cell line 2a3  (Thermo Fisher)

Journal: bioRxiv

Article Title: 3D Droplet-Based Bioprinting of Customized In Vitro Head and Neck Cancer Tumor Microenvironment Models

doi: 10.64898/2026.03.27.714925

Figure Lengend Snippet: FaDu and 2A3 cells were suspended directly into a matrix metalloproteinase (MMP) labile bio-thiol activator which was then printed with its respective 4-arm maleimide PEG bioink to establish hydrogels upon component contact at room temperature. Both cell lines were printed into 8 different matrix conditions (PEG versus PEG fnc in stiffnesses of 0.7, 1.1, 3.0, and 4.8 kPa) for a total of 16 matrix conditions investigated in this study. High-throughput printing was achieved through the use of 96-well plates, with the printing process allowing for spatial control of different cell and gel types to create a customized system based on user-preference.

Article Snippet: FaDu (HTB-43) and 2A3 (CRL-3212) human-derived cell lines were purchased from ATCC, and cultured using ATCC-suggested media for each line.

Techniques: High Throughput Screening Assay, Control

Journal: bioRxiv

Article Title: 3D Droplet-Based Bioprinting of Customized In Vitro Head and Neck Cancer Tumor Microenvironment Models

doi: 10.64898/2026.03.27.714925

Figure Lengend Snippet: Viability stains demonstrate high viability of cell clusters over time. Hydrogel samples were collected at 1, 4, and 7 days of culture, stained to assess viability, and imaged via confocal microscopy as z-stacks. (a) Images of FaDu cells, in a PEG hydrogel of 1.1 kPa stiffness, at 1, 4, and 7 days of culture, show high cell viability (green) and few dead cells (red). The development of multicellular clusters over time tracks with observations from light microscopy in . (b) Final images of each cell type and gel condition, recorded at day 7 of culture, reinforce the varied cell response to each condition. Scale: x and y dimensions reflect 826 × 826 μm, and z dimension reflects 200 μm depth. Blue: DAPI (nuclei), Green: calcein-AM (live cells), Red: ethidium homodimer (dead cell nuclei); (c) Quantified viability from reconstructed z-stacks for FaDu (left) and 2A3 (right) cells, printed in each hydrogel stiffness, either in PEG or peptide-functionalized PEG hydrogels. Each bar reflects triplicate specimens, error bars reflect 95% CI. Comparisons were conducted for cell viability between PEG and PEG fnc matrices at each stiffness, * = p < 0.05. For Day 7 samples at the end of the experiment, letters identify statistically different results across four stiffnesses within the PEG (a, b) matrix or the PEG fnc (a’, b’) matrix, α=0.05.

Article Snippet: FaDu (HTB-43) and 2A3 (CRL-3212) human-derived cell lines were purchased from ATCC, and cultured using ATCC-suggested media for each line.

Techniques: Staining, Confocal Microscopy, Light Microscopy

Journal: bioRxiv

Article Title: 3D Droplet-Based Bioprinting of Customized In Vitro Head and Neck Cancer Tumor Microenvironment Models

doi: 10.64898/2026.03.27.714925

Figure Lengend Snippet: Hydrogels enable cell survival and rapid proliferation to form multicellular clusters. Light microscopy images of each cell type in each hydrogel condition were captured over 7 days to assess cell proliferation and morphology changes. (a) Images of FaDu cells, encapsulated as single cells in a PEG hydrogel of 1.1 kPa stiffness, at 0, 2, 4, and 6 days of culture show progressive cell proliferation and formation of multicellular clusters. (b) Final images of each cell type and gel condition, recorded at day 7 of culture, demonstrate the varied cell response to each condition. All samples were seeded initially as single-cell suspensions under identical cell concentrations. Corresponding day 0 images for panel (b) are provided in Supporting Information Figure SI-1. Scale bar = 100 μm. (c) Mean cluster counts for FaDu and 2A3 cells in each matrix, normalized to the day 1 count. Each point represents triplicate repeats, and error bars reflect the 95% confidence interval. Despite some variation, cluster counts are statistically indistinguishable for all conditions.

