Abstract
Melanoma is the most dangerous type of skin cancer and is responsible for 75% of deaths from skin cancers. For an accurate evaluation of potential treatment efficacy, it is important to use study models as close as possible to the in vivo conditions. A 3D model consisting of B16F10 spheroids was developed using liquid overlay technique on plates coated with 1% agarose, in the presence of 1% methylcellulose and L929-conditioned medium. The model is suitable and can be further used for more complex in vitro drug testing than the classical 2D approach. For exemplification, the behavior of a well-known cytostatic, doxorubicin (DOX), was evaluated in spheroids as compared to classical 2D culture conditions. Fluorescence imaging was used to visualize DOX uptake by B16F10 spheroids at different periods of time. The results showed that a much higher DOX concentration is necessary to produce similar effects compared with the monolayer. The fluorescence images revealed that at least 4 h of stimulation is needed for a sufficient DOX uptake. The 3D model developed in this study was suitable to investigate drug penetration in time. Our findings may explain the decrease of the doxorubicin therapeutical effect, suggesting the need of maintaining the drug concentration at the tumoral place for at least 2 h upon administration. Similar or more advanced studies can lead to a better understanding of drug delivery kinetics and distribution upon administration, conducing toward a better performance in designing suitable delivery systems for obtaining the optimum dose–response effect.
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References
AAT Bioquest, Inc. (2020, September 30). Quest Graph™ IC50 Calculator (v.1). Retrieved from https://www.aatbio.com/tools/ic50-calculator-v1
Beaumont KA, Mohana-Kumaran N, Haass NK (2013) Modeling melanoma in vitro and in vivo. Healthcare (Basel) 2(1):27–46. https://doi.org/10.3390/healthcare2010027
Bresciani G, Hofland LJ, Dogan F, Giamas G, Gagliano T, Zatelli MC (2016) Evaluation of spheroid 3D culture methods to study a pancreatic neuroendocrine neoplasm cell line. Front Endocrinol 10:682. https://doi.org/10.3389/fendo.2019.00682
Breslin S, O'Driscoll L (2013) Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today 18:240–249
Carlsson J, Nilsson K, Westermark B, Ponten J, Sundstrom C, Larsson E, Bergh J, Pahlman S, Busch C, Collins VP (1998) Formation and growth of multicellular spheroids of human origin. Int J Cancer 31:523–533
Carvalho MP, Costa EC, Miguel SP, Correia IJ (2016) Tumor spheroid assembly on hyaluronic acid-based structures: a review. Carbohydr Polym 150:139–148
Chatterjee K, Zhang J, Honbo N, Karliner JS (2010) doxorubicin cardiomyopathy. Cardiology 115(2):155–162. https://doi.org/10.1159/000265166
Costa EC, de Melo-Diogo D, Moreira AF, Carvalho MP, Correia IJ (2018) Spheroids formation on non-adhesive surfaces by liquid overlay technique: considerations and practical approaches. Biotechnol J 13(1). https://doi.org/10.1002/biot.201700417
Couto GK, Segatto NV, Oliveira TL, Seixas FK, Schachtschneider KM, Collares T (2019) The melding of drug screening platforms for melanoma. Front Oncol 9:512. https://doi.org/10.3389/fonc.2019.00512
Cui X, Hartanto Y, Zhang H (2017) Advances in multicellular spheroids formation. J R Soc Interface 14(127):0877–2016. https://doi.org/10.1098/rsif.2016.0877
Edmondson R, Broglie JJ, Adcock AF, Yang L (2014) Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 12:207–218. https://doi.org/10.1089/adt.2014.573
Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J (2013) Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol 31:108–115
Friedric J, Seidel C, Ebner R, Kunz-Schughart LA (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4:309–324
Froehlich K, Haeger JD, Heger J, Pastuschek J, Photini SM, Yan Y, Lupp A, Pfarrer C, Mrowka R, Schleussner E, Markert UR, Schmidt A (2016) Generation of multicellular breast cancer tumor spheroids: comparison of different protocols. J Mammary Gland Biol Neoplasia 21:89–98
Hattermann K, Held-Feindt J, Mentlein R (2011) Spheroid confrontation assay: a simple method to monitor the three-dimensional migration of different cell types in vitro. Ann Anat 193:181–184
Henriksen PA (2018) Anthracycline cardiotoxicity: an update on mechanisms, monitoring and prevention. Heart 104(12):971–977. https://doi.org/10.1136/heartjnl-2017-312103
P. Horvath, N. Aulner, M. Bickle, A.M. Davies, E.D. Nery, D. Ebner, M.C. Montoya, P. Östling, V. Pietiäinen, L.S. Price, et al. Screening out irrelevant cell-based models of disease. Nat Rev Drug Discov, 15: 751-769, 2019
https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2020/cancer-facts-and-figures-2020.pdf, accesed september 2020
https://www.rxlist.com/consumer_doxorubicin_adriamycin/drugs-condition.html (n.d.)
