Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small noncoding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.

Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro- or ex vivo-isolated EVs has considerably contributed to the understanding of biological functions of EV transfer. However, the study of EV release and uptake in an in vivo setting has remained challenging, because cells that take up EVs could not be discriminated from cells that do not take up EVs. Recently, a technique based on the Cre-loxP system was developed to fluorescently mark Cre-reporter cells that take up EVs released by Cre recombinase-expressing cells in various in vitro and in vivo settings. Here we describe a detailed protocol for the generation of Cre(+) cells and reporter(+) cells, which takes ∼ 6 weeks, and subsequent assays with these lines to study functional EV transfer in in vitro and in vivo (mouse) settings, which take up to ∼ 2 months.

The mammary gland consists of an adipose tissue that, in a process called branching morphogenesis, is invaded by a ductal epithelial network comprising basal and luminal epithelial cells. Stem and progenitor cells drive mammary growth, and their proliferation is regulated by multiple extracellular cues. One of the key regulatory pathways for these cells is the β-catenin-dependent, canonical wingless-type MMTV integration site family (WNT) signaling pathway; however, the role of noncanonical WNT signaling within the mammary stem/progenitor system remains elusive. Here, we focused on the noncanonical WNT receptors receptor tyrosine kinase-like orphan receptor 2 (ROR2) and receptor-like tyrosine kinase (RYK) and their activation by WNT5A, one of the hallmark noncanonical WNT ligands, during mammary epithelial growth and branching morphogenesis. We found that WNT5A inhibits mammary branching morphogenesis in vitro and in vivo through the receptor tyrosine kinase ROR2. Unexpectedly, WNT5A was able to enhance mammary epithelial growth, which is in contrast to its next closest relative WNT5B, which potently inhibits mammary stem/progenitor proliferation. We found that RYK, but not ROR2, is necessary for WNT5A-mediated promotion of mammary growth. These findings provide important insight into the biology of noncanonical WNT signaling in adult stem/progenitor cell regulation and development. Future research will determine how these interactions go awry in diseases such as breast cancer. mammary stem cells | noncanonical Wnt signaling | receptor tyrosine kinase | epithelial morphogenesis

Recent data showed that cancer cells from different tumor subtypes with distinct metastatic potential influence each other's metastatic behavior by exchanging biomolecules through extracellular vesicles (EVs). However, it is debated how small amounts of cargo can mediate this effect, especially in tumors where all cells are from one subtype, and only subtle molecular differences drive metastatic heterogeneity. To study this, we have characterized the content of EVs shed in vivo by two clones of melanoma (B16) tumors with distinct metastatic potential. Using the Cre‐LoxP system and intravital microscopy, we show that cells from these distinct clones phenocopy their migratory behavior through EV exchange. By tandem mass spectrometry and RNA sequencing, we show that EVs shed by these clones into the tumor microenvironment contain thousands of different proteins and RNAs, and many of these biomolecules are from interconnected signaling networks involved in cellular processes such as migration. Thus, EVs contain numerous proteins and RNAs and act on recipient cells by invoking a multi‐faceted biological response including cell migration.