Quantitative LA-ICP-MS Imaging of Elemental Distributions with Protein Correlation in 3D Tumor Models

Therapeutic efficacy in complex tissues depends on microscale drug and elemental distributions, yet quantitative mapping of these distributions in three-dimensional (3D) biological systems remains technically challenging. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) enables sensitive elemental imaging, but quantitative application in heterogeneous 3D models is limited by preparation artifacts, calibration challenges and lack of validated integration with biological markers. Here we establish a validated workflow for quantitative LA-ICP-MS imaging in tumor spheroids, enabling spatial correlation with immunohistochemistry using consecutive sections. Systematic evaluation of preparation revealed substantial analyte redistribution under conventional conditions. Optimized cryo-embedding in 2% carboxymethyl cellulose combined with freeze-drying reduced peripheral boron leaching by ∼53% compared with gelatin-based media and ∼84% compared with OCT. Matrix-matched calibration (r2 > 0.99) with endogenous 31P normalization enabled reproducible pixel-level quantification consistent with bulk ICP-MS measurements (p > 0.05). Consecutive section analysis showed protein expression varied by <20% between adjacent 30 μm sections, with radial profiles showing strong correlations across proteins and cell lines (Pearson r = 0.558-0.996), supporting spatial correlation of elemental and protein distributions. Applied to boron as a stringent low-mass analyte, the workflow achieved 10 μm spatial resolution with ∼7.4 ng g-1 detection limits. This approach provides a reproducible platform (Pearson r = 0.865-0.961, n = 3, p < 0.0001) for quantitative elemental imaging and cross modal spatial analysis in heterogeneous 3D biological systems.