chemotherapeutic and Nano Material Cellular Penetration

We present a flexible and highly reproducible method using three-dimensional (3D) multicellular tumor spheroids to quantify chemotherapeutic and nanoparticle penetration properties in vitro. We generated HeLa cell-derived spheroids using the liquid overlay method. To properly characterize HeLa spheroids, scanning electron microscopy, transmission electron microscopy, and multiphoton microscopy were used to obtain high-resolution 3D images of HeLa spheroids. Next, pairing high-resolution optical characterization techniques with flow cytometry, we quantitatively compared the penetration of doxorubicin, quantum dots, and synthetic micelles into 3D HeLa spheroid versus HeLa cells grown in a traditional two-dimensional culturing system. Our data revealed that 3D cultured HeLa cells acquired several clinically relevant morphologic and cellular characteristics (such as resistance to chemotherapeutics) often found in human solid tumors. These characteristic, however, could not be captured using conventional two-dimensional cell culture techniques. This study demonstrated the remarkable versatility of HeLa spheroid 3D imaging. In addition, our results revealed the capability of HeLa spheroids to function as a screening tool for nanoparticles or synthetic micelles that, due to their inherent size, charge, and hydrophobicity, can penetrate into solid tumors and act as delivery vehicles for chemotherapeutics. The development of this image-based, reproducible, and quantifiable in vitro HeLa spheroid screening tool will greatly aid future exploration of chemotherapeutics and nanoparticle delivery into solid tumors.

Live-cell 3D super-resolution imaging in thick biological samples

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.

Probing cellular mechanobiology in three-dimensional culture with collagen–agarose matrices

The study of how cell behavior is controlled by the biophysical properties of the extracellular matrix (ECM) is limited in part by the lack of three-dimensional (3D) scaffolds that combine the biofunctionality of native ECM proteins with the tunability of synthetic materials. Here, we introduce a biomaterial platform in which the biophysical properties of collagen I are progressively altered by adding agarose. We find that agarose increases the elasticity of 3D collagen ECMs over two orders of magnitude with modest effect on collagen fiber organization. Surprisingly, increasing the agarose content slows and eventually stops invasion of glioma cells in a 3D spheroid model. Electron microscopy reveals that agarose forms a dense meshwork between the collagen fibers, which we postulate slows invasion by structurally coupling and reinforcing the collagen fibers and introducing steric barriers to motility. This is supported by time lapse imaging of individual glioma cells and multicellular spheroids, which shows that addition of agarose promotes amoeboid motility and restricts cell-mediated remodeling of individual collagen fibers. Our results are consistent with a model in which agarose shifts ECM dissipation of cell-induced stresses from non-affine deformation of individual collagen fibers to bulk-affine deformation of a continuum network.

Three-dimensional holographic imaging of living tissue using a highly sensitive photorefractive polymer device

Photorefractive materials are dynamic holographic storage media that are highly sensitive to coherent light fields and relatively insensitive to a uniform light background. This can be exploited to effectively separate ballistic light from multiply-scattered light when imaging through turbid media. We developed a highly sensitive photorefractive polymer composite and incorporated it into a holographic optical coherence imaging system. This approach combines the advantages of coherence-domain imaging with the benefits of holography to form a high-speed wide-field imaging technique. By using coherence-gated holography, image-bearing ballistic light can be captured in real-time without computed tomography. We analyzed the implications of Fourier-domain and image-domain holography on the field of view and image resolution for a transmission recording geometry, and demonstrate holographic depth-resolved imaging of tumor spheroids with 12 microm axial and 10 microm lateral resolution, achieving a data acquisition speed of 8 x 10(5) voxels/s.

Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy

Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stably transfected with a vector constructed with multiple copies of the HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1α (hypoxia-inducible factor-1α) is stabilized, the binding of HIF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfected monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1α expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (<100 μm) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities.

Fourier-domain holographic optical coherence imaging of tumor spheroids and mouse eye

Fourier-domain holography (FDH) has several advantages over image-domain holography for optical coherence imaging of tissue. Writing the hologram in the Fourier plane significantly reduces background arising from reference light scattered from the photorefractive holographic film. The ability to use FDH is enhanced by the use of a diffuse target, such as scattering tissue, rather than specular targets, because the broader angular distribution from diffuse targets is transformed into a relatively uniform distribution in the Fourier plane. We demonstrate significantly improved performance for Fourier-domain optical coherence imaging on rat osteogenic sarcoma tumor spheroids and mouse eye. The sensitivity is documented at -95 dB.

Holographic optical coherence imaging of rat osteogenic sarcoma tumor spheroids.

Holographic optical coherence imaging is a full-frame variant of coherence-domain imaging. An optoelectronic semiconductor holographic film functions as a coherence filter placed before a conventional digital video camera that passes coherent (structure-bearing) light to the camera during holographic readout while preferentially rejecting scattered light. The data are acquired as a succession of en face images at increasing depth inside the sample in a fly-through acquisition. The samples of living tissue were rat osteogenic sarcoma multicellular tumor spheroids that were grown from a single osteoblast cell line in a bioreactor. Tumor spheroids are nearly spherical and have radial symmetry, presenting a simple geometry for analysis. The tumors investigated ranged in diameter from several hundred micrometers to over 1 mm. Holographic features from the tumors were observed in reflection to depths of 500-600 microm with a total tissue path length of approximately 14 mean free paths. The volumetric data from the tumor spheroids reveal heterogeneous structure, presumably caused by necrosis and microcalcifications characteristic of some human avascular tumors.

Holographic optical coherence imaging of tumor spheroids

We present depth-resolved coherence-domain images of living tissue using a dynamic holographic semiconductor film. An AlGaAs photorefractive quantum-well device is used in an adaptive interferometer that records coherent backscattered (image-bearing) light from inside rat osteogenic sarcoma tumor spheroids up to 1 mm in diameter in vitro. The data consist of sequential holographic image frames at successive depths through the tumor represented as a visual video “fly-through.” The images from the tumor spheroids reveal heterogeneous structures presumably caused by necrosis and microcalcifications characteristic of human tumors in their early avascular growth.

Use of a high frequency ultrasound microscope to image the action of 2-nitroimidazoles in multicellular spheroids.

A system was designed to allow imaging of control and drug treated multicellular spheroids with a high frequency backscatter ultrasound microscope. It allowed imaging of individual spheroids under good growth conditions. Since little data were available on cellular toxicity of ultrasound at these high frequencies (80 MHz), studies were undertaken to evaluate effects on cell survival, using a colony forming assay. No toxicity was observed on cell monolayers subjected to pulsed ultrasound at the intensities used for imaging experiments. Spheroids were also subjected to pulsed ultrasound and no growth delay was observed when exposed spheroids were compared with mock-exposed spheroids. Imaging studies were performed and pictures of untreated spheroids were obtained in which the necrotic and viable regions are clearly distinguishable. When the hypoxic cell cytotoxin 1-methyl-2-nitroimidazole (INO2) was added to the spheroid, dramatic changes were observed in the backscatter signal. The interior viable cells of the spheroid were selectively affected. Changes in the backscatter signal were also observed when the reduction product 1-methyl-2-nitrosoimidazole (INO) was added to spheroids. With INO however, the changes were located at the periphery of the spheroid, presumably due to the high reactivity of INO which limits diffusion of the drug into the spheroid. The present work demonstrates the potential usefulness of ultrasound backscatter microscopy in following the action of selected drugs in this in vitro tumour model.