Probes for INS

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.

Wnt signals at the base of mammalian crypts play a pivotal role in intestinal stem cell (ISC) homeostasis, whereas aberrant Wnt activation causes colon cancer. Precise control of Wnt signal strength is governed by a number of negative inhibitory mechanisms acting at distinctlevels of the cascade. Here, we identify the Wnt negative regulatory role of Sh3bp4 in the intestinal crypt. We show that the loss of Sh3bp4 increases ISC and Paneth cell numbers in murine intestine and accelerates adenoma development in Apcmin mice. Mechanistically, human SH3BP4 inhibits Wnt signaling downstream of β-catenin phosphorylation and ubiquitination. This Wnt inhibitory role is dependent on the ZU5 domain of SH3BP4. We further demonstrate that SH3BP4 is expressed at the perinuclear region to restrict nuclear localization of β-catenin. Our data uncover the tumor-suppressive role of SH3BP4 that functions as a negative feedback regulator of Wnt signaling through modulating β-catenin's subcellular localization.

Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.

Colon tumours are hierarchically organized and contain multipotent self-renewing cells, called Cancer Stem Cells (CSCs). We have previously shown that the Notch1 receptor is expressed in Intestinal Stem Cells (ISCs); given the critical role played by Notch signalling in promoting intestinal tumourigenesis, we explored Notch1 expression in tumours. Combining lineage tracing in two tumour models with transcriptomic analyses, we found that Notch1+ tumour cells are undifferentiated, proliferative and capable of indefinite self-renewal and of generating a heterogeneous clonal progeny. Molecularly, the transcriptional signature of Notch1+ tumour cells highly correlates with ISCs, suggestive of their origin from normal crypt cells. Surprisingly, Notch1+ expression labels a subset of CSCs that shows reduced levels of Lgr5, a reported CSCs marker. The existence of distinct stem cell populations within intestinal tumours highlights the necessity of better understanding their hierarchy and behaviour, to identify the correct cellular targets for therapy.

The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.

The intestine is maintained by stem cells, marked by LGR5 expression, located at the base of crypts. Genetically engineered mouse models have provided information about marker genes and stem cell pathways. Less is known about human intestinal stem cells due to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas, and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC) associated genes. Normal and neoplastic colon tissue organoids were analyzed for LGR5 expression by immunohistochemistry. LGR5-positive cells were isolated from 4 adenoma organoid lines and analyzed by RNA-sequencing. LGR5 expression in epithelium and stroma was associated with tumor stage. Integrating functional experiments with RNA-seq data from LGR5-positive adenoma organoid cells and normal colon, we associated expression of CRC-specific genes, including DKK4, with LGR5 expression. This system can be used to study LGR5-expressing cells in human tissue homeostasis and carcinogenesis.

The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.