Leveraging and manufacturing in vitro multicellular spheroid-based tumor cell model as a preclinical tool for translating dysregulated tumor metabolism into clinical targets and biomarkers

Abstract

The grand challenge now and in the near future for the pharmaceutical industry is how to efficiently improve R&D productivity. Currently, the approval rate of the entire clinical drug development process is extremely low, and the high attrition in the phase I clinical trial is up to 95%; 67% and 33% of all drugs that enter Phase II and Phase III clinical trials fail to transit into the next stage, respectively. To achieve a higher success rate in clinical trials, developing efficient drug screening method based on more in vivo like tumor tissue is an urgent need to predict the toxicity and efficacy of candidate drugs. In comparison to 2D planar tumor model, the 3D multicellular tumor spheroid (MTS) can better simulate the spatial structure, hypoxia and nutrient gradient, extracellular matrix (ECM) deposition and drug resistance mechanism of tumor in vivo. Thus, such model can be applied for high-throughput drug screening and evaluation, and also can be utilized to initiate a series of fundamental research areas regarding oncogenesis, tumor progression and invasion, pharmacokinetics, drug metabolism, gene therapy and immune mechanism. This review article discusses the abnormal metabolism of cancer cells and highlights the potential role of MTSs as being used as efficient preclinical models. Also, the key features and preparation protocols of MTSs as well as the tools and techniques used for their analysis were summarized and the application of 3D tumor spheroid in specific drug screening and in the elucidation of drug resistance mechanism was also provided. Despite the great knowledge gap within biological sciences and bioengineering, the grand blueprint for adaptable stirred-tank culture strategies for large-scale production of MTSs is envisioned.

Keywords

Multicellular tumor spheroids / In vitro models / Tumor metabolism / Preclinical evaluation / Cancer therapy / Precision medicine

References

[1] Abecasis B, Aguiar T, Arnault E, Costa R, Gomes-Alves P, Aspegren A, Serra M, Alves PM. Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: bioprocess intensification and scaling-up approaches. J Biotechnol, 2017, 246: 81-93.

[2] Achilli T-M, Meyer J, Morgan JR. Advances in the formation, use and understanding of multi-cellular spheroids. Expert Opin Biol Th, 2012, 12: 1347-1360.

[3] Ahn J, Lee HJ, Oh SJ, Kim W, Mun SJ, Lee JH, Jung CR, Cho HS, Kim DS, Son MJ, Chung KS. Developing scalable cultivation systems of hepatic spheroids for drug metabolism via genomic and functional analyses. Biotechnol Bioeng, 2019, 116: 1496-1508.

[4] Alberghina L, Gaglio D. Redox control of glutamine utilization in cancer. Cell Death Dis, 2014, 5: 1561.

[5] Alkan HF, Walter KE, Luengo A, Madreiter-Sokolowski CT, Stryeck S, Lau AN, Al-Zoughbi W, Lewis CA, Thomas CJ, Hoefler G, Graier WF, Madl T, Vander Heiden MG, Bogner-Strauss JG. Cytosolic aspartate availability determines cell survival when glutamine is limiting. Cell Metab, 2018, 28: 706-720.e706.

[6] Alvarez-Pérez J, Ballesteros P, Cerdán S. Microscopic images of intraspheroidal pH by1H magnetic resonance chemical shift imaging of pH sensitive indicators. Magma, 2005, 18: 293-301.

[7] Ananieva E. Targeting amino acid metabolism in cancer growth and anti-tumor immune response. World J Biol Chem, 2015, 6: 281-289.

[8] Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang J, Shen M, Bellinger G, Sasaki AT, Locasale JW, Auld DS, Thomas CJ, Heiden MGV, Cantley LC. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science, 2011, 334: 1278-1283.

[9] Apicella M, Giannoni E, Fiore S, Ferrari KJ, Fernandez-Perez D, Isella C, Granchi C, Minutolo F, Sottile A, Comoglio PM, Medico E, Pietrantonio F, Volante M, Pasini D, Chiarugi P, Giordano S, Corso S. Increased lactate secretion by cancer cells sustains non-cell-autonomous adaptive resistance to MET and EGFR targeted therapies. Cell Metab, 2018, 28(848–865): e846.

[10] Arsenis C, Gordon JS, Touster O. Degradation of nucleic acid by lysosomal extracts of rat liver and Ehrlich ascites tumor cells. J Biol Chem, 1970, 245: 205-211.

[11] Asghar W, El Assal R, Shafiee H, Pitteri S, Paulmurugan R, Demirci U. Engineering cancer microenvironments for in vitro 3-D tumor models. Mater Today, 2015, 18: 539-553.

[12] Azarin SM, Yi J, Gower RM, Aguado BA, Sullivan ME, Goodman AG, Jiang EJ, Rao SS, Ren Y, Tucker SL, Backman V, Jeruss JS, Shea LD. In vivo capture and label-free detection of early metastatic cells. Nat Commun, 2015, 6: 8094.

[13] Badea MA, Balas M, Hermenean A, Ciceu A, Herman H, Ionita D, Dinischiotu A. Influence of matrigel on single- and multiple-spheroid cultures in breast cancer research. SLAS Discov, 2019, 24: 563-578.

[14] Barcellos-Hoff MH, Aggeler J, Ram TG, Bissell MJ. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development, 1989, 105: 223-235.

[15] Basan M, Hui S, Okano H, Zhang Z, Shen Y, Williamson JR, Hwa T. Overflow metabolism in Escherichia coli results from efficient proteome allocation. Nature, 2015, 528: 99-104.

[16] Bennett CF, Swayze EE. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol, 2010, 50: 259-293.

[17] Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, Bensimon A, Zamir G, Shewach DS, Kerem B. Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell, 2011, 145: 435-446.

[18] Bhise NS, Ribas J, Manoharan V, Zhang YS, Polini A, Massa S, Dokmeci MR, Khademhosseini A. Organ-on-a-chip platforms for studying drug delivery systems. J Control Release, 2014, 190: 82-93.

[19] Birsoy K, Possemato R, Lorbeer FK, Bayraktar EC, Thiru P, Yucel B, Wang T, Chen WW, Clish CB, Sabatini DM. Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides. Nature, 2014, 508: 108-112.

[20] Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat Immunol, 2010, 11: 889-896.

[21] Bodur C, Karakas B, Timucin AC, Tezil T, Basaga H. AMP-activated protein kinase couples 3-bromopyruvate-induced energy depletion to apoptosis via activation of FoxO3a and upregulation of proapoptotic Bcl-2 proteins. Mol Carcinogen, 2016, 55: 1584-1597.

[22] Breslin S, O’Driscoll L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today, 2013, 18: 240-249.