Article Snippet: FaDu (HTB-43) and 2A3 (CRL-3212) human-derived cell lines were purchased from ATCC, and cultured using ATCC-suggested media for each line.

Techniques: Light Microscopy, Single Cell

Journal: bioRxiv

Article Title: 3D Droplet-Based Bioprinting of Customized In Vitro Head and Neck Cancer Tumor Microenvironment Models

doi: 10.64898/2026.03.27.714925

Figure Lengend Snippet: Cluster morphology measures and modeling at day 7 timepoint identify impacts of cell type and matrix parameters. 3D reconstructions of calcein-AM green-stained cell clusters for each cell type, matrix stiffness, and composition permutation were analyzed in Imaris to obtain per-cluster measurements of (a) surface area and (b) volume, and generate (c) complexity as a measure of invasiveness. (a-c) Graphs show the median of each value, ± 95% CI, n= 2-3 replicate gels (red, blue, green, offset for visibility) per condition, and n∼50-200 clusters per gel replicate. (d) Population data were modeled using lmer to generate plots of predicted cluster area, volume, and complexity as a function of stiffness. Cluster area and volume measures at day 7 varied with both stiffness and cell type and were cross-correlated. Area and volume magnitudes were correlated with matrix composition at day 7, but their change with stiffness was independent of matrix. Predicted complexity values aligned for FaDu cells in both matrices and for 2A3 cells in PEG fnc , but with unique behavior for 2A3 cells in PEG.

Article Snippet: FaDu (HTB-43) and 2A3 (CRL-3212) human-derived cell lines were purchased from ATCC, and cultured using ATCC-suggested media for each line.

Techniques: Staining

Journal: bioRxiv

Article Title: HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

doi: 10.1101/2025.10.26.684626

Figure Lengend Snippet: (A-D) Venn diagrams of differentially expressed genes in FaDu (A-B) and 2A3 (C-D) cell lines. Venn diagrams illustrate the number of significantly regulated genes under three conditions: hypoxia (green), irradiation (yellow), and the combination of hypoxia and irradiation (blue). Numbers in the overlapping areas indicate genes commonly regulated under the corresponding conditions. Genes were considered differentially expressed if they had an adjusted p-value (padj) <0.1. (E) Biological processes differentially regulated in FaDu and 2A3 HNSCC cell lines under hypoxia and combined hypoxia with gamma irradiation. The figure illustrates biological processes significantly upregulated (red) or downregulated (green) in FaDu and 2A3 cells in response to hypoxia alone or in combination with gamma radiation. The analysis was performed using the WebGestalt 2024 platform and included only genes that were consistently detected across all three independent biological replicates and met the inclusion criteria of adjusted p < 0.1 and |log 2 FC| ≥ 2.

Article Snippet: The 2A3 cell line (CRL-3212, ATCC, Manassas, VA, USA) was generated by transfecting FaDu cells with the E6 and E7 genes of HPV-16.

Techniques: Irradiation

Journal: bioRxiv

Article Title: HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

doi: 10.1101/2025.10.26.684626

Figure Lengend Snippet: (A) FaDu, 2A3, and Detroit-562 cell lines were cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions. Cell numbers were determined 48 and 72 hours after plating, and doubling times were calculated as described in Methods. Data represent the mean ± SD from four independent experiments (n = 4). Statistical analysis was performed using an unpaired t-test (**p < 0.01). (B) Heatmap of the top 20 DEGs. Averaged data from three independent experiments are shown. The color bar is showing the values of z-score for each gene after library size normalization via DESeq2 software. Cell cycle distribution was analyzed 24 hours after 6 Gy gamma-irradiation in (C) FaDu, (D) 2A3, and (E) Detroit-562 cells cultured under normoxic or hypoxic (1% O 2 ) conditions. Results are presented as the percentage of cells in G0/1, S, or G2/M phases. Data represent the mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The 2A3 cell line (CRL-3212, ATCC, Manassas, VA, USA) was generated by transfecting FaDu cells with the E6 and E7 genes of HPV-16.

