Alterations of the cytoskeleton are commonly associated with tumor genesis and cancer progression. For this reason, the characterization of cytoskeletonassociated functions and properties is important to optimize the outcomes to classical and more recent therapeutic approaches, such as chemotherapy, radiotherapy and cancer nanomedicine. In such context, this work investigated the synergy between cancer nanomedicine and radiotherapy. In particular, the eﬀects over time (24 and 48h) of two diﬀerent doses of X-rays (2 and 10Gy) on spreading area, morphological parameters and the internalization mechanism of carboxylated nanoparticles in mammary epithelial cells and mammary adenocarcinoma cells were investigated

Arrichiello, Cecilia

di Gennaro, Gioia

Federico, Costabile

Panzetta, Valeria

Pugliese, Mariagabriella

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DOI 10.1393/ncc/i2018-18204-7Colloquia: SIRR 2018IL NUOVO CIMENTO 41 C (2018) 204X-rays affect cytoskeleton assembly and nanoparticle uptake:Preliminary resultsValeria Panzetta(1), Mariagabriella Pugliese(2), Gioia di Gennaro(1),Costabile Federico(1), Cecilia Arrichiello(3), Paolo Muto(3),Paolo A. Netti(1)(2)(3)(4) and Sabato Fusco(1)(1) Interdisciplinary Research Centre on Biomaterials (CRIB), Università degli Studidi Napoli “Federico II” - Piazzale Tecchio 80, 80125 Naples, Italy(2) Dipartimento di Fisica, Università degli Studi di Napoli “Federico II” and INFN, Sezionedi Napoli - Monte S. Angelo, Via Cintia, 80126 Naples, Italy(3) Radiotherapy Unit, Istituto Nazionale Tumori-IRCCS-Fondazione “G. Pascale”Via Semmola, 53, 80131 Naples, Italy(4) Center for Advanced Biomaterials for Healthcare@CRIB, Istituto Italianodi Tecnologia (IIT) - Largo Barsanti e Matteucci 53, 80125 Naples, Italyreceived 4 December 2018Summary. — Alterations of the cytoskeleton are commonly associated with tumorgenesis and cancer progression. For this reason, the characterization of cytoskeleton-associated functions and properties is important to optimize the outcomes to clas-sical and more recent therapeutic approaches, such as chemotherapy, radiotherapyand cancer nanomedicine. In such context, this work investigated the synergy be-tween cancer nanomedicine and radiotherapy. In particular, the effects over time (24and 48 h) of two different doses of X-rays (2 and 10 Gy) on spreading area, morpho-logical parameters and the internalization mechanism of carboxylated nanoparticlesin mammary epithelial cells and mammary adenocarcinoma cells were investigated.1. – IntroductionThe recent literature has demonstrated that the cytoskeleton (CSK) acts as an epi-genetic determinant of several cell functions, such as adhesion, motility, proliferation,intracellular trafficking and differentiation [1]. Much attention has been paid to therole of the CSK in many diseases genesis and progression and, for example, it is nowwidely recognized that cancer and metastatic cells are generally softer than their healthycounterpart [2-4]. In this context, the characterization of CSK-associated functions andproperties is important to develop alternative diagnostic tools able to understand theCreative Commons Attribution 4.0 License (http://creativecommons.org/licenses/by/4.0) 12 VALERIA PANZETTA et al.response to classical and more recent therapeutic approaches, such as chemotherapy, ra-diotherapy and cancer nanomedicine, and, specifically, to optimize their outcomes alsoby their simultaneous combination.In previous studies it has been observed that ionizing radiations can have appreciableeffects both on the migration capability of cells and the structural properties of theCSK [5-9]. In particular, in some of the aforementioned works the authors found outthat X-rays are able to induce a significant increase of adhesion and the consequentdecrease of the migratory ability of transformed tumor-like fibroblasts.Furthermore, it is now recognized the key role of nanomedicine in