The Size of Glioblastoma Spheroids Influences Patterns of Invasion and Temozolomide Efficacy

Glioblastoma (GBM) is one of the most common malignant brain tumors, with patient mortality driven by invasion into the surrounding brain microenvironment and drug resistance. Multicellular spheroids are an increasingly common model to study GBM invasion and drug response in engineered biomaterials. However, a key design feature of tumor spheroid studies is the size of each spheroid (number of cells, diameter). Given the heterogeneous growth of GBM cells at the surgical margin, spheroids of different sizes may also have clinical relevance. Here, we define shifts in behavior and drug response of wild-type (WT) and temozolomide (TMZ)-resistant GBM spheroids as a function of initial spheroid size. GBM spheroids ranging from 1,000 to 10,000 cells in size were embedded into a methacrylamide-functionalized gelatin hydrogel. GBM spheroid size had an inverse relationship with the number of apoptotic cells. We observed significant spheroid-size-dependent effects on TMZ efficacy for both TMZ-resistant and WT cells. Interestingly, high single doses of TMZ were more effective in reducing three-dimensional migration from smaller spheroids than metronomic dosing, while high single dose and metronomic dosing were equally effective in reducing invasion for large TMZ-resistant spheroids. Our study highlights the importance of considering and reporting spheroid size for cancer tissue engineering studies considering invasion and drug resistance. It also informs future studies of residual GBM at the tumor margins most responsible for patient relapse and mortality.