Techniques: Cell Culture, Software, Irradiation

Journal: bioRxiv

Article Title: HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

doi: 10.1101/2025.10.26.684626

Figure Lengend Snippet: (A) Cleaved caspase-3 levels in FaDu, 2A3, and Detroit-562 cells cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions, assessed 48 hours after 6 Gy gamma-irradiation. Results are shown as fold change relative to non-irradiated normoxic controls. Data represent mean ± SD from three independent experiments (n = 3). Statistical analysis: two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001. (B) Heatmap of the top 20 DEGs. Averaged data from three independent experiments are shown. The color bar is showing the values of z-score for each gene after library size normalization via DESeq2 software. (C) Representative western blots of N-cadherin, E-cadherin, and vimentin in FaDu and 2A3 cells. (D) Quantification of protein levels expressed as mean optical density (O.D.) ± SD, normalized to β-actin, in FaDu and 2A3 cells under normoxic or hypoxic conditions. (E–G) Effects of hypoxia and irradiation on N-cadherin, E-cadherin, and vimentin levels in FaDu and 2A3 cells under normoxic and hypoxic conditions. Data represent mean ± SD from three independent experiments (n = 3). Statistical analysis: unpaired t-test (*p < 0.05).

Article Snippet: The 2A3 cell line (CRL-3212, ATCC, Manassas, VA, USA) was generated by transfecting FaDu cells with the E6 and E7 genes of HPV-16.

Techniques: Cell Culture, Irradiation, Software, Western Blot

Journal: bioRxiv

Article Title: HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

doi: 10.1101/2025.10.26.684626

Figure Lengend Snippet: (A) Heatmap of the top 20 DEGs. Averaged data from three independent experiments are shown. The color bar is showing the values of z-score for each gene after library size normalization via DESeq2 software. (B) Cytokine/chemokine production in response to hypoxia, gamma-irradiation, and their combination. Conditioned media from FaDu, 2A3, and Detroit-562 cells were collected 48 hours after gamma-irradiation under normoxic or hypoxic conditions. Media from three independent experiments per cell line were pooled for analysis. Results are presented as z-scores of log 10 -transformed normalized data. n.d. = not detected. (C) Cell numbers of FaDu, 2A3, and Detroit-562 cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions, assessed 48 hours after 6 Gy gamma-irradiation. Data represent mean ± SD from four independent experiments (n = 4). Statistical analysis: two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001. Levels of IL-8 (D), MIF (E), and Serpin E1 (F) in supernatants from FaDu, 2A3, and Detroit-562 cells cultured under normoxic or hypoxic conditions, assessed 48 hours after gamma-irradiation. Concentrations were normalized to cell numbers per condition. Data represent mean ± SD from three to four independent experiments (n = 3–4). Statistical analysis: two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The 2A3 cell line (CRL-3212, ATCC, Manassas, VA, USA) was generated by transfecting FaDu cells with the E6 and E7 genes of HPV-16.

Techniques: Software, Irradiation, Transformation Assay, Cell Culture

Journal: British Journal of Cancer

Article Title: Combined treatment of the experimental human papilloma virus-16-positive cervical and head and neck cancers with cisplatin and radioimmunotherapy targeting viral E6 oncoprotein

doi: 10.1038/bjc.2013.43

Figure Lengend Snippet: Lactate dehydrogenase assay of CasKi and 2A3 cells treated in vitro with various concentrations of cisplatin.

Article Snippet: The HPV16+ 2A3 cell line was produced by stably transfecting the FaDu cell line from the American Type Culture Collection with the LXSN 16E6/E7 as described in Harris et al (2011) .