cancer treat-ment [10]. In fact, nanoparticles (NPs) can be used as vehicles for efficient anti-cancerdrug delivery and, at the same time, recent investigations have also highlighted an activeinvolvement of the CSK assembly in the mechanism of internalization of NPs [11,12].In such context, this work investigated the synergy between cancer nanomedicine andradiotherapy. In particular, the effects over time (24 and 48 h) of two different dosesof X-rays (2 and 10 Gy) on spreading area, morphological parameters and the internal-ization mechanism of carboxyl-functionalized NPs (uptake) in mammary epithelial cells,MCF10A, and mammary adenocarcinoma cells, MCF7, were investigated.2. – Materials and methods2.1. Cell cultures and X-ray irradiation. – Experiments were performed on mammaryepithelial cells, MCF10A, and mammary adenocarcinoma cells, MCF-7. MCF10A cellswere grown in a nutrient-rich MEBM medium along with the additives obtained fromLonza/Clonetics Corporation as a kit (MEGM, Kit Catalog No. CC-3150) and MCF-7cells in a EMEM medium supplemented with 10% fetal bovine serum (FBS), 1% peni-cillin/streptomycin and 1% L-glutamine at 37 ◦C with 5% CO2.Cell lines were exposed to X-rays (photon beams) delivered by the LINAC SYNERGYAGILITY (ELEKTA) with field size (20 × 20) cm2, using the high endpoint energy of6 MeV, at the National Cancer Institute “PASCALE” of Naples. The two cell lines wereirradiated with two different doses of 2 Gy and 10 Gy.2.2. Cell proliferation. – MCF10A and MCF7 cells were cultured at a concentrationof 2000 cells/cm2. Cell proliferation was evaluated by image analysis. Cells were fixedwith 4% paraformaldehyde for 20 min, stained with Hoechst 33342 (Sigma) at 72 h fromirradiation. Samples were imaged using an inverted microscope system (Cell-R, Olympus,Japan) equipped with a 4× objective. Images of blue nuclei were captured from cellsamples. Fluorescent images were imported into ImageJ software (NIH, USA) for post-processing and analysis. Images were converted to grayscale (8-bit or 16-bit grayscale)and then to binary images by using manual thresholding. The menu command “Analyzeparticle” was used to count the cell number in each image.2.3. Cell spreading . – MCF10A and MCF7 cells were cultured at a density of2000 cells/cm2, respectively. Cells were fixed with 4% paraformaldehyde for 20 min,stained with Alexa 568-phalloidin to quantify the spreading area in control conditionand at 24 and 72 h from irradiation.2.4. Quantification of NP cellular uptake. – Cells were incubated with red NPs atthe final concentration of 3.6 × 109 NPs mL−1 for 1 h and 6 h. After incubation, cellswere rinsed five times with PBS to remove non-internalized nanoparticles, fixed with4% paraformaldehyde for 20 min, and the cells were stained with Alexa 488-phalloidinX-RAYS AFFECT CYTOSKELETON ASSEMBLY AND NANOPARTICLE UPTAKE ETC. 3Fig. 1. – Nuclei were counterstained with Hoechst 33342 and counted in 6 images for each cellline and each condition. The data of proliferation are presented as mean ± SEM. ∗: P < 0.05;∗∗: P < 0.01. Scale bar, 1 mm.(Molecular Probes). Z-sectioning images of cells incubated with fluorescent NPs werecollected with a confocal laser scanning microscope (Zeiss LSM 710) equipped with anargon and He–Ne laser lines at the wavelengths of 488 nm and 543 nm, respectively,using a 40× oil immersion objective (NA 1.30). The pinhole size was 90 μm and Z-stacksspaced by 0.5μm covering the total cell volume were recorded. The images resolution wasfixed at 2048× 2048 squared pixels (0.05μm/px). The image analysis of fluorescent NPsinternalized by cells was performed by NIH software (Fiji ImageJ). Briefly, a maximumprojection image for both color channels was constructed from the consecutive focalplanes. Green images were used to extract individual cell outlines using ImageJ ROImanager tool and COOH-NP internalization at each time-point was evaluated in termsof integrated fluorescence