Document resource
Background Monocytes are a versatile innate immune cell population capable of both promoting and suppressing anti-tumor immune responses. They are highly infiltrative into the tumor microenvironment and their increased presence has been associated with poor responses to immunotherapy in solid tumors. While much of current research focuses on their immune suppressive roles, monocytes also have the potential to contribute to immune response through immune cell recruitment, antigen presentation, and direct cell death induction. We have found trogocytosis, a process of intercellular material exchange, to be a method of monocyte-tumor cell interaction impacting monocyte activation status and tumor cell viability. Foundational work in our lab has begun to shed light on resultant transcriptional changes in trogocytic monocytes, notably upregulation of genes involved in antigen presentation, integrin signaling, and wound healing response. Despite its role in immune regulation, the pathways and molecules dictating trogocytic function in monocytes remains understudied. Here, we present a genome-wide CRISPR/Cas9 screen and mechanistic validation studies of the genetic drivers of monocyte-tumor cell trogocytosis.Methods We utilized a flow cytometry-based co-culture assay to capture trogocytic events between the THP-1 monocytic cell line and fluorescently-labeled human tumor cells opsonized with isotype or tumor-targeting antibodies. CRISPR/Cas9 was used to generate a pooled knock-out library of THP-1 cells to conduct a genome-wide screen as well as single gene knock-outs (KOs) of identified regulators of trogocytosis in THP-1 cells. Single gene KOs were confirmed by western blot and flow cytometry and evaluated for their effects on trogocytosis of tumor cells and downstream monocyte functions.Results Our genome-wide CRISPR/Cas9 screen identified members of Rho-GTPase signaling, actin remodeling, and mitochondrial ion transport pathways – all crucial for plasma membrane dynamics – to be significant regulators of monocyte trogocytosis of tumor cells. Genes involved in chromatin remodeling and protein translational regulation were also represented in our top hits. Notably, our results did not significantly overlap with results from genome-wide screens interrogating monocyte phagocytosis, indicating distinct processes governing trogocytosis. Targets of interest from these prominent pathways as well as controls from literature were selected and further evaluated using THP-1 single gene KOs. KOs of positive regulators successfully decreased levels of trogocytosis while KOs of negative regulators increased trogocytosis. Current studies focus on characterizing downstream immunological impacts of modulating this process.Conclusions These studies provide a basis for our understanding of the key regulatory pathways mediating monocyte-tumor trogocytosis. This insight opens novel avenues to modulate monocyte function and improve cancer immunotherapies.