Cancer stem cells impact heterogeneity and uncertainty pdf

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cancer stem cells impact heterogeneity and uncertainty pdf

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It has been proposed that a subpopulation of tumour cells with stem cell-like characteristics, known as cancer stem cells CSCs , drives tumour initiation and generates tumour heterogeneity, thus leading to cancer metastasis, recurrence, and drug resistance. Although there has been substantial progress in CSC research into many solid tumour types, an understanding of the biology of CSCs in lung cancer remains elusive, mainly because of their heterogeneous origins and high plasticity. Here, we demonstrate that engineered lung cancer cells derived from normal human airway basal epithelial cells possessed CSC-like characteristics in terms of multilineage differentiation potential and strong tumour-initiating ability.

Cancer stem cells in the development of liver cancer

It has been proposed that a subpopulation of tumour cells with stem cell-like characteristics, known as cancer stem cells CSCs , drives tumour initiation and generates tumour heterogeneity, thus leading to cancer metastasis, recurrence, and drug resistance.

Although there has been substantial progress in CSC research into many solid tumour types, an understanding of the biology of CSCs in lung cancer remains elusive, mainly because of their heterogeneous origins and high plasticity.

Here, we demonstrate that engineered lung cancer cells derived from normal human airway basal epithelial cells possessed CSC-like characteristics in terms of multilineage differentiation potential and strong tumour-initiating ability. Moreover, we established an in vitro 3D culture system that allowed the in vivo differentiation process of the CSC-like cells to be recapitulated.

This engineered CSC model provides valuable opportunities for studying the biology of CSCs and for exploring and evaluating novel therapeutic approaches and targets in lung CSCs. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: Our institute is the subsidiary of Eisai Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The patent number is WO There are no further patents, products in development, or marketed products to declare.

Lung cancer is the leading cause of cancer-related mortality, resulting in more than one million deaths worldwide annually [ 1 ]. Even in operable cases, the rate of recurrence is high, and overall 5-year survival rates for NSCLC remain low despite advances in early detection and standard treatment [ 3 ].

As in many other cancers, phenotypic and functional heterogeneity among cancer cells within the tumour make curing lung cancer difficult [ 4 ]. It is now widely accepted that this heterogeneity is generated mainly by cancer stem cells CSCs , which have both tumour-initiating ability and differentiation potential [ 5 , 6 ].

However, controversies and uncertainties remain, and no consensus markers for lung CSCs have yet been identified [ 7 ]. This is most likely due to the heterogeneous origins and high plasticity of lung CSCs [ 8 , 9 ]. Previously, we engineered tumorigenic cells from normal human small airway epithelial cells HSAECs via combined expression of multiple defined genetic elements, all of which are known to be highly relevant to lung cancer development.

We used different oncogene combinations to generate various types of tumorigenic cells with different histological features and that exhibited varying degrees of differentiation on subcutaneous transplantation into nude mice [ 10 ]. Each well was checked with an inverted microscope several hours after plating, and wells containing a single cell were marked. The colonies in these marked wells were expanded. The immunostained spheres were analysed using a Nikon A1 confocal microscopy system Nikon, Tokyo, Japan.

In order to minimize any suffering during surgical procedures, animals were anesthetized by isoflurane. Tumors were harvested from specified mice. No mortality occurred prior to the end of the study. In all animal studies, food and water were available ad libitum. Formalin-fixed, paraffin-embedded xenograft tissues were cut into 4-um sections, and the sections were stained with haematoxylin and eosin, and alcian blue, according to standard protocols. Immunohistochemistry was conducted as described previously [ 12 ] using the antibodies listed in S1 Table.

The embedded tissues were sectioned using a cryostat Leica Biosystems, Wetzlar, Germany. On subcutaneous transplantation into nude mice, these cells formed tumours that showed adenocarcinoma-like histology: they exhibited glandular structures with mucin production, revealed by alcian blue staining Fig 1A and 1B.

These glandular structures consisted of heterogeneous cell types in terms of expression of cytokeratins Fig 1C and the basal cell marker p63 Fig 1D ; and were surrounded by stromal cells that stained positive for alpha-SMA and negative for p53, and were therefore supposed to be of mouse origin Fig 1E. Given that ppositive basal cells have been identified as stem cells in mouse trachea and human airway epithelium [ 14 ], we hypothesized that the heterogeneous tumour histology was generated via differentiation of these ppositive cells.

