ECM-integrated hanging drop platform for spatially controlled assessment of immune cell regulated tumour invasion

The tumour immune microenvironment (TIME) plays a crucial role in tumour progression and metastasis. Although spheroids effectively model tumour invasion by mimicking <i>in vivo</i> 3D structures, their formation and subsequent mixing with the matrix make it difficult to control their position in the 3D matrix, leading to deep embedding and hindering the assessment of immune cell-mediated regulation of invasion. This paper introduces an extracellular matrix (ECM)-integrated hanging drop platform that enables simultaneous spheroid formation and matrix incorporation, allowing precise spatial control and direct assessment of immune cell-mediated regulation of invasion. In the presence of microglia (MG), cancer cells rapidly migrate out of the spheroids through the ECM, demonstrating cancer invasion. The cytotoxic effect of natural killer (NK) cells on glioblastoma multiforme (GBM) spheroids is decreased owing to the inhibition of NK cell infiltration in the presence of MG, highlighting the immunosuppressive nature of the TIME. However, inhibiting signal transducer and activator of transcription 3 (STAT3) activation with drugs halts MG-induced immunosuppression and enhances NK cell infiltration. This model enables efficient high-throughput screening and is the first to allow for precise quantification of the effects of the STAT3 inhibitor on tumour invasion, immune cell movement, and behaviour within a physiologically relevant GBM TIME model.