Experimental workflow to evaluate nanoparticles combined with photon and carbon-ion medical irradiations in spheroids

Treatment protocols of cancer combining radiotherapy and nanoparticles are rapidly evolving. To evaluate their efficacy, spheroids provide a 3D in vitro model that better reflects tumor architecture than traditional 2D cell cultures. This article presents a workflow to prepare and characterize spheroids, optimize the protocols for irradiation with medical photon and ion beams and for exposure to nanoparticles, and, finally, to evaluate the effects of radiation, nanoparticles, and their combination on spheroids. Illustrations with HeLa, U-87 MG, and BxPC-3 tumor cell lines and HDFn are reported. Using this workflow, we observed that spheroids exhibit variable cell organization and interstitial spaces, which affect nutrient and oxygen diffusion and, consequently, cell proliferation. These structural differences also affect enzyme diffusion, limiting the applicability of the clonogenic assay in densely packed spheroids, as the assay requires enzymatic disaggregation of the spheroids. The clonogenic assay remains essential for quantitatively comparing spheroid irradiation results with 2D cell culture experiments. It has long been used as the reference method in radiobiology because it assesses mitotic death and long-term proliferative capacity. Protocols were adapted to ensure the feasibility of the clonogenic assay when possible, depending on cell line characteristics. Interstitial spaces also influenced nanoparticle internalization, which was more efficient in spheroids with larger interstitial spaces. This workflow and associated techniques verified the characteristic effect of carbon ion irradiation with a relative biological effectiveness of ~ 3 and demonstrated a ~ 30% radioenhancing effect of platinum nanoparticles at 2 Gy under 6 MV photons.