Contributed equally
Cellular plasticity, the ability of cells to switch from an epitheial phenotype to a mesenchymal one and
Worldwide, colorectal cancer (CRC) is the fourth most common cancer and the fourth leading cause of cancer-related mortality, with 1.4 million estimated cases and 700,000 estimated deaths in 2012 (
The normal gastrointestinal epithelium is organized along a crypt-villus axis, with a pool of colon stem cells and progenitor cells, which are the most undifferentiated cell types which can undergo self-renewal and exhibit pluripotency, residing at the bottom of the crypts. These cells move along the crypt-villus axis while differentiating into all the epithelial colon lineages, such as Paneth cells, goblet cells, enterocytes and enteroendocrine cells. In approximately 14 days, they arrive at the top of the villus, and undergo apoptosis (
It has been suggested that an oncogenic hit in a stem cell generates cancer stem cells (CSCs), which are tumour-initiating cells that represent the only cell type able to promote cancer onset, progression and metastases. CSCs are probably responsible for both tumour relapse and resistance to therapy (
During tumour progression, epithelial cancer cells undergo EMT. Cells that undergo EMT acquire a mesenchymal phenotype, losing their epithelial features and cell polarity and acquiring motility, stem cell-like properties, self-renewal and apoptosis resistance. These cells are also able to degrade the basement membrane and extracellular matrix. Thus, it has been suggested that EMT confers to cancer cells, including CRC cells, many of the features required to develop metastases (
However, the only crucial cell feature conferring to cancer cells the ability to complete the metastatic process, is cellular plasticity. Cellular plasticity is defined as the ability of cells to switch from an epithelial to a mesenchymal phenotype and
Glycogen synthase kinase 3 β (GSK-3β) is a key regulator of the crosstalk between different signalling pathways involved in CRC development, such as WNT/β-catenin, Ras, PI3K/Akt, cMET and vascular endothelial growth factor (VEGF). Data have also been published regarding the role of GSK-3β in CRC cell drug resistance (
Genomic instability facilitates the accumulation of multiple mutations during CRC development; chromosomal instability (CIN) is observed in 85% and microsatellite instability (MSI) is detected in 15% of sporadic CRCs. The molecular mechanisms underlying CRC progression remain poorly understood, particularly as regards CRCs with MSI (
Herein, we investigated the expression and localisation of key markers of EMT and stemness in CRC cells exhibiting both CIN and MIN by establishing a system of adherent primary mesenchymal colon cancer cells and paired tumourspheres. These cells exhibited plasticity. We also observed an atypical nuclear localisation of N-cadherin, CD133 and the v6 splice form of CD44 glycoprotein (CD44v6) in the majority of the mesenchymal cells, suggesting a change in localisation from the plasma membrane to the nucleus, which could allow cell plasticity in CRC progression. Finally, we demonstrated that GSK-3β inhibition reduced cell migration and cell plasticity in our experimental cell model, thus suggesting that GSK-3β may be a target for CRC therapy.
CRC tissues and normal colorectal mucosa were obtained from patients with sporadic CRC, who were operated at the AOU Federico II and Istituto Nazionale dei Tumori (Naples, Italy) and primary cell cultures were established from these tissues. Data regarding tumour stage were recovered from the medical records of each patient, in accordance with the TNM classifications and tumour budding grades.
Samples from all subjects who participated in this study were collected after obtaining authorisation from the Comitato etico per le attività Biomediche - Carlo Romano of the University of Naples Federico II (protocol no. 432/17). Authorisation was granted only once the study had received ethical approval and written informed consent had been obtained from all participants. All methods were performed in accordance with the relevant guidelines and regulations.
