Создание персонализированных клеточных систем на основе тумороидов для оптимизации лекарственного лечения агрессивных форм солидных опухолей

Злокачественная опухоль представляет собой сложно организованную в пространстве многокомпонентную систему, которая имеет индивидуальные особенности для каждого пациента. Адекватные модельные системы должны отражать анатомическую и физиологическую сложность злокачественного новообразования, влияющую на распределение клеточных рецепторов и различных веществ, в том числе молекул лекарственных препаратов. Опухолевые сфероиды, полученные из клеточных линий злокачественных новообразований человека, наглядно показывают, что они являются приемлемой моделью для изучения микроэкологической регуляции физиологии опухолевых клеток и для решения проблем, связанных с метаболическими и пролиферативными градиентами в трехмерном культивировании при исследовании активности новых терапевтических препаратов. В данной работе были получены 3D-модели (тумороиды) из клеток солидных опухолей пациентов, проходивших лечение в НМИЦ онкологии им. Н.Н. Петрова. Была произведена оценка экспрессируемых цитокинов и хемокинов, изучен пролиферативный и инвазивный потенциал и установлена взаимосвязь между эффектом воздействия химиопрепаратов на тумороиды и клиническим ответом на лекарственное лечение у пациентов.

Список сокращений ………………………………………………………………………………………………………..4
Введение ………………………………………………………………………………………………………………………..5
Глава.1 Обзор литературы ………………………………………………………………………………………………7
СОЗДАНИЕ И ПРИМЕНЕНИЕ ТРЕХМЕРНЫХ КЛЕТОЧНЫХ МОДЕЛЕЙ ДЛЯ
РЕШЕНИЯ ЗАДАЧ ФУНДАМЕНТАЛЬНОЙ И ПРИКЛАДНОЙ ОНКОЛОГИИ ……………..7
1.1. Классификация опухолевых трёхмерных систем ……………………………………………………7
1.2. История создания трёхмерных клеточных моделей ……………………………………………….7
1.3. Строение и состав тумороидов ……………………………………………………………………………..9
1.5. Кинетика роста тумороидов ………………………………………………………………………………..12
1.5. Методы формирования тумороидов …………………………………………………………………….14
1.5.1. Технология висячих капель (Hanging drop method) ………………………………………..14
1.5.2. Технология получения сфероидов на низкоадгезивных поверхностях ……………15
(Liquid overlay technique (LOT)) ……………………………………………………………………………..15
1.5.3. Технологии на основе непрерывного движения (Agitation-based technique) …….15
1.6. Скаффолдные технологии …………………………………………………………………………………..16
1.6.1. Скаффолды, полученные из природных полимеров ……………………………………….16
1.6.2. Скаффолды, полученные из синтезированных полимеров ……………………………..18
1.7. Применение трехмерных клеточных моделей для решения задач теоретической и
практической медицины ……………………………………………………………………………………………19
1.8. Заключение…………………………………………………………………………………………………………25
ГЛАВА 2. Материалы и методы ……………………………………………………………………………………27
2.1. Культивирование опухолевых клеток в монослое ………………………………………………..30
2.1.1. Криоконсервация опухолевых клеток ……………………………………………………………31
2.1.2. Разморозка опухолевых клеток ……………………………………………………………………..31
2.2. Культивирование опухолевых клеток в тумороидах …………………………………………….31
2.2.1. Метод висячей капли – Hanging drop method …………………………………………………31
2.2.2. Технология получения сфероидов на низкоадгезивных поверхностях – Liquid
overlay technique…………………………………………………………………………………………………….31
2.3. Конфокальная микроскопия ………………………………………………………………………………..32
2.4. Иммуногисто-/цитохимический анализ ……………………………………………………………….32
2.4.1. Подготовка образцов …………………………………………………………………………………….32
