Lineage Tracing Analysis Research Articles

Segregating neural and mechanosensory fates in the developing ear: patterning, signaling, and transcriptional control.

The vertebrate inner ear is composed of multiple sensory receptor epithelia, each of which is specialized for detection of sound, gravity, or angular acceleration. Each receptor epithelium contains mechanosensitive hair cells, which are connected to the brainstem by bipolar sensory neurons. Hair cells and their associated neurons are derived from the embryonic rudiment of the inner ear epithelium, but the precise spatial and temporal patterns of their generation, as well as the signals that coordinate these events, have only recently begun to be understood. Gene expression, lineage tracing, and mutant analyses suggest that both neurons and hair cells are generated from a common domain of neural and sensory competence in the embryonic inner ear rudiment. Members of the Shh, Wnt, and FGF families, together with retinoic acid signals, regulate transcription factor genes within the inner ear rudiment to establish the axial identity of the ear and regionalize neurogenic activity. Close-range signaling, such as that of the Notch pathway, specifies the fate of sensory regions and individual cell types. We also describe positive and negative interactions between basic helix-loop-helix and SoxB family transcription factors that specify either neuronal or sensory fates in a context-dependent manner. Finally, we review recent work on inner ear development in zebrafish, which demonstrates that the relative timing of neurogenesis and sensory epithelial formation is not phylogenetically constrained.

In Vivo Clonal Analysis Reveals Lineage-Restricted Progenitor Characteristics in Mammalian Kidney Development, Maintenance, and Regeneration

The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

EGFR and Notch signaling respectively regulate proliferative activity and multiple cell lineage differentiation of Drosophila gastric stem cells

Quiescent, multipotent gastric stem cells (GSSCs) in the copper cell region of adult Drosophila midgut can produce all epithelial cell lineages found in the region, including acid-secreting copper cells, interstitial cells and enteroendocrine cells, but mechanisms controlling their quiescence and the ternary lineage differentiation are unknown. By using cell ablation or damage-induced regeneration assays combined with cell lineage tracing and genetic analysis, here we demonstrate that Delta (Dl)-expressing cells in the copper cell region are the authentic GSSCs that can self-renew and continuously regenerate the gastric epithelium after a sustained damage. Lineage tracing analysis reveals that the committed GSSC daughter with activated Notch will invariably differentiate into either a copper cell or an interstitial cell, but not the enteroendocrine cell lineage, and loss-of-function and gain-of-function studies revealed that Notch signaling is both necessary and sufficient for copper cell/interstitial cell differentiation. We also demonstrate that elevated epidermal growth factor receptor (EGFR) signaling, which is achieved by the activation of ligand Vein from the surrounding muscle cells and ligand Spitz from progenitor cells, mediates the regenerative proliferation of GSSCs following damage. Taken together, we demonstrate that Dl is a specific marker for Drosophila GSSCs, whose cell cycle status is dependent on the levels of EGFR signaling activity, and the Notch signaling has a central role in controlling cell lineage differentiation from GSSCs by separating copper/interstitial cell lineage from enteroendocrine cell lineage.

Conversion of differentiated cancer cells into cancer stem-like cells in a glioblastoma model after primary chemotherapy

Glioblastoma multiforme patients have a poor prognosis due to therapeutic resistance and tumor relapse. It has been suggested that gliomas are driven by a rare subset of tumor cells known as glioma stem cells (GSCs). This hypothesis states that only a few GSCs are able to divide, differentiate, and initiate a new tumor. It has also been shown that this subpopulation is more resistant to conventional therapies than its differentiated counterpart. In order to understand glioma recurrence post therapy, we investigated the behavior of GSCs after primary chemotherapy. We first show that exposure of patient-derived as well as established glioma cell lines to therapeutic doses of temozolomide (TMZ), the most commonly used antiglioma chemotherapy, consistently increases the GSC pool over time both in vitro and in vivo. Secondly, lineage-tracing analysis of the expanded GSC pool suggests that such amplification is a result of a phenotypic shift in the non-GSC population to a GSC-like state in the presence of TMZ. The newly converted GSC population expresses markers associated with pluripotency and stemness, such as CD133, SOX2, Oct4, and Nestin. Furthermore, we show that intracranial implantation of the newly converted GSCs in nude mice results in a more efficient grafting and invasive phenotype. Taken together, these findings provide the first evidence that glioma cells exposed to chemotherapeutic agents are able to interconvert between non-GSCs and GSCs, thereby replenishing the original tumor population, leading to a more infiltrative phenotype and enhanced chemoresistance. This may represent a potential mechanism for therapeutic relapse.