The T88 and T93 cells were previously isolated and stabilized
Cell migration was evaluated using
Boyden chamber assays were performed using the Corning® Transwell® polycarbonate membrane cell culture inserts (pore size, 8.0
Total RNA was extracted from the cells using QIAzol reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Following DNase incubation, cDNA was synthesized from 1
Fractionated nuclear and cytosolic protein extracts were isolated from the T88 and T93 cells using the Qproteome Nuclear Protein kit (Qiagen Hilden, Germany), and quantified by using Traditional Bradford kit (Bio-Rad Laboratories Srl, Segrate, Italy), adopting bovine serum albumin standards. A total of 50
Primary CRC cells were seeded and grown in 12-well cultivation chambers with removable microscopy glass slides (ibidi, Martinsried, Germany); cancer spheroids obtained by the hanging drop assay were also sedi-mented in the same chamber. Immunofluorescence analyses were performed as previously described by Di Maio
We first confirmed, by immunofluorescence analyses, that both the T88 and T93 cells (
Furthermore, to evaluate whether the observed nuclear localisation phenotypes were tumour-specific, we isolated, cultured and analysed cells from the healthy mucosae of a sporadic CRC patient (HM110). Through immunofluorescence staining, we demonstrated that these cultures also expressed both epithelial and mesenchymal proteins (N- and E-cadherin, respectively), but found that they did not exhibit the same nuclear localisation of N-cadherin, CD133 and CD44v6, as was observed in the tumour cells (
It is known that primary colon cultures are able to generate in vitro organoids, which are propagated by intestinal stem cells, and thus, represent an organotypic culture system. Furthermore, it has been demonstrated that it is possible to generate intestinal organoids starting from a single sorted leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)-positive intestinal stem cell from dissociated intestinal crypts (
As GSK-3β is overexpressed and functions as an oncogene in CRC, we examined the effects of GSK-3β inhibition on tumour cells using LiCl, a specific inhibitor. We found that LiCl treatment inhibited cell migration using wound healing and Transwell migration assays. As shown in
We confirmed our previous finding showing that LiCl induces MET in T88 and T93 cell cultures (
Thereafter, we evaluated in both the untreated cells and cells incubated with LiCl, the expression of CD44, its splice isoform CD44v6, CD133 and β-catenin, all proteins playing pivotal roles in CRC development and progression. In accordance with our previous data (
We also analysed the expression of the EMT-associated transcription factor, Snail, and the expression of several stem cell-specific markers, including LGR5, Nanog, aldehyde dehydrogenase 1 (ALDH1), octamer-binding transcription factor 4 (Oct4) and sex determining region Y-box 2 (Sox2), in addition to the aforementioned CD133, CD44 and CD44v6, following LiCl treatment. As shown in
Furthermore, we generated tumourspheres from the T88 and T93 cells by the hanging drop assay, thus establishing a system of adherent primary mesenchymal colon cancer cells and paired tumourspheres, which are useful for studying stemness features, cell plasticity and drug responses. It has been reported that only stem cells and/or stem cell-like cells are able to survive and grow in suspension (
When analysed by confocal microscopy, both the epithelial (Cks and E-cadherin) and mesenchymal (Vimentin and N-cadherin) markers were expressed in the untreated tumourspheres (
We further investigated the molecular basis of the GSK-3β-mediated inhibition of the ability of T88 and T93 cells to generate tumourspheres, by the immunofluorescence analysis of epithelial, mesenchymal and stemness markers, on spheroids obtained in medium containing LiCl. In accordance with above-mentioned findings on adherent cells, we observed a drastic downregulation of all stemness and mesenchymal markers analysed. As shown in
To further investigate the plasticity of the T88 and T93 cells, we disaggregated spheroids obtained by hanging drop assays, grown in medium with or without LiCl, (hereinafter referred to as treated and untreated tumourspheres) and cultivated them in adhesion, to examine the ability of the tumourspheres to re-adhere to a matrix and grow in adherent culture. As shown in
In both cell cultures, and under both experimental conditions, we observed the nuclear localisation of E-cadherin, N-cadherin, CD133 and CD44v6 (
EMT and its reverting process, MET, are physiological processes occurring during embryonic development and tissue remodelling that confer plasticity to cancer cells. It has been suggested that EMT and cell plasticity may be responsible for the acquisition of chemotherapeutic resistance and metastasis development in several tumours, including CRCs (
We previously isolated and characterized at a molecular level two primary CRC cell cultures from the tumour tissues of patients 88 and 93 of our bio-bank (the T88 and T93 cultures). As previously described, the T93 cells exhibited a CIN phenotype, while the T88 cells exhibited a MIN one, with high MSI. We demonstrated that the T88 and T93 cells were mesenchymal colon cancer cells that had undergone EMT from epithelial adenocarcinoma cells and simultaneously expressed epithelial (Cks and E-cadherin) and mesenchymal (Vimentin and N-cadherin) markers. High levels of EMT-associated transcription factors (Twist and Snail) and several stemness markers were also found (
In this study, we characterised our experimental system of adherent primary mesenchymal colon cancer cells and their paired tumourspheres more in depth, by analysing the localisation and expression of a larger panel of markers, including E- and N-cadherin, CD133, CD144v6, ALDH1 and LGR5. Furthermore, we explored the effects of LiCl on cell motility and cell plasticity of CRC cell cultures.