[23] Burke AR, Singh RN, Carroll DL, Wood JC, D’Agostino Jr RB, Ajayan PM, Torti FM, Torti SV. The resistance of breast cancer stem cells to conventional hyperthermia and their sensitivity to nanoparticle-mediated photothermal therapy. Biomaterials, 2012, 33: 2961-2970.

[24] Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat Rev Cancer, 2011, 11: 85-95.

[25] Camarda R, Zhou AY, Kohnz RA, Balakrishnan S, Mahieu C, Anderton B, Eyob H, Kajimura S, Tward A, Krings G, Nomura DK, Goga A. Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer. Nat Med, 2016, 22: 427-432.

[26] Cardone RA, Casavola V, Reshkin SJ. The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer, 2005, 5: 786-795.

[27] Carlsson J, Acker H. Relations between pH, oxygen partial pressure and growth in cultured cell spheroids. Int J Cancer, 1988, 42: 715-720.

[28] Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature, 2000, 407: 249-257.

[29] Carracedo A, Cantley LC, Pandolfi PP. Cancer metabolism: fatty acid oxidation in the limelight. Nat Rev Cancer, 2013, 13: 227-232.

[30] Carvalho MA, Zecchin KG, Seguin F, Bastos DC, Agostini M, Rangel ALC, Veiga SS, Raposo HF, Oliveira HC, Loda M. Fatty acid synthase inhibition with Orlistat promotes apoptosis and reduces cell growth and lymph node metastasis in a mouse melanoma model. Int J Cancer, 2008, 123: 2557-2565.

[31] Carver K, Ming X, Juliano RL. Multicellular tumor spheroids as a model for assessing delivery of oligonucleotides in three dimensions. Mol Ther Nucleic Acids, 2014, 3: e153.

[32] Chan HF, Zhang Y, Ho YP, Chiu YL, Jung Y, Leong KW. Rapid formation of multicellular spheroids in double-emulsion droplets with controllable microenvironment. Sci Rep, 2013, 3: 3462.

[33] Chaneton B, Hillmann P, Zheng L, Martin ACL, Maddocks ODK, Chokkathukalam A, Coyle JE, Jankevics A, Holding FP, Vousden KH, Frezza C, O’Reilly M, Gottlieb E. Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature, 2012, 491: 458-462.

[34] Chatzinikolaidou M. Cell spheroids: the new frontiers in in vitro models for cancer drug validation. Drug Discov Today, 2016, 21: 1553-1560.

[35] Chen J, Xie J, Jiang Z, Wang B, Wang Y, Hu X. Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2. Oncogene, 2011, 30: 4297-4306.

[36] Chen B, Dai W, Mei D, Liu T, Li S, He B, He B, Yuan L, Zhang H, Wang X, Zhang Q. Comprehensively priming the tumor microenvironment by cancer-associated fibroblast-targeted liposomes for combined therapy with cancer cell-targeted chemotherapeutic drug delivery system. J Control Release, 2016, 241: 68-80.

[37] Clem BF, O’Neal J, Tapolsky G, Clem AL, Imbert-Fernandez Y, Kerr DA, Klarer AC, Redman R, Miller DM, Trent JO. Targeting 6-phosphofructo-2-kinase (PFKFB3) as a therapeutic strategy against cancer. Mol Cancer Ther, 2013, 12: 1461-1470.

[38] Correia AL, Bissell MJ. The tumor microenvironment is a dominant force in multidrug resistance. Drug Resist Updat, 2012, 15: 39-49.

[39] Correia C, Koshkin A, Duarte P, Hu D, Carido M, Sebastião MJ, Gomes-Alves P, Elliott DA, Domian IJ, Teixeira AP. 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes. Biotechnol Bioeng, 2018, 115: 630-644.

[40] Costa EC, Gaspar VM, Coutinho P, Correia IJ. Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models. Biotechnol Bioeng, 2014, 111: 1672-1685.

[41] Costa EC, Moreira AF, de Melo-Diogo D, Gaspar VM, Carvalho MP, Correia IJ. 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnol Adv, 2016, 34: 1427-1441.

[42] Costa EC, de Melo-Diogo D, Moreira AF, Carvalho MP, Correia IJ. Spheroids Formation on Non-Adhesive Surfaces by Liquid Overlay Technique: considerations and Practical Approaches. Biotechnol J, 2018, 13(1): 1700417.

[43] Costa EC, Silva DN, Moreira AF, Correia IJ. Optical clearing methods: an overview of the techniques used for the imaging of 3D spheroids. Biotechnol Bioeng, 2019, 116: 2742-2763.

[44] Courtnay R, Ngo DC, Malik N, Ververis K, Tortorella SM, Karagiannis TC. Cancer metabolism and the Warburg effect: the role of HIF-1 and PI3K. Mol Biol Rep, 2015, 42: 841-851.

[45] Craig AJ, von Felden J, Garcia-Lezana T, Sarcognato S, Villanueva A. Tumour evolution in hepatocellular carcinoma. Nat Rev Gastro Hepat., 2019, 2: 1-14.

[46] Currie E, Schulze A, Zechner R, Walther TC, Farese Jr RV. Cellular fatty acid metabolism and cancer. Cell Metab, 2013, 18: 153-161.

[47] Daemen A, Peterson D, Sahu N, McCord R, Du X, Liu B, Kowanetz K, Hong R, Moffat J, Gao M, Boudreau A, Mroue R, Corson L, O’Brien T, Qing J, Sampath D, Merchant M, Yauch R, Manning G, Settleman J, Hatzivassiliou G, Evangelista M. Metabolite profiling stratifies pancreatic ductal adenocarcinomas into subtypes with distinct sensitivities to metabolic inhibitors. Proc Natl Acad Sci USA, 2015, 112: E4410-4417.

[48] Dalton WS. The tumor microenvironment as a determinant of drug response and resistance. Drug Resist Update, 1999, 2: 285-288.

[49] Däster S, Amatruda N, Calabrese D, Ivanek R, Turrini E, Droeser RA, Zajac P, Fimognari C, Spagnoli GC, Iezzi G, Mele V, Muraro MG. Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment. Oncotarget, 2017, 8: 1725-1736.

[50] Deep G, Schlaepfer IR. Aberrant lipid metabolism promotes prostate cancer: role in cell survival under hypoxia and extracellular vesicles biogenesis. Int J Mol Sci, 2016, 17: 1061.

[51] Doke SK, Dhawale SC. Alternatives to animal testing: a review. Saudi Pharm J, 2015, 23: 223-229.

[52] Dolznig H, Rupp C, Puri C, Haslinger C, Schweifer N, Wieser E, Kerjaschki D, Garin-Chesa P. Modeling colon adenocarcinomas in vitro a 3D co-culture system induces cancer-relevant pathways upon tumor cell and stromal fibroblast interaction. Am J Pathol, 2011, 179: 487-501.

