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γδ T cells represent a substantial fraction of intestinal lymphocytes at homeostasis, but they also constitute a major lymphocyte population infiltrating colorectal cancers (CRCs); however, their temporal contribution to CRC development or progression remains unclear. Using human CRC samples and murine CRC models, we found that most γδ T cells in premalignant or nontumor colons exhibit cytotoxic markers, whereas tumor-infiltrating γδ T cells express a protumorigenic profile. These contrasting T cell profiles were associated with distinct T cell receptor (TCR)-Vγδ gene usage in both humans and mice. Longitudinal intersectional genetics and antibody-dependent strategies targeting murine γδ T cells enriched in the epithelium at steady state led to heightened tumor development, whereas targeting γδ subsets that accumulate during CRC resulted in reduced tumor growth. Our results uncover temporal pro- and antitumor roles for γδ T cell subsets.

Barrier epithelia depend upon resident stem cells for homeostasis, defense, and repair. Epithelial stem cells of small and large intestines (ISCs) respond to their local microenvironments (niches) to fulfill a continuous demand for tissue turnover. The complexity of these niches and underlying communication pathways are not fully known. Here, we report a lymphatic network at the intestinal crypt base that intimately associates with ISCs. Employing in vivo loss of function and lymphatic:organoid cocultures, we show that crypt lymphatics maintain ISCs and inhibit their precocious differentiation. Pairing single-cell and spatial transcriptomics, we apply BayesPrism to deconvolve expression within spatial features and develop SpaceFold to robustly map the niche at high resolution, exposing lymphatics as a central signaling hub for the crypt in general and ISCs in particular. We identify WNT-signaling factors (WNT2, R-SPONDIN-3) and a hitherto unappreciated extracellular matrix protein, REELIN, as crypt lymphatic signals that directly govern the regenerative potential of ISCs., Competing Interests: Declaration of interests D.P. is on the scientific advisory board of Insitro. B.J.M. is an advisor to and recipient of an investigator-initiated research award from PureTech Corp. and recipient of an investigator-initiated research award from Regeneron. E.F. served on the scientific advisory boards of Arsenal Biosciences and L’Oreal., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tumors have evolved mechanisms to escape anti-tumor immunosurveillance. They limit humoral and cellular immune activities in the stroma and render tumors resistant to immunotherapy. Sensitizing tumor cells to immune attack is an important strategy to revert immunosuppression. However, the underlying mechanisms of immune escape are still poorly understood. Here we discover Indoleamine-2,3-dioxygenase-1 (IDO1) + Paneth cells in the stem cell niche of intestinal crypts and tumors, which promoted immune escape of colorectal cancer (CRC). Ido1 expression in Paneth cells was strictly Stat1 dependent. Loss of IDO1 + Paneth cells in murine intestinal adenomas with tumor cell-specific Stat1 deletion had profound effects on the intratumoral immune cell composition. Patient samples and TCGA expression data suggested corresponding cells in human colorectal tumors. Thus, our data uncovered an immune escape mechanism of CRC and identify IDO1 + Paneth cells as a target for immunotherapy.

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts 1 . Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types 2,3 . However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E 2 (PGE 2 ). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE 2 drives the expansion οf a population of Sca-1 + reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1 + cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE 2 promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1 + cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE 2 -Ptger4. Analyses of patient-derived organoids established that PGE 2 -PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE 2 -Ptger4-Yap signalling axis.