2.4.2. Иммуногисто-/цитохимический метод выявления антигенов …………………………33
2.5. Анализ продукции альдегиддегидрогеназы (ALDH) с помощью проточной
цитофлуорометрии ……………………………………………………………………………………………………34
2.6. Иммуноферментный анализ (ИФА)……………………………………………………………………..35
2.7. Мультиплексный анализ ……………………………………………………………………………………..36
2.8. Исследование цитотоксического действия химиопрепаратов на монослойные
культуры и сфероиды ………………………………………………………………………………………………..37
2.8.1. MTT-тест ……………………………………………………………………………………………………..38
2.9. Исследование инвазивых свойств тумороидов в матригеле с помощью
автоматической аналитической системы Cell-IQ ………………………………………………………..39
2.10. Статистическая обработка данных …………………………………………………………………….40
3.1. Морфологические характеристики опухолевых культур ………………………………………41
3.3. Визуализация структурных особенностей тумороидов с помощью конфокальной
микроскопии …………………………………………………………………………………………………………….46
3.4. Иммуногисто-/цитохимическое исследование тумороидов …………………………………..48
3.5. Сравнителная оценка альдегиддегидрогеназной активности в 2D- и 3D-системах с
помощью проточной цитофлуориметрии …………………………………………………………………..53
3.6. Оценка продукции цитокинов и хемокинов 2D- и 3D-системами с помощью
мультиплексного анализа ………………………………………………………………………………………….54
3.7. Иммуноферментный анализ продукции иммуносупрессирующих факторов MICA и
TGF-β 2D- и 3D-клеточными системами ……………………………………………………………………56
3.8. Оценка цитотоксического действия химиопрепаратов на 2D- и 3D-системы с
помощью MTT-теста …………………………………………………………………………………………………58
3.9. Исследование инвазивых свойств тумороидов в матригеле под влиянием
цитостатиков с помощью автоматической аналитической системы Cell-IQ………………..64
ОБСУЖДЕНИЕ ……………………………………………………………………………………………………………77
ВЫВОДЫ …………………………………………………………………………………………………………………….84
СПИСОК ЛИТЕРАТУРЫ ……………………………………………………………………………………………..85

1.Alfarouk, K.O., Verduzco, D., Rauch, C., Muddathir, A.K., Bashir, A.H.H.,
Elhassan, G.O., Ibrahim, M.E., Orozco, J.D.P., Cardone, R.A., Reshkin, S.J., et al. (2014).
Glycolysis, tumor metabolism, cancer growth and dissemination. A new pH-based
etiopathogenic perspective and therapeutic approach to an old cancer question. Oncoscience 1,
777–802.
2.Baek, N., Seo, O.W., Lee, J., Hulme, J., and An, S.S.A. (2016a). Real-time
monitoring of cisplatin cytotoxicity on three-dimensional spheroid tumor cells. Drug Des. Devel.
Ther. 10, 2155–2165.
3.Baek, N.H., Seo, O.W., Kim, M.S., Hulme, J., and An, S.S.A. (2016b).
Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time. Onco. Targets.
Ther. 9, 7207–7218.
4.Bai, C., Yang, M., Fan, Z., Li, S., Gao, T., and Fang, Z. (2015). Associations of
chemo- and radio-resistant phenotypes with the gap junction, adhesion and extracellular matrix
in a three-dimensional culture model of soft sarcoma. J. Exp. Clin. Cancer Res. 34, 1–10.
5.Bartling, B., Hofmann, H.S., Silber, R.E., and Simm, A. (2008). Differential
impact of fibroblasts on the efficient cell death of lung cancer cells induced by paclitaxel and
cisplatin. Cancer Biol. Ther. 7, 1250–1261.
6.Benien, P., and Swami, A. (2014). 3D tumor models: History, advances and future
perspectives. Futur. Oncol. 10, 1311–1327.
7.Benton, G., George, J., Kleinman, H.K., and Arnaoutova, I.P. (2009). Advancing
science and technology via 3D culture on basement membrane matrix. J. Cell. Physiol. 221, 18–
25.
8.Benton, G., Arnaoutova, I., George, J., Kleinman, H.K., and Koblinski, J. (2014).
