Core Of Spheroids Research Articles

Remodeling of Adhesion Network within Cancer Spheroids via Cell-Polymer Interaction.

3D spheroids are considered as the improved in vitro model to mimic the distinct arrangements of the cells in vivo. To date, low-attachment surfaces have been most widely used to induce the spontaneous aggregation of cells in suspension by simply tuning the relative strength of the cell-cell adhesion over cell-substrate adhesion. However, aggregating cancer cells into 3D clusters should mean more than just adjoining the cells in the physical proximity. The tumor cell functionality is strongly affected by the adhesion networks between cancer cells and extracellular matrix (ECM). Here, we performed an in-depth analysis of how the nonmetastatic breast cancer cells (MCF7) can be transformed to gain invasive phenotypes through compact aggregation into 3D spheroids on a functional polymer film surface, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4). By comparing the adhesion networks and invasion dynamics between 3D spheroids cultured on the pV4D4 surface with those cultured on conventional ultra-low-attachment (ULA) dishes, we report that only spheroids on the pV4D4 display active and sporadic cell-surface binding activities via dynamic protrusions, which correlates strongly with an increase in integrin β1. Moreover, localized laminin expression at the core of the pV4D4-cultured spheroids confirms the prominence of the intimate integrin-laminin interactions prompted by the exposure to pV4D4. This study suggests that structurally and functionally dissimilar 3D spheroids can be generated from the same type of cells on the surfaces of different physicochemical properties without any chemical treatment or genetic manipulation.

Abstract 6317: 3D multicellular coculture platform enables rapid engineering of tumor-homing bacteria

Abstract Engineering tumor-homing bacteria to locally deliver therapeutics is an emerging approach for next-generation living therapy for cancer. However, the lack of physiologically-relevant in vitro testing environments to rapidly and quantitatively characterize complex multicellular interactions of bacteria and cancer cells is a major bottleneck for clinical translation. We recently developed a 3D multicellular coculture platform that grow various bacteria within the necrotic core of multicellular spheroids, recapitulating selective bacterial growth in solid tumor in vivo. Tumor spheroids were generated from CT26 colorectal cell lines in a round-bottom low-adhesion 96 well plate. Bacteria were inoculated into the media containing tumor spheroids and gentamicin, a poorly diffusible antimicrobial agent, were added to confine bacterial growth to spheroid cores. Time-lapse microscopy was utilized to track growth dynamics and spatial profiles. To validate efficacy, mice with hind flank tumors were treated with engineered bacteria and measured for the therapeutic effect on tumor size. The multicellular coculture platform allowed for parallel and continuous culture of bacteria in tumor spheroids. Notably, bacteria stably grew within the spheroid necrotic cores, representing tumor colonization in animals and patients. Leveraging the high throughput nature of the platform, we screened a library of ten cytotoxic molecules delivered by clinically relevant tumor-homing S. typhimurium. We validated similarities in efficacies using a syngeneic mouse model and identified novel therapeutic candidates that reduced tumor growth significantly. Additionally, we utilized the tumor spheroid to construct bacterial biosensors. As tumor microenvironment such as oxygen, pH, and metabolite gradients are recapitulated in the 3D spheroid setting, we tested hypoxia, acid and lactate sensors and confirmed their activity in tumor environment. Constructing an AND-gate logic circuit for bacterial growth using synthetic biology tools, we rapidly tested the bacterial containment capability to the spheroid cores. This strain demonstrated a 100-fold reduction in off-target colonization compared to the wildtype strain in an animal model. Rapid and quantitative characterization of dynamical bacteria-cancer cell interactions can predict therapeutic performance more accurately. We hope this scalable approach serves to accelerate future clinical development of bacterial applications for cancer therapy. Citation Format: Tetsuhiro Harimoto, Zakary Singer, Oscar Velazquez, Joanna Zhang, Samuel Castro, Taylor Hinchliffe, William Mather, Tal Danino. 3D multicellular coculture platform enables rapid engineering of tumor-homing bacteria [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6317.

