3D multicellular spheroids as high throughput platforms to screen novel combinatory therapies for treatment of pancreatic cancer

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Establishment of 2D Cell Cultures Derived From 3D MCF‐7 Spheroids Displaying a Doxorubicin Resistant Profile

Publication . Nunes, Ana S.; Costa, Elisabete; Barros, Andreia; Diogo, Duarte de Melo; Correia, I.J.

In vitro 3D cancer spheroids generally exhibit a drug resistance profile similar to that found in solid tumors. Due to this property, these models are an appealing for anticancer compounds screening. Nevertheless, the techniques and methods aimed for drug discovery are mostly standardized for cells cultured in 2D. The development of 2D cell culture models displaying a drug resistant profile is required to mimic the in vivo tumors, while the equipment, techniques, and methodologies established for conventional 2D cell cultures can continue to be employed in compound screening. In this work, the response of 3D-derived MCF-7 cells subsequently cultured in 2D in medium supplemented with glutathione (GSH) (antioxidant agent found in high levels in breast cancer tissues and a promoter of cancer cells resistance) to Doxorubicin (DOX) is evaluated. These cells demonstrated a resistance toward DOX closer to that displayed by 3D spheroids, which is higher than that exhibited by standard 2D cell cultures. In fact, the 50% inhibitory concentration (IC50 ) of DOX in 3D-derived MCF-7 cell cultures supplemented with GSH is about eight-times higher than that obtained for conventional 2D cell cultures (cultured without GSH), and is only about two-times lower than that attained for 3D MCF-7 spheroids (cultured without GSH). Further investigation revealed that this improved resistance of 3D-derived MCF-7 cells may result from their increased P-glycoprotein (P-gp) activity and reduced production of intracellular reactive oxygen species (ROS).

2019-04-10Journal article

Open access

Spheroids formation on non‐adhesive surfaces by Liquid Overlay Technique: considerations and practical approaches

Publication . Costa, Elisabete C.; Diogo, Duarte Miguel de Melo; Moreira, André; Carvalho, Marco António Paulo de; Correia, Ilídio Joaquim Sobreira

Scalable and reproducible production of 3D cellular spheroids is highly demanded, by pharmaceutical companies, for drug screening purposes during the pre‐clinical evaluation phase. These 3D cellular constructs, unlike the monolayer culture of cells, can mimic different features of human tissues, including cellular organization, cell–cell and cell‐extracellular matrix (ECM) interactions. Up to now, different techniques (scaffold‐based and ‐free) have been used for spheroids formation, being the Liquid Overlay Technique (LOT) one of the most explored methodologies, due to its low cost and easy handling. Additionally, during the last few decades, this technique has been widely investigated in order to enhance its potential for being applied in high‐throughput analysis. Herein, an overview of the LOT advances, practical approaches, and troubleshooting is provided for those researchers that intend to produce spheroids using LOT, for drug screening purposes. Moreover, the advantages of the LOT over the other scaffold‐free techniques used for the spheroids formation are also addressed.

2018Journal article

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Hyaluronic acid functionalized nanoparticles loaded with IR780 and DOX for cancer chemo-photothermal therapy

Publication . Alves, Cátia; Diogo, Duarte Miguel De Melo; Sousa, Ana Rita Lima; Costa, Elisabete; Correia, Ilidio

