Email this article From 2D to 3D in vitro models of nasal septal squamous cell carcinoma: tumor-associated gene expression under normal conditions and after neutron exposure
- Authors: Soboleva A.G.1,2, Arutyunyan I.V.1,2, Balchir D.V.2, Saburov V.O.3, Lositskii G.A.1,2, Aleksandrova S.A.1,2, Makarov A.V.2, Elchaninov A.V.1,2,4
-
Affiliations:
- Petrovsky National Research Centre of Surgery
- RUDN University
- National Medical Research Radiological Centre
- National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov
- Issue: Vol 30, No 3 (2025)
- Pages: 194-205
- Section: Original Study Articles
- URL: https://ogarev-online.ru/1028-9984/article/view/366001
- DOI: https://doi.org/10.17816/onco691685
- EDN: https://elibrary.ru/UJAUME
- ID: 366001
Cite item
Open Access
Access granted
Subscription Access
Abstract
BACKGROUND: Nasal cancer is one of the most challenging cancers for dosimetric planning in radiotherapy. The RPMI-2650 cell line is the most available for modeling in this squamous cell carcinoma. However, there are few published studies on the biological effects of irradiation in RPMI-2650-based models.
AIM: The work aimed to examine changes in tumor-associated gene expression when switching from a 2D to 3D human model of nasal septal squamous cell carcinoma and assess the response of tumor cells to a single neutron exposure.
METHODS: The phenotype of RPMI-2650 cells was assessed by immunocytochemical staining. 3D spheroids were formed using ultra-low attachment plates. An NG-14 neutron generator was used for neutron exposure of 2D and 3D models. The expression of tumor-associated genes was assessed using real-time reverse transcription polymerase chain reaction.
RESULTS: The RPMI-2650 cell line had a keratin 17+ vimentin+ phenotype, which is typical of cell lines isolated from primary tumor metastases. There was a 6.9-fold increase in keratin 17 and keratin 10 gene silencing, along with an increase in the relative expression of CDH1 by 4.9 times, CD44 by 4.4 times, VIM by 12.4 times, TP63 by 3.2 times, PIK3CA by 2.7 times, TGFB1 by 3.8 times, MMP2 by 13.1 times, and TIMP2 by 35 times. CDKN2A expression increased in both 2D and 3D models 24 hours after neutron exposure (4.7 and 6.7 times, respectively). Furthermore, KRT10 and TIMP1 expression increased (5 and 4.5 times, respectively; spheroids only), as did TIMP2, TP63, and CD44 expression (6.8, 6.5, and 9.3 times, respectively; monolayer culture only). Vimentin gene expression increased 22 times in the exposed 2D model and reduced 7 times in the cancer 3D model.
CONCLUSION: Switching from 2D to 3D RPMI-2650 models was associated with decreased expression of genes encoding tumor cell resistance to therapy, with a simultaneous increase in the expression of genes responsible for progression, metastasis, and drug resistance in squamous cell carcinoma of head and neck. A single neutron exposure in a monolayer culture increased the expression of genes associated with an unfavorable outcome. In the 3D model, neutron exposure induced a more complex response, including cell cycle regulation, fibrosis, and specific cytoskeleton remodeling.
About the authors
Anna G. Soboleva
Petrovsky National Research Centre of Surgery; RUDN University
Email: annasobo@mail.ru
ORCID iD: 0000-0002-9158-1933
SPIN-code: 2582-5511
Cand. Sci. (Biology)
Russian Federation, Moscow; MoscowIrina V. Arutyunyan
Petrovsky National Research Centre of Surgery; RUDN University
Author for correspondence.