Incorporation of Gelatin Microspheres into HepG2 Human Hepatocyte Spheroids for Functional Improvement through Improved Oxygen Supply to Spheroid Core

The multicellular spheroid three-dimensional cell culture system can be used as a formulation for cell-based therapy. However, the viability and functions of the cells in the core region of the spheroid tend to decrease because of limited oxygen supply. In this study, we incorporated gelatin microspheres (GMS) into HepG2 human hepatocyte spheroids to allow oxygen to reach the spheroid core. GMS with an approximate diameter of 37 µm were fabricated by water-in-oil emulsification followed by freeze drying. GMS-containing HepG2 spheroids (GMS/HepG2 spheroids) were prepared by incubation of the cells with GMS at various mixing ratios in agarose gel-based microwells. Increasing the GMS ratio increased the diameter of the spheroids, and few spheroids formed with excess GMS. HepG2 cells in the GMS/HepG2 spheroids were more oxygenated than those in the GMS-free spheroids. GMS incorporation increased the viability of HepG2 cells in the spheroids and increased the CYP1A1 activity of the cells to metabolize 7-ethoxyresorufin, although mRNA expression of the CYP1A1 gene was hardly affected by GMS incorporation. These results indicate that incorporating GMS into HepG2 spheroids improves the hypoxic microenvironment in the spheroids and increases cell viability and CYP1A1 metabolic activity.

Dark matter annihilation in ω Centauri: Astrophysical implications derived from the MWA radio data

We present an analysis of Murchison Widefield Array radio telescope data from $\omega$ Cen, possibly a stripped dwarf spheroidal galaxy core captured by our Galaxy. Recent interpretations of Fermi-LAT $\gamma$-ray data by Brown {\it et al.} (2019) and Reynoso-Cordova {\it et al.} (2019) suggest that $\omega$ Cen may contain significant Dark Matter. We utilise their best-fit Dark Matter annihilation models, and an estimate of the magnetic field strength in $\omega$ Cen, to calculate the expected radio synchrotron signal from annihilation, and show that one can usefully rule out significant parts of the magnetic field - diffusion coefficient plane using our current observational limits on the radio emission. Improvement by a factor of 10-100 on these limits could constrain the models even more tightly.

Characterization of Uveal Melanoma Cell Lines and Primary Tumor Samples in 3D Culture.

PurposeUveal melanoma (UM) typically spreads to the liver, where it is incurable, as there are limited therapeutic interventions available. This study aimed to standardize laboratory methods for generating three-dimensional (3D) spheroids using UM cell lines and primary UM (PUM) samples for use in drug screening.MethodsSix UM cell lines and nine PUM, of differing genetic characteristics were cultured in two dimensions (2D) and three dimensions. 3D spheroid formation and growth were time monitored, and ImageJ software was used to calculate cross-sectional areas. PUM spheroids underwent immunohistochemistry for melanoma markers, nuclear BAP1, and cell proliferation. Chromosomal alterations in patient UM biopsies were compared with the corresponding 3D spheroid. In vitro drug assays testing doxorubicin and selumetinib assessed drug penetration and toxicity after 48 hours using imaging and the CellTiter-Glo 3D Cell Viability Assay.ResultsAll six UM cell lines formed spheroids of varying sizes and compactness; six of the nine PUM samples (67%) also formed spheroids, composed of MelanA+ proliferating melanocytes and admixed macrophages. PUM spheroids were genetically identical to the original sampled tumor. In vitro drug assays showed varying penetrations into UM cell line spheroids, with doxorubicin passing into the spheroid core and selumetinib having an effect largely on peripheral cells. Both drugs caused a dose-dependent reduction in viability of 3D spheroid cells.ConclusionsUM cell lines and PUM samples can successfully generate uniform 3D spheroids. PUM spheroids retain histological and genetic characteristics of the primary tumor. 3D spheroids are an important system for use in future high-throughput drug testing.Translational RelevanceThe use of 3D spheroids allows early-phase drug screening and is an important first step toward treatment personalization for UM patients.