[53] Duman C, Yaqubi K, Hoffmann A, Acikgöz AA, Korshunov A, Bendszus M, Herold-Mende C, Liu H-K, Alfonso J. Acyl-CoA-binding protein drives glioblastoma tumorigenesis by sustaining fatty acid oxidation. Cell Metab, 2019, 30: 274-289.

[54] Duvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, Triantafellow E, Ma Q, Gorski R, Cleaver S, Vander Heiden MG, MacKeigan JP, Finan PM, Clish CB, Murphy LO, Manning BD. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell, 2010, 39: 171-183.

[55] El Sayed S, El-Magd RA, Shishido Y, Chung S, Diem T, Sakai T, Watanabe H, Kagami S, Fukui K. 3-Bromopyruvate antagonizes effects of lactate and pyruvate, synergizes with citrate and exerts novel anti-glioma effects. J Bioenerg Biomembr, 2012, 44: 61-79.

[56] Erecińska M, Deas J, Silver I. The effect of pH on glycolysis and phosphofructokinase activity in cultured cells and synaptosomes. J Neurochem, 1995, 65: 2765-2772.

[57] Ertel A, Verghese A, Byers SW, Ochs M, Tozeren A. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells. Mol Cancer, 2006, 5: 55.

[58] Fan J, Ye J, Kamphorst JJ, Shlomi T, Thompson CB, Rabinowitz JD. Quantitative flux analysis reveals folate-dependent NADPH production. Nature, 2014, 510: 298-302.

[59] Fan TW-M, El-Amouri SS, Macedo JK, Wang QJ, Song H, Cassel T, Lane AN. Stable isotope-resolved metabolomics shows metabolic resistance to anti-cancer selenite in 3D spheroids versus 2D cell cultures. Metabolites, 2018, 8: 40.

[60] Fane M, Weeraratna AT. How the ageing microenvironment influences tumour progression. Nat Rev Cancer., 2019, 20: 1-18.

[61] Fang Y, Eglen RM. Three-dimensional cell cultures in drug discovery and development. SLAS Discov, 2017, 22: 456-472.

[62] Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol, 2013, 31: 108-115.

[63] Ferreira LP, Gaspar VM, Mano JF. Design of spherically structured 3D in vitro tumor models -Advances and prospects. Acta Biomater, 2018, 75: 11-34.

[64] Field CS, Baixauli F, Kyle RL, Puleston DJ, Cameron AM, Sanin DE, Hippen KL, Loschi M, Thangavelu G, Corrado M, Edwards-Hicks J, Grzes KM, Pearce EJ, Blazar BR, Pearce EL. Mitochondrial integrity regulated by lipid metabolism is a cell-intrinsic checkpoint for treg suppressive function. Cell Metab, 2019, 31(2): 422-437.

[65] Fitzgerald KA, Malhotra M, Curtin CM, O’Brien FJ, O’Driscoll CM. Life in 3D is never flat: 3D models to optimise drug delivery. J Control Release, 2015, 10(215): 39-54.

[66] Flavell RA, Sanjabi S, Wrzesinski SH, Licona-Limon P. The polarization of immune cells in the tumour environment by TGFbeta. Nat Rev Immunol, 2010, 10: 554-567.

[67] Follain G, Herrmann D, Harlepp S, Hyenne V, Osmani N, Warren S C, Timpson P, Goetz J G (2019) Fluids and their mechanics in tumour transit: shaping metastasis. Nat Rev Cancer:1–18

[68] Friedrich J, Eder W, Castaneda J, Doss M, Huber E, Ebner R, Kunz-Schughart LA. A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay. J Biomol Screen, 2007, 12: 925-937.

[69] Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA. Spheroid-based drug screen: considerations and practical approach. Nat Protoc, 2009, 4: 309-324.

[70] Froehlich K, Haeger JD, Heger J, Pastuschek J, Photini SM, Yan Y, Lupp A, Pfarrer C, Mrowka R, Schleussner E, Markert UR, Schmidt A. Generation of multicellular breast cancer tumor spheroids: comparison of different protocols. J Mammary Gland Biol Neoplasia, 2016, 21: 89-98.

[71] Galateanu B, Hudita A, Negrei C, Ion R-M, Costache M, Stan M, Nikitovic D, Hayes AW, Spandidos DA, Tsatsakis AM, Ginghina O. Impact of multicellular tumor spheroids as an in vivo-like tumor model on anticancer drug response. Int J Oncol, 2016, 48: 2295-2302.

[72] Gill BJ, Gibbons DL, Roudsari LC, Saik JE, Rizvi ZH, Roybal JD, Kurie JM, West JL. A synthetic matrix with independently tunable biochemistry and mechanical properties to study epithelial morphogenesis and EMT in a lung adenocarcinoma model. Cancer Res, 2012, 72: 6013-6023.

[73] Gilmore AP, Metcalfe AD, Romer LH, Streuli CH. Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization. J Cell Biol, 2000, 149: 431-446.

[74] Glimelius B, Norling B, Nederman T, Carlsson J. Extracellular matrices in multicellular spheroids of human glioma origin: increased incorporation of proteoglycans and fibronectin as compared to monolayer cultures. Apmis, 1988, 96: 433-444.

[75] Godugu C, Patel AR, Desai U, Andey T, Sams A, Singh M. AlgiMatrix based 3D cell culture system as an in vitro tumor model for anticancer studies. PLoS ONE, 2013, 8: e53708.

[76] Gong X, Lin C, Cheng J, Su J, Zhao H, Liu T, Wen X, Zhao P. Generation of multicellular tumor spheroids with microwell-based agarose scaffolds for drug testing. PLoS ONE, 2015, 10: e0130348.

[77] Gonzalez PS, O’Prey J, Cardaci S, Barthet VJA, Sakamaki JI, Beaumatin F, Roseweir A, Gay DM, Mackay G, Malviya G, Kania E, Ritchie S, Baudot AD, Zunino B, Mrowinska A, Nixon C, Ennis D, Hoyle A, Millan D, McNeish IA, Sansom OJ, Edwards J, Ryan KM. Mannose impairs tumour growth and enhances chemotherapy. Nature, 2018, 563: 719-723.

[78] Goodwin T, Prewett T, Wolf DA, Spaulding G. Reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity. J Cell Biochem, 1993, 51: 301-311.

[79] Graf BW, Boppart SA. Carloni V. Imaging and analysis of three-dimensional cell culture models. Live cell imaging, 2010, Berlin: Springer.

[80] Granchi C. ATP citrate lyase (ACLY) inhibitors: an anti-cancer strategy at the crossroads of glucose and lipid metabolism. Eur J Med Chem, 2018, 157: 1276-1291.

