Airway organoids as models of human disease.

Studies developing and applying organoid technology have greatly increased in volume and visibility over the past decade. Organoids are three-dimensional structures that are established from pluripotent stem cells (PSCs) or adult tissue stem cells (ASCs). They consist of organ-specific cell types that self-organize through cell sorting and spatially restricted lineage commitment to generate architectural and functional characteristics of the tissue of interest. The field of respiratory development and disease has been particularly productive in this regard. Starting from human cells (PSCs or ASCs), models of the two segments of the lung, the airways and the alveoli, can be built. Such organoids allow the study of development, physiology and disease and thus bridge the gap between animal models and clinical studies. This review discusses current developments in the pulmonary organoid field, highlighting the potential and limitations of current models.

Comparative proteomics of colon cancer stem cells and differentiated tumor cells identifies BIRC6 as a potential therapeutic target.

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that "cell death " regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.

APC, signal transduction and genetic instability in colorectal cancer.

Colorectal cancer arises through a gradual series of histological changes, each of which is accompanied by a specific genetic alteration. In general, an intestinal cell needs to comply with two essential requirements to develop into a cancer: it must acquire selective advantage to allow for the initial clonal expansion, and genetic instability to allow for multiple hits in other genes that are responsible for tumour progression and malignant transformation. Inactivation of APC--the gene responsible for most cases of colorectal cancer--might fulfil both requirements.

Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.

Mutations of the genes encoding APC or beta-catenin in colon carcinoma induce the constitutive formation of nuclear beta-catenin/Tcf-4 complexes, resulting in activated transcription of Tcf target genes. To study the physiological role of Tcf-4 (which is encoded by the Tcf7/2 gene), we disrupted Tcf7/2 by homologous recombination. Tcf7/2-/- mice die shortly after birth. A single histopathological abnormality was observed. An apparently normal transition of intestinal endoderm into epithelium occurred at approximately embryonic day (E) 14.5. However, no proliferative compartments were maintained in the prospective crypt regions between the villi. As a consequence, the neonatal epithelium was composed entirely of differentiated, non-dividing villus cells. We conclude that the genetic program controlled by Tcf-4 maintains the crypt stem cells of the small intestine. The constitutive activity of Tcf-4 in APC-deficient human epithelial cells may contribute to their malignant transformation by maintaining stem-cell characteristics.

Directed Differentiation of Murine and Human Small Intestinal Organoids Toward All Mature Lineages.

Intestinal organoids are three-dimensional structures derived from tissue-resident adult stem cells. These organoids recapitulate key aspects of epithelial biology and can be used to study homeostatic turnover of the corresponding tissue. Organoids can be enriched for the various mature lineages which allows studies of the respective differentiation processes and of the diverse cellular functions. Here we describe mechanisms of intestinal fate specification and how these can be exploited to drive mouse and human small intestinal organoids into each of the functionally mature lineages.

Mutations in the APC tumour suppressor gene cause chromosomal instability.

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.

The drug resistance-related protein LRP is the human major vault protein.

Multidrug-resistant cancer cells frequently overexpress the 110-kD LRP protein (originally named Lung Resistance-related Protein). LRP overexpression has been found to predict a poor response to chemotherapy in acute myeloid leukaemia and ovarian carcinoma. We describe the cloning and chromosome localization of the gene coding for this novel protein. The deduced LRP amino acid sequence shows 87.7% identity with the 104-kD rat major vault protein. Vaults are multi-subunit structures that may be involved in nucleo-cytoplasmic transport. The LRP gene is located on chromosome 16, close to the genes coding for multidrug resistance-associated protein and protein kinase C-beta, and may mediate drug resistance, perhaps via a transport process.

Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.

