2a3  (Bio X 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: 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: Cellular and Molecular Life Sciences: CMLS

Article Title: ALDH2 inhibits head and neck tumorigenesis through RAS signaling suppression, transactivation of TGM2 , and synergy with ALDH6A1

doi: 10.1007/s00018-025-06027-7

Figure Lengend Snippet: ALDH2 functions as a tumor suppressor in vitro. (A-D) Quantitative RT-PCR and immunoblot analyses were performed to evaluate ALDH2 mRNA and protein levels. Actin beta ( ACTB ) and tubulin alpha-1B chain (TUBA1B) served as internal controls for quantitative RT-PCR and immunoblotting, respectively. (A) Endogenous ALDH2 mRNA and (B) ALDH2 protein levels in four HNSC-derived cell lines. (C) Stable knockdown of ALDH2 in Detroit 562 and SCC-25 cells using replication-incompetent lentiviruses carrying two distinct shRNA clones targeting ALDH2 . (D) Stable overexpression of ALDH2 in 2A3 and FaDu cells using lentiviruses packaged from pLenti-ALDH2-Myc-DDK-P2A-Puro plasmid. (E-P) Functional assays, including soft agar colony formation and MTT viability assays, immunoblotting, transwell migration and invasion, ELISA, and the tube formation assay using HUVECs, were conducted following ALDH2 knockdown or overexpression in the four cell lines. (E) Anchorage-independent cell growth. (F) Expression levels of cell cycle-related proteins. (G) Cell migration and invasion. (H) Expression levels of epithelial-mesenchymal transition (EMT) markers. (I) VEGFA levels in conditioned media. (J) In vitro angiogenesis in HUVECs treated with conditioned media from ALDH2 -knockdown Detroit 562 and SCC-25 cells. (K-P) Opposing phenotypes observed in ALDH2 -overexpressing 2A3 and FaDu cells, confirmed by functional assays, immunoblotting, and ELISA. Data are presented as mean ± SD. Statistical significance: * p < 0.05, *** p < 0.001

Article Snippet: We utilized four HNSC-derived cell lines: 2A3 (pharynx, HPV-positive, CRL-3212TM, ATCC, Manassas, VA, USA), Detroit 562 (pharynx, HPV-negative, CCL-138TM, ATCC), FaDu (hypopharyngeal, HPV-negative, HTB-43TM, ATCC), and SCC-25 (tongue, CRL-1628TM, Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Derivative Assay, Knockdown, shRNA, Clone Assay, Over Expression, Plasmid Preparation, Functional Assay, Migration, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALDH2 inhibits head and neck tumorigenesis through RAS signaling suppression, transactivation of TGM2 , and synergy with ALDH6A1

doi: 10.1007/s00018-025-06027-7

Figure Lengend Snippet: ALDH2 suppresses tumorigenesis in vitro by inhibiting the RAS-AKT signaling. (A , B) Immunoblot analysis of RAS-AKT pathway components, including NR4A1, in ALDH2 -knockdown Detroit 562 and ALDH2 -overexpressing FaDu cells. (C) RAS pull-down activation assay using Raf-RBD glutathione beads, selectively captured GTP-bound RAS (active form), followed by immunoblotting with pan-RAS antibody to assess RAS activation status in ALDH2 -knockdown Detroit 562 and ALDH2-overexpressing FaDu cells. (D) Schematic of the experimental design of co-overexpression of ALDH2 and HRAS(G12D) in 2A3 and FaDu cells. (E) Transient transfection of pHRAS(G12D)-HaloTag plasmid induced expression of the HRAS(G12D)-HaloTag fusion protein (~ 54 kDa) in 2A3 and FaDu cells. (F-H) Functional assays, including soft agar colony formation, MTT viability, transwell migration and invasion, and HUVEC tube formation (calcein AM staining, quantified via ImageJ) were performed using conditioned media from four groups: Lenti/pHaloTag (control), ALDH2-/pHaloTag-transfected, Lenti/pHRAS(G12D)-transfected, and ALDH2-/pHRAS(G12D)-transfected cells. (I) Immunoblot analysis of RAS-AKT pathway proteins, including NR4A1, following ALDH2 and/or HRAS(G12D) overexpression in 2A3 and FaDu cells. (J) HRAS protein and HRAS mRNA levels were assessed by immunoblot and quantitative RT-PCR after HRAS knockdown using three distinct shRNA clones in Detroit 562 and ALDH2 -knockdown Detroit 562 cells. (K) Schematic of a dual-knockdown strategy targeting ALDH2 and HRAS in Detroit 562 and SCC-25 cells. (L-N) Functional assays, including soft agar/MTT, transwell migration and invasion, and HUVEC tube formation by treatments with conditioned media from HRAS -knockdown and ALDH2/HRAS dual knockdown Detroit 562 and SCC-25 cells. (O) Immunoblot analysis of RAS-AKT pathway components, including NR4A1, in Detroit 562 and SCC-25 cells following HRAS -knockdown and ALDH2/HRAS dual knockdown. Data are presented as mean ± SD. Statistical significance: ** p < 0.01, *** p < 0.001

