Meta-Data Informed Microphysiological System Replicates in vivo Macrophage Regulation of CD8+ T Cell Migration in Renal Cell Carcinoma 2254287

Introduction CD8+ T cells are abundant in clear cell renal cell carcinoma (ccRCC) yet are often retained at the tumor margin, a hallmark of immune exclusion that limits immunotherapy efficacy. Tumor-associated macrophages (TAMs) may regulate this trafficking, but the specific myeloid subsets affecting post-extravasation T-cell migration remain poorly defined. Standard in vitro assays rarely capture the full sequence from T cell transendothelial migration to directional movement toward tumor and macrophage-mediated rejection, and these dynamics are hard to monitor in vivo. Improved in vitro models that are physiologically relevant, are needed to dissect macrophage control of T-cell homing in ccRCC. Methods To build a patient metadata-informed ccRCC microphysiological system (MPS),we performed the first ccRCC single-cell meta-analysis (>1 million cells) and applied Monocle3 pseudotime to define myeloid states and trajectories, identifying a transitional macrophage population. Guided by these insights, we engineered a T cell migration MPS consisting of a perfusable HUVEC-lined lumen in 3D collagen, primary ccRCC spheroids, ex vivo generated transitional macrophages, and CD8+ T cells. The platform quantifies CD8+ T-cell transendothelial and interstitial migration. Results Our myeloid meta-analysis identified an IL10/IL1B-high transitional state between monocytes and pro-inflammatory TAMs, upstream of all other immunosuppressive TAMs by pseudotime. We generated IL10/IL1B-high—like TAMs through tumor co-culture. When incorporated into the MPS, these TAMs reduced CD8+ T-cell extravasation and curtailed interstitial migration, suggesting differentiation towards TREM2+ immunosuppressive TAMs. Conclusion We defined and recreated a novel IL10/IL1B-high transitional state between monocytes and TAMs. Integrating meta-analysis with ex vivo MPS, we enable trajectory reconstruction from tumor-recruited monocytes toward immunosuppressive TAMs for mechanistic study and therapeutic targets discovery in ccRCC.