Use of optimized single-cell RNA flow cytometry protocol identifies monocytes as main producers of type I interferon in mouse syngeneic tumors.

The tumor microenvironment (TME) consists of complex interactions between cellular and extracellular components, among which the immune system is known to play an integral role in disease progression and response to therapy. Cytokines and chemokines are cell signaling proteins used by immune cells to communicate with each other as well as with other cell types in the body. These proteins control systemic and local immune responses and levels of cytokines/chemokines in the TME have been associated with tumor outcomes. However, cytokines and chemokines have varied expression across cell types, tumors, and host conditions. Therefore, approaches to effectively study the production of these proteins at the single-cell level in the TME are needed to fully elucidate the mechanisms governing the anti-cancer immune response. Here, we detail a protocol to assess the production of cytokines/chemokines across leukocyte populations in mouse tumors using RNA flow cytometry. Importantly, this method can be adapted with minimal changes to study various mouse and human tumors, other RNA analytes, and non-tumor tissues. With this approach, we characterize single-cell production of