Lgr5-positive Cells Research Articles

NOX1-Dependent mTORC1 Activation via S100A9 Oxidation in Cancer Stem-like Cells Leads to Colon Cancer Progression

Cancer stem cells (CSCs) are associated with the refractory nature of cancer, and elucidating the targetable pathways for CSCs is crucial for devising innovative antitumor therapies. We find that the proliferation of CSC-enriched colon spheroids from clinical specimen is dependent on mTORC1 kinase, which is activated by reactive oxygen species (ROS) produced by NOX1, an NADPH oxidase. In the spheroid-derived xenograft tumors, NOX1 is preferentially expressed in LGR5-positive cells. Dependence on NOX1 expression or mTOR kinase activity is corroborated in the xenograft tumors and mouse colon cancer-derived organoids. NOX1 co-localizes with mTORC1 in VPS41-/VPS39-positive lysosomes, where mTORC1 binds to S100A9, a member of S100 calcium binding proteins, in a NOX1-produced ROS-dependent manner. S100A9 is oxidized by NOX1-produced ROS, which facilitates binding to mTORC1 and its activation. We propose that NOX1-dependent mTORC1 activation via S100A9 oxidation in VPS41-/VPS39-positive lysosomes is crucial for colon CSC proliferation and colon cancer progression.

Skin Epidermis and Adnexa Regrowth Induced by Treatment With Artificial Dermal Template After Full-Thickness Skin Wound.

Full-thickness skin wounds are common accidents. Although healing can be achieved by treatments like autologous skin grafts, donor site morbidity is hardly evitable. In this article, we provide compelling evidence demonstrating that artificial dermal template (ADT)-treated wound healing is achieved by regrowth of skin epidermis as well as adnexa without skin grafts by use of rodent models. First, by fixating a chamber to the wound edge, we confirmed that wound healing was achieved by regeneration instead of contracture. We found highly proliferative cells in adnexa in the newly formed skin. In the distal edge of newly formed skin, we identified immature hair follicles at early developing stages, suggesting they were newly regenerated. Second, we observed that the Lgr5-positive hair follicle stem cells contributed to formation of new hair follicles through a lineage tracing model. Also, Lgr6-positive cells were enriched in distal edge of newly developed skin. Finally, WNT signaling pathway mediators were highly expressed in the new skin epidermis and adnexa, implying a potential role of WNT signaling during ADT treatment-stimulated skin regrowth. Taken together, our findings demonstrated that full skin regrowth can be induced by ADT treatment alone, thus arguing for its wide clinical application in skin wound treatment.

Immunohistochemical Expression of CD133 and LGR5 in Ulcerative Colitis-associated Colorectal Cancer and Dysplasia.

Cluster of differentiation 133 (CD133) and leu cine-rich orphan G-protein-coupled receptor 5 (LGR5) are the most putative stem cell markers for colorectal cancer (CRC), and are associated with poor prognosis of patients with CRC. However, the role of CD133 and LGR5 in the inflammation-dysplasia-carcinoma sequence has not been fully elucidated. We examined the expression of CD133 and LGR5 in ulcerative colitis-associated CRC (UC-CRC; n=20) and UC-associated colorectal dysplasia (n=16) by immunohistochemistry. The rate of CD133-positive cases in UC-CRC was significantly higher than that in dysplasia (p=0.026), but that of LGR5 expression was not. Moreover, LGR5 expression was significantly positively associated with p53 expression (p=0.03), whereas CD133 expression positively correlated with p53 expression, but not significantly (p=0.10). CD133 may play an important role in tumor development in the context of the inflammation-dysplasia-carcinoma sequence. LGR5-positive cancer stem cells may play a critical role in the development of UC-CRC, particularly upon loss of p53 function.