intensity of NPs within individual cell boundaries.3. – Results3.1. The effects of X-rays on cell proliferation. – In order to evaluate the MCF10Aand MCF-7 reproductive activity, cells were plated at equal densities and recounted 72 hafter irradiation. In control condition, MCF10A cells showed a significantly reducedproliferation activity in comparison with MCF7 cells. In fact, after 72 h the number ofcells increased by 6 and 20 times for MCF10A and MCF-7, respectively. Furthermore, theresults showed that only 10 Gy delivered dose inhibited the growth and the proliferationof MCF10A, while MCF-7 proliferation ability was markedly reduced by both X-raysdoses (fig. 1). In particular, the MCF10A growth was halved after irradiation with 10 Gy4 VALERIA PANZETTA et al.Fig. 2. – The morphology and cytoskeleton in MCF10A and MCF-7 cells are compared before((a),(f)) and after X-irradiation with doses of 2 Gy ((b),(c),(g),(h)) and 10Gy ((d),(e),(i)(j)).The spreading area data are presented as mean ± SEM. ∗: P < 0.05; ∗∗∗: P < 0.001. Scale bar,100 μm.doses, while MCF-7 proliferation was reduced by 40% and 70%, when irradiated withdoses of 2 and 10 Gy, compared to control cells.3.2. The effects of X-rays on cell adhesion. – The morphology and the cytoskeletonin MCF10A and MCF-7 cells are compared in fig. 2(a)–(j). In control conditions, thecellular shape of MCF10A cells appeared quite elongated and completely flattened, whileMCF-7 cells showed a polygonal cell shape (fig. 2(a) and (f)). Both cell lines tend togrow attached to the plastic plates in discrete patches, even if cultured at low density.In addition, in MCF10A cells actin stress fibers were evident throughout the cell bodyand the perinuclear actin cap, wrapping the nucleus, was observable. In MCF-7 cellsit is possible to observe cortical actin stress fibers around the cell boundary. Thesedifferences in the morphology and cytoskeleton structure reflected in the spreading areaX-RAYS AFFECT CYTOSKELETON ASSEMBLY AND NANOPARTICLE UPTAKE ETC. 5which appeared slightly higher in MCF10A (847μm2) than in MCF-7 cells (729μm2).Twenty-four hours after irradiation, MCF10A and MCF7 resulted to be, respectively, lessand more spread compared to control cells when irradiated (fig. 2(a),(b),(d),(f),(g),(i)).The effect seems to be maintained at 48 h for both cell lines and reinforced for tumorcells, which responded at X-irradiation in a dose-dependent manner (fig. 2(a),(c),(e)–(f),(h),(j)). Cells irradiated by X-rays presented actin filament accumulation, which, inturn, promotes a more flattened and spread morphology in the case of MCF-7 cells. Theincrease of tumor cell spreading at values significantly greater than those of normal cellsin control condition could be explained by considering that MCF-7 cells are characterizedby a cell volume remarkably higher than MCF10A cells [13]3.3. The effects of X-rays on the NPs uptake. – To investigate the effects of X-rayson the carboxyl-functionalized NPs uptake, cells were incubated with NPs at 37 ◦C andtwo time points (1 and 6 h) in control conditions and after 24 and 48 h from irradia-tion. In particular, in MCF10A cells the image analysis showed that the NPs uptake wassignificantly higher in healthy cells than in tumor counterparts, probably because of thegreater structuration of the CSK of MCF10A cells. As reported in fig. 3, the X-irradiationplayed a key role also in the internalization of NPs. Twenty-four hours after irradiation,COOH-NPs uptake at 1 h was impaired in MCF10A cells as a consequence of their re-duced adhesion, while the ability to internalize the NPs was not significantly changed at6 h. Over longer periods (48 h) the effect of the low dose was reduced and the nanoparti-cles uptake did not result significantly decreased in irradiated cells. On the other hand,the cells irradiated with a dose of 10 Gy continued to internalize a lower amount of NPsat 48 h, as a consequence of the more persistent