Previously, hepatic iron overload resembling that in hereditary hemachromatosis (HH) has been found in beta 2-microglobulin knockout (beta 2m-/-) mice. We have now characterized iron metabolism in beta 2m-/- mice. The mutant mice fail to limit the transfer of iron from mucosal cells into the plasma. Transferrin saturation is abnormally high. Pathologic iron depositions occur predominantly in liver parenchymal cells. Reconstitution with normal hematopoietic cells redistributes the iron from parenchymal to Kupffer cells, but does not correct the mucosal defect. We conclude that (a) iron metabolism is defective in the gut mucosa as well as the liver of beta 2m-/- mice; and (b) a beta 2m-dependent gene product is involved in iron homeostasis. Recently, a novel gene of the major histocompatibility complex class I family, HLA-H, has been found to be mutated in a large proportion of HH patients. Our data provide functional support for the proposed causative role of HLA-H mutations in HH.

Cloning of murine TCF-1, a T cell-specific transcription factor interacting with functional motifs in the CD3-epsilon and T cell receptor alpha enhancers.

CD3-epsilon gene expression is confined to the T cell lineage. We have recently identified and cloned a human transcription factor, TCF-1, that binds to a functional element in the T lymphocyte-specific enhancer of CD3-epsilon. In a panel of human cell lines, TCF-1 expression was restricted to T lineage cells. TCF-1 belonged to a novel family of genes that contain the so-called high mobility group 1 (HMG) box. Here we report the cloning of murine TCF-1. Two splice alternatives were identified that were not previously observed in human TCF-1. Murine and human TCF-1 displayed a 95.5% overall amino acid homology. Recombinant murine and human TCF-1 recognized the same sequence motif in the CD3-epsilon enhancer as judged by gel retardation and methylation interference assays. With the murine cDNA clones several aspects of TCF-1 were analyzed. First, deletion analysis revealed that a region of TCF-1 containing the HMG box was sufficient for sequence-specific binding. Second, by high stringency Northern blotting and in situ hybridization, TCF-1 expression was shown to be confined to the thymus and to the T cell areas of the spleen. Third, TCF-1 bound specifically to a functional T cell-specific element in the T cell receptor alpha (TCR-alpha) enhancer. The T lineage-specific expression and the affinity for functional motifs in the TCR-alpha and CD3-epsilon enhancers imply an important role for TCF-1 in the establishment of the mature T cell phenotype.

The epithelial cellular adhesion molecule (Ep-CAM) is a ligand for the leukocyte-associated immunoglobulin-like receptor (LAIR).

Human leukocyte-associated immunoglobulin-like receptor (LAIR)-1 is expressed on many cells of the immune system and is predicted to mediate inhibitory functions based on the presence of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic domain. Although the role of LAIR-1 in the regulation of immune responses in vivo is unknown, LAIR-1 cross-linking by monoclonal antibody inhibits various immune cell functions in vitro. Here, we identify the colon carcinoma-associated epithelial cellular adhesion molecule (Ep-CAM) as a ligand for LAIR-1 and LAIR-2, a related soluble LAIR-1 family member. Ep-CAM interacts with the LAIR molecules through its first epidermal growth factor domain; Ep-CAM--specific antibodies can abrogate the binding. Intraepithelial T lymphocytes express LAIR-1 and thus may interact with Ep-CAM present on human intestinal epithelium. We propose that LAIR-1--Ep-CAM interaction may contribute to mucosal tolerance and that LAIR-2 possibly modulates this function.

Correction: Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia.

[This corrects the article DOI: 10.1371/journal.pone.0231588.].

Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia.

We have recently established a protocol to grow wildtype human oral mucosa organoids. These three-dimensional structures can be maintained in culture long-term, do not require immortalization, and recapitulate the multilayered composition of the epithelial lining of the oral mucosa. Here, we validate the use of this model to study the effect of Leucovorin (LV) on Methotrexate (MTX)-induced toxicity. MTX is a chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia. Although effective, the use of MTX often results in severe side-effects, including oral mucositis, which is characterized by epithelial cell death. Here, we show that organoids are sensitive to MTX, and that the addition of LV reduces MTX toxicity, in both a concentration- and timing-dependent manner. Additionally, we show that a 24 hour 'pretreatment' with LV reduces MTX-induced cell death, suggesting that such a pretreatment could decrease mucositis in patients. Taken together, we provide the first in vitro model to study the effect of MTX on wildtype oral mucosa cells. Our findings underscore the relevance of the clinically applied LV regimen and highlight the potential of this model to further optimize modifications in dosing and timing of Leucovorin on oral mucosa cells.

Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma.

The adenomatous polyposis coli (APC) tumor suppressor protein binds to beta-catenin, a protein recently shown to interact with Tcf and Lef transcription factors. The gene encoding hTcf-4, a Tcf family member that is expressed in colonic epithelium, was cloned and characterized. hTcf-4 transactivates transcription only when associated with beta-catenin. Nuclei of APC-/- colon carcinoma cells were found to contain a stable beta-catenin-hTcf-4 complex that was constitutively active, as measured by transcription of a Tcf reporter gene. Reintroduction of APC removed beta-catenin from hTcf-4 and abrogated the transcriptional transactivation. Constitutive transcription of Tcf target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium.

Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by beta-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of beta-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of beta-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or beta-catenin.

Synergy between tumor suppressor APC and the beta-catenin-Tcf4 target Tcf1.

Mutations in APC or beta-catenin inappropriately activate the transcription factor Tcf4, thereby transforming intestinal epithelial cells. Here it is shown that one of the target genes of Tcf4 in epithelial cells is Tcf1. The most abundant Tcf1 isoforms lack a beta-catenin interaction domain. Tcf1(-/-) mice develop adenomas in the gut and mammary glands. Introduction of a mutant APC allele into these mice substantially increases the number of these adenomas. Tcf1 may act as a feedback repressor of beta-catenin-Tcf4 target genes and thus may cooperate with APC to suppress malignant transformation of epithelial cells.

Dysfunctional AMPK activity, signalling through mTOR and survival in response to energetic stress in LKB1-deficient lung cancer.

LKB1, mutated in Peutz-Jeghers and in sporadic lung tumours, phosphorylates a group of protein kinases named AMP-activated protein kinase (AMPK)-related kinases. Among them is included the AMPK, a sensor of cellular energy status. To investigate the relevance of LKB1 in lung carcinogenesis, we study several lung cancer cells with and without LKB1-inactivating mutations. We report that LKB1-mutant cells are deficient for AMPK activity and refractory to mTOR inhibition upon glucose depletion but not growth-factor deprivation. The requirement for wild-type LKB1 to properly activate AMPK is further demonstrated in genetically modified cancer cells. In addition, LKB1-deficient lung primary tumours had diminished AMPK activity, assessed by complete absence or low level of phosphorylation of its critical substrate, acetyl-CoA carboxylase. We also demonstrate that LKB1 wild-type cells are more resistant to cell death upon glucose withdrawal than their mutant counterparts. Finally, modulation of AMPK activity did not affect PI3K/AKT signalling, an advantage for the potential use of AMPK as a target for cancer therapy in LKB1 wild-type tumours. Thus, sustained abrogation of cell energetic checkpoint control, through alterations at key genes, appear to be an obligatory step in the development of some lung tumours.

Interplay between VHL/HIF1alpha and Wnt/beta-catenin pathways during colorectal tumorigenesis.

Activation of the Wnt signaling pathway initiates the transformation of colorectal epithelial cells, although the transition to metastatic cancer requires angiogenesis. We have investigated the expression of the von Hippel-Lindau (VHL) tumor suppressor in the intestines from humans and mice. Here, we show that VHL expression is regulated by TCF4 and is restricted to the proliferative compartment at the bottom of intestinal crypts. Accordingly, VHL is completely absent from the proliferative intestinal pockets of Tcf4(-/-) perinatal mice. We observed complementary staining of the hypoxia-inducible factor (HIF) 1alpha to VHL in normal intestinal epithelium as well as in all stages of colorectal cancer (CRC). To the best of our knowledge, this is the first report demonstrating the presence of nuclear HIF1alpha in normoxic healthy adult tissue. Although we observed upregulated levels of VHL in very early CRC lesions from sporadic and familial adenomatous polyposis patients - presumably due to activated Wnt signaling - a clear reduction of VHL expression is observed in later stages of CRC progression, coinciding with stabilization of HIF1alpha. As loss of VHL in later stages of CRC progression results in stabilization of HIF, these data provide evidence that selection for VHL downregulation provides a proangiogenic impulse for CRC progression.