PERK is essential for proliferation of intestinal stem cells in mice

Protein kinase RNA-like Endoplasmic Reticulum Kinase (PERK) is an endoplasmic reticulum stress sensor that possesses pro-survival capability and contributes to cell homeostasis and survival. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) has been recognized as a stem cell marker in intestinal epithelial cells. To determine whether PERK modulates the proliferation of intestinal stem cells, we investigated the effects of PERK knock-down on intestinal Lgr5-positive stem cells in mice. Lgr5-EGFP knock-in mice were fed with lentivirus-PERK shRNA twice a day for three days. Isolated intestinal Lgr5-positive stem cells were treated with lentivirus-PERK shRNA. The number of Lgr5-positive cells, the proliferation and apoptotic indices, several biomarkers for proliferation and differentiation, and Akt expression in intestinal stem cells were detected in vivo, in vitro and in two intestinal epithelial injury models caused by radiotherapy and sepsis. PERK knock-down could significantly diminish the number and proliferation of Lgr5-positive cells, induce the low expression of several proliferation markers and the high expression of several differentiation markers in Lgr5-positive cells, enhance the apoptotic Lgr5-positive cells, and reduce the Akt expression in intestinal Lgr5-positive stem cells. Similar results were observed in radiotherapy- and sepsis-induced intestinal injuries. Moreover, PERK inhibition markedly decreased the survival of mice in response to radiation and sepsis. These results suggest a critical role for PERK in the proliferation and survival of intestinal stem cells in mice.

BHLHA15-Positive Secretory Precursor Cells Can Give Rise to Tumors in Intestine and Colon in Mice

The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated. We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apcflox/flox, LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5-positive cells, were irradiated, or were given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues, we analyzed the fate of progeny that expressed Bhlha15. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using The Cancer Genome Atlas data set. Bhlha15 identified Paneth cells andshort-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15+ cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15+ secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15+ secretory precursors to long-lived enterocyte progenitors. Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with enterocyte progenitors, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15+ precursors toward those of ISCs. Bhlha15+ enterocyte progenitors with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells after dextran sodium sulfate-induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon. In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. Thesecells could mediate tumor formation in addition to traditional ISCs.

A Simple System for Differentiation of Functional Intestinal Stem Cell-like Cells from Bone Marrow Mesenchymal Stem Cells

Disruption of normal barrier function is a fundamental factor in the pathogenesis of inflammatory bowel disease, and intestinal stem cell (ISC) transplantation may be an optional treatment for patients. However, it is complicated and inefficient to isolate ISCs from the intestine, which hampers its wide application in clinic. We developed a two-step protocol in which mesenchymal stem cells (MSCs) were first induced into Sox17- or Foxa2-positive definitive endoderm cells by activin A treatment and then into Lgr5-positive ISC-like cells by miR-17 and FGF2 treatment. Furthermore, these Lgr5-positive cells could differentiate into enterocyte-like cells following induction with EGF. The results from an in vivo experiment showed that the MSC-derived Lgr5-positive cells were able to protect against dextran sulfate sodium-induced colitis. Taken together, our work might provide a new source of autologous ISCs.

Research progress of Lgr5-positive stem cells in the formation of organoid in 3D culture

Stem cell is critical to regeneration of tissue or organ of human. How to promote repair or regeneration in the tissues/organ using its pluripotency is always an important issue. Lgr5-possitive cell is one type of the stem cell-like cells capable of pluripotent differentiation in various tissues/organs of both humans and mice. Current study showed that single or small amount Lgr5-possitive stem cells can grow and form a plurality of organs in 3D culture system, and some organs can present similar biological and physiological properties with the progenitor they were derived. These studies provided new insight into future orientation, for example, Lgr5-possitive inner ear cells were confirmed as inner ear pluripotent cells population, the experiences obtained from organoid studies of Lgr5-possitive cells have certainly showed potential in the future study of inner ear stem cells. This review will focus on the recent progress associated with Lgr 5-positive stem cells forming organoids in the 3D culture.

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

The Coordinated Activities of nAChR and Wnt Signaling Regulate Intestinal Stem Cell Function in Mice.