effect of X-rays also on the adhesivenessof MCF10A cells. On the contrary, the increased adhesion and the more structured CSKof MCF7 cells after irradiation enhanced significantly the internalization of NPs in tumorcells already at 24 h. This outcome was strengthened at 48 h in a dose-dependent way.4. – DiscussionCancer onset, progression and aggression are deeply associated to changes in thecytoskeletal structure, cellular shape, differentiation, proliferation, adhesion, and migra-tion [1-4, 14]. These alterations are responsible of the decreased stiffness of cancer cellsand the extent of the cell compliance correlates with aggressiveness and metastatic po-tential of cancer cells [2, 3, 8]. The knowledge of the physical properties of tumor cellsand their microenvironment is fundamental to understand the molecular pathways whichdrive the cell transformation and cancer progression, but also to develop effective thera-peutics to treat cancer and to verify their effectiveness. In this work we have evaluatedthe effects over time (24 and 48 h) of two different doses of X-rays (2 and 10 Gy) onthe adhesiveness and cytoskeleton structure of mammary epithelial cells(MCF10A) andmammary adenocarcinoma cells (MCF-7). As already observed in previous works, radi-ations had important consequences on the cell adhesiveness, evaluated by measuring thecell spreading area. Indeed, healthy cells reduced their spreading area already at 24 hand this effect was partially recovered at 48 h for the low dose and slightly reinforcedfor the high one. On the contrary, irradiated tumor cells showed a more spread areathan control cells already at 24 h and the increase of adhesiveness was also confirmed bythe augmentation of stress fibers consequent to the irradiation. Over longer periods, thespreading area of MCF-7 cells was further increased in a dose-dependent manner.6 VALERIA PANZETTA et al.Fig. 3. – MCF10A and MCF-7 cells were incubated with COOH-NPs for 1 and 6 h after 24 and48 h from irradiation. The NPs uptake was found to be affected by X-rays in an opposite way inMCF10A and MCF-7 cells. The NPs uptake was evaluated in terms of integrated fluorescenceintensity and the data are presented as mean ± SEM. ∗: P < 0.05; ∗∗: P < 0.01; ∗∗∗: P < 0.001.Scale bar, 20 μm.It is interesting to highlight the opposite behaviour of the two cell lines and the abil-ity of normal cells to partially recover their initial physical properties (both average andp-value of spreading increased passing from 24 to 48 h) at least when low doses wereadministered. The consequences of the increased adhesion of irradiated tumor cells areX-RAYS AFFECT CYTOSKELETON ASSEMBLY AND NANOPARTICLE UPTAKE ETC. 7controversial. Indeed, some works report an increased cell adhesion together with in-creased motility and invasiveness of surviving cancer cells [15]. Nevertheless, we havepreviously observed that in normal and transformed fibroblasts the growth of the cellspreading area was associated to the stiffening of the CSK, to the enhanced expres-sion of adhesive surface molecules and, in particular, to the reduced motility of bothcell lines [7-9]. Anyway, the cell’s intrinsic response to ionizing radiations can be usedto improve the efficiency of classical and more recent therapeutic approaches, such aschemotherapy and cancer nanomedicine, when they are used in combination with radio-therapy. In particular, nanomedicine is running fast in developing new and promisingtumor-targeted drug delivery systems which are able to enhance antitumor efficacy andto attenuate drug toxicity. For these reasons, the effects of X-rays on the internaliza-tion mechanism of carboxyl-functionalized NPs with diameters of 100 nm were analyzedand explained by taking into consideration the pivotal role of the cell cytoskeleton. TheCOOH-NPs uptake was incubated with both cell lines in control conditions and afterirradiation and, assuming that the fluorescence intensity is