Spheroid versus monolayer culture: proteomic insights into divergent pathways in myxofibrosarcoma

Abstract

Aim: Myxofibrosarcoma (MFS) is characterized by high local recurrence and complex microenvironmental interactions. Although three-dimensional (3D) culture systems better mimic in vivo tumor architecture than conventional two-dimensional (2D) monolayer cultures, the global proteomic consequences of dimensionality in MFS remain incompletely defined.

Methods: We performed quantitative mass spectrometry-based proteomic profiling of eight independently established patient-derived MFS cell lines cultured under 2D monolayer and 3D spheroid conditions. Differential protein expression and pathway enrichment analyses were conducted to delineate dimensionality-driven molecular programs.

Results: Culture dimensionality emerged as the principal determinant of proteomic variation across all cell lines. Compared with monolayers, 3D spheroids exhibited significant enrichment of hypoxia response, autophagy-related processes, extracellular matrix organization, and PI3K-Akt signaling pathways. In contrast, 2D cultures preferentially upregulated DNA replication, RNA processing, and cell-cycle-associated pathways. These findings indicate that 3D architecture alone is sufficient to induce coordinated stress-adaptive and survival-oriented proteomic reprogramming in MFS cells.

Conclusions: This study provides a comprehensive proteomic atlas defining dimensionality-dependent molecular states in MFS. While functional validation was beyond the scope of this work, the pathway rewiring identified here establishes a mechanistic framework for future hypothesis-driven investigations targeting autophagy- and PI3K-Akt-associated vulnerabilities in 3D MFS models.

Keywords

myxofibrosarcoma (MFS) / tissue culture / spheroids / 3D models / protein quantification / mass spectrometry / cell-based model / proteomic analysis

References

[1] Yu K, Chen B, Aran D, Charalel J, Yau C, Wolf DM, et al. Comprehensive transcriptomic analysis of cell lines as models of primary tumors across 22 tumor types. Nat Commun. 2019; 10: 3574.

[2] Gonçalves E, Poulos RC, Cai Z, Barthorpe S, Manda SS, Lucas N, et al. Pan-cancer proteomic map of 949 human cell lines. Cancer Cell. 2022; 40: 835-49.

[3] Breslin S, O’Driscoll L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today. 2013; 18: 240-9.

[4] Garnique AdMB, Parducci NS, de Miranda LBL, de Almeida BO, Sanches L, Machado-Neto JA. Two-Dimensional and Spheroid-Based Three-Dimensional Cell Culture Systems: Implications for Drug Discovery in Cancer. Drugs Drug Candidates. 2024; 3: 391-409.

[5] Yue X, Lukowski JK, Weaver EM, Skube SB, Hummon AB. Quantitative Proteomic and Phosphoproteomic Comparison of 2D and 3D Colon Cancer Cell Culture Models. J Proteome Res. 2016; 15: 4265-76.

[6] Tidwell TR, Røsland GV, Tronstad KJ, Søreide K, Hagland HR. Metabolic flux analysis of 3D spheroids reveals significant differences in glucose metabolism from matched 2D cultures of colorectal cancer and pancreatic ductal adenocarcinoma cell lines. Cancer Metab. 2022; 10: 9.

[7] Juarez-Moreno K, Chávez-García D, Hirata G, Vazquez-Duhalt R. Monolayer (2D) or spheroids (3D) cell cultures for nanotoxicological studies? Comparison of cytotoxicity and cell internalization of nanoparticles. Toxicol In Vitro. 2022; 85: 105461.

[8] Shannon AE, Boos CE, Hummon AB. Co-culturing multicellular tumor models: Modeling the tumor microenvironment and analysis techniques. Proteomics. 2021; 21: e2000103.

[9] Vanni S, De Vita A, Gurrieri L, Fausti V, Miserocchi G, Spadazzi C, et al. Myxofibrosarcoma landscape: diagnostic pitfalls, clinical management and future perspectives. Ther Adv Med Oncol. 2022; 14: 17588359221093973.

[10] Nakahashi N, Emori M, Takada K, Murahashi Y, Shimizu J, Murase K, et al. Establishment and characterization of the novel myxofibrosarcoma cell line, SMU-MFS. Human Cell. 2024; 38: 25.

[11] Adachi Y, Noguchi R, Osaki J, Ono T, Iwata S, Akiyama T, et al. Establishment and characterization of two novel patient-derived cell lines from myxofibrosarcoma: NCC-MFS7-C1 and NCC-MFS8-C1. Hum Cell. 2024; 37: 1742-50.

[12] Yoshimatsu Y, Noguchi R, Sin Y, Tsuchiya R, Ono T, Akiyama T, et al. Establishment and characterization of NCC-MFS6-C1: a novel patient-derived cell line of myxofibrosarcoma. Hum Cell. 2022; 35: 1993-2001.

[13] Tsuchiya R, Yoshimatsu Y, Noguchi R, Sin Y, Ono T, Akiyama T, et al. Establishment and Characterization of NCC-MFS5-C1: A Novel Patient-Derived Cell Line of Myxofibrosarcoma. Cells. 2022; 11: 207.