Cholinergic signaling, which modulates cell activities via nicotinic and muscarinic acetylcholine receptors (n- and mAChRs) in response to internal or external stimuli, has been demonstrated in mammalian non-neuronal cells that synthesize acetylcholine (ACh). One of the major pathways of excitatory transmission in the enteric nervous system (ENS) is mediated by cholinergic transmission, with the transmitter ACh producing excitatory potentials in postsynaptic effector cells. In addition to ACh-synthesizing and ACh-metabolizing elements in the ENS, the presence of non-neuronal ACh machinery has been reported in epithelial cells of the small and large intestines of rats and humans. However, little is known about how non-neuronal ACh controls physiological function in the intestine. Here, experiments using crypt–villus organoids that lack nerve and immune cells in culture suggest that endogenous ACh is synthesized in the intestinal epithelium to drive organoid growth and differentiation through activation of nAChRs. Treatment of organoids with nicotine enhanced cell growth and the expression of marker genes for stem and epithelial cells. On the other hand, the nAChR antagonist mecamylamine strongly inhibited the growth and differentiation of organoids, suggesting the involvement of nAChRs in the regulation of proliferation and differentiation of Lgr5-positive stem cells. More specifically, RNA sequencing analysis revealed that Wnt5a expression was dramatically upregulated after nicotine treatment, and Wnt5a rescued organoid growth and differentiation in response to mecamylamine. Taken together, our results indicate that coordinated activities of nAChR and Wnt signaling maintain Lgr5-positive stem cell activity and balanced differentiation. Furthermore, we could clearly separate the two groups, neuronal ACh in the ENS and non-neuronal ACh in the intestinal epithelium. Dysfunction of the non-neuronal cholinergic system is involved in the pathogenesis of disease. The data will increase our understanding of the cholinergic properties of non-neuronal cells and lead to optimization of drug therapy.

BP-C3 POLYPHENOLIC COMPOSITION INFLUENCE ON DYNAMICS OF 5-FLUOROURACIL INDUCED DAMAGE IN SMALL INTESTINE EPITHELIUM IN MICE

Chemotherapeutic drugs negatively influence on normal tissues with high proliferative activity such as intestinal epithelium. The damage of chemotherapy to healthy tissues significantly reduces oncological patients’ quality of life. Drugs based on polyphenolic natural compounds are a perspective mean of adjuvant therapy due to their antioxidant and antiinflammatory properties. In this study we have showed that a single intragastric administration of BPC3 polyphenolic composition 2 hours before 5-fluorouracil treatment alleviates small intestine damage in C57Bl/6 mice after 3.5, 24 or 72 hours. BPC3 effect manifested in reduced apoptosis rate, increased mitotic activity of the epithelium in the regenerative phase and protection of LGR5-positive jejunum stem cells against 5-fluorouracil induced damage.

Dynamics of Proliferative and Quiescent Stem Cells in Liver Homeostasis and Injury

Adult liver stem cells are usually maintained in a quiescent/slow-cycling state. However, a proliferative population, marked by leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), was recently identified as an important liver stem cell population. We aimed to investigate the dynamics and functions of proliferative and quiescent stem cells in healthy and injured livers. We studied LGR5-positive stem cells using diphtheria toxin receptor and green fluorescent protein (GFP) knock-in mice. In these mice, LGR5-positive cells specifically coexpress diphtheria toxin receptor and the GFP reporter. Lineage-tracing experiments were performed in mice in which LGR5-positive stem cells and their daughter cells expressed a yellow fluorescent protein/mTmG reporter. Slow-cycling stem cells were investigated using GFP-based, Tet-on controlled transgenic mice. We studied the dynamics of both stem cell populations during liver homeostasis and injury induced by carbon tetrachloride. Stem cells were isolated from mouse liver and organoid formation assays were performed. We analyzed hepatocyte and cholangiocyte lineage differentiation in cultured organoids. We did not detect LGR5-expressing stem cells in livers of mice at any stage of a lifespan, but only following liver injury induced by carbon tetrachloride. In the liver stem cell niche, where the proliferating LGR5+ cells are located, we identified a quiescent/slow-cycling cell population, called label-retaining cells (LRCs). These cells were present in the homeostatic liver, capable of retaining the GFP label over 1 year, and expressed a panel of progenitor/stem cell markers. Isolated single LRCs were capable of forming organoids that could be carried in culture, expanded for months, and differentiated into hepatocyte and cholangiocyte lineages invitro, demonstrating their bona fide stem cell properties. More interestingly, LRCs responded to liver injury and gave rise to LGR5-expressing stem cells, as well as other potential progenitor/stem cell populations, including SOX9- and CD44-positive cells. Proliferative LGR5 cells are an intermediate stem cell population in the liver that emerge only during tissue injury. In contrast, LRCs are quiescent stem cells that are present in homeostatic liver, respond to tissue injury, and can give rise to LGR5 stem cells, as well as SOX9- and CD44-positive cells.