Three-dimensional structure of the Sagittarius dwarf spheroidal core from RR Lyrae

ABSTRACT We obtain distances to a sample of RR Lyrae in the central core of the Sagittarius dwarf spheroidal galaxy from OGLE data. We use these distances, along with RR Lyrae from Gaia DR2, to measure the shape of the stellar distribution within the central ∼2 kpc. The best-fitting stellar distribution is triaxial, with axis ratios 1 : 0.76 : 0.43. A prolate-spheroid model is ruled out at high statistical significance relative to the triaxial model. The major axis is aligned nearly parallel to the sky plane as seen by an Earth-based observer and is nearly perpendicular to the direction of the Galactic Centre. This result may be compared to cosmological simulations which generally predict that the major axis of the dark matter distribution of subhalos is aligned with the Galactic Centre. The triaxial structure that we obtain can provide important constraints on the Sagittarius progenitor, as well as the central dark matter distribution under the assumption of dynamical equilibrium.

Ultrasmall gold nanoparticles (2 nm) can penetrate and enter cell nuclei in an in vitro 3D brain spheroid model

The neurovascular unit (NVU) is a complex functional and anatomical structure composed of endothelial cells and their blood-brain barrier (BBB) forming tight junctions. It represents an efficient barrier for molecules and drugs. However, it also prevents a targeted transport for the treatment of cerebral diseases. The uptake of ultrasmall nanoparticles as potential drug delivery agents was studied in a three-dimensional co-culture cell model (3D spheroid) composed of primary human cells (astrocytes, pericytes, endothelial cells). Multicellular 3D spheroids show reproducible NVU features and functions. The spheroid core is composed mainly of astrocytes, covered with pericytes, while brain endothelial cells form the surface layer, establishing the NVU that regulates the transport of molecules. After 120 h cultivation, the cells self-assemble into a 350 µm spheroid as shown by confocal laser scanning microscopy. The passage of different types of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm) both into the spheroid and into three constituting cell types was studied by confocal laser scanning microscopy. Three kinds of covalently fluorophore-conjugated gold nanoparticles were used: One with fluorescein (FAM), one with Cy3, and one with the peptide CGGpTPAAK-5,6-FAM-NH2. In 2D cell co-culture experiments, it was found that all three kinds of nanoparticles readily entered all three cell types. FAM- and Cy3-labelled nanoparticles were able to enter the cell nucleus as well. The three dissolved dyes alone were not taken up by any cell type. A similar situation evolved with 3D spheroids: The three kinds of nanoparticles entered the spheroid, but the dissolved dyes did not. The presence of a functional blood-brain barrier was demonstrated by adding histamine to the spheroids. In that case, the blood-brain barrier opened, and dissolved dyes like a FITC-labelled antibody and FITC alone entered the spheroid. In summary, our results qualify ultrasmall gold nanoparticles as suitable carriers for imaging or drug delivery into brain cells (sometimes including the nucleus), brain cell spheroids, and probably also into the brain. Statement of significance3D brain spheroid model and its permeability by ultrasmall gold nanoparticles.We demonstrate that ultrasmall gold nanoparticles can easily penetrate the constituting cells and sometimes even enter the cell nucleus. They can also enter the interior of the blood-brain barrier model. In contrast, small molecules like fluorescing dyes are not able to do that. Thus, ultrasmall gold nanoparticles can serve as carriers of drugs or for imaging inside the brain.

Identification of lipid biomarkers of metastatic potential and gene expression (HER2/p53) in human breast cancer cell cultures using ambient mass spectrometry.