[81] Granchi C, Calvaresi EC, Tuccinardi T, Paterni I, Macchia M, Martinelli A, Hergenrother PJ, Minutolo F. Assessing the differential action on cancer cells of LDH-A inhibitors based on the N-hydroxyindole-2-carboxylate (NHI) and malonic (Mal) scaffolds. Org Biomol Chem, 2013, 11: 6588-6596.

[82] Gu C, Du Y, Gao Y, Yao Z, Gu X, Zhang Q, Xu J, Deng W. Anti-CD44 mAb remodels biological behaviors of spheroid cells with stemness from human ovarian cancer cell line SKOV-3. Chinese Sci Bull, 2012, 57: 1288-1297.

[83] Gunness P, Mueller D, Shevchenko V, Heinzle E, Ingelman-Sundberg M, Noor F. 3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. Toxicol Sci, 2013, 133: 67-78.

[84] Hamilton GA, Westmoreland C, George E. Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers. Vitro Cell Dev-An, 2001, 37: 656.

[85] Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell, 2012, 21: 309-322.

[86] Harjes U, Kalucka J, Carmeliet P. Targeting fatty acid metabolism in cancer and endothelial cells. Crit Rev Oncol Hemat, 2016, 97: 15-21.

[87] Harmon AW, Patel YM. Naringenin inhibits glucose uptake in MCF-7 breast cancer cells: a mechanism for impaired cellular proliferation. Breast Cancer Res Tr, 2004, 85: 103-110.

[88] Hauptmann S, Zwadlo-Klarwasser G, Jansen M, Klosterhalfen B, Kirkpatrick CJ. Macrophages and multicellular tumor spheroids in co-culture: a three-dimensional model to study tumor-host interactions. Evidence for macrophage-mediated tumor cell proliferation and migration. Am J Pathol, 1993, 143: 1406-1415.

[89] Heiden MGV, Cantley LC, Thompson CB. Understanding the warburg effect: the metabolic requirements of cell proliferation. Science, 2009, 324: 1029-1033.

[90] Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA. Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol, 2010, 148: 3-15.

[91] Hirt C, Papadimitropoulos A, Mele V, Muraro MG, Mengus C, Iezzi G, Terracciano L, Martin I, Spagnoli GC. “In vitro” 3D models of tumor-immune system interaction. Adv Drug Deliv Rev, 2014, 79–80: 145-154.

[92] Hoffmann OI, Ilmberger C, Magosch S, Joka M, Jauch KW, Mayer B. Impact of the spheroid model complexity on drug response. J Biotechnol, 2015, 205: 14-23.

[93] Horie M, Saito A, Yamaguchi Y, Ohshima M, Nagase T. Three-dimensional co-culture model for tumor-stromal interaction. JoVE., 2015, 20: e52469.

[94] Huang BW, Gao JQ. Application of 3D cultured multicellular spheroid tumor models in tumor-targeted drug delivery system research. J Control Release, 2018, 270: 246-259.

[95] Huang K, Ma H, Liu J, Huo S, Kumar A, Wei T, Zhang X, Jin S, Gan Y, Wang PC. Size-dependent localization and penetration of ultrasmall gold nanoparticles in cancer cells, multicellular spheroids, and tumors in vivo. ACS Nano, 2012, 6: 4483-4493.

[96] Huang H, Ding Y, Sun XS, Nguyen TA. Peptide hydrogelation and cell encapsulation for 3D culture of MCF-7 breast cancer cells. PLoS ONE, 2013, 8: e59482.

[97] Hui S, Ghergurovich JM, Morscher RJ, Jang C, Teng X, Lu W, Esparza LA, Reya T, Le Z, Yanxiang Guo J, White E, Rabinowitz JD. Glucose feeds the TCA cycle via circulating lactate. Nature, 2017, 551: 115-118.

[98] Ivascu A, Kubbies M. Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen, 2006, 11: 922-932.

[99] Jain M, Nilsson R, Sharma S, Madhusudhan N, Kitami T, Souza AL, Kafri R, Kirschner MW, Clish CB, Mootha VK. Metabolite profiling identifies a key role for glycine in rapid cancer cell proliferation. Science, 2012, 336: 1040-1044.

[100] Jang M, Kim SS, Lee J. Cancer cell metabolism: implications for therapeutic targets. Exp Mol Med, 2013, 45: e45.

[101] Jarrar MH, Baranova A. PPARγ activation by thiazolidinediones (TZDs) may modulate breast carcinoma outcome: the importance of interplay with TGFβ signalling. J Cell Mol Med, 2007, 11: 71-87.

[102] Jiang L, Boufersaoui A, Yang C, Ko B, Rakheja D, Guevara G, Hu Z, DeBerardinis RJ. Quantitative metabolic flux analysis reveals an unconventional pathway of fatty acid synthesis in cancer cells deficient for the mitochondrial citrate transport protein. Metab Eng, 2017, 43: 198-207.

[103] Jo S, Son M, Koh H, Lee S, Song I, Kim Y, Lee Y, Jeong K, Kim WB, Park J. Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase. J Biol Chem, 2001, 276: 16168-16176.

[104] Jo EB, Hong D, Lee YS, Lee H, Park JB, Kim SJ. Establishment of a Novel PDX Mouse Model and Evaluation of the Tumor Suppression Efficacy of Bortezomib Against Liposarcoma. Transl Oncol, 2019, 12: 269-281.

[105] Johansson HJ, Socciarelli F, Vacanti NM, Haugen MH, Zhu Y, Siavelis I, Fernandez-Woodbridge A, Aure MR, Sennblad B, Vesterlund M. Breast cancer quantitative proteome and proteogenomic landscape. Nat Commun, 2019, 10: 1-14.

[106] Jung HR, Kang HM, Ryu JW, Kim DS, Noh KH, Kim ES, Lee HJ, Chung KS, Cho HS, Kim NS, Im DS, Lim JH, Jung CR. Cell spheroids with enhanced aggressiveness to mimic human liver cancer in vitro and in vivo. Sci Rep, 2017, 7: 10499.

[107] Jyoti A, Fugit KD, Sethi P, McGarry RC, Anderson BD, Upreti M. An in vitro assessment of liposomal topotecan simulating metronomic chemotherapy in combination with radiation in tumor-endothelial spheroids. Sci Rep, 2015, 5: 15236.

[108] Kamphorst JJ, Cross JR, Fan J, de Stanchina E, Mathew R, White EP, Thompson CB, Rabinowitz JD. Hypoxic and Ras-transformed cells support growth by scavenging unsaturated fatty acids from lysophospholipids. Proc Natl Acad Sci USA, 2013, 110: 8882-8887.

[109] Kang A, Park J, Ju J, Jeong GS, Lee SH. Cell encapsulation via microtechnologies. Biomaterials, 2014, 35: 2651-2663.

[110] Kang A, Seo HI, Chung BG, Lee SH. Concave microwell array-mediated three-dimensional tumor model for screening anticancer drug-loaded nanoparticles. Nanomedicine, 2015, 11: 1153-1161.