Matrigel: From discovery and ECM mimicry to assays and models for cancer research. Adv.
Drug Deliv. Rev. 79, 3–18.
9.Bjerkvig, R., Tonnesen, A., Laerum, O.D., and Backlund, E.O. (1990).
Multicenter tumor spheroids from human gliomas maintained in organ culture. J. Neurosurg. 72,
463–475.
10.Blanco, E., Shen, H., and Ferrari, M. (2015). Principles of nanoparticle design for
overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941–951.
11.Breslin, S., and O’Driscoll, L. (2013). Three-dimensional cell culture: The
missing link in drug discovery. Drug Discov. Today 18, 240–249.
12.Bryce, N.S., Zhang, J.Z., Whan, R.M., Yamamoto, N., and Hambley, T.W.
(2009). Accumulation of an anthraquinone and its platinum complexes in cancer cell spheroids:
The effect of charge on drug distribution in solid tumour models. Chem. Commun. 2673–2675.
13.Byrne, H.M. (2010). Dissecting cancer through mathematics: From the cell to the
animal model. Nat. Rev. Cancer 10, 221–230.
14.Carrel, A. (1912). On the permanent life of tissues outside of the organism. J. Exp.
Med. 15, 516–528.
15.Carvalho, K.C., Cunha, I.W., Rocha, R.M., Ayala, F.R., Cajaíba, M.M., Begnami,
M.D., Vilela, R.S., Paiva, G.R., Andrade, R.G., and Soares, F.A. (2011). GLUT1 expression in
malignant tumors and its use as an immunodiagnostic marker. Clinics 66, 965–972.
16.Chou, M.J., Hsieh, C.H., Yeh, P.L., Chen, P.C., Wang, C.H., and Huang, Y.Y.
(2013). Application of open porous poly(D, L -lactide-co-glycolide) microspheres and the
strategy of hydrophobic seeding in hepatic tissue cultivation. J. Biomed. Mater. Res. – Part A
101, 2862–2869.
17.Costa, E.C., Gaspar, V.M., Coutinho, P., and Correia, I.J. (2014). Optimization of
liquid overlay technique to formulate heterogenic 3D co-cultures models. Biotechnol. Bioeng.
111, 1672–1685.
18.Costa, E.C., Moreira, A.F., de Melo-Diogo, D., Gaspar, V.M., Carvalho, M.P.,
and Correia, I.J. (2016). 3D tumor spheroids: an overview on the tools and techniques used for
their analysis. Biotechnol. Adv. 34, 1427–1441.
19.Courau, T., Bonnereau, J., Chicoteau, J., Bottois, H., Remark, R., Assante
Miranda, L., Toubert, A., Blery, M., Aparicio, T., Allez, M., et al. (2019). Cocultures of human
colorectal tumor spheroids with immune cells reveal the therapeutic potential of MICA/B and
NKG2A targeting for cancer treatment. J. Immunother. Cancer 7, 1–14.
20.Colella G., Fazioli F., Gallo M., De Chiara A., Apice G., Ruosi C., Cimmino A.,
de Nigris F. 2018. Sarcoma Spheroids and Organoids-Promising Tools in the Era of Personalized
Medicine. Int J Mol Sci. 19(2), E615
21.St. Croix, B., Man, S., and Kerbel, R.S. (1998). Reversal of intrinsic and acquired
forms of drug resistance by hyaluronidase treatment of solid tumors. Cancer Lett. 131, 35–44.
22.Cui, X., Hartanto, Y., and Zhang, H. (2017). Advances in multicellular spheroids
formation. J. R. Soc. Interface 14.
23.Danilov A.O., Larin S.S., Danilova A.B., Moiseenko V.M., Balduyeva I.A.,
Kiselev S.L., Tourkevich Ye.A., Bartchuk A.S., Anisimov V.V., Gaftov G.I., Kochnev V.A.,
Hanson K.P. (2004) Аn improved procedure for autologous gene-modified vaccine preparation
for active specific immunotherapy of disseminated solid tumors. Voprosy onkologii. 50(2), 219–
227.
