A 3D Tumor Spheroid Model with Robust T Cell Infiltration for Evaluating Immune Cell Engagers

A strong interest in drugs targeting the tumor microenvironment (TME) necessitates new experimental systems that incorporate key TME components. Compared to traditional 2D cell lines, 3D ex vivo spheroids from patient-derived xenograft (PDX) materials may better capture patient tumor characteristics. We developed and validated 3D tumor spheroid model from non-small cell lung cancer (NSCLC) PDXs to enable T cell infiltration. Histologic and transcriptomic analysis suggested that tumor spheroids closely recapitulate their source PDX tumor tissue. Consistent T cell infiltration into tumor spheroids was achieved using a well-established magnetic nanoparticle technology, which maintained T cell function and tumor-killing activity. Drug treatment studies with immunotherapy agents also demonstrated the potential scalability of 3D tumor-T cell spheroids in assessing drug activity, including tumor viability and cytokine secretion. This platform provides a useful tool for evaluating drug candidates that can be translated to patient tumor responses related to both tumor intrinsic and TME factors. Overall design: To understand whether our PDX-derived spheroids recapitulate in vivo tumors in their gene expression profile, we performed RNA-seq on isolated human tumor cells from five PDX tumor models and their matched ex vivo spheroids. This included LU450, LU481, LU554, LU211, LU120. Samples were collected in triplicates. Principal component analysis (PCA) revealed that samples strongly cluster by patient of origin, regardless of whether samples came from freshly dissociated PDXs or PDX-derived spheroids. Some PDX models had RNA collected from multiple passages (LU450, LU481, LU554, LU211), where we saw that samples of different passages still clustered closely together by PDX lineage. In addition, we collected spheroid samples (LU120, LU481, LU554) without magnetic nanoparticles to perform comparisons with spheroids generated with magnetic nanoparticles, where we found no significant differences between the two conditions.