When subcutaneously transplanted into nude mice, six out of seven of these clones formed tumours with adenocarcinoma-like histology containing heterogeneous cell types as did the parental cells, with one exceptional case forming poorly differentiated tumor. Results of histological examination of three representative clones were shown in Fig 2. Immunofluorescence analysis of the xenograft tumour derived from one of these clones revealed cells expressing the Clara cell marker SCGB1A1, and cells expressing the goblet cell marker MUC5AC [ 15 ], in addition to ppositive basal cells in the tumour tissue Fig 3A and 3B.

Given that both the Clara and goblet cell linages are reported to originate from basal cells [ 14 ], these results clearly indicate that the heterogeneity observed in the xenograft tumour tissue resulted from differentiation of single—cell-derived clones with stem cell characteristics. To gain more direct evidence for the differentiation of ppositive cells, we attempted to establish an in vitro culture system in which the in vivo differentiation process could be recapitulated.

To this end, we used a previously reported 3D culture developed for the functional assay of stem cells in the adult mouse lung [ 11 ]. Immunostaining these spheres with antibodies against keratin 5 K5 and keratin 8 K8 revealed that the peripheral ppositive cells were also positive for K5, as reported previously, whereas the pnegative cells in the central region were positive for K8 Fig 3C.

This staining pattern was consistent with that of the in vivo xenografts Fig 3A. To our knowledge, this is the first report of generation of cells possessing CSC-like characteristics from normal human lung epithelial cells. A number of studies have previously reported the isolation and characterisation of lung CSCs from both established cell lines and clinical specimens [ 7 ]. For example, Meng et al. Such conflicting results are likely attributable to the heterogeneous origins and high plasticity of lung CSCs [ 8 , 9 ].

Considering the inconsistencies in the markers identified for lung CSCs, it is preferable to define CSCs based on their biological functions. HSAECs, which we used as the starting cell type in this study, are thought to be distal airway basal cells with intrinsic multipotent differentiation capacity [ 24 , 25 ]. By introducing several defined genetic elements hTERT, Cdk4, a dominant negative p53 mutant, K-rasV12, and cyclin D1 , we conferred the HSAECs with immortality and tumourigenicity without removing their capacity to function as multipotent progenitors.

Airway basal cells are generally thought to be the cellular origin of lung squamous cell carcinoma, whereas AT2 cells are the cellular origin of lung adenocarcinoma [ 26 , 27 ]. More recently, it has been reported that not only AT2 cells, but also Clara cells and bronchioalveolar stem cells, can be the cellular origin of lung adenocarcinoma, depending on conditions, in several genetically engineered mouse models [ 28 — 30 ].

Malkoski et al. It is now increasingly recognized that basal cells exhibit increased plasticity and multipotency in response to injury, and play pivotal roles as stem cells in the process of tissue regeneration [ 32 ], and therefore are at high risk for oncogenic transformation.

Moreover, through the analysis of three independent data sets of clinical samples, Fukui et al. Although it seems ideal to use CSCs isolated from clinical samples to study CSC biology, there are several problems with this approach.

First, as mentioned previously, no definitive markers of lung CSCs have yet been identified, and therefore it is currently technically very challenging to purify CSCs from clinical lung cancer samples [ 7 ]. Second, clinical tumour samples harbour many genetic changes, with high variability between patients.

Sequencing data from cancer genome projects has revealed approximately mutations per tumour on average in lung cancer [ 34 ], and the combinations of mutations differ between patients, resulting in huge diversity. Thus, it is difficult to identify common molecular mechanisms underlying the nature of CSCs against a background of such extensive variation. It is therefore a very powerful approach to engineer surrogate CSC models that can recapitulate CSC biology with the minimum of essential genetic changes necessary to confer CSC properties on normal cells.

As we previously reported, the combination of the genetic changes used in the current study is minimally sufficient to induce tumorigenic transformation in HSAECs [ 10 ], and all of which are well recognized to have considerable clinical relevance. Despite the lack of reports characterizing the human lung cancer stem cells in relation to the mutated oncogenes so far, it has been demonstrated in genetically engineered mouse lung adenocarcinoma model that the biological properties of cancer stem cells differ significantly depending on the driver oncogenes [ 37 ].

