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Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis

ABSTRACT: Purpose: to determine cellular heterogeneity of the murine colonic epithelium and mesenchyme; Summary: We determined 16 disctinct sub-populations of colonic epithelial and mesenchymal cells. Further we found 3 distinct sub-populations of colonic PdgfraEGFP-positive fibroblasts.

ORGANISM(S): Mus musculus

PROVIDER: GSE151257 | GEO | 2020/11/19

REPOSITORIES: GEO

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Project description:In human colon cancer, the malignant component of tumor tissues often contains multiple sub-types of cancer cells, whose transcriptional profiles and surface marker phenotypes correspond to those of the epithelial lineages that are found in normal colonic crypts (e.g., goblet cells, enterocytes, LGR5+ columnar basal cells). Among those various sub-types of malignant cells, those characterized by a surface marker phenotype that is characteristic of epithelial stem/progenitor cells residing at the bottom of colonic crypts (EpCAM+, CD44+, CD166+) are enriched in cells with "cancer stem cell" (CSC) properties, such as self-renewal (i.e., the capacity to sustain the formation of new tumors upon serial xeno-transplantation in immune-deficient animals) and multi-lineage differentiation (i.e., the capacity to sustain the formation of other cell-types, thus reconstituting the heterogeneous population of the parent tumors from which they have been isolated). Cell populations with "cancer stem cell" (CSC) properties are known to be preferentially resistant to several cytotoxic agents used in conventional chemotherapy, but the molecular mechanisms underpinning this property remain poorly understood. In this dataset, we report the analysis by gene-expression microarrays of the transcriptional profile of two sub-populations of human colon cancer cells: 1) cells with a "bottom-of-the-crypt" phenotype (EpCAM+, CD44+, CD166+), which are known to be enriched in "cancer stem cells" (CSCs); and 2) cells with a "top-of-the-crypt" phenotype (EpCAM+, CD44neg, CD166neg), which are known to be non-tumorigenic upon xeno-transplantation in immune-deficient mice. The two populations were purified in parallel by fluorescence activated cell sorting (FACS), starting from solid tumors established by sub-cutaneous (s.c.) engraftment in immune-deficient mice of a patient-derived xenograft (PDX) line representative of a moderately differentiated (G2) primary colon carcinoma. To identify genes whose expression is modulated by in vivo exposure to chemotherapy (and thus potentially involved in the mechanistic regulation of chemo-resistance), we compared the transcriptional profile of cells purified from tumors that were exposed to 4 weeks of in vivo treatment with either irinotecan (CPT-11) or a placebo control (saline solution).

Project description:Abstract: The intestinal epithelium is replaced weekly by non-quiescent stem cells with kinetics that rely on a rapid loss of stemness and choice for secretory or absorptive lineage differentiation. To determine how the cellular transcriptome and proteome changes during these transitions, we developed a new cell sorting method to purify stem cells, secretory and absorptive progenitor cells, and mature, differentiated cells. Transcriptome analyses revealed that as stem cells transition to the progenitor stage, alternative mRNA splicing and polyadenylation dominate changes in the transcriptome. In contrast, as progenitors differentiate into mature cell types, alterations in gene expression and mRNA levels drive the changes. RNA processing targets mRNAs encoding regulators of cell cycle, RNA regulators, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, carrier-assisted mass spectrometry of sorted cell populations detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt. Sorting Protocol: To create a high-resolution profile of colon crypt stem cells and their daughter cells we developed a new flow sorting protocol using freshly dissected, wild-type C57Bl/6N mouse colons and antibodies to validated intestinal cell surface markers including Cd44. Upon discovery that Cd44 is highly sensitive to TrypLE, and other commonly used proteases, we developed a dissociation protocol that uses only EDTA and mechanical force. This change enabled a 10-fold greater range of Cd44 antigen surface expression and therefore higher resolution for cell sorting. Using additional commonly used cell surface markers, six cryptal populations were purified. A previously validated intestinal stem cell signature of Cd44 high, Cd24 low, and cKit negative was used to identify and isolate an abundant fraction of stem cells, a cell population that directly overlapped with Lgr5-EGFP+ cells from Lgr5-EGFP-IRES-creERT2 mice, confirming their stem cell identity. In addition to the stem cell population, five additional Epcam positive populations were collected, and the biological replicate samples of the six populations were processed for bulk RNA-seq. The clearly identified populations are stem cells, two distinct populations of progenitor cells (absorptive and secretory), and three mature, differentiated populations (enterocytes, Tuft cells and Enteroendocrine (EEC) cells). Thus, the new protocol for crypt isolation and the greater range of Cd44 surface expression it preserves, enables a significant improvement in the resolution and sorting of stem cells away from their daughter cells and differentiated progeny. Specifically, it is now possible to distinguish stem cells from Absorptive Progenitor cells (AbsPro; Cd44Med) and from mature Enterocytes (Ent; Cd44Low/-) because of the higher resolution of Cd44 surface expression. Secretory progenitors are identified as SecPDG as this population contains a majority of Secretory Progenitors and Deep Crypt Secretory cells, with a possible minor contribution of Goblet cells, a cell type that is largely missing in this procedure. Finally, preserved Cd44 expression (and cKit expression) enabled resolution of two rare Epcam+/Cd24high populations identified as Tuft cells and Enteroendocrine cells (EEC), mature cell types from the secretory lineage (EEC are predominantly Enterochromaffin; Tuft, comprise both Tuft-1 and Tuft-2 subtypes).