Techniques: Lactate Dehydrogenase Assay, In Vitro

Journal: British Journal of Cancer

Article Title: Combined treatment of the experimental human papilloma virus-16-positive cervical and head and neck cancers with cisplatin and radioimmunotherapy targeting viral E6 oncoprotein

doi: 10.1038/bjc.2013.43

Figure Lengend Snippet: Tumour uptake of E6-binding mAb 188 Re-C1P5 in nude mice-bearing CasKi and 2A3 tumours post treatment with cisplatin. Mice were treated with various doses of cisplatin for 3 days, 188 Re-C1P5 mAb was administered 24 h after the last dose of cisplatin, and the tumour uptake was assessed 24 h post 188 Re-C1P5 administration: ( A ) CasKi; ( B ) 2A3; ( C , D ) TUNEL staining of the untreated and cisplatin-treated tumours. Apoptotic cells stained brown. Original magnification × 400: ( C ) CasKi tumours—untreated (left panel) and treated with 5 mg kg −1 cisplatin (right panel); ( D ) 2A3 tumours—untreated (left panel) and treated with 5 mg kg −1 cisplatin (right panel).

Article Snippet: The HPV16+ 2A3 cell line was produced by stably transfecting the FaDu cell line from the American Type Culture Collection with the LXSN 16E6/E7 as described in Harris et al (2011) .

Techniques: Binding Assay, TUNEL Assay, Staining

Journal: British Journal of Cancer

Article Title: Combined treatment of the experimental human papilloma virus-16-positive cervical and head and neck cancers with cisplatin and radioimmunotherapy targeting viral E6 oncoprotein

doi: 10.1038/bjc.2013.43

Figure Lengend Snippet: Therapy with cisplatin and RIT with E6-binding mAb 188 Re-C1P5 of nude mice-bearing 2A3 ( A ) and CasKi ( B ) tumours. T n—tumour volume on the day of measurement; T 0 —tumour volume on day 0. The mice with 2A3 HNSCC tumours were treated IP with: 400 μ Ci 188 Re-C1P5 mAb on day 0; or 5 mg kg −1 per day cisplatin on days 0, 1, 2 followed by 400 μ Ci 188 Re-C1P5 mAb on day 3; or 5 mg kg −1 cisplatin alone on days 0, 1, 2; or left untreated. The mice with CasKi cervical tumours were treated IP with: 200 μ Ci 188 Re-C1P5 mAb on day 0; or 5 mg kg −1 per day cisplatin on days 0, 1, 2 followed by 200 μ Ci 188 Re-C1P5 mAb on day 3; or 5 mg kg −1 cisplatin alone on days 0. 1, 2; or left untreated.

Article Snippet: The HPV16+ 2A3 cell line was produced by stably transfecting the FaDu cell line from the American Type Culture Collection with the LXSN 16E6/E7 as described in Harris et al (2011) .

Techniques: Binding Assay

Journal: British Journal of Cancer

Article Title: Combined treatment of the experimental human papilloma virus-16-positive cervical and head and neck cancers with cisplatin and radioimmunotherapy targeting viral E6 oncoprotein

doi: 10.1038/bjc.2013.43

Figure Lengend Snippet: 18 F-FDG microPET/CT images of 2A3 tumour-bearing on day 15 post treatment: left panel—untreated mouse, middle panel—the mouse treated with cisplatin alone, right panel—the mouse treated with cisplatin and RIT. Green arrows are pointing to the tumours.

Article Snippet: The HPV16+ 2A3 cell line was produced by stably transfecting the FaDu cell line from the American Type Culture Collection with the LXSN 16E6/E7 as described in Harris et al (2011) .

Techniques:

Journal: British Journal of Cancer

Article Title: Combined treatment of the experimental human papilloma virus-16-positive cervical and head and neck cancers with cisplatin and radioimmunotherapy targeting viral E6 oncoprotein

doi: 10.1038/bjc.2013.43

Figure Lengend Snippet: Immunohistochemical evaluation of E6 and E7 oncogenes expression in RIT-treated CasKi and 2A3 cells. ( A ) E6 in CasKi cells, ( B ) E7 in CasKi cells; ( C ) E6 in 2A3 cells; ( D ) E7 in 2A3 cells. Left panels show untreated cells and right panels RIT-treated cells. Cells positive for E6 and E7 oncogenes stained brown. Original magnification × 400.

Article Snippet: The HPV16+ 2A3 cell line was produced by stably transfecting the FaDu cell line from the American Type Culture Collection with the LXSN 16E6/E7 as described in Harris et al (2011) .

Techniques: Immunohistochemical staining, Expressing, Staining