24.Debnath, J., Mills, K.R., Collins, N.L., Reginato, M.J., Muthuswamy, S.K., and
Brugge, J.S. (2002). The role of apoptosis in creating and maintaining luminal space within
normal and oncogene-expressing mammary acini. Cell 111, 29–40.
25.Van Dijk, M., Göransson, S.A., and Strömblad, S. (2013). Cell to extracellular
matrix interactions and their reciprocal nature in cancer. Exp. Cell Res. 319, 1663–1670.
26.Doix, B., Bastien, E., Rambaud, A., Pinto, A., Louis, C., Grégoire, V., Riant, O.,
and Feron, O. (2018). Preclinical evaluation of white led-activated non-porphyrinic
photosensitizer OR141 in 3D tumor spheroids and mouse skin lesions. Front. Oncol. 8, 1–9.
27.Důra, M., Němejcová, K., Jakša, R., Bártů, M., Kodet, O., Tichá, I., Michálková,
R., and Dundr, P. (2019). Expression of Glut-1 in Malignant Melanoma and Melanocytic Nevi:
an Immunohistochemical Study of 400 Cases. Pathol. Oncol. Res. 25, 361–368.
28.Enmon, R.M., O’Connor, K.C., Lacks, D.J., Schwartz, D.K., and Dotson, R.S.
(2001). Dynamics of spheroid self-assembly in liquid-overlay culture of DU 145 human prostate
cancer cells. Biotechnol. Bioeng. 72, 579–591.
29.Everitt B.S., Pickles A. (2004) Statistical Aspects of the Design and Analysis of
Clinical Trials. Imperial College Press, London
30.Freshney R.I. Culture of animal cells: a manual of basic technique and specialized
applications / R.I.Freswhney. – 6th ed., 2010. – John WileySons, Inc., Hoboken, New Jersey,
USA. – 732p.;
31.Friedrich, J., Seidel, C., Ebner, R., and Kunz-Schughart, L.A. (2009). Spheroid-
based drug screen: Considerations and practical approach. Nat. Protoc. 4, 309–324.
32.Geraldo, S., Simon, A., and Vignjevic, D.M. (2013). Revealing the cytoskeletal
organization of invasive cancer cells in 3D. J. Vis. Exp. 2–7.
33.Ghajar, C.M., and Bissell, M.J. (2008). Extracellular matrix control of mammary
gland morphogenesis and tumorigenesis: Insights from imaging. Histochem. Cell Biol. 130,
1105–1118.
34.Green, S.K., Karlsson, M.C.I., Ravetch, J. V., and Kerbel, R.S. (2002). Disruption
of cell-cell adhesion enhances antibody-dependent cellular cytotoxicity: Implications for
antibody-based therapeutics of cancer. Cancer Res. 62, 6891–6900.
35.Gu, J., Zhao, Y., Guan, Y., and Zhang, Y. (2015). Effect of particle size in a
colloidal hydrogel scaffold for 3D cell culture. Colloids Surfaces B Biointerfaces 136, 1139–
1147.
36.Günther, S., Ruhe, C., Derikito, M.G., Böse, G., Sauer, H., and Wartenberg, M.
(2007). Polyphenols prevent cell shedding from mouse mammary cancer spheroids and inhibit
cancer cell invasion in confrontation cultures derived from embryonic stem cells. Cancer Lett.
250, 25–35.
37.Haq, S., Samuel, V., Haxho, F., Akasov, R., Leko, M., Burov, S. V.,
Markvicheva, E., and Szewczuk, M.R. (2017). Sialylation facilitates self-assembly of 3D
multicellular prostaspheres by using cyclo-RGDFK(TPP) peptide. Onco. Targets. Ther. 10,
2427–2447.
38.Harris, A.L. (2002). Hypoxia – A key regulatory factor in tumour growth. Nat.
Rev. Cancer 2, 38–47.