In breast cancer, overexpression of human epidermal growth factor receptor 2 (HER2) correlates with overactivation of lipogenesis, mutation of tumor suppressor p53, and increased metastatic potential. The mechanisms through which lipids mediate p53, HER2, and metastatic potential are largely unknown. We havedeveloped a desorption electrospray ionization mass spectrometry (DESI-MS) method to identify lipid biomarkers of HER2/p53 expression, metastatic potential, and disease state (viz. cancer vs. non-cancerous) in monolayer and suspension breast cancer cell cultures (metastatic potential: MCF-7, T-47D, MDA-MB-231; HER2/p53: HCC2218 (HER2+++/p53+), HCC1599 (HER2-/p53-), HCC202 (HER2++/p53-), HCC1419 (HER2+++/p53-) HCC70 (HER2-/p53+++); non-cancerous: MCF-10A). Unsupervised principal component analysis(PCA) of DESI-MS spectra enabled identification of twelve lipid biomarkers of metastatic potential and disease state, as well as ten lipids that distinguish cell lines based on HER2/p53 expression levels (> 200 lipids were identified per cell line). In addition, we developed a DESI-MS imaging (DESI-MSI) method for mapping the spatial distribution of lipids in metastatic spheroids (MDA-MB-231). Of the twelve lipids that correlate with changes in themetastatic potentialof monolayercell cultures, three were localized to the necrotic coreof spheroids, indicating a potential role in promoting cancer cell survival in nutrient-deficient environments. One lipid species, which was not detected in monolayer MDA-MB-231 cultures, was spatially localized to the periphery of the spheroid, suggesting a potential role in invasion and/or proliferation. These results demonstrate thatcombiningDESI-MS/PCA of monolayer and suspension cell cultures with DESI-MSI of spheroids isa promising approach for identifying lipid biomarkers of specific genotypes and phenotypes, as well as elucidating the potential functionof these biomarkers in breast cancer. Graphical Absract.

Compaction Dynamics during Progenitor Cell Self-Assembly Reveal Granular Mechanics

Summary We study the self-assembly dynamics of human progenitor cells in agarose microwells that are used for production of chondrogenic organoids. Using image analysis on time-lapse microscopy, we estimate the aggregate area in function of time for a large number of aggregates. In control conditions, the aggregate radius follows an exponential relaxation that is consistent with the dewetting dynamics of a liquid film. Upon introducing Y-27632 Rho kinase inhibitor, the compatibility with the liquid model is lost, and slowed-down relaxation dynamics are observed. We demonstrate that these aggregates behave as granular piles undergoing compaction, with density relaxation that follows a stretched exponential. Using simulations with an individual cell-based model, we construct a phase diagram of cell aggregates that suggests that the aggregate in presence of Rho kinase inhibitor approaches glass transition.

Effects of microtubule-inhibiting small molecule and antibody-drug conjugate treatment on differentially-sized A431 squamous carcinoma spheroids

Multicellular tumor spheroids have been increasingly used by researchers to produce more physiologically relevant experimental environments. However, tracking of spheroid growth and treatment-induced volume reduction has not been readily adopted. Here, squamous carcinoma cells were seeded at different starting cell numbers with growth and reduction kinetics monitored using live cell imaging. Following the initial growth phase, spheroids were treated with auristatin as small molecule (MMAE) or as antibody-drug conjugate containing non-cleavable auristatin drug payload (033-F). Compared to cells in monolayers, 033-F had notably weaker potency against spheroids despite potency levels of MMAE being similar against monolayers and spheroids. Accumulation of released payload from 033-F was reduced in higher volume spheroids, likely contributing to the potency differences. Despite lowered potency towards spheroids with 033-F, spheroid volume was still readily reduced by 033-F in a dose-dependent fashion, with >85% volume reductions at the highest concentrations for all spheroid sizes. Additionally, the core of the larger spheroids showed more resiliency towards microtubule inhibition. Overall, this work highlights how various in-vivo ‘features’ such as tumor penetration, cell interactions, and increased resistance to therapeutics can be integrated into a spheroid model and tracked over time by automated imaging technology.