Skin Progenitor Cells Contribute to Bleomycin‐Induced Skin Fibrosis

The origin of the cells that contribute to skin fibrosis is unclear. We undertook the present study to assess the contribution of Sox2-expressing skin progenitor cells to bleomycin-induced scleroderma. Scleroderma was induced, by bleomycin administration, in wild-type mice and in mice in which CCN2 was deleted from Sox2-expressing cells. Lineage tracing analysis was performed to assess whether cells expressing Sox2 are recruited to fibrotic lesions in response to bleomycin-induced scleroderma. In response to bleomycin, Sox2-positive/α-smooth muscle actin-positive cells were recruited to fibrotic tissue. CCN2-conditional knockout mice in which CCN2 was deleted from Sox2-expressing cells exhibited resistance to bleomycin-induced skin fibrosis. Collectively, these results indicate that CCN2 is required for the recruitment of progenitor cells and that CCN2-expressing progenitor cells are essential for bleomycin-induced skin fibrosis. Lineage tracing analysis using mice in which a tamoxifen-dependent Cre recombinase was expressed under the control of the Sox2 promoter confirmed that progenitor cells were recruited to the fibrotic lesion in response to bleomycin, and that this did not occur in CCN2-knockout mice. The ability of serum to induce α-smooth muscle actin expression in skin progenitor cells required the presence of CCN2. Sox2-positive skin progenitor cells are required in order for bleomycin-induced skin fibrosis to occur, and CCN2 is required for the recruitment of these cells to the fibrotic lesion. Targeting stem cell recruitment or CCN2 may therefore represent a useful therapeutic approach in combating fibrotic skin disease.

Alveolar progenitor and stem cells in lung development, renewal and cancer

Alveoli are gas-exchange sacs lined by squamous alveolar type (AT) 1 cells and cuboidal, surfactant-secreting AT2 cells. Classical studies suggested that AT1 arise from AT2 cells, but recent studies propose other sources. Here we use molecular markers, lineage tracing and clonal analysis to map alveolar progenitors throughout the mouse lifespan. We show that, during development, AT1 and AT2 cells arise directly from a bipotent progenitor, whereas after birth new AT1 cells derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. This stem-cell function is broadly activated by AT1 injury, and AT2 self-renewal is selectively induced by EGFR (epidermal growth factor receptor) ligands in vitro and oncogenic Kras(G12D) in vivo, efficiently generating multifocal, clonal adenomas. Thus, there is a switch after birth, when AT2 cells function as stem cells that contribute to alveolar renewal, repair and cancer. We propose that local signals regulate AT2 stem-cell activity: a signal transduced by EGFR-KRAS controls self-renewal and is hijacked during oncogenesis, whereas another signal controls reprogramming to AT1 fate.

Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling.

The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.

Regeneration of Liver After Extreme Hepatocyte Loss Occurs Mainly via Biliary Transdifferentiation in Zebrafish

The liver has high regenerative capacity, but it is not clear whether most biliary cells (particularly larger cholangiocytes) transdifferentiate into hepatocytes in regenerating liver. We investigated how this process might contribute to liver regeneration in zebrafish. Zebrafish transgenic lines were generated using the standard I-SceI meganuclease transgenesis technique. Hepatocytes of the Tg(lfabp:mCherry-NTR)(cq2) animals were ablated by the administration of metronidazole. We investigated transdifferentiation of biliary cells to hepatocytes and expression of markers using whole mount antibody staining, fluorescent in situ hybridization, and Cre/loxP-based genetic lineage tracing analyses. The role of biliary cells in hepatocyte regeneration was explored using zebrafish larvae with defects in biliary cell development. After extreme loss of hepatocytes, nearly all the biliary cells steadily lost their tubular morphology, proliferated, and expressed hepatocyte-specific markers. Cre/loxP-based inducible lineage tracing showed that new hepatocytes mainly arose from transdifferentiation of biliary cells; this process required Notch signaling and, in turn, activation of Sox9b in cholangiocytes. Activation of early endoderm and hepatoblast markers in most of the cholangiocytes indicated that biliary transdifferentiation includes a step of dedifferentiation into a bipotential intermediate. Defects in development of biliary cells impaired hepatocyte regeneration. Using our zebrafish liver regeneration model, we found that biliary cells can transdifferentiate into hepatocytes and are the major contributors to hepatocyte regeneration after extreme hepatocyte loss.

