Unveiling immune cell response disparities in human primary cancer-associated fibroblasts between two- and three-dimensional cultures.

Cancer-associated fibroblasts (CAFs) play pivotal roles in solid tumor initiation, growth, and immune evasion. However, the optimal biomimetic modeling conditions remain elusive. In this study, we investigated the effects of 2D and 3D culturing conditions on human primary CAFs integrated into a modular tumor microenvironment (TME). Using single-nucleus RNA sequencing (snRNAseq) and Proteomics' Proximity Extension Assays, we characterized CAF transcriptomic profiles and cytokine levels. Remarkably, when cultured in 2D, CAFs exhibited a myofibroblast (myCAF) subtype, whereas in 3D tumor spheroid cultures, CAFs displayed a more inflammatory (iCAF) pathological state. By integrating single-cell gene expression data with functional interrogations of critical TME-related processes [natural killer (NK)-mediated tumor killing, monocyte migration, and macrophage differentiation], we were able to reconcile form with function. In 3D TME spheroid models, CAFs enhance cancer cell growth and immunologically shield cells from NK cell-mediated cytotoxicity, in striking contrast with their 2D TME counterparts. Notably, 3D CAF-secreted proteins manifest a more immunosuppressive profile by enhancing monocyte transendothelial migration and differentiation into M2-like tumor-associated macrophages (TAMs). Our findings reveal a more immunosuppressive and clinically relevant desmoplastic TME model that can be employed in industrial drug discovery campaigns to expand the cellular target range of chemotherapeutics.