Metallicity and α-Element Abundance Gradients along the Sagittarius Stream as Seen by APOGEE

Abstract Using 3D positions and kinematics of stars relative to the Sagittarius (Sgr) orbital plane and angular momentum, we identify 166 Sgr stream members observed by the Apache Point Observatory Galactic Evolution Experiment (APOGEE) that also have Gaia DR2 astrometry. This sample of 63/103 stars in the Sgr trailing/leading arm is combined with an APOGEE sample of 710 members of the Sgr dwarf spheroidal core (385 of them newly presented here) to establish differences of 0.6 dex in median metallicity and 0.1 dex in [α/Fe] between our Sgr core and dynamically older stream samples. Mild chemical gradients are found internally along each arm, but these steepen when anchored by core stars. With a model of Sgr tidal disruption providing estimated dynamical ages (i.e., stripping times) for each stream star, we find a mean metallicity gradient of 0.12 ± 0.03 dex Gyr−1 for stars stripped from Sgr over time. For the first time, an [α/Fe] gradient is also measured within the stream, at 0.02 ± 0.01 dex Gyr−1 using magnesium abundances and at 0.04 ± 0.01 dex Gyr−1 using silicon, which imply that the Sgr progenitor had significant radial abundance gradients. We discuss the magnitude of those inferred gradients and their implication for the nature of the Sgr progenitor within the context of the current family of Milky Way satellite galaxies, and we suggest that more sophisticated Sgr models are needed to properly interpret the growing chemodynamical detail we have on the Sgr system.

Spheroid Culture System Methods and Applications for Mesenchymal Stem Cells.

Owing to the importance of stem cell culture systems in clinical applications, researchers have extensively studied them to optimize the culture conditions and increase efficiency of cell culture. A spheroid culture system provides a similar physicochemical environment in vivo by facilitating cell–cell and cell–matrix interaction to overcome the limitations of traditional monolayer cell culture. In suspension culture, aggregates of adjacent cells form a spheroid shape having wide utility in tumor and cancer research, therapeutic transplantation, drug screening, and clinical study, as well as organic culture. There are various spheroid culture methods such as hanging drop, gel embedding, magnetic levitation, and spinner culture. Lately, efforts are being made to apply the spheroid culture system to the study of drug delivery platforms and co-cultures, and to regulate differentiation and pluripotency. To study spheroid cell culture, various kinds of biomaterials are used as building forms of hydrogel, film, particle, and bead, depending upon the requirement. However, spheroid cell culture system has limitations such as hypoxia and necrosis in the spheroid core. In addition, studies should focus on methods to dissociate cells from spheroid into single cells.

Enhancement of Radiosensitization by Silver Nanoparticles Functionalized with Polyethylene Glycol and Aptamer As1411 for Glioma Irradiation Therapy.

BackgroundThe efficacy of radiotherapy for glioma is often limited by the radioresistance of glioma cells. The radiosensitizing effects of silver nanoparticles (AgNPs) on glioma were found in the previous studies of our group. In order to enhance the radiosensitivity of tumor cells and selectively kill them while reducing the side effects of irradiation therapy, targeted modification of AgNPs is urgently needed.Materials and methodsIn the present study, AgNPs functionalized with polyethylene glycol (PEG) and aptamer As1411 (AsNPs) were synthesized and subsequently characterized by transmission electron microscopy, ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Then the targeting property of AsNPs was evaluated by dark-field imaging, confocal microscopy and in vivo imaging. Both colony formation assay and glioma-bearing mouse model were employed to study the radiosensitizing effect of AsNPs.ResultsThe characterization results revealed a spherical shape of AgNPs with an average diameter of 18 nm and the successful construction of AsNPs. AsNPs were confirmed to specifically target C6 glioma cells, but not normal human microvascular endothelial cells. Moreover, AsNPs could not only internalize into tumor cells, but also penetrate into the core of tumor spheroids. In vitro experiments showed that AsNPs exhibited a better radiosensitizing effect than AgNPs and PEGylated AgNPs (PNPs), inducing a higher rate of apoptotic cell death. In vivo imaging demonstrated that Cy5-AsNPs preferentially accumulated at the tumor site, and the ratio of fluorescence intensity of Cy5-AsNPs to that of Cy5-PNPs reached the maximum at 6 h post-systemic administration. Furthermore, the combination of AsNPs with irradiation significantly prolonged the median survival time of C6 glioma-bearing mice.ConclusionOur results indicated that AsNPs could be an effective nano-radiosensitizer for glioma targeting treatment.