The engineered CSC model described in this study represents a valuable experimental system for the study of basic CSC biology of KRAS -mutated lung adenocarcinoma, including the molecular mechanisms governing self-renewal and differentiation; and also should greatly contribute to the discovery and evaluation of novel therapeutic approaches and targets in CSCs of this intractable lung cancer. Antibodies used in this study are listed with the clone names if applicable , suppliers and dilution conditions.

Conceptualization: KS TA. Project administration: KS TA. Supervision: KS TA. Writing — original draft: KS TA. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract It has been proposed that a subpopulation of tumour cells with stem cell-like characteristics, known as cancer stem cells CSCs , drives tumour initiation and generates tumour heterogeneity, thus leading to cancer metastasis, recurrence, and drug resistance.

Introduction Lung cancer is the leading cause of cancer-related mortality, resulting in more than one million deaths worldwide annually [ 1 ]. Histological analysis and immunohistochemistry Formalin-fixed, paraffin-embedded xenograft tissues were cut into 4-um sections, and the sections were stained with haematoxylin and eosin, and alcian blue, according to standard protocols. Download: PPT. Fig 2. Fig 3. Expression of differentiation markers in both in vivo xenografts and in vitro differentiation cultures.

Fig 4. Expression of p63 in in vitro differentiation cultures. Discussion To our knowledge, this is the first report of generation of cells possessing CSC-like characteristics from normal human lung epithelial cells.

Supporting information. S1 Table. Antibody information. References 1. Global cancer statistics. CA: a cancer journal for clinicians. View Article Google Scholar 2. Lung cancer. N Engl J Med. Cancer statistics, View Article Google Scholar 4. Lung cancer stem cells: tools and targets to fight lung cancer. Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer cell. Kreso A, Dick JE. Evolution of the cancer stem cell model. Cell stem cell. Cancer stem cells in lung cancer: Evidence and controversies.

Cancer stem cells: progress and challenges in lung cancer. Stem cell investigation. Lung cancer stem cells-characteristics, phenotype.

A novel model of liver cancer stem cells developed from induced pluripotent stem cells

Review Series Free access Phone: Find articles by Yamashita, T. Find articles by Wang, X. Published May 1, - More info. Liver cancer is an aggressive disease with a poor outcome.


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Cancer stem cells: impact, heterogeneity, and uncertainty.

The cancer stem cell CSC hypothesis has been disproved in many cancers. Hepatocytes gave rise to HCC. Several groups have demonstrated that oval cells or liver progenitor cells give rise to TICs. The definition of CSCs includes pluripotency, while TICs do not have to have pluripotency and only need to have bi- or multipotential to give rise to diverse tumor types and tumor initiation potential in mouse models.

Cancer stem cells CSCs are subpopulations of cells with stem cell characteristics that produce both cancerous and non-tumorigenic cells in tumor tissues. The literature reports that CSCs are closely related to the development of hepatocellular carcinoma HCC and promote the malignant features of HCC such as high invasion, drug resistance, easy recurrence, easy metastasis, and poor prognosis. This review discusses the origin, molecular, and biological features, functions, and applications of CSCs in HCC in recent years; the goal is to clarify the importance of CSCs in treatment and explore their potential value in HCC-targeted therapy. CSCs have the capacity to self-renew and differentiate into heterogeneous tumor cells, which are responsible for the maintenance and propagation of the tumor Batlle and Clevers, Basing on this breakthrough, CSCs were subsequently found in a variety of hematopoietic cancer and solid tumors.

Tissues with defined cellular hierarchies in development and homeostasis give rise to tumors with cellular hierarchies, suggesting that tumors recapitulate specific tissues and mimic their origins. Glioblastoma GBM is the most prevalent and malignant primary brain tumor and contains self-renewing, tumorigenic cancer stem cells CSCs that contribute to tumor initiation and therapeutic resistance. As normal stem and progenitor cells participate in tissue development and repair, these developmental programs re-emerge in CSCs to support the development and progressive growth of tumors.

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