Article Snippet: We utilized four HNSC-derived cell lines: 2A3 (pharynx, HPV-positive, CRL-3212TM, ATCC, Manassas, VA, USA), Detroit 562 (pharynx, HPV-negative, CCL-138TM, ATCC), FaDu (hypopharyngeal, HPV-negative, HTB-43TM, ATCC), and SCC-25 (tongue, CRL-1628TM, Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: In Vitro, Western Blot, Knockdown, Activation Assay, Over Expression, Transfection, Plasmid Preparation, Expressing, Functional Assay, Migration, Staining, Control, Quantitative RT-PCR, shRNA, Clone Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALDH2 inhibits head and neck tumorigenesis through RAS signaling suppression, transactivation of TGM2 , and synergy with ALDH6A1

doi: 10.1007/s00018-025-06027-7

Figure Lengend Snippet: ALDH2 transactivates TGM2 to induce apoptosis in HNSC-derived cells. (A) Immunoblotting and CASP3/7 activity assay (colorimetric) assessed apoptosis-related protein levels and caspase activity in ALDH2 -knockdown Detroit 562 and ALDH2-overexpressing FaDu cells. (B , C) Quantitative RT-PCR and immunoblot analysis measured TGM2 mRNA and protein levels in ALDH2-overexpressing 2A3 and FaDu cells, and ALDH2 -knockdown Detroit and SCC-25 cells. (D) Apoptotic cells were quantified via flow cytometry using annexin V-FITC/PI staining after 72 h of transfection with pTGM2-HaloTag plasmids. (E) TGM2 promoter activity was assessed using the Dual-Luciferase ® Reporter Assay with pGL4.17 (control), pGL4.17-A TGM2 (−900 to +100 ), and pGL4.17-B TGM2 (−600 to +100) constructs cotransfected with pRL Renilla vector (14:1) into Detroit 562, ALDH2-overexpressing FaDu, and ALDH2 -knockdown Detroit cells. (F , G) Immunoblot analysis was performed to evaluate TGM2 protein levels in 2A3 and FaDu cell lysates from Fig. D, encompassing Lenti/pHaloTag, HaloTag/ALDH2 overexpression, Lenti/HRAS(G12D)-HaloTag overexpression, and dual overexpression of ALDH2/HRAS(G12D)-HaloTag, and from Fig. k, containing shLuc, shALDH2#2, shHRAS#3, and double knockdown shALDH2#2/shHRAS#3. (H , I) Stable overexpression of ALDH2(WT), ALDH2(E504K), ALDH2(T261A), ALDH2(S488A), and ALDH2(T261A/S488A) was achieved via transduction of replication-incompetent lentiviral vectors carrying the specific target gene into FaDu and ALDH2 -knockdown Detroit 562 cells. TGM2 mRNA and protein levels were measured using quantitative RT-PCR and immunoblot analysis, respectively. (J-O) Functional assays following TGM2 overexpression in ALDH2 -knockdown Detroit 562 cells. (J) Experimental design. (K) Immunoblotting confirmed pTGM2-HaloTag transfection and expression of ~ 110 kDa fusion protein. (L-N) Soft agar/anchorage-independent cell growth/MTT, transwell migration and invasion, and HUVEC tube formation (calcein AM staining/ImageJ) using conditioned media were assessed from control (shLuc/pHaloTag), shLuc/pTGM2-HaloTag, shALDH2#2/pHaloTag, and shALDH2#2/pTGM2-HaloTag groups. (O) Flow cytometry with annexin V-FITC/PI staining evaluated apoptosis across groups. (P) Immunblotting and CASP3/7 assay quantified apoptosis-related proteins and caspase activity. (Q) TGM2 knockdown in SCC-25 cells using three shRNA clones was validated by quantitative RT-PCR and immunoblotting. (R-W) Functional and apoptosis marker analyses were performed in shLuc/Lenti (control), shLuc/ALDH2-overexpressing, shTGM2#2/Lenti, and shTGM2#2/ALDH2-overexpressing SCC-25 cells. Data are presented as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: We utilized four HNSC-derived cell lines: 2A3 (pharynx, HPV-positive, CRL-3212TM, ATCC, Manassas, VA, USA), Detroit 562 (pharynx, HPV-negative, CCL-138TM, ATCC), FaDu (hypopharyngeal, HPV-negative, HTB-43TM, ATCC), and SCC-25 (tongue, CRL-1628TM, Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: Derivative Assay, Western Blot, Activity Assay, Knockdown, Quantitative RT-PCR, Flow Cytometry, Staining, Transfection, Luciferase, Reporter Assay, Control, Construct, Plasmid Preparation, Over Expression, Transduction, Functional Assay, Expressing, Migration, shRNA, Clone Assay, Marker

