Transition from 2D to 3D in vitro model of squamous cell carcinoma of the nasal septum: changes in the expression profile of tumor-associated genes under normal conditions and after neutron irradiation

Background: Nasal cavity tumors are considered one of the most challenging locations in terms of dosimetric planning in radiation therapy. The RPMI-2650 cell line is most readily available for modeling this type of squamous cell carcinoma, yet very few studies have been published on the biological effects of irradiation in models based on these cells.

Aim: To evaluate the change in the expression profile of tumor-associated genes upon transition from a 2D to a 3D model of human nasal septum squamous cell carcinoma and to compare the response of tumor cells in the models to a single-dose neutron irradiation.

Materials and methods: Phenotypic characterization of RPMI-2650 cells was performed using immunocytochemical staining. 3D spheroid formation was performed in ultra-low-adhesion culture vessels. An NG-14 neutron generator was used to irradiate 2D and 3D models. Tumor-associated gene expression was assessed using real-time RT-PCR.

Results: RPMI-2650 cells exhibited a keratin 17+ vimentin+ phenotype, characteristic of cell lines isolated from primary tumor metastases. When switching from 2D to 3D culture conditions, the relative expression of the KRT5, KRT7, KRT8, KRT18, KRT19, YAP1, HIF1A, PTEN, NOTCH1, TIMP1, MMP9, TP53, MKI67, CDKN2A, and TGFBR1 genes did not change significantly. However, downregulation of the keratin 17 and 10 genes was observed, along with increased expression of the CDH1, CD44, VIM, TP63, PIK3CA, TGFB1, MMP2, and TIMP2 genes. Twenty-four hours after neutron irradiation, an increase in CDKN2A was detected in both 2D and 3D models, along with increased KRT10 and TIMP1 expression only in spheroids and an increase in TIMP2, TP63, and CD44 only in monolayer culture. Vimentin gene expression increased in the irradiated 2D and decreased in the 3D tumor model.

Conclusion: The transition from a 2D to a 3D RPMI-2650 model was accompanied by a decrease in the expression of genes that influence tumor cell resistance to immunotherapy and radiotherapy, while simultaneously increasing the expression of genes that mediate progression, metastasis, and drug resistance in head and neck squamous cell carcinoma. A single-dose neutron irradiation of a monolayer culture resulted in an increase in the expression of genes associated with a negative prognosis, while in the 3D model, neutrons induced a more complex response, including cell cycle regulation, fibrosis development, and specific cytoskeletal rearrangements.