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Background <p>Three-dimensional (3D) spheroid models better replicate the in vivo tumor microenvironment than conventional two-dimensional (2D) cultures, making them valuable tools for preclinical drug screening in oral squamous cell carcinoma (OSCC).</p> Methods <p>Scaffold-free spheroids were generated from CAL 27 and OECM-1 OSCC cell lines using three approaches: methylcellulose suspension, agarose-coated plates, and ultra-low attachment (ULA) plates. Spheroid morphology and size uniformity were assessed microscopically. Functional characterization included viability assays and histological evaluation for necrosis. Molecular profiling of cancer hallmark genes was performed using qRT-PCR. Drug response assays for cisplatin and doxorubicin were compared between 2D and 3D cultures.</p> Results <p>Compact, uniform spheroids (~ 150–200&#xa0;μm) formed within 72&#xa0;h in both cell lines, with ULA plates producing the highest reproducibility and structural integrity. Histological analysis revealed central necrosis with viable peripheral cell layers. Gene expression analysis confirmed the modulation of hallmark cancer-associated genes in spheroid associated with hypoxia, angiogenesis, stress-response, adhesion and EMT-related pathways. Drug assays demonstrated significantly higher resistance to cisplatin and doxorubicin in 3D spheroids than in 2D monolayers (<i>p</i> &lt; 0.05).</p> Conclusion <p>ULA-based 3D spheroids provide a reproducible, structurally stable, and scalable model that closely mimics OSCC tumor biology and chemoresistance. These findings support their application as a robust platform for high-throughput drug screening and translational cancer research.</p>

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Optimization of OSCC spheroid generation for high-throughput drug screening

  • 期刊论文

  • 发表日期：

    2025年10月1日

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  • Mansi Dobariya,
  • Seena Sunil,
  • Sai Swetha Uppalapati,
  • Ravindra Taware,
  • Vinal Upadhyay,
  • Amit Mandoli

摘要

Background

Three-dimensional (3D) spheroid models better replicate the in vivo tumor microenvironment than conventional two-dimensional (2D) cultures, making them valuable tools for preclinical drug screening in oral squamous cell carcinoma (OSCC).

Methods

Scaffold-free spheroids were generated from CAL 27 and OECM-1 OSCC cell lines using three approaches: methylcellulose suspension, agarose-coated plates, and ultra-low attachment (ULA) plates. Spheroid morphology and size uniformity were assessed microscopically. Functional characterization included viability assays and histological evaluation for necrosis. Molecular profiling of cancer hallmark genes was performed using qRT-PCR. Drug response assays for cisplatin and doxorubicin were compared between 2D and 3D cultures.

Results

Compact, uniform spheroids (~ 150–200 μm) formed within 72 h in both cell lines, with ULA plates producing the highest reproducibility and structural integrity. Histological analysis revealed central necrosis with viable peripheral cell layers. Gene expression analysis confirmed the modulation of hallmark cancer-associated genes in spheroid associated with hypoxia, angiogenesis, stress-response, adhesion and EMT-related pathways. Drug assays demonstrated significantly higher resistance to cisplatin and doxorubicin in 3D spheroids than in 2D monolayers (p < 0.05).

Conclusion

ULA-based 3D spheroids provide a reproducible, structurally stable, and scalable model that closely mimics OSCC tumor biology and chemoresistance. These findings support their application as a robust platform for high-throughput drug screening and translational cancer research.