39.Heimdal, J.H., Aarstad, H.J., Olsnes, C., and Olofsson, J. (2001). Human
autologous monocytes and monocyte-derived macrophages in co-culture with carcinoma F-
spheroids secrete IL-6 by a non-CD 14-dependent pathway. Scand. J. Immunol. 53, 162–170.
40.Heredia-Soto V., Redondo A., Kreilinger J.J.P., Martínez-Marín V., Berjón A.,
Mendiola M. (2019). 3D culture modelling: An emerging approach for Translational Cancer
Research in Sarcomas. Curr Med Chem.
41.Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., and
Kunz-Schughart, L.A. (2010). Multicellular tumor spheroids: An underestimated tool is catching
up again. J. Biotechnol. 148, 3–15.
42.Holtfreter, J. (1943). A study of the mechanics of gastrulation. Part I. J. Exp. Zool.
94, 261–318.
43.Humphreys, T. (1963). Chemical Dissolution of Sponge and Cell Adhesions. Dev.
Biol. 8, 27–47.
44.Ivascu, A., and Kubbies, M. (2007). Diversity of cell-mediated adhesions in breast
cancer spheroids. Int. J. Oncol. 31, 1403–1413.
45.Jang, S.H., Wientjes, M.G., and Au, J.L.S. (2001). Determinants of paclitaxel
uptake, accumulation and retention in solid tumors. Invest. New Drugs 19, 113–123.
46.Jensen C, Teng Y. (2020) Is It Time to Start Transitioning From 2D to 3D Cell
Culture?Front Mol Biosci. 7, 33.
47.Jo V.Y. (2014) WHO classification of soft tissue tumours: An update based on the
2013 (4th) edition / V. Y. Jo, C. D. M. Fletcher // Pathology. 46(2). – 95–104.
48.Kim, J.W., and Dang, C. V. (2006). Cancer’s molecular sweet tooth and the
warburg effect. Cancer Res. 66, 8927–8930.
49.Knuchel, R., Hofstadter, F., Jenkins, W.E.A., and Masters, J.R.W. (1989).
Sensitivities of Monolayers and Spheroids of the Human Bladder Cancer Cell Line MGH-U1 to
the Drugs Used for Intravesical Chemotherapy. Cancer Res. 49, 1397–1401.
50.Konstantinovsky, S., Davidson, B., and Reich, R. (2012). Ezrin and
BCAR1/p130Cas mediate breast cancer growth as 3-D spheroids. Clin. Exp. Metastasis 29, 527–
540.
51.Konur, A., Kreutz, M., Knüchel, R., Krause, S.W., and Andreesen, R. (1996).
Three-dimensional co-culture of human monocytes and macrophages with tumor cells: Analysis
of macrophage differentiation and activation. Int. J. Cancer 66, 645–652.
52.Kostarelos, K., Emfietzoglou, D., Papakostas, A., Yang, W.-H., Ballangrud, Å.M.,
and Sgouros, G. (2005). Engineering Lipid Vesicles of Enhanced Intratumoral Transport
Capabilities: Correlating Liposome Characteristics with Penetration into Human Prostate Tumor
Spheroids. J. Liposome Res. 15, 15–27.
53.Kozin, S. V., and Gerweck, L.E. (1998). Cytotoxicity of weak electrolytes after
the adaptation of cells to low pH: Role of the transmembrane pH gradient. Br. J. Cancer 77,
1580–1585.
54.L’Espérance, S., Bachvarova, M., Tetu, B., Mes-Masson, A.M., and Bachvarov,
D. (2008). Global gene expression analysis of early response to chemotherapy treatment in
ovarian cancer spheroids. BMC Genomics 9, 1–21.
55.Lee, B.H., Kim, M.H., Lee, J.H., Seliktar, D., Lay, N.J.C., and Tan, P. (2015).
Modulation of Huh7.5 spheroid formation and functionality using modified peg-based hydrogels
of different stiffness. PLoS One 10, 1–20.
56.Lin, R.Z., and Chang, H.Y. (2008). Recent advances in three-dimensional
multicellular spheroid culture for biomedical research. Biotechnol. J. 3, 1172–1184.