[111] Karacali B, Vamvakidou AP, Tozeren A. Automated recognition of cell phenotypes in histology images based on membrane- and nuclei-targeting biomarkers. BMC Med Imaging, 2007, 7: 7.

[112] Katt ME, Placone AL, Wong AD, Xu ZS, Searson PC. In vitro tumor models: advantages, disadvantages, variables, and selecting the right platform. Front Bioeng Biotechnol, 2016, 4: 12.

[113] Kelm JM, Fussenegger M. Microscale tissue engineering using gravity-enforced cell assembly. Trends Biotechnol, 2004, 22: 195-202.

[114] Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng, 2003, 83: 173-180.

[115] Khaitan D, Chandna S, Arya MB, Dwarakanath BS. Establishment and characterization of multicellular spheroids from a human glioma cell line; Implications for tumor therapy. J Transl Med, 2006, 4: 12.

[116] Kimura H, Sakai Y, Fujii T. Organ/body-on-a-chip based on microfluidic technology for drug discovery. Drug Metab Pharmacokinet, 2018, 33: 43-48.

[117] Koudan EV, Gryadunova AA, Karalkin PA, Korneva JV, Meteleva NY, Babichenko II, Volkov AV, Rodionov SA, Parfenov VA, Pereira FD. Multiparametric analysis of tissue spheroids fabricated from different types of cells. Biotechnol J., 2020, 15: 1900217.

[118] Kunz-Schughart LA, Freyer JP, Hofstaedter F, Ebner R. The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J Biomol Screen, 2004, 9: 273-285.

[119] Labuschagne CF, van den Broek NJ, Mackay GM, Vousden KH, Maddocks OD. Serine, but not glycine, supports one-carbon metabolism and proliferation of cancer cells. Cell Rep, 2014, 7: 1248-1258.

[120] Lamichhane SP, Arya N, Kohler E, Xiang S, Christensen J, Shastri VP. Recapitulating epithelial tumor microenvironment in vitro using three dimensional tri-culture of human epithelial, endothelial, and mesenchymal cells. BMC Cancer, 2016, 16: 581.

[121] Lazzari G, Couvreur P, Mura S. Multicellular tumor spheroids: a relevant 3D model for the in vitro preclinical investigation of polymer nanomedicines. Polym Chem, 2017, 8: 4947-4969.

[122] Lazzari G, Nicolas V, Matsusaki M, Akashi M, Couvreur P, Mura S. Multicellular spheroid based on a triple co-culture: a novel 3D model to mimic pancreatic tumor complexity. Acta Biomater, 2018, 78: 296-307.

[123] Lee SY, Jeong E, Jeon HM, Kim CH, Kang HS. Implication of necrosis-linked p53 aggregation in acquired apoptotic resistance to 5-FU in MCF-7 multicellular tumour spheroids. Oncol Rep, 2010, 24: 73-79.

[124] Lee SW, Hong S, Jung B, Jeong SY, Byeon JH, Jeong GS, Choi J, Hwang C. In vitro lung cancer multicellular tumor spheroid formation using a microfluidic device. Biotechnol Bioeng, 2019, 116: 3041-3052.

[125] Li W, Liu J, Jackson K, Shi R, Zhao Y. Sensitizing the therapeutic efficacy of taxol with shikonin in human breast cancer cells. PloS ONE, 2014, 9: e94079.

[126] Liao J, Qian F, Tchabo N, Mhawech-Fauceglia P, Beck A, Qian Z, Wang X, Huss WJ, Lele SB, Morrison CD, Odunsi K. Ovarian cancer spheroid cells with stem cell-like properties contribute to tumor generation, metastasis and chemotherapy resistance through hypoxia-resistant metabolism. PLoS ONE, 2014, 9: e84941.

[127] Liberti MV, Locasale JW. The Warburg effect: how does it benefit cancer cells?. Trends Biochem Sci, 2016, 41: 211-218.

[128] Lim W, Park S, Bazer FW, Song G. Naringenin-Induced Apoptotic Cell Death in Prostate Cancer Cells Is Mediated via the PI3K/AKT and MAPK Signaling Pathways. J Cell Biochem, 2017, 118: 1118-1131.

[129] Lin RZ, Chang HY. Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol J, 2008, 3: 1172-1184.

[130] Liu XQ, Kiefl R, Roskopf C, Tian F, Huber RM. Interactions among lung cancer cells, fibroblasts, and macrophages in 3D Co-Cultures and the impact on MMP-1 and VEGF expression. PLoS ONE, 2016, 11: e0156268.

[131] Liu X, Cooper DE, Cluntun AA, Warmoes MO, Zhao S, Reid MA, Liu J, Lund PJ, Lopes M, Garcia BA, Wellen KE, Kirsch DG, Locasale JW. Acetate production from glucose and coupling to mitochondrial metabolism in mammals. Cell, 2018, 175: 502-513.

[132] Locasale JW. Serine, glycine and one-carbon units: cancer metabolism in full circle. Nat Rev Cancer, 2013, 13: 572-583.

[133] Lukina MM, Dudenkova VV, Ignatova NI, Druzhkova IN, Shimolina LE, Zagaynova EV, Shirmanova MV. Metabolic cofactors NAD(P)H and FAD as potential indicators of cancer cell response to chemotherapy with paclitaxel. BBA-Gen Subjects, 2018, 1862: 1693-1700.

[134] Ma H, Jiang Q, Han S, Wu Y, Tomshine JC, Wang D, Gan Y, Zou G, Liang X-J. Multicellular tumor spheroids as an in vivo-like tumor model for three-dimensional imaging of chemotherapeutic and nano material cellular penetration. Mol imaging, 2012, 11(7290): 00012.

[135] Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul, 2001, 41: 189-207.

[136] Majety M, Pradel LP, Gies M, Ries CH. Fibroblasts influence survival and therapeutic response in a 3D Co-Culture model. PLoS ONE, 2015, 10: e0127948.

[137] Mao S, Zhang W, Huang Q, Khan M, Li H, Uchiyama K, Lin JM. In Situ Scatheless Cell Detachment reveals correlation between adhesion strength and viability at single-cell resolution. Angew Chem Int Ed Engl, 2018, 57: 236-240.

[138] Mao X, McManaway S, Jaiswal JK, Patel PB, Wilson WR, Hicks KO, Bogle G. An agent-based model for drug-radiation interactions in the tumour microenvironment: hypoxia-activated prodrug SN30000 in multicellular tumour spheroids. PLoS Comput Biol, 2018, 14: e1006469.

[139] Mari M, Morales A, Colell A, Garciaruiz C, Fernandezcheca JC. Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Sign, 2009, 11: 2685-2700.