Thus, we confirmed the epithelial/mesenchymal features of these cells and demonstrated that they were characterised by the nuclear localisation of several stemness markers, including Nanog, Oct4, Sox2, LGR5, ALDH1, CD133 and CD44v6.
Of note, we observed atypical nuclear N-cadherin, CD133 and Cd44v6 localisation in mesenchymal CRC cells. N-cadherin is a crucial protein during EMT, cancer progression and invasion, and an elevated N-cadherin expression is often associated with a poor prognosis (
Data regarding nuclear CD133 localisation are rarely described. To the best of our knowledge, there are only three studies that have reported nuclear CD133 localisation, one in triple-negative breast cancer (
CD44 is a plasma membrane glycoprotein that is a receptor for hyaluronan and many other extracellular matrix components. Thus, it transduces signals to membrane-associated cytoskeletal proteins or to the nucleus, to regulate the expression of genes related to cell-matrix adhesion, cell migration, proliferation, differentiation and survival. Previous data have indicated that CD44, particularly CD44v isoforms, are specific CSC markers. CD44 proteins integrate environmental and cellular signalling to regulate cancer stemness, they are also involved in EMT regulation (
Several cell surface proteins are known to migrate into the nucleus as intact polypeptides or proteolytic fragments, where they function as transcription factors. It was previously described that the canonical isoform of CD44 is imported into the nucleus through the nuclear pore complex, thus promoting cell proliferation (
GSK-3β is a multitasking serine-threonine kinase that can function as a pro-apoptotic or anti-apoptotic factor (
We have also observed that LiCl affects stemness features, abolishing the expression of all mesenchymal and stemness markers, thus altering the dynamics of tumoursphere formation and cell plasticity. As previously described (
Cellular plasticity plays an important role in cancer development and progression; only cancer cells that are able to switch from epithelial to mesenchymal phenotypes and
Finally, we observed that when cells were disaggregated from tumourspheres performed in LiCl, only the T93 cells, which exhibited a microsatellite stable (MSS) and CIN phenotype, were able to re-grow, while the T88 cells, which exhibited an MSI-high phenotype, appeared quiescent. This observation is in agreement with findings in the literature, which indicates a less aggressive phenotype of MSI CRC compared with MSS subtypes (
In conclusion, in this study, we established a system of adherent primary mesenchymal colon cancer cells and paired tumourspheres, which are useful for studying the mechanisms underlying CRC progression and drug response. In light of our finding, we suggest that LiCl, a specific GSK-3β inhibitor, could represent a drug candidate and that GSK-3β inhibition may be a promising direction for future cancer therapy that needs to be better elucidated. As recently demonstrated for other cancer types, such as glioma (
epithelial-to-mesenchymal transition
mesenchymal-to-epithelial transition
colorectal cancer
chromosomal instability
microsatellite instability
healthy colon mucosa
The authors would like to thank Dr James P. Mahaffey from Edanz Group (
This study was supported by: Regione Campania, LR5/2002-2014; 'Fondo Straordinario di Ateneo-2017-Università di Napoli Federico II'.
All data generated or analysed during this study are included in this published article. No datasets were generated or analysed during the current study.
MDR and PI designed the study; MDR, MT, VC and AC, performed research; PD, DR, UP, CAD and MM provided sample collection and clinical support; MDR, PI and MT contributed to data interpretation. MDR and MT wrote the manuscript, and FD and RL critically revised the manuscript and participated in the analysis and interpretation of the data. All authors reviewed, edited and approved the final version of the manuscript.