57.Lin, R.Z., Chou, L.F., Chien, C.C.M., and Chang, H.Y. (2006). Dynamic analysis
of hepatoma spheroid formation: Roles of E-cadherin and β1-integrin. Cell Tissue Res. 324,
411–422.
58.Liu, Z., and Vunjak-Novakovic, G. (2016). Modeling tumor microenvironments
using custom-designed biomaterial scaffolds. Curr. Opin. Chem. Eng. 11, 94–105.
59.Liu, J., Tan, Y., Zhang, H., Zhang, Y., Xu, P., Chen, J., Poh, Y., Tang, K., Wang,
N., and Huang, B. (2012). Soft fibrin gels promote selection and growth of tumorigenic cells.
Nat. Mater. 11, 734–741.
60.Lu, S., Zhao, F., Zhang, Q., and Chen, P. (2018). Therapeutic peptide amphiphile
as a drug carrier with ATP-triggered release for synergistic effect, improved therapeutic index,
and penetration of 3D cancer cell spheroids. Int. J. Mol. Sci. 19, 1–14.
61.Lv, X., Li, J., Zhang, C., Hu, T., Li, S., He, S., Yan, H., Tan, Y., Lei, M., Wen,
M., et al. (2017). The role of hypoxia-inducible factors in tumor angiogenesis and cell
metabolism. Genes Dis. 4, 19–24.
62.Madsen, S.J., Sun, C.-H., Tromberg, B.J., Yeh, A.T., Sanchez, R., and
Hirschberg, H. (2002). Effects of Combined Photodynamic Therapy and Ionizing Radiationon
Human Glioma Spheroids¶. Photochem. Photobiol. 76, 411.
63.Majety, M., Pradel, L.P., Gies, M., and Ries, C.H. (2015). Fibroblasts influence
survival and therapeutic response in a 3D co-culture model. PLoS One 10, 1–18.
64.Martin, A.R., Ronco, C., Demange, L., and Benhida, R. (2017). Hypoxia
inducible factor down-regulation, cancer and cancer stem cells (CSCs): ongoing success stories.
Medchemcomm 8, 21–52.
65.Mayer, B., Klement, G., Kaneko, M., Man, S., Jothy, S., Rak, J., and Kerbel, R.S.
(2001). Multicellular gastric cancer spheroids recapitulate growth pattern and differentiation
phenotype of human gastric carcinomas. Gastroenterology 121, 839–852.
66.Mehta, G., Hsiao, A.Y., Ingram, M., Luker, G.D., and Takayama, S. (2012).
Opportunities and challenges for use of tumor spheroids as models to test drug delivery and
efficacy. J. Control. Release 164, 192–204.
67.Menakuru, S.R., Brown, N.J., Staton, C.A., and Reed, M.W.R. (2008).
Angiogenesis in pre-malignant conditions. Br. J. Cancer 99, 1961–1966.
68.Minchinton, A.I., and Tannock, I.F. (2006). Drug penetration in solid tumours.
Nat. Rev. Cancer 6, 583–592.
69.Miranti, C.K., and Brugge, J.S. (2002). Sensing the environment: a historical
perspective on integrin signal transduction Early studies on the regulation of cell behaviour by
adhesion. Nat. Cell Biol. 4.
70.Mori, Y., Yamawaki, K., Ishiguro, T., Yoshihara, K., Ueda, H., Sato, A., Ohata,
H., Yoshida, Y., Minamino, T., Okamoto, K., et al. (2019). ALDH-Dependent Glycolytic
Activation Mediates Stemness and Paclitaxel Resistance in Patient-Derived Spheroid Models of
Uterine Endometrial Cancer. Stem Cell Reports 13, 730–746.
71.Moscona, A., and Moscona, H. (1952). The dissociation and aggregation of cells
from organ rudiments of the early chick embryo. J. Anat. 86, 287–301.
72.Mueller-Klieser, W. (1984). Method for the determination of oxygen consumption
rates and diffusion coefficients in multicellular spheroids. Biophys. J. 46, 343–348.