Email: labrosta@yandex.ru
ORCID iD: 0000-0002-4344-8943
SPIN-code: 5220-1893
Cand. Sci. (Biology)
Russian Federation, Moscow; MoscowDorzhu V. Balchir
RUDN University
Email: dbalchir@mail.ru
SPIN-code: 2264-5333
Russian Federation, Moscow
Vyacheslav O. Saburov
National Medical Research Radiological Centre
Email: vosaburov@gmail.com
ORCID iD: 0000-0002-6907-2753
SPIN-code: 7212-1328
Russian Federation, Obninsk
George A. Lositskii
Petrovsky National Research Centre of Surgery; RUDN University
Email: glosierror404@gmail.com
ORCID iD: 0009-0001-8822-8645
Russian Federation, Moscow; Moscow
Sofia A. Aleksandrova
Petrovsky National Research Centre of Surgery; RUDN University
Email: sofia.aleksa@mail.ru
Russian Federation, Moscow; Moscow
Andrey V. Makarov
RUDN University
Email: anvitmak@yandex.ru
ORCID iD: 0000-0003-2133-2293
SPIN-code: 3534-3764
MD, Cand. Sci. (Medicine)
Russian Federation, MoscowAndrey V. Elchaninov
Petrovsky National Research Centre of Surgery; RUDN University; National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov
Email: elchandrey@yandex.ru
MD, Dr. Sci. (Medicine), Assistant Professor
Russian Federation, Moscow; Moscow; MoscowReferences
- Binazzi A, Ferrante P, Marinaccio A. Occupational exposure and sinonasal cancer: a systematic review and meta-analysis. BMC Cancer. 2015;15:49. doi: 10.1186/s12885-015-1042-2
- Nishikawa A, Nagano K, Kojima H, Fukushima S, Ogawa K. Pathogenesis of chemically induced nasal cavity tumors in rodents: contribution to adverse outcome pathway. J Toxicol Pathol. 2024;37(1):11–27. doi: 10.1293/tox.2023-0098
- Mesia R, Iglesias L, Lambea J, et al. SEOM clinical guidelines for the treatment of head and neck cancer (2020). Clin Transl Oncol. 2021;23(5):913–921. doi: 10.1007/s12094-020-02533-1
- Sroussi HY, Epstein JB, Bensadoun RJ, et al. Common oral complications of head and neck cancer radiation therapy: mucositis, infections, saliva change, fibrosis, sensory dysfunctions, dental caries, periodontal disease, and osteoradionecrosis. Cancer Med. 2017;6(12):2918–2931. doi: 10.1002/cam4.1221
- Dutta R, Dubal PM, Svider PF, et al. Sinonasal malignancies: A population-based analysis of site-specific incidence and survival. Laryngoscope. 2015;125(11):2491–7. doi: 10.1002/lary.25465
- Kim SA, Chung YS, Lee BJ. Recurrence patterns of sinonasal cancers after a 5-year disease-free period. Laryngoscope. 2019;129(11):2451–2457. doi: 10.1002/lary.27866
- Smyk DI, Gulidov IA, Gordon KB, et al. Reirradiation using proton therapy for the local recurrence of nasal cavity cancer. Problems in oncology. 2023;69(1):127–134. doi: 10.37469/0507-3758-2023-69-1-127-134 EDN: EIIHQS
- Patel SH, Wang Z, Wong WW, et al. Charged particle therapy versus photon therapy for paranasal sinus and nasal cavity malignant diseases: a systematic review and meta-analysis. Lancet Oncol. 2014;15(9):1027–38. doi: 10.1016/S1470-2045(14)70268-2
- Saito T, Nakayama M, Ohnishi K, et al. Proton beam therapy in multimodal treatment for locally advanced squamous cell carcinoma of the nasal cavity and paranasal sinus. Radiat Oncol. 2023;18(1):106. doi: 10.1186/s13014-023-02296-3
- Lin CJ, Grandis JR, Carey TE, et al. Head and neck squamous cell carcinoma cell lines: established models and rationale for selection. Head Neck. 2007;29(2):163–88. doi: 10.1002/hed.20478
- Moore GE, Sandberg AA. Studies of a human tumor cell line with a diploid karyotype. Cancer. 1964;17:170–5. doi: 10.1002/1097-0142(196402)17:2<170::aid-cncr2820170206>3.0.co;2-n
- Zhan J, Qi Y, Fu Y, et al. LncRNA ZFAS1 Alleviated NLRP3 Inflammasome-Mediated Pyroptosis through Regulating miR-96-5p/Smad7 Signaling in Allergic Rhinitis. Int Arch Allergy Immunol. 2024:185(7):704–717. doi: 10.1159/000535646
- Lewandowska-Polak A, Brauncajs M, Jarzębska M, et al. Parainfluenza virus infection enhances NSAIDs-induced inhibition of PGE2 generation and COX-2 expression in human airway epithelial cells. Adv Med Sci. 2019;64(2):338–343. doi: 10.1016/j.advms.2019.04.004