[140] Markovitz-Bishitz Y, Tauber Y, Afrimzon E, Zurgil N, Sobolev M, Shafran Y, Deutsch A, Howitz S, Deutsch M. A polymer microstructure array for the formation, culturing, and high throughput drug screening of breast cancer spheroids. Biomaterials, 2010, 31: 8436-8444.

[141] Martinez-Monge I, Albiol J, Lecina M, Liste-Calleja L, Miret J, Sola C, Cairo JJ. Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures. Biotechnol Bioeng, 2019, 116: 388-404.

[142] Mashima T, Sato S, Okabe S, Miyata S, Matsuura M, Sugimoto Y, Tsuruo T, Seimiya H. Acyl-CoA synthetase as a cancer survival factor: its inhibition enhances the efficacy of etoposide. Cancer Sci, 2010, 100: 1556-1562.

[143] Mashimo T, Pichumani K, Vemireddy V, Hatanpaa KJ, Singh DK, Sirasanagandla S, Nannepaga S, Piccirillo SG, Kovacs Z, Foong C, Huang Z, Barnett S, Mickey BE, DeBerardinis RJ, Tu BP, Maher EA, Bachoo RM. Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Cell, 2014, 159: 1603-1614.

[144] Matsubara S, Ding Q, Miyazaki Y, Kuwahata T, Tsukasa K, Takao S. mTOR plays critical roles in pancreatic cancer stem cells through specific and stemness-related functions. Sci Rep, 2013, 3: 3230.

[145] Mayers JR, Wu C, Clish CB, Kraft P, Torrence ME, Fiske BP, Yuan C, Bao Y, Townsend MK, Tworoger SS, Davidson SM, Papagiannakopoulos T, Yang A, Dayton TL, Ogino S, Stampfer MJ, Giovannucci EL, Qian ZR, Rubinson DA, Ma J, Sesso HD, Gaziano JM, Cochrane BB, Liu S, Wactawski-Wende J, Manson JE, Pollak MN, Kimmelman AC, Souza A, Pierce K, Wang TJ, Gerszten RE, Fuchs CS, Vander Heiden MG, Wolpin BM. Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development. Nat Med, 2014, 20: 1193-1198.

[146] Mayers JR, Torrence ME, Danai LV, Papagiannakopoulos T, Davidson SM, Bauer MR, Lau AN, Ji BW, Dixit PD, Hosios AM, Muir A, Chin CR, Freinkman E, Jacks T, Wolpin BM, Vitkup D, Vander Heiden MG. Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers. Science, 2016, 353: 1161-1165.

[147] McBrayer SK, Mayers JR, DiNatale GJ, Shi DD, Khanal J, Chakraborty AA, Sarosiek KA, Briggs KJ, Robbins AK, Sewastianik T, Shareef SJ, Olenchock BA, Parker SJ, Tateishi K, Spinelli JB, Islam M, Haigis MC, Looper RE, Ligon KL, Bernstein BE, Carrasco RD, Cahill DP, Asara JM, Metallo CM, Yennawar NH, Vander Heiden MG, Kaelin Jr WG. Transaminase inhibition by 2-Hydroxyglutarate impairs glutamate biosynthesis and redox homeostasis in glioma. Cell, 2018, 175(101–116): e125.

[148] McIntyre A, Hulikova A, Ledaki I, Snell C, Singleton D, Steers G, Seden P, Jones D, Bridges E, Wigfield S. Disrupting hypoxia-induced bicarbonate transport acidifies tumor cells and suppresses tumor growth. Cancer Res, 2016, 76: 3744-3755.

[149] McMahon KM, Volpato M, Chi HY, Musiwaro P, Poterlowicz K, Peng Y, Scally AJ, Patterson LH, Phillips RM, Sutton CW. Characterization of changes in the proteome in different regions of 3D multicell tumor spheroids. J Proteome Res, 2012, 11: 2863-2875.

[150] Mehta G, Hsiao AY, Ingram M, Luker GD, Takayama S. Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J Control Release, 2012, 164: 192-204.

[151] Minchinton AI, Tannock IF. Drug penetration in solid tumours. Nat Rev Cancer, 2006, 6: 583-592.

[152] Molenaar RJ, Botman D, Smits MA, Hira VV, van Lith SA, Stap J, Henneman P, Khurshed M, Lenting K, Mul AN. Radioprotection of IDH1-mutated cancer cells by the IDH1-mutant inhibitor AGI-5198. Cancer Res, 2015, 75: 4790-4802.

[153] Moorst MV, Dass CR. Methods for co-culturing tumour and endothelial cells: systems and their applications. J Pharma Pharmacol, 2011, 63(12): 1513-1521.

[154] Mullarky E, Lucki NC, Zavareh RB, Anglin JL, Gomes AP, Nicolay BN, Wong JCY, Christen S, Takahashi H, Singh PK. Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers. Proc Natl Acad Sci USA, 2016, 113: 1778-1783.

[155] Nath S, Devi GR. Three-dimensional culture systems in cancer research: focus on tumor spheroid model. Pharmacol Ther, 2016, 163: 94-108.

[156] Nielsen J. Systems biology of metabolism: a driver for developing personalized and precision medicine. Cell Metab, 2017, 25: 572-579.

[157] Nishikawa T, Tanaka Y, Kusamori K, Mizuno N, Mizukami Y, Ogino Y, Shimizu K, Konishi S, Takahashi Y, Takakura Y. Using size-controlled multicellular spheroids of murine adenocarcinoma cells to efficiently establish pulmonary tumors in mice. Biotechnol J, 2017, 12: 1600513.

[158] Nunes AS, Barros AS, Costa EC, Moreira AF, Correia IJ. 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs. Biotechnol Bioeng, 2019, 116: 206-226.

[159] Odell ID, Cook D. Immunofluorescence techniques. J Invest Dermatol, 2013, 133: e4.

[160] Orita H, Coulter J, Lemmon C, Tully E, Vadlamudi A, Medghalchi SM, Kuhajda FP, Gabrielson E. Selective inhibition of fatty acid synthase for lung cancer treatment. Clin Cancer Res, 2007, 13: 7139-7145.

[161] Palm W, Thompson CB. Nutrient acquisition strategies of mammalian cells. Nature, 2017, 546: 234-242.

[162] Parker WB. Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev, 2009, 109: 2880-2893.

[163] Patra KC, Wang Q, Bhaskar PT, Miller L, Wang Z, Wheaton W, Chandel N, Laakso M, Muller WJ, Allen EL, Jha AK, Smolen GA, Clasquin MF, Robey B, Hay N. Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer. Cancer Cell, 2013, 24: 213-228.

[164] Patra B, Peng CC, Liao WH, Lee CH, Tung YC. Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device. Sci Rep, 2016, 6: 21061.