Ippolito AN, Digioia AM, Atwater BL., DiFelice LA Optimization of spheroid formation: the case of Mn9D cells as a novel tool to assess cytotoxicity and neurodegeneration. 2014. Retrieved from https://digitalcommons.wpi.edu/mqp-all/1176
Jensen C, Teng Y (2020) Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci 7:33. https://doi.org/10.3389/fmolb.2020.00033
Jeong SY, Lee JH, Shin Y, Chung S, Kuh HJ (2016) Co-culture of tumor spheroids and fibroblasts in a collagen matrix-incorporated microfluidic chip mimics reciprocal activation in solid tumor microenvironment. PLoS One 11(7):e0159013. https://doi.org/10.1371/journal.pone.0159013
Kalal BS, Upadhya D, Pai VR (2017) Chemotherapy resistance mechanisms in advanced skin cancer. Oncol Rev 11(1):326. https://doi.org/10.4081/oncol.2017.326
Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev Cancer 6:392–401
Klicks J, Maßlo C, Kluth A et al (2019) A novel spheroid-based co-culture model mimics loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced selection of ABCB5-expressing cells. BMC Cancer 19:402. https://doi.org/10.1186/s12885-019-5606-4
Knight E, Przyborski S (2015) Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. J Anat 227(6):746–756. https://doi.org/10.1111/joa.12257
Kumar A, White J, James Christie R, Dimasi N, Gao C (2017) Antibody-drug conjugates. Platform technologies in drug discovery and validation. Annu Rep Med Chem:441–480. https://doi.org/10.1016/bs.armc.2017.08.002
Kuroda Y, Wakao S, Kitada M, Murakami T, Nojima M, Dezawa M (2013) Isolation. culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nat Protoc 8:1391–1415
Longati P, Jia X, Eimer J, Wagman A, Witt M-R, Rehnmark S, Verbeke C, Toftgård R, Löhr M, Heuchel RL (2012) 3D pancreatic carcinoma spheroids induce a matrix-rich. chemoresistant phenotype offering a better model for drug testing. BMC Cancer 13:95
Ma H, Jiang Q, Han S, Wu Y, Tomshine JC, Wang D, Gan Y, Zou G, Liang X-J (2012) Multicellular tumor spheroids as an in vivo-like tumor model for three-dimensional imaging of chemotherapeutic and nano material cellular penetration. Mol Imaging 11:487–498
Nagelkerke A, Bussink J, Sweep FC, Span PN (2013) Generation of multicellular tumor spheroids of breast cancer cells: how to go three-dimensional. Anal Biochem 437(1):17–19. https://doi.org/10.1016/j.ab.2013.02.004
Nunes AS, Barros AS, Costa EC, Moreira AF, Correia IJ (2019) 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs. Biotechnol Bioeng 116:206–226. https://doi.org/10.1002/bit.26845
Picollet Dha-han N, Dolega ME, Liguori L, Marquette C, LeGac S, Gidrol X, Martin DK (2016) A 3D toolbox to enhance physiological relevance of human tissue models. Trends Biotechnol 34:757–769
Rimann M, Graf-Hausner U (2012) Synthetic 3D multicellular systems for drug development. Curr Opin Biotechnol 23:803–809
Shah S, Chandra A, Kaur A, Sabnis N, Lacko A, Gryczynski Z, Fudala R, Gryczynski I (2011) Fluorescence properties of doxorubicin in PBS buffer and PVA films. J Photochem Photobiol B Biol 170:65–69. https://doi.org/10.1016/j.jphotobiol.2017.03.024
Tiago M, De Oliveira E, Brohem CA, Pennacchi P, Paes RD, Haga RB, Campa A, Barros SBDM, Smalley KS, Maria-Engler SS (2014) Fibroblasts protect melanoma cells from the cytotoxic effects of doxorubicin. Tissue Eng Part A 20:2412–2421
Uphoff CC, Drexler HG (2014) Detection of Mycoplasma contamination in cell cultures. Curr Protoc Mol Biol 106:28.4.1–28.414. https://doi.org/10.1002/0471142727.mb2804s106
Van Meerloo J, Kaspers GJL, Cloos J (2011) Cell sensitivity assays: the MTT Assay. Chapter 20 in: Cancer cell culture. In: Cree IA (ed) Methods in molecular biology (methods and protocols). Humana Press, pp 237–245
Varshney N, Sahi AK, Vajanthri KY et al (2014) Culturing melanocytes and fibroblasts within three-dimensional macroporous PDMS scaffolds: towards skin dressing material. Cytotechnology 71:287–303. https://doi.org/10.1007/s10616-018-0285-6
Yao JC, Pavel M, Lombard-Bohas C, Van Cutsem E, Voi M, Brandt U et al (2016) Everolimus for the treatment of advanced pancreatic neuroendocrine tumors: overall survival and circulating biomarkers from the randomized, phase III RADIANT-3 study. J Clin Oncol 34:3906–3913. https://doi.org/10.1200/JCO.2016.68.0702
Zanoni M, Piccinini F, Arienti C, Zamagni A, Santi S, Polico R, Bevilacqua A, Tesei A (2016) 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep 6:19–103
Acknowledgements
This study was supported by the Romanian National Authority for Scientific Research, UEFISCDI, project no. 63PCCDI (PN-III-P1-1.2-PCCDI2017-0728).
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Baciu, D.D., Dumitrașcu, A.M., Vasile, V. et al. Generation of a 3D melanoma model and visualization of doxorubicin uptake by fluorescence imaging. In Vitro Cell.Dev.Biol.-Animal 58, 44–53 (2022). https://doi.org/10.1007/s11626-021-00636-9
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DOI: https://doi.org/10.1007/s11626-021-00636-9