proportional to the numberof internalized NPs, the fluorescence within individual cells was used as a parameterto evaluate the efficiency of cellular uptake. Compared to tumor cells, MCF10A cellsinternalized NPs in a greater amount both at 1 and 6 h as a consequence of the morestructured actin of CSK. Indeed, as previously reported, there exists a positive corre-lation between cell adhesion and NPs uptake which depends on the direct involvementof CSK in endocytic pathways [11]. Similarly, the changes in the spreading area andin the structure of the CSK, found here for normal and tumor cells after irradiation,affected the amount of internalized NPs. Indeed, the uptake process resulted to be sig-nificantly impaired in MCF10A cells and, as observed for the spreading area, this effectpersisted for the dose of 10 Gy, while a partial recovery was measured for the low dose.On the contrary, it has been observed that the radiations significantly increase the NPsuptake in tumor cells, particularly at 48 h from irradiation, activating an internalizationpathway directly correlated to the dynamics of the CSK. In fact, as ascertained, X-raysinduce in MCF-7 cells structural variations of CSK, which resulted in increased adhe-siveness and augmented CSK structuration. The formation of a more structured CSKafter X-irradiation, in turn, promotes the internalization of NPs in a larger amount. Itis necessary to underline that the NPs used in this work were free of surface conjugationwith specific ligand molecules. Then, in the future, NPs able to activate a tumor specificpathway directly involving the cytoskeleton could be designed and fabricated. Furtherand more detailed studies on the effects of X-rays on the expression of adhesion and sig-naling molecules strictly related to the CSK, such as integrins, could provide new insightsinto the design of promising ligand-based targeting approaches. Finally, these findingscan give fundamental information for the development of new strategies for combinationsand timings of radiotherapy and cancer nanomedicine.5. – ConclusionsIn conclusion, it was found that X-rays increased significantly tumor cell adhesiveness,evaluated by spreading area, CSK structure and COOH-NPs uptake. On the contrary,ionizing radiations reduced the cell spreading area and NPs uptake of normal cells, evenif these effects resulted to be less persistent in MCF10A cells when the low dose was used.Future studies could give a deeper comprehension of the role of cytoskeletal and surfaceadhesive molecules involved in these processes. More importantly, this knowledge could8 VALERIA PANZETTA et al.be used for the development of new drug-radiotherapy combinations and for the designof specific ligand-targeted nanocarriers.REFERENCES[1] Peyton S. R. et al., Cell Biochem. Biophys., 47 (2007) 300.[2] Guck J. et al., Biophys. J., 88 (2005) 2689.[3] Cross S. E. et al., Nanotechnol., 19 (2008) 384003.[4] Panzetta V. et al., Acta Biomater., 57 (2017) 34.[5] Woloschak G. E. et al., Mol. Carcinog., 3 (1990) 374.[6] Moncharmont C. et al., Crit. Rev. Oncol. Hematol., 92 (2014) 133.[7] Panzetta V. et al., J. Mech. Med. Biol., 15 (2015) 1540022.[8] Panzetta V. et al., Cytoskeleton, 74 (2017) 40.[9] Panzetta V. et al., ENBENG 2017 - IEEE 5th Portuguese Meeting on Bioengineering,Proceedings, Vol. 16 (2017) p. 7889448.[10] Kim W. et al., J. Control. Release, 200 (2015) 138.[11] Panzetta V. et al., ACS Biomater. Sci. Eng., 3 (2017) 1586.[12] He L. et al., Sci. Rep., 8 (2018) 7318.[13] Geltmeier A. et al., PLoS ONE, 10 (2015) e0134999.[14] Bosch-Fortea M. et al., Curr. Opin. Cell Biol., 50 (2018) 42.[15] Tsutsumi K. et al., Cell Struct. Funct., 34 (2009) 89.

X-rays aﬀect cytoskeleton assembly and nanoparticle uptake: Preliminary results

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X-rays aﬀect cytoskeleton assembly and nanoparticle uptake: Preliminary results

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