- Ladel S, Schlossbauer P, Flamm J, et al. Improved In Vitro Model for Intranasal Mucosal Drug Delivery: Primary Olfactory and Respiratory Epithelial Cells Compared with the Permanent Nasal Cell Line RPMI 2650. Pharmaceutics. 2019;11(8):367. doi: 10.3390/pharmaceutics11080367
- Barlang LA, Weinbender K, Merkel OM, Popp A. Characterization of critical parameters using an air-liquid interface model with RPMI 2650 cells for permeability studies of small molecules. Drug Deliv Transl Res. 2024;14(6):1601–1615. doi: 10.1007/s13346-023-01474-w
- Sibinovska N, Žakelj S, Trontelj J, Kristan K. Applicability of RPMI 2650 and Calu-3 Cell Models for Evaluation of Nasal Formulations. Pharmaceutics. 2022;14(2):369. doi: 10.3390/pharmaceutics14020369
- Deruyver L, Rigaut C, Gomez-Perez A, et al. In vitro Evaluation of Paliperidone Palmitate Loaded Cubosomes Effective for Nasal-to-Brain Delivery. Int J Nanomedicine. 2023;18:1085–1106. doi: 10.2147/IJN.S397650
- Danielsson A, Karlsson E, Delle U, Helou K, Mercke C. The biological effect of pentoxifylline on the survival of human head and neck cancer cells treated with continuous low and high dose-rate irradiation. J Cancer Res Clin Oncol. 2005;131(7):459–67. doi: 10.1007/s00432-004-0665-5
- Emer C, Hildebrand LS, Friedrich B, et al. In Vitro Analysis of Superparamagnetic Iron Oxide Nanoparticles Coated with APTES as Possible Radiosensitizers for HNSCC Cells. Nanomaterials (Basel). 2023;13(2):330. doi: 10.3390/nano13020330
- Zhu Y, Xu C, Li Z, et al. Nanostructured organic sheets sequestering small extracellular vesicles and reactive species to protect against radiation-induced mucositis. Nat Commun. 2025;16(1):6120. doi: 10.1038/s41467-025-61236-9
- Arutyunyan I, Jumaniyazova E, Makarov A, Fatkhudinov T. In Vitro Models of Head and Neck Cancer: From Primitive to Most Advanced. J Pers Med. 2023;13(11):1575. doi: 10.3390/jpm13111575
- Manduca N, Maccafeo E, De Maria R, Sistigu A, Musella M. 3D cancer models: One step closer to in vitro human studies. Front Immunol. 2023;14:1175503. doi: 10.3389/fimmu.2023.1175503
- Lan YJ, Chen H, Chen JQ, et al. Immunolocalization of vimentin, keratin 17, Ki-67, involucrin, β-catenin and E-cadherin in cutaneous squamous cell carcinoma. Pathol Oncol Res. 2014;20(2):263–6. doi: 10.1007/s12253-013-9690-5
- Thul PJ, Lindskog C. The human protein atlas: A spatial map of the human proteome. Protein Sci. 2018;27(1):233–244. doi: 10.1002/pro.3307
- Melissaridou S, Wiechec E, Magan M, et al. The effect of 2D and 3D cell cultures on treatment response, EMT profile and stem cell features in head and neck cancer. Cancer Cell Int. 2019;19:16. doi: 10.1186/s12935-019-0733-1
- Gheytanchi E, Naseri M, Karimi-Busheri F, et al. Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines. Cancer Cell Int. 2021;21(1):204. doi: 10.1186/s12935-021-01898-9
- Fontana F, Raimondi M, Marzagalli M, et al. Epithelial-To-Mesenchymal Transition Markers and CD44 Isoforms Are Differently Expressed in 2D and 3D Cell Cultures of Prostate Cancer Cells. Cells. 2019;8(2):143. doi: 10.3390/cells8020143
- Jiang Y, Zhou T, Shi Y, Feng W, Lyu T. A SMYD3/ITGB6/TGFβ1 Positive Feedback Loop Promotes the Invasion and Adhesion of Ovarian Cancer Spheroids. Front Oncol. 2021;11:690618. doi: 10.3389/fonc.2021.690618
- Jang M, Koh I, Lee SJ, Cheong JH, Kim P. Droplet-based microtumor model to assess cell-ECM interactions and drug resistance of gastric cancer cells. Sci Rep. 2017;7:41541. doi: 10.1038/srep41541
- Zhang H, Zhang Y, Xia T, et al. The Role of Keratin17 in Human Tumours. Front Cell Dev Biol. 2022;10:818416. doi: 10.3389/fcell.2022.818416
- Nair RR, Hsu J, Jacob JT, et al. A role for keratin 17 during DNA damage response and tumor initiation. Proc Natl Acad Sci U S A. 2021;118(13):e2020150118. doi: 10.1073/pnas.2020150118
- Wang W, Lozar T, Golfinos AE, et al. Stress Keratin 17 Expression in Head and Neck Cancer Contributes to Immune Evasion and Resistance to Immune-Checkpoint Blockade. Clin Cancer Res. 2022;28(13):2953–2968. doi: 10.1158/1078-0432.CCR-21-3039