[14] Yoshimatsu Y, Noguchi R, Tsuchiya R, Sin Y, Ono T, Sugaya J, et al. Establishment and characterization of NCC-MFS4-C1: a novel patient-derived cell line of myxofibrosarcoma. Hum Cell. 2021; 34: 1911-8.

[15] Tsuchiya R, Yoshimatsu Y, Noguchi R, Sin Y, Ono T, Sei A, et al. Establishment and characterization of NCC-MFS3-C1: a novel patient-derived cell line of myxofibrosarcoma. Hum Cell. 2021; 34: 1266-73.

[16] Noguchi R, Yoshimatsu Y, Ono T, Sei A, Hirabayashi K, Ozawa I, et al. Establishment and characterization of NCC-MFS2-C1: a novel patient-derived cancer cell line of myxofibrosarcoma. Hum Cell. 2021; 34: 246-53.

[17] Kito F, Oyama R, Sakumoto M, Shiozawa K, Qiao Z, Toki S, et al. Establishment and characterization of a novel cell line, NCC-MFS1-C1, derived from a patient with myxofibrosarcoma. Hum Cell. 2019; 32: 214-22.

[18] Lou R, Shui W. Acquisition and analysis of DIA-based proteomic data: a comprehensive survey in 2023. Mol Cell Proteomics. 2024; 23: 100712.

[19] Kitata RB, Yang JC, Chen YJ. Advances in data-independent acquisition mass spectrometry towards comprehensive digital proteome landscape. Mass Spectrom Rev. 2023; 42: 1260-86.

[20] Krasny L, Huang PH. Data-independent acquisition mass spectrometry (DIA-MS) for proteomic applications in oncology. Mol Omics. 2021; 17: 29-42.

[21] Minami F, Sasaki N, Shichi Y, Gomi F, Michishita M, Ohkusu-Tsukada K, et al. Morphofunctional analysis of human pancreatic cancer cell lines in 2- and 3-dimensional cultures. Sci Rep. 2021; 11: 6775.

[22] Noguchi S, Wakita K, Matsuda F, Shimizu H. ¹³C metabolic flux analysis clarifies distinct metabolic phenotypes of cancer cell spheroid mimicking tumor hypoxia. Metab Eng. 2022; 73: 192-200.

[23] Levy JMM, Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer. 2017; 17: 528-42.

[24] Daskalaki I, Gkikas I, Tavernarakis N. Hypoxia and selective autophagy in cancer development and therapy. Front Cell Dev Biol. 2018; 6: 104.

[25] Amaravadi R, Kimmelman AC, White E. Recent insights into the function of autophagy in cancer. Genes Dev. 2016; 30: 1913-30.

[26] Towers CG, Thorburn A. Therapeutic Targeting of Autophagy. EBioMedicine. 2016; 14: 15-23.

[27] Choi AMK, Ryter SW, Levine B. Autophagy in human health and disease. N Engl J Med. 2013; 368: 651-62.

[28] Vinci M, Box C, Eccles SA. Three-dimensional (3D) tumor spheroid invasion assay. J Vis Exp. 2015; 99: e52686.

[29] Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA. Spheroid-based drug screen: considerations and practical approach. Nat Protoc. 2009; 4: 309-24.

[30] Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol. 2014; 12: 207-18.

[31] Chiarugi P, Giannoni E. Anoikis: a necessary death program for anchorage-dependent cells. Biochem Pharmacol. 2008; 76: 1352-64.

[32] Ivanov DP, Parker TL, Walker DA, Alexander C, Ashford MB, Gellert PR, et al. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres. PLoS One. 2014; 9: e103817.

[33] Leight JL, Wozniak MA, Chen S, Lynch ML, Chen CS. Matrix rigidity regulates a switch between TGF-β1-induced apoptosis and epithelial-mesenchymal transition. Mol Biol Cell. 2012; 23: 781-91.

[34] Ulrich TA, Jain A, Tanner K, MacKay JL, Kumar S. Probing cellular mechanobiology in three-dimensional culture with collagen-agarose matrices. Biomaterials. 2010; 31: 1875-84.

[35] Humphrey JD, Dufresne ER, Schwartz MA. Mechanotransduction and extracellular matrix homeostasis. Nat Rev Mol Cell Biol. 2014; 15: 802-12.

[36] Olsen JV, Mann M. Status of large-scale analysis of post-translational modifications by mass spectrometry. Mol Cell Proteomics. 2013; 12: 3444-52.

[37] Pogkas A, Reichardt P, Tunn PU, Niethard M, Werner M, Ghani S. Localized myxofibrosarcoma: a retrospective analysis of primary therapy and prognostic factors in 134 patients in a single institution. Oncologist. 2024; 29: e544-52.

[38] Feodoroff M, Mikkonen P, Arjama M, Murumägi A, Kallioniemi O, Potdar S, et al. Protocol for 3D drug sensitivity and resistance testing of patient-derived cancer cells in 384-well plates. SLAS Discov. 2023; 28: 36-41.

[39] Giesen C, Wang HAO, Schapiro D, Zivanovic N, Jacobs A, Hattendorf B, et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods. 2014; 11: 417-22.