Transcriptomic and functional analyses of 3D placental extravillous trophoblast spheroids

Placental extravillous trophoblast (EVT) invasion is essential in establishing proper blood supply to the fetus during pregnancy. However, traditional 2D in vitro systems do not model the in vivo invasion process in an anatomically-relevant manner. Our objectives were to develop a 3D spheroid model that would allow better emulation of placental invasion in vitro and to characterize the transcriptomic and functional outcomes. HTR8/SVneo EVT cells were self-assembled into 3D spheroids using ultra-low attachment plates. Transcriptomic profiling followed by gene set enrichment and gene ontology analyses revealed major global transcriptomic differences, with significant up-regulations in EVTs cultured as 3D spheroids in canonical pathways and biological processes such as immune response, angiogenesis, response to stimulus, wound healing, and others. These findings were further validated by RT-qPCR, showing significant up-regulations in genes and/or proteins related to epithelial-mesenchymal transition, cell-cell contact, angiogenesis, and invasion/migration. A high-throughput, spheroid invasion assay was applied to reveal the dynamic invasion of EVTs away from the spheroid core into extracellular matrix. Lastly, lipopolysaccharide, dexamethasone, or Δ9-tetrahydrocannabinol exposure was found to impact the invasion of EVT spheroids. Altogether, we present a well-characterized, 3D spheroid model of EVT invasion and demonstrate its potential use in drug and toxin screening during pregnancy.

Light sheet fluorescence microscopy versus confocal microscopy: in quest of a suitable tool to assess drug and nanomedicine penetration into multicellular tumor spheroids

We recently constructed a multicellular spheroid model of pancreatic tumor based on a triple co-culture of cancer cells, fibroblasts and endothelial cells and characterized by the presence of fibronectin, an important component of the tumor extracellular matrix. By combining cancer cells and stromal components, this model recreates in vitro the three-dimensional (3D) architecture of solid tumors. In this study, we used these hetero-type spheroids as a tool to assess the penetration of doxorubicin (used as a model drug) through the whole tumor mass either in a free form or loaded into polymer nanoparticles (NPs), and we investigated whether microscopy images, acquired by Confocal Laser Scanning Microscopy (CLSM) and Light Sheet Fluorescence Microscopy (LSFM), would be best to provide reliable information on this process. Results clearly demonstrated that CLSM was not suitable to accurately monitor the diffusion of small molecules such as the doxorubicin. Indeed, it only allowed to scan a layer of 100 µm depth and no information on deeper layers could be available because of a progressive loss of the fluorescence signal. On the contrary, a complete 3D tomography of the hetero-type multicellular tumor spheroids (MCTS) was obtained by LSFM and multi-view image fusion which revealed that the fluorescent molecule was able to reach the core of spheroids as large as 1 mm in diameter. However, no doxorubicin-loaded polymer nanoparticles were detected in the spheroids, highlighting the challenge of nanomedicine delivery through biological barriers. Overall, the combination of hetero-type MCTS and LSFM allowed to carry out a highly informative microscopic assessment and represents a suitable approach to precisely follow up the drug penetration in tumors. Accordingly, it could provide useful support in the preclinical investigation and optimization of nanoscale systems for drug delivery to solid tumors.