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALDH2 inhibits head and neck tumorigenesis through RAS signaling suppression, transactivation of TGM2 , and synergy with ALDH6A1

doi: 10.1007/s00018-025-06027-7

Figure Lengend Snippet: Cooperative interaction between ALDH2 and ALDH6A1 enhances suppression of tumorigenetic traits in vitro. (A) Immunoblotting revealed ALDH6A1 protein expression across various HNSC-derived cell lines. Notably, transfection with the pALDH6A1 plasmid for 48 h led to a marked increase in ALDH6A1-HaloTag fusion protein (~ 91 kDa) in ALDH2-overexpressing 2A3 and FaDu cells. (B) ALDH6A1-HaloTag fusion protein levels were notably elevated following pALDH6A1-HaloTag transfection in ALDH2-overexpressing 2A3 and FaDu cells. (C-F) 2A3 and FaDu cells were subjected to the following transfection conditions: control (Lenti/pHaloTag), ALDH2 overexpression (ALDH2/pHaloTag), ALDH6A1 overexpression (Lenti/pALDH6A1-HaloTag), and co-overexpression of ALDH2 and ALDH6A1 (ALDH2/pALDH6A1-HaloTag). A series of functional assays was used to analyze the phenotypic alterations, including anchorage-independent cell growth (soft agar colony formation/MTT), cell motility and invasiveness (transwell migration and invasion), and angiogenic potential (HUVEC tube formation with calcein AM staining following treatments with conditioned media collected from different groups). (G-J) Detroit 562 and SCC-25 cells were transfected with pALDH6A1-HaloTag plasmids under ALDH2 -knockdown conditions to assess the phenotypic impact of ALDH6A1 overexpression. Four experimental groups were established: control (shLuc/pHaloTag), ALDH6A1 overexpression (shLuc/pALDH6A1-HaloTag), ALDH2 knockdown (shALDH2#2/pHaloTag), and ALDH6A1 overexpression in ALDH2 -knockdown background (shALDH2#2/pALDH6A1-HaloTag). Functional assays were performed to evaluate anchorage-independent cell growth via soft agar colony formation/MTT, cell motility, and invasiveness using transwell migration and invasion assays, followed by calcein AM staining after treatment with conditioned media from each group. (K) Protein-protein docking using ClusPro (Piper algorithm) predicted interaction interfaces between ALDH2 and ALDH6A1. PyMOL visualization identified contact residues: ALDH2 (green) and ALDH6A1 (cyan), with atoms color-coded (red: oxygen, blue: nitrogen, yellow: sulfur, white: hydrogen). (L) PyMOL further highlighted polar interactions using yellow dashed lines. (M) Pull-down assays employing anti-ALDH2 antibodies, followed by immunoblotting with anti-HaloTag, confirmed physical interaction between ALDH2 and ALDH6A1 in ALDH2-overexpressing FaDu cells transfected with pALDH6A1-HaloTag. Mutually, pull-down using anti-HaloTag antibodies, followed by immunoblotting with anti-ALDH2, further validated this interaction. (N) Reciprocal co-immunoprecipitation (Co-IP) assays using anti-ALDH2 and anti-ALDH6 antibodies validated endogenous interaction in Detroit 562 and SCC-25. Anti-GFP probing served as a negative control. (O , P) Immunoblot analysis confirmed expression of the ALDH6-HaloTag fusion protein (~ 91 kDa) following transient transfection of pALDH6A1-HaloTag plasmids into FaDu and shALDH2#2 Detroit 562 cells overexpressing ALDH2(WT) or one of five ALDH2 mutants. (Q) Reciprocal pull-down assays using anti-HaloTag and anti-ALDH2 antibodies confirmed that ALDH6A1 interacts with the E504K and phosphorylation-deficiency mutants, similar to its interaction with ALDH2(WT). Data are presented as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: We utilized four HNSC-derived cell lines: 2A3 (pharynx, HPV-positive, CRL-3212TM, ATCC, Manassas, VA, USA), Detroit 562 (pharynx, HPV-negative, CCL-138TM, ATCC), FaDu (hypopharyngeal, HPV-negative, HTB-43TM, ATCC), and SCC-25 (tongue, CRL-1628TM, Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: In Vitro, Western Blot, Expressing, Derivative Assay, Transfection, Plasmid Preparation, Control, Over Expression, Functional Assay, Migration, Staining, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Phospho-proteomics