Samples from all subjects who participated in this study were collected after obtaining authorisation from the Comitato etico per le attività Biomediche - Carlo Romano of the University of Naples Federico II (protocol no. 432/17). Authorisation was granted only once the study had received ethical approval and written informed consent had been obtained from all participants. All methods were performed in accordance with the relevant guidelines and regulations.
Not applicable.
The authors declare that they have no competing interests.
Nuclear N-cadherin, CD133 and CD44v6 localisation in T88 and T93 mesenchymal colorectal cancer (CRC) cells. (A) Brightfield images of T88 and T93 cells at ×10 magnification. (B) Confocal images of T88 and T93 primary colon cancer cells stained for anti-Vimentin (red)/anti-pan-CK (green), anti-N-cadherin (red)/anti-E-cadherin (green) and anti-CD133 (red)/anti-CD44v6 (green) antibodies. Nuclei were counterstained with DAPI (blue). (C) Confocal images of HT29 and RKO cells stained with anti-E-cadherin (green) antibody. Nuclei were counterstained with DAPI (blue). (D) Western blot analysis of cytoplasmic 'C'' and nuclear 'N' extracts from T88 and T93 cells using anti-CD133, anti-N-cadherin, anti-Cd44v6, anti-actin and anti-histone H1 antibodies. Histone H1 and actin were used as nuclear and cytoplasmic marker proteins, respectively. Each lane was loaded with 50
Primary epithelial-mesenchymal colon cancer cells generate
LiCl inhibits the migration of epithelial-mesenchymal colon cancer cells. (A) Brightfield images at ×4 magnification of T88 and T93 cells during wound healing assays performed with cells incubated (LiCl) or not incubated (NT) with LiCl. Wound closures were imaged immediately after scratching (0 h) and 24, 48 and 72 h later. (B) Transwell migration assay. Brightfield images of crystal violet staining are shown on the left, while on the right are shown epifluorescence images of nuclei stained with DAPI (blue). In both images, magnification was ×4.
Pan-cytokeratin, Vimentin, E-cadherin and N-cadherin expression following LiCl treatment in adherent 2D T88 and T93 primary cell cultures. Confocal images of untreated T88 and T93 primary colon cancer cells and cells after 10 days (10 d) of LiCl incubation. Cells were dual-stained with anti-Vimentin (red)/anti-pan-CK (green), anti-N-cadherin (red)/anti-E-cadherin (green) and anti-CD133 (red)/anti-CD44v6 (green) antibodies. Nuclei were counterstained with DAPI (blue).
Effect of LiCl on the expression of β-catenin, CD44, CD133 and CD44v6 proteins in adherent 2D T88 and T93 primary cell cultures. Confocal images of untreated T88 and T93 primary colon cancer cells and cells after 10 days (10 d) of LiCl incubation. Cells were dual-stained with anti-β-catenin (red)/anti-CD44 (green) and anti-CD133 (red)/anti-CD44v6 (green) antibodies. Nuclei were counterstained with DAPI (blue).
Effect of LiCl on the expression of Nanog, ALDH1, Snail, LGR5 and Oct4 proteins, in adherent 2D T88 and T93 primary cell cultures. Confocal images of untreated T88 and T93 primary colon cancer cells and cells after 10 days (10 d) of LiCl incubation. Cells were dual-stained with anti-Nanog (red)/anti-ALDH1 (green), anti-Snail (red)/anti-LGR5 (green) and anti-Oct4 (red)/anti-Sox2 (green) antibodies. Nuclei were counterstained with DAPI (blue). Nanog (yellow arrows) exhibited a higher nuclear expression in T88 cells than in T93 cells, while Oct4 (white arrows) exhibited an opposite trend.
LiCl inhibits tumoursphere formation. (A) Brightfield images at ×4 and ×20 magnification of hanging drop assays on T88 (images on left panels) and T93 (images on right panels) cells obtained in medium containing (LiCl) or not containing (untreated) LiCl. (B) Confocal images of the tumourspheres dual-stained with anti-vimentin (red)/anti-pan-CK (green) and anti-N-cadherin (red)/anti-E-cadherin (green) antibodies. Nuclei were counterstained with DAPI (blue).
