Colon stem cell characterization in normal and tumoral tissues: description of a novel feed-forward circuit of Msi-1 regulation
Year: 2013
Type: article
Abstract: Normal tissues are organized in a hierarchical fashion, where rare somatic cells endowed with stem-like properties give origin to a population of differentiated cells forming the bulk of tissue. These self-maintaining cells, called stem cells, are characterized by 4 fundamental properties: longevity, multipotency, quiescence, and asymmetric cell division. \nWe decided to develop a new in vitro system for separation of stem-like epithelial cell based not on surface marker-driven selection, but by simply taking advantage of their slow proliferation rate in the absence of serum. \nDissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKHpos population survived in culture and formed spheroids; this population included subsets with slow (PKHhigh) and rapid (PKHlow) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr5 in PKHhigh cells; by cytofluorimetric analysis, Msi-1+/Lgr5+ cells were only found within PKHhigh cells, whereas Msi-1+/Lgr5- cells were also observed in the PKHlow population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1+/Lgr5+ cells. While CK20 expression was mainly found in PKHlow and PKHneg cells, a small PKHhigh subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin-1, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define a hierarchy of cellular subsets at different maturation stages: PKHhigh/Lgr5+/Msi-1+/CK20-, PKHhigh/Lgr5-/Msi-1+/CK20+, PKHlow/Lgr5-/Msi-1+/Ck20-, and PKHlow/ Lgr5-/Msi-1-/ CK20+ cells. \nWe next addressed the issue of cancer stem cells in colorectal cancer (CRC). Isolated cells from five primary colon tumors and 15 CRC metastatic samples were stained with PKH26 and cultured in PhEMA-treated plate in the absence of serum. As in normal samples, in tumors we identified a slowly cycling population able to persist in vitro in serum-free condition. On the contrary, in metastatic samples we could not document an in vitro selection of a PKHhigh population; actually PKHlow cells seemed to remain constant over several weeks. Cytofluorimetric analysis revealed that, at variance with normal samples, in metastatic samples Lgr5+/Msi-1+ cells were not only present in the slowly cycling PKHhigh population but also in PKHlow cells. No Lgr5 or Msi-1 expression was recorded in PKHneg cells. Both PKHhigh and PKHlow cells were able to form spheroids; in the presence of 10% FCS in uncoated plates they adhered to the plate and differentiated assuming an epithelial morphology. They eventually lost Msi-1 and Lgr5 expression after 14 days of culture in the presence of serum and adhesion conditions, and they also acquired CK20 expression. \nFinally, we investigated the possible involvement of Notch signaling on regulation of Msi-1 levels in the metastatic cell line MICOL-14tum and primary metastatic samples. Seventy two hours of treatment with the Notch ligand DLL4 increased Msi-1 mRNA and protein levels. This phenomenon was largely prevented by the addition of an antibody neutralizing Notch2/3, whereas no reduction was detected after incubation with anti-Notch1. Since Msi-1 regulates Numb levels, its reduction led to increased Numb protein, as demonstrated by Western Blot analysis of MICOL-14tum and primary metastatic cells. Furthermore, after four days of treatment, formation of spheroids was significantly reduced by incubation with anti-Notch2/3 antibody in primary samples as well as in MICOL-14tum cells. \nIn conclusion, our data showed the possibility of deriving in vitro from normal colon tissue, without any selection strategy, an array of cellular subsets differentially expressing the most common stemness and differentiation markers of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in an anatomical position compatible with stem-like cells. \nFurthermore in metastatic cell line and primary samples we described a novel feed-forward circuit involving Msi-1, Numb, Notch3 and Notch1. In particular, we demonstrated that Msi-1 could be considered a target of Notch3, and Notch3 could act as a positive regulator of Notch1 levels through Msi-1/Numb circuit. \n
Author: Anna Pastò
Language: English
Topic: Cancer Cells and Metastasis
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2275022203