73.Nath, S., and Devi, G.R. (2016). Three-dimensional culture systems in cancer
research: Focus on tumor spheroid model. Pharmacol. Ther. 163, 94–108.
74.Oudar, O. (2000). Spheroids: Relation between tumour and endothelial cells. Crit.
Rev. Oncol. Hematol. 36, 99–106.
75.Pathi, S.P., Kowalczewski, C., Tadipatri, R., and Fischbach, C. (2010). A novel 3-
D mineralized tumor model to study breast cancer bone metastasis. PLoS One 5, 1–10.
76.Quail, D.F., and Joyce, J.A. (2013). Microenvironmental regulation of tumor
progression and metastasis. Nat. Med. 19, 1423–1437.
77.Rama-Esendagli, D., Esendagli, G., Yilmaz, G., and Guc, D. (2014). Spheroid
formation and invasion capacity are differentially influenced by co-cultures of fibroblast and
macrophage cells in breast cancer. Mol. Biol. Rep. 41, 2885–2892.
78.De Ridder, L., Cornelissen, M., and De Ridder, D. (2000). Autologous spheroid
culture: A screening tool for human brain tumour invasion. Crit. Rev. Oncol. Hematol. 36, 107–
122.
79.Rimann, M., and Graf-Hausner, U. (2012). Synthetic 3D multicellular systems for
drug development. Curr. Opin. Biotechnol. 23, 803–809.
80.Rodríguez-Enríquez, S., Gallardo-Pérez, J.C., Avilés-Salas, A., Marín-Hernández,
A., Carreño-Fuentes, L., Maldonado-Lagunas, V., and Moreno-Sánchez, R. (2008). Energy
metabolism transition in multi-cellular human tumor spheroids. J. Cell. Physiol. 216, 189–197.
81.Sachs, N., and Clevers, H. (2014). Organoid cultures for the analysis of cancer
phenotypes. Curr. Opin. Genet. Dev. 24, 68–73.
82.Shao H, Moller M, Wang D, Ting A, Boulina M, Liu ZJ. (2020) A Novel Stromal
Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and
Drug Discovery. J Vis Exp. 156.
83.Shi, C., Cao, H., He, W., Gao, F., Liu, Y., and Yin, L. (2015). Novel drug delivery
liposomes targeted with a fully human anti-VEGF165 monoclonal antibody show superior
antitumor efficacy in vivo. Biomed. Pharmacother. 73, 48–57.
84.Smyrek, I., Mathew, B., Fischer, S.C., Lissek, S.M., Becker, S., and Stelzer,
E.H.K. (2019). E-cadherin, actin, microtubules and FAK dominate different spheroid formation
phases and important elements of tissue integrity. Biol. Open 8.
85.Sutherland, R.M., and Mccredie, J.A. (1971). Growth of Multicell Spheroids in
Tissue Culture as a Model of Nodular Carcinomas2.
JNCI J. Natl. Cancer Inst. 113–120.
86.Sutherland, R.M., Inch, W.R., McCredie, J.A., and Kruuv, J. (1970). A multi-
component radiation survival curve using an in vitro tumour model. Int. J. Radiat. Biol. 18, 491–
495.
87.T Christensen, J Moan, T Sandquist, L.S. (1984). Multicellular Spheroids as an in
Vitro Model System for Photoradiation Therapy in the Presence of Hpd – PubMed.
88.Talukdar, S., and Kundu, S.C. (2012). A non-mulberry silk fibroin protein based
3D in vitro tumor model for evaluation of anticancer drug activity. Adv. Funct. Mater. 22, 4778–
4788.
89.Tannock, I.F., and Rotin, D. (1989). Acid pH in Tumors and Its Potential for
Therapeutic Exploitation. Cancer Res. 49, 4373–4384.
90.Themistocleous, G.S., Katopodis, H., Sourla, A., Lembessis, P., Doillon, C.J.,
Soucacos, P.N., and Koutsilieris, M. (2004). Three-dimensional type I collagen cell culture
systems for the study of bone pathophysiology. In Vivo (Brooklyn). 18, 687–696.