Ionizable lipid nanoparticles are effective at penetrating the core of epithelial ovarian cancer spheroids

Ionizable lipid nanoparticles are effective at penetrating the core of epithelial ovarian cancer spheroids

1041 Development of human skin spheroid system for psoriatic disease modeling and drug development

Psoriasis is a complex chronic immune-mediated inflammatory cutaneous disease that affects 2–3% of the global population. Various 3D skin models have been developed to mimic psoriatic pathogenesis and help develop drugs, but these models are very low throughput and expensive. Here, we established a cost-effective, high throughput system for disease modeling and drug development, using spheroid approach. We plated 1x105 cells primary fibroblasts in 96-well plates for 2-5 days before addition of 5x104 primary keratinocytes. The culture medium contained DMEM/Ham’s F12, (3 : 1) 10% FBS, 0.18 mM adenine , 0.5 μg/ml hydrocortisone , 5 μg/ml insulin, 10-10 M cholera toxin, and10 ng/ml EGF. Similarly, we generated spheroids with iPSC-derived fibroblasts and keratinocytes. Immuno-staining and fluorescent dyes showed that fibroblasts formed a spheroid core surrounded with keratinocytes on the outer layers. More importantly, RT-PCR revealed that treatment with 30 ng/ml of IL-17 for 2-5 days upregulated a panel of psoriatic signature genes such as kRT16, S100A7, S100A12, DEFB4A and IL-8. To facilitate high throughput screening, DEFB4A/GFP reporters will be incorporated into keratinocytes with CRISPR technology. The availability of such cost-effective high throughput systems will greatly accelerate drug development to treat patients. Similar approaches can be used to generate models of other diseases, such as atopic dermatitis.

Penetration and Uptake of Nanoparticles in 3D Tumor Spheroids.

Animal models are effective for assessing tumor localization of nanosystems but difficult to use for studying penetration beyond the vasculature. Here, we have used well-characterized HCT116 colorectal cancer spheroids to study the effect of nanoparticle (NP) physicochemical properties on penetration and uptake. Incubation of spheroids with Hoechst 33342 resulted in a dye gradient, which facilitated discrimination between the populations of cells in the core and at the periphery of spheroids by flow cytometry. This approach was used to compare doxorubicin and liposomal doxorubicin (Caelyx) and a range of model poly(styrene) nanoparticles of different sizes (30 nm, 50 nm, 100 nm) and with different surface chemistries (50 nm uniform plain, carboxylated, aminated and a range of NPs and polyethylene glycol modified NPs prepared from a promising new functionalized biodegradable polymer (poly(glycerol-adipate), PGA). Unmodified poly(styrene) nanoparticles (30 nm/50 nm) were able to penetrate to the core of HCT116 spheroids more efficiently than larger poly(styrene) nanoparticles (100 nm). Surprisingly, penetration of 30 and 50 nm particles was as good as clinically relevant doxorubicin concentrations. However, penetration was reduced with higher surface charge. PGA NPs of 100 nm showed similar penetration into spheroids as 50 nm poly(styrene) nanoparticles, which may be related to polymer flexibility. PEG surface modification of polymeric particles significantly improved penetration into the spheroid core. The new model combining the use of spheroids Hoechst staining and flow cytometry was a useful model for assessing NP penetration and gives useful insights into the effects of NPs' physical properties when designing nanomedicines.

Pulmonary delivery of transferrin receptors targeting peptide surface-functionalized liposomes augments the chemotherapeutic effect of quercetin in lung cancer therapy.