Periosteum

The bone-regenerative potentials of the periosteum have been explored as early as the 17th century. Over the past few years, however, much has been discovered in terms of the molecular and cellular mechanisms that control the periosteal contribution to bone regeneration. Lineage tracing analyses and knock-in transgenic mice have helped define the relative contributions of the periosteum and endosteum to bone regeneration. Additional studies have shed light on the critical roles that BMP, FGF, Hedgehog, Notch, PDGF, Wnt, and inflammation signaling have or may have in periosteal-mediated bone regeneration, fostering the path to novel approaches in bone-regenerative therapy. Thus, by examining the role that each pathway has in periosteal-mediated bone regeneration, in this review we analyze the status of the current research on the regenerative potential of the periosteum. The provided analysis aims to inform both clinician-scientists who may have interest in the current studies about the biology of the periosteum as well as dental surgeons who may find this review useful to perform periosteal-harnessing bone-regenerative procedures.

Abstract IA10: Using lineage tracing and clonal analysis to unravel mammary gland expansion and cancer initiation

Abstract We developed novel lineage tracing approaches in mice to decipher the cellular hierarchy of the mammary epithelium during development, adulthood, pregnancy and lactation and to study the cell of origin of different types of breast cancers. Knowing that luminal cells specifically express the keratins K8 and K18, while myoepithelial cells express K5 an K14, we generated two inducible CRE recombinase (CREER) mice expressed in the luminal cells, under the control of K8 and K18 promoters to target luminal cells. To target myoepithelial cells, we used mice expressing the CREER under the control of K5 promoter, as well as the K14rtTA-TetOCRE mice. We demonstrated that the mammary gland initially develops from multipotent embryonic progenitors expressing K14, which give rise upon differentiation to both myoepithelial and luminal cells. However, during puberty and homeostasis, as well as during pregnancy and lactation, the expansion and the maintenance of luminal cells is ensured by the presence of luminal unipotent SCs, while the expansion and maintenance of myoepithelial cells is ensured by the presence of myoepithelial unipotent SCs, rather than being maintained by rare multipotent SCs. We also demonstrated that transplantation assays, while extremely informative about the differentiation potential of tissue specific SCs, can be misleading in extrapolating the differentiation potential of SCs under physiological conditions. While the different genes implicated in inherited and sporadic breast cancers have been identified, the cellular origin of the different types of breast cancers remains elusive. Mutations in the cancer suppressor genes BRCA (BRCA1 and BRCA2) account for the majority of inherited breast cancers, and are associated with basal-like tumors. Oncogenic mutations in PIK3CA are found in 20 to 40% of invasive breast cancers and are associated with luminal tumors. To determine the cell of origin of breast cancers, we use the K8CREER mice to introduce oncogenic mutations specifically in luminal cells, while we use the K5CREER mice to introduce the mutations in basal cells. We target deletion of Brca1 and p53 to recapitulate basal-like breast cancers, while expression of the oncogene PIK3CA is used to recapitulate luminal-like breast cancers. Our results show that the differentiation of breast tumors can be misleading to extrapolate their cellular origin and that cell fate changes occur during breast cancer initiation. Citation Format: Alexandra Van Keymeulen, May Yin Lee, Marielle Ousset, Cédric Blanpain. Using lineage tracing and clonal analysis to unravel mammary gland expansion and cancer initiation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA10.

WT1 Maintains Adrenal-Gonadal Primordium Identity and Marks a Population of AGP-like Progenitors within the Adrenal Gland

Adrenal glands and gonads share a common primordium (AGP), but the molecular events driving differentiation are poorly understood. Here we demonstrate that the Wilms tumor suppressor WT1 is a key factor defining AGP identity by inhibiting the steroidogenic differentiation process. Indeed, ectopic expression of WT1 precludes differentiation into adrenocortical steroidogenic cells by locking them into a progenitor state. Chromatin immunoprecipitation experiments identify Tcf21 and Gli1 as direct targets of WT1. Moreover, cell lineage tracing analyses identify a long-living progenitor population within the adrenal gland, characterized by the expression of WT1, GATA4, GLI1, and TCF21, that can generate steroidogenic cells invivo. Strikingly, gonadectomy dramatically activates these WT1(+) cells and leads to their differentiation into gonadal steroidogenic tissue. Thus, our data describe a mechanism of response to organ loss by recreating hormone-producing cells at a heterotopic site.