IR780 is a near infrared (NIR) dye with a huge potential to be applied in cancer phototherapy and imaging. However, IR780 poor water solubility and acute cytotoxicity limit its direct use in cancer theragnostic. Herein, a novel Hyaluronic acid (HA)-based amphiphilic polymer was used, for the first time, in the preparation of polymeric nanoparticles (HPN) encapsulating IR780 aimed to be applied in breast cancer therapy. Furthermore, HPN co-encapsulating IR780 and Doxorubicin (DOX) were also produced in order to further enhance the therapeutic effectiveness of this nanoformulation. The results revealed that HPN were able to successfully encapsulate IR780 (IR-HPN) and the IR780-DOX combination (IR/DOX-HPN). Furthermore, the encapsulation of IR780 in HPN improved its absorption at 808 nm by about 2.2-fold, thereby enhancing its photothermal potential, as well as its cytocompatibility. The 2D in vitro cell uptake studies demonstrated that the nanostructures displayed a higher internalization by breast cancer cells than by normal cells. In addition, the assays performed in 3D in vitro models of breast cancer revealed that HPN can penetrate into spheroids. Furthermore, the 3D in vitro studies also demonstrated that the combined application of IR-HPN and NIR light was unable to induce cytotoxicity on spheroids. In contrast, IR/DOX-HPN produced a decrease on spheroids cells' viability, and their combination with NIR light induced an even stronger therapeutic effect, thus revealing the potential of these nanoparticles for cancer chemo-phototherapy.

2019-04Journal article

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Assembly of breast cancer heterotypic spheroids on hyaluronic acid coated surfaces

Publication . Carvalho, Marco António Paulo de; Costa, Elisabete C.; Correia, I.J.

Drug screening is currently demanding for realistic models that are able to reproduce the structural features of solid tumors. 3D cell culture systems, namely spheroids, emerged as a promising approach to provide reliable results during drug development. So far, liquid overlay technique (LOT) is one of the most used methods for spheroids assembly. It comprises cellular aggregation due to their limited adhesion to certain biomaterials, like agarose. However, researchers are currently improving this technique in order to obtain spheroids on surfaces that mimic cancer extracellular matrix (ECM), since cell–ECM interactions modulate cells behavior and their drug resistance profile. Herein, hyaluronic acid (HA) coated surfaces were used, for the first time, for the production of reproducible heterotypic breast cancer spheroids. The obtained results revealed that it is possible to control the size, shape, and number of spheroids gotten per well by changing the HA concentration and the number of cells initially seeded in each well.

2017-05-25Journal article

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Bioreducible poly(2-ethyl-2-oxazoline)–PLA–PEI-SS triblock copolymer micelles for co-delivery of DNA minicircles and Doxorubicin

Publication . Gaspar, Vítor Manuel Abreu; Baril, Patrick; Costa, Elisabete C.; Diogo, Duarte Miguel de Melo; Foucher, Frédéric; Queiroz, João; Sousa, Fani; Pichon, Chantal; Correia, I.J.

The co-delivery of minicircle DNA (mcDNA) and small anti-cancer drugs via stimuli-sensitive nanocarriers is a promising approach for combinatorial cancer therapy. However, the simultaneous loading of drugs and DNA in nanosized delivery systems is remarkably challenging. In this study we describe the synthesis of triblock copolymer micelles based on poly(2-ethyl-2-oxazoline)–poly(L-lactide) grafted with bioreducible polyethylenimine (PEOz–PLA-g–PEI-SS) for co-delivery of supercoiled (sc) mcDNA vectors and Doxorubicin (Dox). These amphiphilic carriers take advantage of non-fouling oxazolines to confer biological stability, of PLA to provide a hydrophobic core for drug encapsulation and of bioreducible PEI-SS to provide mcDNA complexation and an on-demand stimuli-responsive release. The obtained results show that mcDNA-loaded micelleplexes penetrate into in vitro tumor spheroid models with specific kinetics and exhibit a higher gene expression when compared to non-bioreducible nanocarriers. Moreover, in vivo bioluminescence imaging showed that gene expression is detected up to 8 days following mcDNA-micelles intratumoral administration. Furthermore, drug–gene co-delivery in PEOz–PLA-g–PEI-SS carriers was verified by successful encapsulation of both Dox and mcDNA with high efficacy. Moreover, dual-loaded micelleplexes presented significant uptake and a cytotoxic effect in 2D cultures of cancer cells. The co-delivery of mcDNA-Dox to B16F10-Luciferase tumor bearing mice resulted in a reduction in tumor volume and cancer cells viability. Overall, such findings indicate that bioreducible triblock micelles are efficient for focal delivery in vivo and have potential for future application in combinatorial DNA-drug therapy.

2015-07-13Journal article

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