Exosome-targeted xmot-shRNA as a modular platform to dissect signaling pathways driving tolerogenic dendritic cells in the tumor microenvironment

Dendritic cells (DCs) orchestrate tumor-targeted immunity and are essential for immune checkpoint inhibitor (ICI) efficacy, but ICI responses often fail when tumors drive DCs into a pro-tolerogenic state exhibiting an impaired capacity to stimulate CD8 + T cell activation and an enhanced ability to drive CD4 + FoxP3 + regulatory T cell (Treg) differentiation. Despite the potent impact of DC tolerization on the generation of anti-tumor immunity, the pathways involved in their development in vivo are poorly understood. We have previously demonstrated that tolerogenic DCs in the tumor microenvironment maintain increased fatty acid oxidation (FAO) and this metabolic program fuels indoleamine-2,3-dioxygenase 1 (IDO1) activation and downstream Treg development. A pivotal check point in this pathway is the rate limiting enzyme, carnitine palmitoyltransferase 1a (CPT1a). Tumor-derived extracellular vesicles (EVs) are abundantly released in response to stress and can transfer regulatory RNAs to infiltrating immune cells. DCs are highly proficient at engulfing these EVs within tumors. A defined 3′ exosome-enriched motif (xmot) sequence can selectively load oligonucleotides into these vesicles, providing an opportunity to exploit tumor EV biogenesis for shRNA delivery. Here, we aimed to utilize the xmot-shRNA platform to selectively silence Cpt1a in DCs to suppress FAO and prevent Treg development. Cpt1a -targeted shRNA is fused to a 3′ exosome-enrichment trafficking motif and utilized to generate a xmot-shCpt1a-expressing BRAF V600E -PTEN -/- melanoma cell line. The tumor EVs were confirmed to harbor Cpt1a -shRNA by qrt-PCR and found to suppress both DC Cpt1a expression and DC oxidative phosphorylation in vitro . In order to track selective DC targeting in vivo , the platform was equipped with an EV-enriched CD81-mEmerald marker protein. Using this approach, mEmerald + DCs infiltrating xmot-shCpt1a-expressing BRAF V600E -PTEN -/- melanomas were isolated by fluorescence-activated cell sorting and found to exhibit suppressed Cpt1a expression levels. By comparing with control BRAF V600E -PTEN -/- melanomas, this work demonstrated xmot-shCpt1a-expression to inhibit intra-tumoral CD4 + FoxP3 + Treg accumulation, enhance tumor CD8 + T cell infiltration and activation, and suppress BRAF V600E -PTEN ⁻/⁻ melanoma progression. Collectively, this study serves as a proof-of-principle for the xmot-shRNA/CD81-mEmerald platform as a modular delivery system for dissecting signaling pathways contributing to the development of tolerized DCs within the tumor microenvironment. This approach is now being utilized to probe additional regulatory circuits contributing to DC tolerization in vivo .