APKCλ controls epidermal homeostasis and stem cell fate through regulation of division orientation

The atypical protein kinase C (aPKC) is a key regulator of polarity and cell fate in lower organisms. However, whether mammalian aPKCs control stem cells and fate in vivo is not known. Here we show that loss of aPKCλ in a self-renewing epithelium, the epidermis, disturbed tissue homeostasis, differentiation, and stem cell dynamics, causing progressive changes in this tissue. This was accompanied by a gradual loss of quiescent hair follicle bulge stem cells and a temporary increase in proliferating progenitors. Lineage tracing analysis showed that loss of aPKCλ altered the fate of lower bulge/hair germ stem cells. This ultimately led to loss of proliferative potential, stem cell exhaustion, alopecia, and premature aging. Inactivation of aPKCλ produced more asymmetric divisions in different compartments, including the bulge. Thus, aPKCλ is crucial for homeostasis of self-renewing stratifying epithelia, and for the regulation of cell fate, differentiation, and maintenance of epidermal bulge stem cells likely through its role in balancing symmetric and asymmetric division.

Rapid Transition of NESTIN‐Expressing Dividing Cells from PROP1‐Positive to PIT1‐Positive Advances Prenatal Pituitary Development

We recently reported that the quantitative and qualitative transition of stem/progenitor cells occurs by the acquisition of a novel mechanism in the terminal differentiation during postnatal development of the anterior pituitary. We hypothesised that this novel mechanism is an alteration of a cell supply system accompanying proliferation of the progenitor cells. In the present study, we examined the proliferation activities of progenitor cells by indication of the expression of Nestin, a marker of rapidly dividing progenitor cells, aiming to verify our hypothesis and to resolve another outstanding issue regarding whether the Nestin gene is expressed in the pituitary. We found that NESTIN-positive dividing cells certainly exist in the pituitary through all stages of development. Almost all of the PROP1-positive progenitor cells express Nestin in early embryonic pituitary development. Thereafter, Nestin-expressing dividing cells involved in the cell supply system transfer from PROP1-positive progenitor cells to committed progenitor cells, such as PIT1-positive cells, on neonatal pituitary development. Furthermore, our data, together with the findings of previous studies on cell lineage tracing analyses using Nestin-Cre mice derived by the central nervous system (CNS)-specific Nestin promoter, suggest that at least two regulation systems for Nestin-expression exist in the pituitary, with the majority of these not being CNS-specific.

ETV4 promotes metastasis in response to activation of PI3-kinase and Ras signaling in a mouse model of advanced prostate cancer

Combinatorial activation of PI3-kinase and RAS signaling occurs frequently in advanced prostate cancer and is associated with adverse patient outcome. We now report that the oncogenic Ets variant 4 (Etv4) promotes prostate cancer metastasis in response to coactivation of PI3-kinase and Ras signaling pathways in a genetically engineered mouse model of highly penetrant, metastatic prostate cancer. Using an inducible Cre driver to simultaneously inactivate Pten while activating oncogenic Kras and a fluorescent reporter allele in the prostate epithelium, we performed lineage tracing in vivo to define the temporal and spatial occurrence of prostate tumors, disseminated tumor cells, and metastases. These analyses revealed that though disseminated tumors cells arise early following the initial occurrence of prostate tumors, there is a significant temporal lag in metastasis, which is temporally coincident with the up-regulation of Etv4 expression in primary tumors. Functional studies showed that knockdown of Etv4 in a metastatic cell line derived from the mouse model abrogates the metastatic phenotype but does not affect tumor growth. Notably, expression and activation of ETV4, but not other oncogenic ETS genes, is correlated with activation of both PI3-kinase and Ras signaling in human prostate tumors and metastases. Our findings indicate that ETV4 promotes metastasis in prostate tumors that have activation of PI3-kinase and Ras signaling, and therefore, ETV4 represents a potential target of therapeutic intervention for metastatic prostate cancer.