[165] Perche F, Patel NR, Torchilin VP. Accumulation and toxicity of antibody-targeted doxorubicin-loaded PEG-PE micelles in ovarian cancer cell spheroid model. J Control Release, 2012, 164: 95-102.

[166] Pike LS, Smift AL, Croteau NJ, Ferrick DA, Wu M. Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells. Biochim Biophys Acta, 2011, 1807: 726-734.

[167] Pilotte L, Larrieu P, Stroobant V, Colau D, Dolušić E, Frédérick R, De Plaen E, Uyttenhove C, Wouters J, Masereel B. Reversal of tumoral immune resistance by inhibition of tryptophan 2, 3-dioxygenase. Proc Natl Acad Sci USA, 2012, 109: 2497-2502.

[168] Pollak N, Dolle C, Ziegler M. The power to reduce: pyridine nucleotides—small molecules with a multitude of functions. Biochem J, 2007, 402: 205-218.

[169] Rebelo SP, Pinto C, Martins TR, Harrer N, Estrada MF, Loza-Alvarez P, Cabecadas J, Alves PM, Gualda EJ, Sommergruber W, Brito C. 3D-3-culture: a tool to unveil macrophage plasticity in the tumour microenvironment. Biomaterials, 2018, 163: 185-197.

[170] Richter Michael. Functional diversity of organic molecule enzyme cofactors. Nat Prod Rep, 2013, 30: 1324.

[171] Riedl A, Schlederer M, Pudelko K, Stadler M, Walter S, Unterleuthner D, Unger C, Kramer N, Hengstschlager M, Kenner L, Pfeiffer D, Krupitza G, Dolznig H. Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses. J Cell Sci, 2017, 130: 203-218.

[172] Riss T, Moravec R, Niles A, Duellman S, Benink H, Worzella T, Minor L (2004) Assay guidance manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, MD, 2004

[173] Rodrigues T, Kundu B, Silva-Correia J, Kundu SC, Oliveira JM, Reis RL, Correlo VM. Emerging tumor spheroids technologies for 3D in vitro cancer modeling. Pharmacol Ther, 2018, 184: 201-211.

[174] Rusz M, Rampler E, Keppler BK, Jakupec MA, Koellensperger G. Single spheroid metabolomics: optimizing sample preparation of three-dimensional multicellular tumor spheroids. Metabolites, 2019, 9: 304.

[175] Saglam-Metiner P, Gulce-Iz S, Biray-Avci C. Bioengineering-inspired three-dimensional culture systems: organoids to create tumor microenvironment. Gene, 2019, 686: 203-212.

[176] Sakalem ME, Ribeiro-Paes JT. New methodologies for old problems: tridimensional gastrointestinal organoids and guts-on-a-chip. J Coloproctol, 2018, 38: 90-93.

[177] Sakthivel K, O’Brien A, Kim K, Hoorfar M. Microfluidic analysis of heterotypic cellular interactions: a review of techniques and applications. TrAC Trends Anal Chem, 2019, 117: 166-185.

[178] Salamanca-Cardona L, Shah H, Poot AJ, Correa FM, Di Gialleonardo V, Lui H, Miloushev VZ, Granlund KL, Tee SS, Cross JR, Thompson CB, Keshari KR. In vivo imaging of glutamine metabolism to the oncometabolite 2-Hydroxyglutarate in IDH1/2 mutant tumors. Cell Metab, 2017, 26(830–841): e833.

[179] Samudio I, Harmancey R, Fiegl M, Kantarjian H, Konopleva M, Korchin B, Kaluarachchi K, Bornmann W, Duvvuri S, Taegtmeyer H. Pharmacologic inhibition of fatty acid oxidation sensitizes human leukemia cells to apoptosis induction. J Clin Invest, 2010, 120: 142-156.

[180] Sant S, Johnston PA. The production of 3D tumor spheroids for cancer drug discovery. Drug Discov Today Technol, 2017, 23: 27-36.

[181] Santagiuliana R, Stigliano C, Mascheroni P, Ferrari M, Decuzzi P, Schrefler BA. The role of cell lysis and matrix deposition in tumor growth modeling. Adv Model Simul Eng Sci, 2015, 2(1): 19.

[182] Santo VE, Estrada MF, Rebelo SP, Abreu S, Silva I, Pinto C, Veloso SC, Serra AT, Boghaert E, Alves PM, Brito C. Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models. J Biotechnol, 2016, 221: 118-129.

[183] Santo VE, Rebelo SP, Estrada MF, Alves PM, Boghaert E, Brito C. Drug screening in 3D in vitro tumor models: overcoming current pitfalls of efficacy read-outs. Biotechnol J, 2017, 12(1): 1600505.

[184] Schmidt M, Dringen R. Differential effects of iodoacetamide and iodoacetate on glycolysis and glutathione metabolism of cultured astrocytes. Front Neuroenerge, 2009, 1: 1.

[185] Schnell M, Mittal S, Falahkheirkhah K, Mittal A, Yeh K, Kenkel S, Kajdacsy-Balla A, Carney PS, Bhargava R. All-digital histopathology by infrared-optical hybrid microscopy. Proc Natl Acad Sci USA, 2020, 117: 3388-3396.

[186] Shield K, Ackland ML, Ahmed N, Rice GE. Multicellular spheroids in ovarian cancer metastases: biology and pathology. Gynecol Oncol, 2009, 113: 143-148.

[187] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin, 2016, 66: 7-30.

[188] Simian M, Hirai Y, Navre M, Werb Z, Lochter A, Bissell MJ. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development, 2002, 128: 3117-3131.

[189] Spinelli JB, Yoon H, Ringel AE, Jeanfavre S, Clish CB, Haigis MC. Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass. Science, 2017, 358: 941-946.

[190] Sultana N, Hassan MI, Lim MM. Composite synthetic scaffolds for tissue engineering and regenerative medicine, 2015, Berlin: Springer

[191] Sutherland RM, Mccredie JA, Inch WR. Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. J Natl Cancer I, 1971, 46: 113-120.

[192] Svensson RU, Parker SJ, Eichner LJ, Kolar MJ, Wallace M, Brun SN, Lombardo PS, Van Nostrand JL, Hutchins A, Vera L. Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical models. Nat Med, 2016, 22: 1108-1119.

[193] Taylor CR, Shi S-R, Barr N, Wu N. Techniques of immunohistochemistry: principles, pitfalls, and standardization. Diagn Immunohistochem, 2013, 2: 1-42.

[194] Tevis KM, Colson YL, Grinstaff MW. Embedded spheroids as models of the cancer microenvironment. Adv Biosyst, 2017, 1: 1700083.

[195] Thapa M, Dallmann G. Role of coenzymes in cancer metabolism. Semin Cell Dev Biol, 2020, 98: 44-53.