91.Trédan, O., Galmarini, C.M., Patel, K., and Tannock, I.F. (2007). Drug resistance
and the solid tumor microenvironment. J. Natl. Cancer Inst. 99, 1441–1454.
92.Trikha, M., Zhou, Z., Nemeth, J.A., Chen, Q., Sharp, C., Emmell, E., Giles-
Komar, J., and Nakada, M.T. (2004). CNTO 95, a fully human monoclonal antibody that inhibits
αv integrins, has antitumor and antiangiogenic activity in vivo. Int. J. Cancer 110, 326–335.
93.Vogel, S., Peters, C., Etminan, N., Börger, V., Schimanski, A., Sabel, M.C., and
Sorg, R. V (2013). Biochemical and Biophysical Research Communications Migration of
mesenchymal stem cells towards glioblastoma cells depends on hepatocyte-growth factor and is
enhanced by aminolaevulinic acid-mediated photodynamic treatment. Biochem. Biophys. Res.
Commun. 431, 428–432.
94.Weiswald, L.B., Richon, S., Validire, P., Briffod, M., Lai-Kuen, R., Cordelières,
F.P., Bertrand, F., Dargere, D., Massonnet, G., Marangoni, E., et al. (2009). Newly characterised
ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness.
Br. J. Cancer 101, 473–482.
95.Weiswald, L.B., Bellet, D., and Dangles-Marie, V. (2015). Spherical Cancer
Models in Tumor Biology. Neoplasia (United States) 17, 1–15.
96.Weng, K.C., Hashizume, R., Noble, C.O., Serwer, L.P., Drummond, D.C.,
Kirpotin, D.B., Kuwabara, A.M., Chao, L.X., Chen, F.F., James, C.D., et al. (2013). Convection-
enhanced delivery of targeted quantum dot-immunoliposome hybrid nanoparticles to intracranial
brain tumor models. Nanomedicine 8, 1913–1925.
97.West, C.M.L., and Moore, J. V. (1989). Flow Cytometric Analysis of Intracellular
Hematoporphyrin Derivative in Human Tumor Cells and Multicellular Spheroids. Photochem.
Photobiol. 50, 665–669.
98.Wu T, Dai Y. Tumor Microenvironment and Therapeutic Response. (2017).
Cancer Lett . 387, 61–68.
99.Ying, X., Wen, H., Lu, W.L., Du, J., Guo, J., Tian, W., Men, Y., Zhang, Y., Li,
R.J., Yang, T.Y., et al. (2010). Dual-targeting daunorubicin liposomes improve the therapeutic
efficacy of brain glioma in animals. J. Control. Release 141, 183–192.
100.Yokoi, K., Tanei, T., Godin, B., van de Ven, A.L., Hanibuchi, M., Matsunoki, A.,
Alexander, J., and Ferrari, M. (2014). Serum biomarkers for personalization of nanotherapeutics-
based therapy in different tumor and organ microenvironments. Cancer Lett. 345, 48–55.
101.Young, S.R., Saar, M., Santos, J., Nguyen, H.M., Vessella, R.L., and Peehl, D.M.
(2013). Establishment and serial passage of cell cultures derived from LuCaP xenografts.
Prostate 73, 1251–1262.
102.Yuhas, J.M., Tarleton, A.E., and Molzen, K.B. (1978). Multicellular Tumor
Spheroid Formation by Breast Cancer Cells Isolated from Different Sites. Cancer Res. 38, 2486–
2491.
103.Zhu, C., Sempkowski, M., Holleran, T., Linz, T., Bertalan, T., Josefsson, A.,
Bruchertseifer, F., Morgenstern, A., and Sofou, S. (2017). Alpha-particle radiotherapy: For large
solid tumors diffusion trumps targeting. Biomaterials 130, 67–75.
104.Ziskin, M.C. (1983). Growth of Mammalian Multicellular Tumor Spheroids.
Cancer Res. 43, 556–560.