Adjuvant Gemcitabine Therapy Improves Survival in A Locally Induced, R0-Resectable Model of Metastatic Intrahepatic Cholangiocarcinoma

Complete surgical tumor resection (R0) for treatment of intrahepatic cholangiocarcinoma (ICC) is potentially curative, but the prognosis remains dismal due to frequent tumor recurrence and metastasis after surgery. Adjuvant therapies may improve the outcome, but clinical studies for an adjuvant approach are difficult and time-consuming for rare tumor entities. Therefore, animal models reflecting the clinical situation are urgently needed to investigate novel adjuvant therapies. To establish a mouse model of resectable cholangiocarcinoma including the most frequent genetic alterations of human ICC, we electroporated Sleeping Beauty-based oncogenic transposon plasmids into the left liver lobe of mice. KRas-activation in combination with p53-knockout in hepatocytes resulted in formation of a single ICC nodule within 3-5 weeks. Lineage tracing analyses confirmed the development of ICC by transdifferentiation of hepatocytes. Histologic examination demonstrated that no extrahepatic metastases were detectable during primary tumor progression. However, formation of tumor satellites close to the primary tumor and vascular invasion were observed, indicating early invasion into normal tissue adjacent to the tumor. After R0-resection of the primary tumor, we were able to prolong median survival, thereby observing tumor stage-dependent local recurrence, peritoneal carcinomatosis, and lung metastasis. Adjuvant gemcitabine chemotherapy after R0-resection significantly improved median survival of treated animals. We have developed a murine model of single, R0-resectable ICC with favorable characteristics for the study of recurrence patterns and mechanisms of metastasis after resection. This model holds great promise for preclinical evaluation of novel multimodal or adjuvant therapies to prevent recurrence and metastasis after R0-resection.

Coordination of heart and lung co-development by a multipotent cardiopulmonary progenitor

SUMMARYCo-development of the cardiovascular and pulmonary systems is a recent evolutionary adaption to terrestrial life that couples cardiac output with the gas exchange function of the lung 1. In this report, we show that the pulmonary vasculature develops even in the absence of lung development. We have identified a population of multi-potent cardiopulmonary mesoderm progenitors (CPPs) within the posterior pole of the heart that are marked by the expression of Wnt2/Gli1/Isl1. We show that CPPs arise from cardiac progenitors prior to lung development. Lineage tracing and clonal analysis demonstrates that CPPs generate the mesoderm lineages within the cardiac inflow tract and lung including cardiomyocytes, pulmonary vascular and airway smooth muscle, proximal vascular endothelium, and pericyte-like cells. CPPs are regulated by hedgehog expression from the foregut endoderm, which is required for connection of the pulmonary vasculature to the heart. Together, these studies identify a novel population of multipotent cardiopulmonary progenitors that coordinates heart and lung co-development that is required for adaptation to terrestrial existence.

The Interfollicular Epidermis of Adult Mouse Tail Comprises Two Distinct Cell Lineages that Are Differentially Regulated by Wnt, Edaradd, and Lrig1

Current models of how mouse tail interfollicular epidermis (IFE) is maintained overlook the coexistence of two distinct terminal differentiation programs: parakeratotic (scale) and orthokeratotic (interscale). Lineage tracing and clonal analysis revealed that scale and interscale are maintained by unipotent cells in the underlying basal layer, with scale progenitors dividing more rapidly than interscale progenitors. Although scales are pigmented and precisely aligned with hair follicles, melanocytes and follicles were not necessary for scale differentiation. Epidermal Wnt signaling was required for scale enlargement during development and for postnatal maintenance of scale-interscale boundaries. Loss of Edaradd inhibited ventral scale formation, whereas loss of Lrig1 led to scale enlargement and fusion. In wild-type skin, Lrig1 was not expressed in IFE but was selectively upregulated in dermal fibroblasts underlying the interscale. We conclude that the different IFE differentiation compartments are maintained by distinct stem cell populations and are regulated by epidermal and dermal signals.

The Touch Dome Defines an Epidermal Niche Specialized for Mechanosensory Signaling

In mammalian skin, Merkel cells are mechanoreceptor cells that are required for the perception of gentle touch. Recent evidence indicates that mature Merkel cells descend from the proliferative layer of skin epidermis; however, the stem cell niche for Merkel cell homeostasis has not been reported. Here, we provide genetic evidence for maintenance of mature Merkel cells during homeostasis by Krt17+ stem cells located in epidermal touch domes of hairy skin and in the tips of the rete ridges of glabrous skin. Lineage tracing analysis indicated that the entire pool of mature Merkel cells is turned over every 7-8 weeks in the adult epidermis and that Krt17+ stem cells also maintain squamous differentiation in the touch dome and in glabrous skin. Finally, selective genetic ablation of Krt17+ touch-dome keratinocytes indicates that these cells, and not mature Merkel cells, are primarily responsible for maintaining innervation of the Merkel cell-neurite complex.

