Extracellular vesicles (EVs) are membrane-surrounded structures that transmit biologically important molecules from the releasing to target cells, thus providing a novel intercellular communication mechanism. Since EVs carry their cargo in a protected form and their secretion is generally increased in tumorigenesis, EVs hold a great potential for early cancer diagnosis. By 3D culturing, we provide evidence that colorectal cancer (CRC) patient-derived organoids, representing a state-of-the-art established and essential approach for studying human CRC, is a suitable model for EV analysis. When testing the effects of major factors promoting CRC progression on EV release in the organoid model, we observed that Apc mutation, leading to uncontrolled Wnt activation and thus to tumorigenesis in the vast majority in CRC patients, critically induces EV release by activating the Wnt pathway. Furthermore, the extracellular matrix component collagen, known to accumulate in tumorigenesis, enhances EV secretion as well. Importantly, we show that fibroblast-derived EVs induce colony formation of CRC organoid cells under hypoxia. In contrast, there was no major effect of tumor cell-derived EVs on the activation of fibroblasts. Collectively, our results with CRC and Apc -mutant adenoma organoids identify Apc mutation and collagen deposition as critical factors for increasing EV release from tumors. Furthermore, we provide evidence that stromal fibroblast-derived EVs contribute to tumorigenesis under unfavorable conditions in CRC. Electronic supplementary material The online version of this article (10.1007/s00018-019-03052-1) contains supplementary material, which is available to authorized users.

Intestinal homeostasis is maintained by tightly controlled proliferation and differentiation of tissue-resident multipotent stem cells during aging and regeneration, which ensures organismal adaptation. Here we show that autophagy is required in Drosophila intestinal stem cells to sustain proliferation, and preserves the stem cell pool. Autophagy-deficient stem cells show elevated DNA damage and cell cycle arrest during aging, and are frequently eliminated via JNK-mediated apoptosis. Interestingly, loss of Chk2, a DNA damage-activated kinase that arrests the cell cycle and promotes DNA repair and apoptosis, leads to uncontrolled proliferation of intestinal stem cells regardless of their autophagy status. Chk2 accumulates in the nuclei of autophagy-deficient stem cells, raising the possibility that its activation may contribute to the effects of autophagy inhibition in intestinal stem cells. Our study reveals the crucial role of autophagy in preserving proper stem cell function for the continuous renewal of the intestinal epithelium in Drosophila.

Extracellular vesicles (EV) are membrane-surrounded vesicles that represent a novel way of intercellular communication by carrying biologically important molecules in a concentrated and protected form. The intestinal epithelium is continuously renewed by a small proliferating intestinal stem cell (ISC) population, residing at the bottom of the intestinal crypts in a specific microenvironment, the stem cell niche. By using 3D mouse and human intestinal organoids, we show that intestinal fibroblast-derived EVs are involved in forming the ISC niche by transmitting Wnt and epidermal growth factor (EGF) activity. With a mouse model that expresses EGFP in the Lgr5+ ISCs, we prove that loss in ISC number in the absence of EGF is prevented by fibroblastderived EVs. Furthermore, we demonstrate that intestinal fibroblast-derived EVs carry EGF family members, such as amphiregulin. Mechanistically, blocking EV-bound amphiregulin inhibited the EV-induced survival of organoids. In contrast, EVs have no role in transporting R-Spondin, a critical niche factor amplifying Wnt signaling. Collectively, we prove the important role of fibroblast-derived EVs as a novel transmission mechanism of factors in the normal ISC niche. K E Y W O R D S amphiregulin, exosomes, extracellular vesicle, fibroblast, intestinal stem cell, Lgr5, organoid, Wnt

Genetic variations of Atg16l1, Slit2 and Rab19 predispose to the development of inflammatory bowel disease (IBD), but the relationship between these mutations is unclear. Here we show that in Drosophila guts lacking the WD40 domain of Atg16, pre-enteroendocrine (pre-EE) cells accumulate that fail to differentiate into properly functioning secretory EE cells. Mechanistically, loss of Atg16 or its binding partner Rab19 impairs Slit production, which normally inhibits EE cell generation by activating Robo signaling in stem cells. Importantly, loss of Atg16 or decreased Slit/Robo signaling triggers an intestinal inflammatory response. Surprisingly, analysis of Rab19 and domainspecific Atg16 mutants indicates that their stem cell niche regulatory function is independent of autophagy. Our study reveals how mutations in these different genes may contribute to IBD.