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.

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.

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.

Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumorigenesis in murine mammary epithelium.

Aberrant Wnt signaling within breast cancer is associated with poor prognosis, but regulation of this pathway in breast tissue remains poorly understood and the consequences of immediate or long-term dysregulation remain elusive. The exact contribution of the Wnt-regulating proteins adenomatous polyposis coli (APC) and APC2 in the pathogenesis of human breast cancer are ill-defined, but our analysis of publically available array data sets indicates that tumors with concomitant low expression of both proteins occurs more frequently in the 'triple negative' phenotype, which is a subtype of breast cancer with particularly poor prognosis. We have used mouse transgenics to delete Apc and/or Apc2 from mouse mammary epithelium to elucidate the significance of these proteins in mammary homeostasis and delineate their influences on Wnt signaling and tumorigenesis. Loss of either protein alone failed to affect Wnt signaling levels or tissue homeostasis. Strikingly, concomitant loss led to local disruption of ß-catenin status, disruption in epithelial integrity, cohesion and polarity, increased cell division and a distinctive form of ductal hyperplasia with 'squamoid' ghost cell nodules in young animals. Upon aging, the development of Wnt activated mammary carcinomas with squamous differentiation was accompanied by a significantly reduced survival. This novel Wnt-driven mammary tumor model highlights the importance of functional redundancies existing between the Apc proteins both in normal homeostasis and in tumorigenesis.

Stem cell CD44v isoforms promote intestinal cancer formation in Apc(min) mice downstream of Wnt signaling.

A gene signature specific for intestinal stem cells (ISCs) has recently been shown to predict relapse in colorectal cancer (CRC) but the tumorigenic role of individual signature genes remains poorly defined. A prominent ISC-signature gene is the cancer stem cell marker CD44, which encodes various splice variants comprising a diverse repertoire of adhesion and signaling molecules. Using Lgr5 as ISC marker, we have fluorescence-activated cell sorting-purified ISCs to define their CD44 repertoire. ISCs display a specific set of CD44 variant isoforms (CD44v), but remarkably lack the CD44 standard (CD44s) isoform. These CD44v also stand-out in transformed human ISCs isolated from microadenomas of familial adenomatous polyposis patients. By employing knock-in mice expressing either CD44v4-10 or CD44s, we demonstrate that the CD44v isoform, but not CD44s, promotes adenoma initiation in Apc(Min/+)mice. Our data identify CD44v as component of the ISCs program critical for tumor initiation, and as potential treatment target in CRC.

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.

The role of APC and beta-catenin in the aetiology of aggressive fibromatosis (desmoid tumors).

BACKGROUND: Aggressive fibromatosis (syn. desmoid tumor) is a sporadically occurring neoplastic proliferation of fibroblasts originating from musculoaponeurotic planes, forming invasively growing masses without the capability to metastasize. The choice of treatment remains surgical resection with or without radiotherapy, and is characterized by high recurrence rates. Better understanding of the aetiology of aggressive fibromatosis is needed to be able to develop new treatment strategies to cope with the high recurrence rates. METHODS: Relevant studies were identified through a search of the electronic databases PubMed/ Medline. The following search terms were used: 'aggressive fibromatosis', 'desmoid tumor', 'adenomatous polyposis coli', 'APC', 'beta-catenin', 'Wnt', 'Wingless' and 'Wnt/Wingless'. Studies were selected for review on the basis of abstract reading. A hand search was performed by checking reference lists in selected articles. RESULTS: The neoplastic nature of aggressive fibromatosis and the role of the adenomatous polyposis coli (APC) and beta-catenin signaling cascade in driving the onset and progression of this disease are discussed. CONCLUSION: Mutations in either the APC or beta-catenin genes are likely to be a major driving force in the formation of these desmoid tumors. More research is needed to develop new treatment strategies.

Very long-term self-renewal of small intestine, colon, and hair follicles from cycling Lgr5+ve stem cells.

The intestinal epithelium and the hair follicle represent examples of rapidly self-renewing tissue in adult mammals. We have recently identified a novel stem cell gene Lgr5 expressed in multiple adult tissues. At the bottoms of crypts in small intestine and colon as well as in hair follicles, Lgr5 marks cycling cells with stem cell properties (Barker et al. 2007; Jaks et al. 2008). Using an inducible Lgr5-Cre knockin allele in conjunction with the Rosa26-LacZ Cre reporter strain, long-term lineage-tracing experiments were performed in adult mice. The Lgr5(+ve) crypt-based cell generated all epithelial lineages during a 14-month period, implying that it represents the stem cell of the small intestine and colon. Similarly, lineage tracing during a 14-month period revealed that Lgr5(+ve) cells located in the bulge of the hair follicle sustained multiple rounds of hair growth. These observations support the counterintuitive notion that Lgr5(+ve) cells are actively cycling, yet represent long-term stem cells of these adult, self-renewing tissues.

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.

Mucosal prolapse in the pathogenesis of Peutz-Jeghers polyposis.

Germline mutations in LKB1 cause the rare cancer prone disorder Peutz-Jeghers syndrome (PJS). Gastrointestinal hamartomatous polyps constitute the major phenotypic trait in PJS. Hamartomatous polyps arising in PJS patients are generally considered to lack premalignant potential although rare neoplastic changes in these polyps and an increased gastrointestinal cancer risk in PJS are well documented. These conflicting observations are resolved in the current hypothesis by providing a unifying explanation for these contrasting features of PJS polyposis. We postulate that a genetic predisposition to epithelial prolapse underlies the formation of the polyps associated with PJS. Conventional sporadic adenomas arising in PJS patients will similarly show mucosal prolapse and carry the associated histological features.

Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD.

The LKB1 gene encodes a serine/threonine kinase mutated in Peutz-Jeghers cancer syndrome. Despite several proposed models for LKB1 function in development and in tumour suppression, the detailed molecular action of LKB1 remains undefined. Here, we report the identification and characterization of an LKB1-specific adaptor protein and substrate, STRAD (STe20 Related ADaptor). STRAD consists of a STE20- like kinase domain, but lacks several residues that are indispensable for intrinsic catalytic activity. Endogenous LKB1 and STRAD form a complex in which STRAD activates LKB1, resulting in phosphorylation of both partners. STRAD determines the subcellular localization of wild-type, but not mutant LKB1, translocating it from nucleus to cytoplasm. One LKB1 mutation previously identified in a Peutz-Jeghers family that does not compromise its kinase activity is shown here to interfere with LKB1 binding to STRAD, and hence with STRAD-dependent regulation. Removal of endogenous STRAD by siRNA abrogates the LKB1-induced G(1) arrest. Our results imply that STRAD plays a key role in regulating the tumour suppressor activities of LKB1.