LiCl downregulates the stem cell markers, CD133 and CD44v6, in tumourspheres. Confocal images of the tumourspheres obtained by the hanging drop assays. Tumourspheres were dual-stained with anti-CD133 (red)/anti-CD44v6 (green) and anti-β-catenin (red)/anti-CD44 (canonical isoform; green) antibodies. Nuclei were counterstained with DAPI (blue).
LiCl downregulates EMT-associated transcription factors and stem cell markers in tumourspheres. Confocal images of the tumourspheres obtained by hanging drop assays. Tumourspheres were dual-stained with anti-Snail (red)/anti-LGR5 (green), anti-Nanog (red)/anti-ALDH1 (green), and anti-Oct4 (red)/anti-Sox2 (green) antibodies. Nuclei were counterstained with DAPI (blue).
LiCl inhibits cell plasticity. Molecular characterisation of survived cells. (A) Brightfield images (×10 magnification) of cells disaggregated from T88 and T93 spheroids obtained in medium containing (LiCl) or not containing (untreated) LiCl. (B) Confocal images of cells dual-stained with anti-N-cadherin (red)/anti-E-cadherin (green) and anti-CD133 (red)/anti-CD44v6 (green) antibodies Nuclei were counterstained with DAPI (blue). Magnification, ×20. (C) Real-time quantification of Snail and Twist1 mRNA expression in T88 and T93 adherent cells before tumoursphere formation (1st step), cells disaggregated from untreated tumourspheres (drop cells) and cells disaggregated from treated tumourspheres (drop cells in LiCl). This experiment was performed two times with similar results. A typical experiment is shown. Numbers on the top of each of the bars represent the precise value of relative expression for each sample, as calculated by the ΔΔCq method.
Expression of Vimentin, Pan-cytokeratin, β-catenin and CD44 in cells disaggregated from T88 and T93 spheroids. Confocal images of cells disaggregated from T88 and T93 spheroids obtained in medium containing (LiCl) or not containing (untreated) LiCl. Cells were dual-stained with anti-Vimentin (red)/anti-Pan-cytokeratin (green) and anti-β-catenin (red)/anti-CD44 (green) antibodies. Nuclei were counterstained with DAPI (blue).
Expression of Nanog, ALDH1, Snail, LGR5, Oct4 and Sox-2 in cells disaggregated from T88 and T93 spheroids. Confocal images of cells disaggregated from T88 and T93 spheroids obtained in medium containing (LiCl) or not containing (untreated) LiCl. Cells were dual-stained with anti-Nanog (red)/anti-ALDH1 (green), anti-Snail (red)/anti-LGR5 (green) and anti-Oct4 (red)/anti-Sox-2 (green) antibodies. Nuclei were counterstained with DAPI (blue).
Antibodies and dilutions used for immunofluorescence staining.
Antibody: Dilution (Cat. no., provider) | Antibody: Dilution (Cat. no., provider) |
---|---|
Pan-cytokeratin (CK): 1:50 (MA5-13203, Invitrogen, Thermo Fisher Scientific) | E-cadherin: 1:50 (ab76055, Abcam) |
Nanog: 1:50 (3580, Cell Signaling Technology) | N-cadherin: 1:50 (ab76057, Abcam) |
Sox2: 1:50 (4900, Cell Signaling Technology) | Snail: 1:150 (ab180714, Abcam) |
Oct4: 1:50 (2840, Cell Signaling Technology) | LGR5: 1:50 (sc135238, Santa Cruz Biotechnology) |
Vimentin: 1:200 (5741, Cell Signaling Technology) | ALDH1: 1:50 (ab24343, Abcam) |
CD44: 1:500 (5640, Cell Signaling Technology) | CD133: 1:50 (ab16518, Abcam) |
β-catenin: 1:100 (9581, Cell Signaling Technology) | CD44v6: 1:50 (BBA13, R&D Systems) |
Sox2, sex determining region Y-box 2; Oct4, octamer-binding transcription factor 4; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; ALDH1, aldehyde dehydrogenase 1.