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Pastò, Anna (2013) Colon stem cell characterization in normal and tumoral tissues: description of a novel feed-forward circuit of Msi-1 regulation. [Tesi di dottorato]

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Abstract (inglese)

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.
We 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.
Dissociated 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.
We 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.
Finally, 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.
In 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.
Furthermore 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.

Abstract (italiano)

I tessuti normali non patologici sono organizzati in una struttura gerarchica all'interno della quale un numero esiguo di cellule origina l'intera totalità della popolazione. Queste cellule presentano longevità, multipotenza, quiescenza e capacità di dividersi in maniera asimmetrica, tutte caratteristiche di staminalità.
Abbiamo deciso di sviluppare un sistema in vitro per separare le cellule staminali epiteliali del colon, non basandoci sull'espressione di specifici marcatori di superficie, ma semplicemente traendo vantaggio dal ridotto tasso di proliferazione che queste cellule presentano quando tenute in assenza di siero.
Da campioni primari di colon le cellule epiteliali sono state dissociate, marcate con PKH26, che permette l'identificazione di distinte sottopopolazioni basandosi sul tasso di proliferazione, e coltivate in vitro in assenza di siero. Sono stati analizzati i livelli di espressione proteica di CK20, Msi-1 e Lgr5 e di mRNA di alcuni geni associati alla staminalità. L'espressione di questi geni è stata confrontata tra la popolazione cellulare in cultura e tre sottopopolazioni isolate dalle cripte di colon attraverso microdissezione laser.
In coltura abbiamo identificato una popolazione PKHpos, in grado di sopravvivere e formare sferoidi, comprendente cellule con un ridotto tasso di proliferazione (PKHhigh) e cellule con rapido tasso (PKHlow). L'analisi citofluorimetrica ha dimostrato che cellule Msi-1+/Lgr5+ si trovano solo nella popolazione PKHhigh, mentre cellule Msi-1+/Lgr5- sono presenti anche nella popolazione PKHlow. Questi risultati sono stati supportati dall'analisi molecolare nelle diverse sottopopolazioni. Nelle cellule Msi-1+/Lgr5+ abbiamo, infatti, riscontrato livelli più alti di espressione di geni associati con la staminalità (Bmi-1, EphB2, EpCAM, ALDH1). Invece l'espressione di citocheratina (CK20) è quasi totalmente ristretta alle cellule PKHlow e PKHneg, le quali esprimono anche Mucin-1 ma non Lgr5.
Questi risultati rispecchiano quelli trovati analizzando le popolazioni isolate dalle diverse porzioni delle cripte attraverso microdissezione, e ci hanno permesso di definire una gerarchia di sottopopolazioni cellulari diverse in differenti fasi di maturazione: PKHhigh/Lgr5+/Msi-1+/CK20-, PKHhigh/Lgr5-/Msi-1+/CK20+, PKHlow/Lgr5-/Msi-1+/Ck20-, e PKHlow/ Lgr5-/Msi-1-/ CK20+.
Per valutare la presenza di diverse sottopopolazioni con caratteristiche di staminalità anche nel tessuto tumorale, da 5 campioni di tumore primario colorettale e da 15 di metastasi, abbiamo isolato le cellule, marcate con PKH26 e mantenute in assenza di siero. Come per i campioni normali, nei tumori abbiamo identificato una popolazione cellulare poco proliferante e in grado di sopravvivere in vitro in assenza di siero. Invece, nei campioni metastatici non siamo stati in grado di definire chiaramente una popolazione PKHhigh; sembra, infatti, che siano le cellule PKHlow in grado di sopravvivere più a lungo in cultura e quindi selezionarsi.
L'analisi citofluorimetrica ha dimostrato che, a differenza dei campioni normali, in quelli metastatici cellule Lgr5+/Msi-1+ non sono presenti solo nella popolazione PKHhigh, ma anche all'interno della sottopopolazione PKHlow. Nelle cellule PKHneg invece non si riscontra l'espressione nè di Lgr5 nè di Msi-1. Entrambe le popolazioni PKHhigh e PKHlow sono state in grado di formare sferoidi e, quando mantenute in presenza del 10% di siero, hanno aderito alle piastre differenziandosi ed assumendo una morfologia epiteliale. Queste cellule perdono conseguentemente l'espressione di Msi-1 e Lgr5 solo dopo 14 giorni in queste condizioni, mentre acquistano l'espressione di CK20.
Vista la ridotta presenza di dati inerenti la regolazione di Msi-1, abbiamo infine indagato il possibile coinvolgimento del pathway di Notch sulla regolazione di Msi-1 nelle cellule di tumore del coloretto. Dopo 72 ore di trattamento con il ligando di Notch (DLL4) i livelli di proteina e mRNA di Msi-1 aumentano. Questo fenomeno è bloccato dall'aggiunta di un anticorpo neutralizzante Notch2/3, mentre nessuna variazione è stata riscontrata dopo il trattamento con anti-Notch1. Poiché Msi-1 regola i livelli di Numb, la sua diminuzione comporta un aumento dei livelli proteici di Numb, come riscontrato dall'analisi Western Blot effettuata nei campioni tumorali primari e nella linea cellulare MICOL-14tum. Inoltre, dopo quattro giorni di trattamento, la formazione di sferoidi appare significativamente ridotta dal trattamento con anti-Notch2/3.
In conclusione, i nostri dati dimostrano la possibilità di derivare in vitro da campioni di colon normale, senza alcuna strategia selettiva, delle sottopopolazioni in differenti fasi di maturazione. Inoltre, nel tessuto metastatico siamo stati in grado di descrivere un possibile circuito di regolazione che coinvolge Msi-1, Numb, Notch3 e Notch1. In particolare, abbiamo dimostrato che Msi-1 può esser considerato un target di Notch3, e Notch3 può agire come un regolatore positivo di Notch1 attraverso il circuito Msi-1/Numb.

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Tipo di EPrint:Tesi di dottorato
Relatore:Amadori, Alberto
Dottorato (corsi e scuole):Ciclo 25 > Scuole 25 > ONCOLOGIA E ONCOLOGIA CHIRURGICA
Data di deposito della tesi:29 Gennaio 2013
Anno di Pubblicazione:29 Gennaio 2013
Parole chiave (italiano / inglese):colon, stem cells, Notch3, Msi-1
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/06 Oncologia medica
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Chirurgiche Oncologiche e Gastroenterologiche
Codice ID:5644
Depositato il:22 Ott 2013 10:03
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