CAR-Engineered Human Hematopoietic Stem Cell Macrophages Control Solid Tumors

Abstract

Chimeric antigen receptor (CAR) T-cells have represented a groundbreaking advance in the control of hematological cancers. However, their efficacy in controlling solid tumors has been rather limited, highlighting the importance of new cell-based therapies strategies to curb the progression of solid cancers. Here, we generated functional macrophages from human umbilical cord blood derived CD34+ hematopoietic stem cells (HSCs) engineered to express CARs. Approximately 50% of the CAR-MacCD34 population expressed anti-HER2 CARs and maintained high viability throughout differentiation. Mass spectrometry (MS) and multiparametric flow cytometry analysis revealed upregulation of proteins associated with phagocytosis, matrix remodeling, and degradation, indicating enhanced tumor infiltration potential. In vitro, CAR-MacCD34 exhibited a significantly higher capacity to phagocytose HER2-positive tumor cells compared to untransduced MacCD34 cells. Additionally, CAR-MacCD34 cells that phagocytosed cancer cells showed increased nuclear translocation of NF-kB, suggesting CAR-mediated intracellular signaling. To assess functionality in a more physiologically relevant context, we used tumor spheroids embedded in a dense 3D collagen matrix. Confocal microscopy and live imaging revealed that CAR-MacCD34 cells exhibited superior infiltration of dense tumor spheroids compared to untransduced MacCD34 cells. Notably, we observed multiple instances of tumor cell phagocytosis by CAR-MacCD34 cells in this 3D model. In addition, we employed in vivo zebrafish larvae models of HER2-positive tumors. We noted that CAR-MacCD34 cells persisted for over 8 days post-injection and demonstrated significantly greater efficacy in controlling tumor growth compared to untransduced MacCD34 cells. Our findings introduce a novel CAR-macrophage therapeutic approach with promising clinical potential, leveraging a renewable and accessible cellular source. Optimizing CAR-MacCD34 functionality in combination with existing therapies may lead to durable and effective anti-tumor responses for patients with solid tumors.