[196] Tian X, Wang W, Zhang Q, Zhao L, Wei J, Xing H, Song Y, Wang S, Ma D, Meng L. Hypoxia-inducible factor-1α enhances the malignant phenotype of multicellular spheroid HeLa cells in vitro. Oncol Lett, 2010, 1: 893-897.

[197] Tong X, Zhao F, Thompson CB. The molecular determinants of de novo nucleotide biosynthesis in cancer cells. Curr Opin Genet Dev, 2009, 19: 32-37.

[198] Tredan O, Galmarini CM, Patel K, Tannock IF. Drug resistance and the solid tumor microenvironment. J Natl Cancer Inst, 2007, 99: 1441-1454.

[199] Vaseva AV, Marchenko ND, Ji K, Tsirka SE, Holzmann S, Moll UM. p53 opens the mitochondrial permeability transition pore to trigger necrosis. Cell, 2012, 149: 1536-1548.

[200] Vinci M, Gowan S, Boxall F, Patterson L, Zimmermann M, Court W, Lomas C, Mendiola M, Hardisson D, Eccles SA. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol., 2012, 10: 29.

[201] Vriens K, Christen S, Parik S, Broekaert D, Yoshinaga K, Talebi A, Dehairs J, Escalona-Noguero C, Schmieder R, Cornfield T, Charlton C, Romero-Perez L, Rossi M, Rinaldi G, Orth MF, Boon R, Kerstens A, Kwan SY, Faubert B, Mendez-Lucas A, Kopitz CC, Chen T, Fernandez-Garcia J, Duarte JAG, Schmitz AA, Steigemann P, Najimi M, Hagebarth A, Van Ginderachter JA, Sokal E, Gotoh N, Wong KK, Verfaillie C, Derua R, Munck S, Yuneva M, Beretta L, DeBerardinis RJ, Swinnen JV, Hodson L, Cassiman D, Verslype C, Christian S, Grunewald S, Grunewald TGP, Fendt SM. Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity. Nature, 2019, 566: 403-406.

[202] Walenta S, Mueller-Klieser W. Differential superiority of heavy charged-particle irradiation to X-Rays: studies on biological effectiveness and side effect mechanisms in multicellular tumor and normal tissue models. Front Oncol, 2016, 6: 30.

[203] Walzl A, Unger C, Kramer N, Unterleuthner D, Scherzer M, Hengstschläger M, Schwanzer-Pfeiffer D, Dolznig H. The resazurin reduction assay can distinguish cytotoxic from cytostatic compounds in spheroid screening assays. J Biomol Screen, 2014, 19: 1047-1059.

[204] Wang F, Weaver VM, Petersen OW, Larabell CA, Dedhar S, Briand P, Lupu R, Bissell MJ. Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology. Proc Natl Acad Sci USA, 1998, 95: 14821-14826.

[205] Wang JZ, Zhu YX, Ma HC, Chen SN, Chao JY, Ruan WD, Wang D, Du FG, Meng YZ. Developing multi-cellular tumor spheroid model (MCTS) in the chitosan/collagen/alginate (CCA) fibrous scaffold for anticancer drug screening. Mater Sci Eng C Mater Biol Appl, 2016, 62: 215-225.

[206] Weaver VM, Lelièvre S, Lakins JN, Chrenek MA, Jones JCR, Giancotti F, Werb Z, Bissell MJ. β4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell, 2002, 2: 205-216.

[207] Weis SM, Cheresh DA. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med, 2011, 17: 1359-1370.

[208] Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E, Peng J, Lin E, Wang Y, Sosman J, Ribas A, Li J, Moffat J, Sutherlin DP, Koeppen H, Merchant M, Neve R, Settleman J. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature, 2012, 487: 505-509.

[209] Wishart DS. Emerging applications of metabolomics in drug discovery and precision medicine. Nat Rev Drug Discov, 2016, 15: 473-484.

[210] Wong AD, Searson PC. Mitosis-mediated intravasation in a tissue-engineered tumor-microvessel platform. Cancer Res, 2017, 77: 6453-6461.

[211] Wu CH, Ho YS, Tsai CY, Wang YJ, Tseng H, Wei PL, Lee CH, Liu RS, Lin SY. In vitro and in vivo study of phloretin-induced apoptosis in human liver cancer cells involving inhibition of type II glucose transporter. Proc Natl Acad Sci USA, 2009, 124: 2210-2219.

[212] Xin X, Yang S-T. A dual fluorescent 3-D multicellular coculture of breast cancer MCF-7 and fibroblast NIH-3T3 cells for high throughput cancer drug screening. Biochem Eng J, 2019, 148: 152-161.

[213] Xin X, Yang H, Zhang F, Yang S-T. 3D cell coculture tumor model: a promising approach for future cancer drug discovery. Process Biochem, 2019, 78: 148-160.

[214] Xing C, LaPorte JR, Barbay JK, Myers AG. Identification of GAPDH as a protein target of the saframycin antiproliferative agents. Proc Natl Acad Sci USA, 2004, 101: 5862-5866.

[215] Yeon SE, da No Y, Lee SH, Nam SW, Oh IH, Lee J, Kuh HJ. Application of concave microwells to pancreatic tumor spheroids enabling anticancer drug evaluation in a clinically relevant drug resistance model. PLoS ONE, 2013, 8: e73345.

[216] Yu L, Chen MC, Cheung KC. Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing. Lab Chip, 2010, 10: 2424-2432.

[217] Yuhas JM, Li AP, Martinez AO, Ladman AJ. A simplified method for production and growth of multicellular tumor spheroids. Cancer Res, 1977, 37: 3639-3643.

[218] Yun J, Rago C, Cheong I, Pagliarini R, Angenendt P, Rajagopalan H, Schmidt K, Willson JK, Markowitz S, Zhou S. Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells. Science, 2009, 325: 1555-1559.

[219] Zauri M, Berridge G, Thézénas ML, Pugh KM, Goldin R, Kessler BM, Kriaucionis S. CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer. Nature, 2015, 524: 114-118.

[220] Zhang WC, Shyh-Chang N, Yang H, Rai A, Umashankar S, Ma S, Soh BS, Sun LL, Tai BC, Nga ME, Bhakoo KK, Jayapal SR, Nichane M, Yu Q, Ahmed DA, Tan C, Sing WP, Tam J, Thirugananam A, Noghabi MS, Pang YH, Ang HS, Mitchell W, Robson P, Kaldis P, Soo RA, Swarup S, Lim EH, Lim B. Glycine decarboxylase activity drives non-small cell lung cancer tumor-initiating cells and tumorigenesis. Cell, 2012, 148: 259-272.

[221] Zhu J, Thompson CB. Metabolic regulation of cell growth and proliferation. Nat Rev Mol Cell Biol, 2019, 20: 436-450.

Funding

National Natural Science Foundation of China(31900073)

Natural Science Foundation of Shanghai(19ZR1413600)