Abstract 5768: Intrahepatic cholangiocarcinoma cells can be converted into functional hepatocytes by inhibition of Wnt signaling pathway

Abstract Background and Aim: Intrahepatic cholangiocarcinoma (IHCC) is the second most prevalent malignancy in the liver, and is one of the most aggressive malignancies characterized by a poor prognosis. It is believed that IHCC is an epithelial cell malignancy arising from cholangiocytes composing the intrahepatic bile ducts. However, patients with viral hepatitis develop IHCC, suggesting that IHCC is derived from transformed hepatocytes. Indeed, recent studies have revealed that IHCC can arise from Notch-mediated conversion of hepatocytes in mice. The new 3D culture system called organoid culture has been developed, which allows long-term expansion of Lgr5-positive stem cells. To clarify whether IHCC cells harbor the potency of conversion into functional hepatocytes, we established organoids derived from human IHCC and cultured them in different culture conditions. Methods: We established organoids derived from human IHCC and cultured these organoids with expansion medium (EM) containing R-spondin 1. Conversion of IHCC cells into hepatocytes was conducted by organoid culture with differentiation medium (DM) containing BMP7, DAPT, FGF19 and dexamethasone. Results: Gene set enrichment analysis (GSEA) showed that genes associated with hepatocyte differentiation signature were significantly enriched in IHCC organoids cultured with DM compared with those cultured with EM. IHCC organoids cultured with DM induced up-regulation of hepatocyte markers albumin, HNF4a and CYP3A4, and acquired mature hepatocyte functions including albumin secretion, bile acid production and increased CYP3A4 activity. Removal of R-spondin 1, a ligand of Wnt signaling pathway, from EM enhanced hepatocyte differentiation. IHCC organoids cultured with DM significantly reduced the malignant potential in vitro and in vivo compared with those cultured with EM. Further studies using thioacetamide (TAA)-induced IHCC model mice showed that TAA-induced inflammation in the liver promoted recruited macrophages to secrete Wnt3a. Conclusions: These findings indicated that IHCC cells can be converted into functional hepatocytes by inhibition of Wnt signaling pathway. Activation of Wnt signaling pathway by recruited macrophages may play a critical role in the malignant transformation of hepatocytes into IHCC, which could be an important therapeutic target for IHCC. Citation Format: Toshiaki Nakaoka, Yoshimasa Saito, Toshihide Muramatsu, Hidenori Ojima, Yae Kanai, Yuko Sugiyama, Masaki Kimura, Takanori Kanai, Toshiro Sato, Hidetsugu Saito. Intrahepatic cholangiocarcinoma cells can be converted into functional hepatocytes by inhibition of Wnt signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5768. doi:10.1158/1538-7445.AM2017-5768

Heterogeneity among Lgr5-positive colon stem cells

ABSTRACTLeucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is considered a representative marker of intestinal stem cells from both non-tumor and tumor tissues. However, it remains unclear whether all or only a fraction of Lgr5-positive cells behave as stem cells. Recently, we reported that Lgr5-positive cells from non-tumor and tumor tissues can be classified into overlapping yet distinct groups and that the tumor-specific groups are associated with tumorigenic capability, suggesting that these cells could represent targets for therapeutic intervention.

Intestinal Stem Cell Pool Regulation in Drosophila.

SummaryIntestinal epithelial renewal is mediated by intestinal stem cells (ISCs) that exist in a state of neutral drift, wherein individual ISC lineages are regularly lost and born but ISC numbers remain constant. To test whether an active mechanism maintains stem cell pools in the Drosophila midgut, we performed partial ISC depletion. In contrast to the mouse intestine, Drosophila ISCs failed to repopulate the gut after partial depletion. Even when the midgut was challenged to regenerate by infection, ISCs retained normal proportions of asymmetric division and ISC pools did not increase. We discovered, however, that the loss of differentiated midgut enterocytes (ECs) slows when ISC division is suppressed and accelerates when ISC division increases. This plasticity in rates of EC turnover appears to facilitate epithelial homeostasis even after stem cell pools are compromised. Our study identifies unique behaviors of Drosophila midgut cells that maintain epithelial homeostasis.