Purpose: Lung cancer has a high incidence rate worldwide with a 5-year survival rate of 18%, and is the leading cause of cancer-related deaths. The aim of this study is to augment therapeutic efficacy of quercetin (QR) for lung cancer therapy by targeting transferrin receptors, which are overexpressed and confined to tumor cells.Methods: In this study, T7 surface-functionalized liposomes loaded with QR (T7-QR-lip) having different T7 peptide densities (0.5%, 1% and 2%) were prepared by the film hydration method. T7 surface-functionalized liposomes were characterized and evaluated in terms of in vitro cytotoxicity and cellular uptake, 3D tumor spheroid penetration and inhibition capabilities, in vivo biodistribution and therapeutic efficacy in mice with orthotopic lung-tumor implantation by fluorescent and bioluminescent imaging via pulmonary administration.Results: In vitro, 2% T7-QR-lip exhibited significantly augmented cytotoxicity (~3-fold), higher apoptosis induction and S-phase cell-cycle arrest. A prominent peak right-shift and enhanced mean fluorescence intensity was observed in A549 cells treated with T7 Coumarin-6 liposomes (T7-Cou6-lip), confirming the target specificity of T7 targeted liposomes; while, after treatment with T7-QR-lip and non-targeted QR-lip, no significant difference was observed in cellular uptake and in vitro cytotoxicity studies in MRC-5 (normal lung fibroblast) cells. T7-Cou6-lip showed higher fluorescence intensity in A549 cells and a significantly deeper penetration depth of 120 µm in the core of the tumor spheroids and T7-QR-lip produced significantly higher tumor-spheroid growth inhibition. The in vivo biodistribution study via pulmonary delivery of T7 1,1’-dioctadecyltetramethyl-indotricarbocyanine iodide liposomes demonstrated liposome accumulation in the lungs and sustained-release behavior up to 96 h. Further, T7-QR-lip significantly enhanced the anticancer activity of QR and lifespan of mice (p<0.01, compared with saline) in orthotopic lung tumor-bearing mice via pulmonary administration.Conclusion: T7 surface-functionalized liposomes provide a potential drug delivery system for a range of anticancer drugs to enhance their therapeutic efficacy by localized (pulmonary) administration and targeted delivery.

Culture of hybrid spheroids composed of calcium phosphate materials and mesenchymal stem cells on an oxygen-permeable culture device to predict in vivo bone forming capability

Three-dimensional (3-D) cell culture can better mimic physiological conditions in which cells interact with adjacent cells and the extracellular matrix than monolayer culture. We have developed a 3-D cell culture device, the Oxy chip, which can be used to generate and supply oxygen to cell spheroids to prevent hypoxia. Here, we used the Oxy chip to generate hybrid spheroids comprising calcium phosphate (CaP) particles (hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) or octacalcium phosphate (OCP)) and mesenchymal stem cells (MSCs, C3H10T1/2 cells or D1 cells) that can be used to analyze cell differentiation mechanisms. We showed that the 3-D cell-cell and cell-material interactions and oxygenation offered by the Oxy chip promoted osteoblastic differentiation of MSCs. We also used histomorphometric analysis of hematoxylin and eosin staining, quality analyses by μCT and collagen orientation observation with picrosirius red staining in bone regeneration following implantation of three CaPs in a critical-sized defect in mouse calvaria. The in vivo bone formation capacity of the three tested CaP materials was OCP ≥ β-TCP > HA: the newly formed bone by OCP had a structure relatively close to that of the calvaria intact bone. When MSCs were 3-D cultured with the CaP materials using the Oxy chip, the in vitro osteogenic capacity of these materials was highly similar to trends observed in vivo. The in vitro alkaline phosphatase activity of D1 cells had the highest correlation with in vivo bone volume (R = 0.900). Chemical and FTIR spectroscopic analyses confirmed that differentiation of D1 cells could be associated with amorphous calcium phosphate (ACP) precipitation concomitant with OCP hydrolysis. Taken together, hybrid spheroid cultures using the Oxy chip can be used to screen and predict bone forming potential of bone substitute materials. Statement of significanceAn oxygen permeable-culture chip (Oxy chip) can be used to induce formation of cell spheroids by mesenchymal stem cells (MSCs). Use of the Oxy chip avoids hypoxia in the spheroid core and enhances MSC osteoblastic differentiation relative to conventional spheroid culture methods. The present study showed that the Oxy chip mimics the in vivo environment associated with bone formation and can be used to generate hybrid spheroids consisting of calcium phosphates and MSCs that are useful for analyzing cell differentiation mechanisms. Bone formation analysis following implantation of calcium phosphate materials in mouse calvaria defects showed positive correlation with the in vitro results. We propose that hybrid spheroids cultured on the Oxy chip can be used to screen and predict the bone forming potential of bone substitute materials.