- Jung EK, Kim SA, Salam SMA, Lee KH, Yoon TM. Role of Keratin-10 in Tumor Progression and Radioresistance of Laryngeal Cancer Cells. Anticancer Res. 2025;45(3):1035–1045. doi: 10.21873/anticanres.17490
- Kulasinghe AJ, Perry C, Boyle G, et al. Epithelial-mesenchymal axis in head and neck cancer cell lines. Journal of Solid Tumors. 2015;6(1):28–37. doi: 10.5430/JST.V6N1P28
- Lian GY, Wang QM, Mak TS, et al. Inhibition of tumor invasion and metastasis by targeting TGF-β-Smad-MMP2 pathway with Asiatic acid and Naringenin. Mol Ther Oncolytics. 2021;20:277–289. doi: 10.1016/j.omto.2021.01.006
- Stuelten CH, DaCosta Byfield S, Arany PR, et al. Breast cancer cells induce stromal fibroblasts to express MMP-9 via secretion of TNF-alpha and TGF-beta. J Cell Sci. 2005;118(Pt 10):2143–53. doi: 10.1242/jcs.02334
- Li Y, Giovannini S, Wang T, et al. p63: a crucial player in epithelial stemness regulation. Oncogene. 2023;42(46):3371–3384. doi: 10.1038/s41388-023-02859-4
- Rezakhani L, Alizadeh M, Alizadeh A. A three dimensional in vivo model of breast cancer using a thermosensitive chitosan-based hydrogel and 4 T1 cell line in Balb/c. J Biomed Mater Res A. 2021;109(7):1275–1285. doi: 10.1002/jbm.a.37121
- Abbas ZN, Al-Saffar AZ, Jasim SM, Sulaiman GM. Comparative analysis between 2D and 3D colorectal cancer culture models for insights into cellular morphological and transcriptomic variations. Sci Rep. 2023;13(1):18380. doi: 10.1038/s41598-023-45144-w
- Compagnone M, Gatti V, Presutti D, et al. ΔNp63-mediated regulation of hyaluronic acid metabolism and signaling supports HNSCC tumorigenesis. Proc Natl Acad Sci U S A. 2017;114(50):13254–13259. doi: 10.1073/pnas.1711777114
- García-Escudero R, Segrelles C, Dueñas M, et al. Overexpression of PIK3CA in head and neck squamous cell carcinoma is associated with poor outcome and activation of the YAP pathway. Oral Oncol. 2018;79:55–63. doi: 10.1016/j.oraloncology.2018.02.014
- Chen X, Cao Y, Sedhom W, et al. Distinct roles of PIK3CA in the enrichment and maintenance of cancer stem cells in head and neck squamous cell carcinoma. Mol Oncol. 2020;14(1):139–158. doi: 10.1002/1878-0261.12584
- Antonelli F. 3D Cell Models in Radiobiology: Improving the Predictive Value of In Vitro Research. Int J Mol Sci. 2023;24(13):10620. doi: 10.3390/ijms241310620
- Wang Y, Zhou C, Li T, Luo J. Prognostic value of CDKN2A in head and neck squamous cell carcinoma via pathomics and machine learning. J Cell Mol Med. 2024;28(9):e18394. doi: 10.1111/jcmm.18394
- Arutyunyan IV, Soboleva AG, Gordon KB, et al. Differential Markers of Subpopulations of Epithelial Cells of the Larynx in Squamous Cell Carcinoma. Bull Exp Biol Med. 2022;173(4):553–559. doi: 10.1007/s10517-022-05588-y EDN: BMWUBE
- Fornieles G, Núñez MI, Expósito J. Matrix Metalloproteinases and Their Inhibitors as Potential Prognostic Biomarkers in Head and Neck Cancer after Radiotherapy. Int J Mol Sci. 2023;25(1):527. doi: 10.3390/ijms25010527
- Yu Z, Xu C, Song B, et al. Tissue fibrosis induced by radiotherapy: current understanding of the molecular mechanisms, diagnosis and therapeutic advances. J Transl Med. 2023;21(1):708. doi: 10.1186/s12967-023-04554-0
- Zamulaeva I, Matchuk O, Selivanova E, et al. Effects of Fractionated Radiation Exposure on Vimentin Expression in Cervical Cancers: Analysis of Association with Cancer Stem Cell Response and Short-Term Prognosis. Int J Mol Sci. 2023;24(4):3271. doi: 10.3390/ijms24043271 EDN: HIHBUI
- Galarza TE, Mohamad NA, Táquez Delgado MA, et al. Histamine prevents radiation-induced mesenchymal changes in breast cancer cells. Pharmacol Res. 2016;111:731–739. doi: 10.1016/j.phrs.2016.07.039
- Akil H, Rouanet J, Viallard C, et al. Targeted Radionuclide Therapy Decreases Melanoma Lung Invasion by Modifying Epithelial-Mesenchymal Transition-Like Mechanisms. Transl Oncol. 2019;12(11):1442–1452. doi: 10.1016/j.tranon.2019.07.015
Supplementary files