The Induction of Selected Wnt Target Genes by Tcf1 Mediates Generation of Tumorigenic Colon Stem Cells

The generation of tumor-initiating cells during colon carcinogenesis is associated with the dysregulation of Wnt signaling, which is known to act on Lgr5-positive intestinal stem cells. Here, using single-cell qPCR analysis, we identified a subset of Lgr5-positive stem cells that emerged during tumorigenesis in a mouse model of colon cancer. These tumor-specific Lgr5-positive cells expressed low levels of Ceacam1 and increased levels of a specific subset of Wnt targets and showed enhanced tumorigenicity. Among the Wnt targets that were specifically expressed, the long isoform of Tcf1 was required forthe proliferation of tumor organoids and drove aunique Wnt target gene expression profile. Tcf1 expression increased at an early stage of colon carcinogenesis and was associated with the nuclear accumulation of β-catenin, underscoring the importance of the induction of Tcf1 expression in generating tumorigenic colon stem cells.

Intestinal cancer stem cells marked by Bmi1 or Lgr5 expression contribute to tumor propagation via clonal expansion

Although the existence of cancer stem cells in intestine tumors has been suggested, direct evidence has not been yet provided. Here, we showed, using the multicolor lineage-tracing method and mouse models of intestinal adenocarcinoma and adenoma that Bmi1- or Lgr5- positive tumorigenic cells clonally expanded in proliferating tumors. At tumor initiation and during tumor propagation in the colon, the descendants of Lgr5-positive cells clonally proliferated to form clusters. Clonal analysis using ubiquitous multicolor lineage tracing revealed that colon tumors derived from Lgr5-positive cells were monoclonal in origin but eventually merged with neighboring tumors, producing polyclonal tumors at the later stage. In contrast, the origin of small intestine tumors was likely polyclonal, and during cancer progression some clones were eliminated, resulting in the formation of monoclonal tumors, which could merge similar to colon tumors. These results suggest that in proliferating intestinal neoplasms, Bmi1- or Lgr5-positive cells represent a population of cancer stem cells, whereas Lgr5-positive cells also function as cells-of-origin for intestinal tumors.

Telomerase Expression in Medaka (Oryzias melastigma) Pharyngeal Teeth

Nonmammalian vertebrates have the capacity of lifelong tooth replacement. In all vertebrates, tooth formation requires contact and interaction between the oral or pharyngeal epithelium and the underlying mesenchyme. To secure lifelong replacement, the presence of odontogenic stem cells has been postulated, particularly in the epithelial compartment. This study uses an advanced teleost fish species, the marine medaka Oryzias melastigma, a close relative to Oryzias latipes, to examine the expression and distribution of telomerase reverse transcriptase (Tert), the catalytic unit of telomerase, in developing pharyngeal teeth and to relate these data to the proliferative activity of the cells. The data are complemented by expression analysis of the pluripotency marker oct4 and bona fide stem cell marker lgr5. Tert distribution and tert expression in developing tooth germs show a dynamic spatiotemporal pattern. Tert is present first in the mesenchyme but is downregulated as the odontoblasts differentiate. In contrast, in the epithelial enamel organ, Tert is absent during early stages of tooth formation and upregulated first in ameloblasts. Later, Tert is expressed and immunolocalized throughout the entire inner enamel epithelium. The pattern of Tert distribution is largely mutually exclusive with that of proliferating cell nuclear antigen (PCNA) immunoreactivity: highly proliferative cells, as revealed by PCNA staining, are negative for Tert; conversely, PCNA-negative cells are Tert-positive. Only the early condensed mesenchyme is both Tert- and PCNA-positive. The absence of tert-positive cells in the epithelial compartment of early tooth germs is underscored by the absence of oct4- and lgr5-positive cells, suggesting ways other than stem cell involvement to secure continuous renewal.

Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells

Death of cochlear hair cells, which do not regenerate, is a cause of hearing loss in a high percentage of the population. Currently, no approach exists to obtain large numbers of cochlear hair cells. Here, using asmall-molecule approach, we show significant expansion (>2,000-fold) of cochlear supporting cells expressing and maintaining Lgr5, an epithelial stem cell marker, in response to stimulation of Wnt signaling by a GSK3β inhibitor and transcriptional activation by a histone deacetylase inhibitor. The Lgr5-expressing cells differentiate into hair cells in high yield. From a single mouse cochlea, we obtained over 11,500 hair cells, compared to less than 200 in the absence of induction. The newly generated hair cells have bundles and molecular machinery for transduction, synapse formation, and specialized hair cell activity. Targeting supporting cells capable of proliferation and cochlear hair cell replacement could lead to the discovery of hearing loss treatments.

Clinical Significance of Disseminated Pluripotent Tumor Cell Signature Expression in the Bone Marrow from Patients with Colorectal Cancer.

Purpose:Disseminated tumor cells (DTCs) are critically involved in tumor relapse and survival in several invasive tumors. We previously showed that the ATP-binding cassette (ABC) transporter, ABCB5, is a chemoresistance mediator expressed on specific cell subsets in colorectal cancer (CRC) and other malignancies. This study evaluated the molecular signature expression and its clinical relevance of DTCs in bone marrow from patients with colon cancer.Methods:This study included 49 consecutive patients (UICC stage I-IV) that underwent curatively intended or palliative surgery for CRC. We analyzed cells from bone marrow aspirates obtained before surgery and derived from patients that had completed minimally a 5-year follow-up. The gene expression of ABCB5 in comparison to CD133 (molecule for identifying cancer initiating cells), Lgr5 (an intestinal stem cell marker) as well as Cytokeratin (CK) 20 (terminally differentiated tumor cells of epithelial origin) in these cells was evaluated.Results:Bone marrow analysis showed differential expression between the analyzed genes. ABCB5 and Lgr5 and to lesser extent CD133 and CK20 genes were significantly expressed in the analyzed cells from bone marrow aspirates while only ABCB5 and Lgr5 were significantly negative associated with tumor progress and overall survival.Conclusion:Overexpression of ABCB5 and Lgr5 in bone marrow negatively influenced patient survival pointing to a specific chemo resistant and pluripotent cell subgroup of DTCs in the bone marrow. ABCB5 like Lgr5 positive cells seem to be involved in limited tumor related patient survival, suggesting that ABCB5- and Lgr5-positive cells may be relevant for specific clinical intervention strategies

Co-expression of Lgr5 and CXCR4 characterizes cancer stem-like cells of colorectal cancer.

Therapies designed to target cancer stem cells (CSCs) in colorectal cancer (CRC) may improve treatment outcomes. Different markers have been used to identify CSCs or CSC-like cells in CRC, but the enrichment of CSCs using these markers has yet to be optimized. We recently reported the importance of Lgr5-positive CRC cells in cancer growth. Here, we studied the possibility of using Lgr5 and CXCR4 as CSC markers for CRC. We detected high Lgr5 and CXCR4 levels in stage IV CRC specimens. Both high Lgr5 and CXCR4 levels were associated with poor prognosis in stage IV CRC patients. In vitro, Lgr5+CXCR4-, CXCR4+Lgr5- and Lgr5+CXCR4+ cells were purified in human CRC cell lines and examined for their CSC properties. We found that compared to the unsorted cells, CXCR4+Lgr5-, Lgr5+CXCR4-, and Lgr5+/CXCR4+ cells showed significantly greater cancer mass after subcutaneous transplantation, greater tumor sphere formation, higher resistance to chemotherapy, and higher incidence of tumor formation after serial adoptive transplantation into NOD/SCID mice. Taken together, our data suggest that the combined use of Lgr5 and CXCR4 may facilitate the enrichment of CSCs in CRC, and that treating Lgr5+/CXCR4+ CRC cells may improve the outcome of CRC therapy.