Abstract 5012: Intestinal tumors are continuously renewed by Dclk1-positive tumor stem cells.

Abstract Backgrounds and aims Many studies have reported the existence of tumor stem cells (TSCs) in solid tumors including colorectal cancer, and targeting TSCs is believed to be a promising cancer therapy. However, previously reported TSC markers are often expressed in normal stem cells (NSCs), and using those markers for cancer therapy would severely affect the normal tissues. Therefore, it is necessary to identify the specific markers for TSCs. To identify a TSC-specific marker, we focused on doublecortin-like kinase 1 (Dclk1). Dclk1 was a putative marker of intestinal NSCs, but it has been debated whether Dclk1 marks NSCs or differentiated cells (e.g., Tuft cells). There have been no reports which elucidate the stemness of Dclk1-positive cells in the intestine and in tumors by lineage tracing. The aim of this study is to examine the role of Dclk1-positive cells in both the normal intestines and intestinal tumors. Methods We generated the knock-in mice with the creERT2-IRES-EGFP cassette under the Dclk1 promoter (Dclk1-creERT2 mice). These mice were crossed with Rosa26-LacZ mice, and the stemness of Dclk1-positive cells was examined by lineage tracing analyses in the intestines under normal homeostasis and experimental injury conditions (chemically-induced colitis and radiation-induced enteritis). Next, the stemness of Dclk1-positive tumor cells was also evaluated by lineage tracing in the polyps of ApcMin/+ mice. Character of Dclk1-positve tumor cells was investigated by immunohistochemistory and FACS analyses. Results In the normal intestines of Dclk1-creERT2; Rosa26-LacZ mice, scattered LacZ-labeled Dclk1-positive cells were observed 1 day after tamoxifen injection. However, these cells did not produce progeny, and disappeared within a few weeks. Stemness of Dclk1-positive cells was also strictly limited even under experimental injury conditions. On the contrary, lineage tracing in the polyps of ApcMin/+; Dclk1-creERT2; Rosa26-LacZ mice showed that Dclk1-positive tumor cells continuously produce tumor progeny and maintained the polyps over the prolonged period. Immunohistochemistory showed that a population of Dclk1-positive cells at the tumor base expressed proliferation markers and Lgr5. FACS analyses showed that the Dclk1- and Lgr5- double positive fraction showed the strongest expression of CD44, CD133, and Bcl-2 compared to other cell fractions. Conclusions Our lineage tracing experiments suggested that Dclk1 can be used as a marker which distinguishes TSCs from NSCs in the intestine. These results support the development of novel therapeutic strategies in colorectal cancer through the targeting Dclk1-positive TSCs. Citation Format: Yuki Nakanishi, Hiroshi Seno, Taro Ueo, Tsutomu Chiba. Intestinal tumors are continuously renewed by Dclk1-positive tumor stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5012. doi:10.1158/1538-7445.AM2013-5012

Abstract 5021: Temozolomide-induced conversion of glioma cells into glioma “stem-like” cells with enhanced invasion properties.

Abstract Glioblastoma multiforme patients present a poor prognosis due to therapeutic resistance and tumor recurrence after surgical resection. It has been suggested that gliomas are driven by a rare subset of tumor cells, known as cancer stem cells (CSCs). These tumor-initiating cells are considered to be responsible for sustaining long-term tumor growth and disease recurrence. Today, two popular hypotheses try to explain the origin of CSCs: one postulates that CSCs represent an adult stem cell population with tumorigenic alterations, while the second argues that CSCs were once differentiated tumor cells that have re-initiated a self-renewal program. In this study, we first show that exposure to the therapeutic dose of 5 μM (the peak concentration in patient's cerebral spinal fluid) or 50 μM (the concentration in patient's blood) of temozolamide (TMZ), the most commonly used alkylating agent to treat GBM patients, consistently increase the CSC pool over time both in vitro and in vivo. Second, lineage-tracing analysis of genetically tagged glioma cells suggests that TMZ induces a phenotypic shift in the non-CSC population to mimic that of CSCs. These newly formed CSC-like cells co-express markers of pluripotency and stemness (CD133, CD15, Sox-2, Oct-4 and Nestin), a classical characteristic of CSCs. We further show that intracranial implantation of TMZ-treated non-CSC cells in athymic nude mice result in more efficient grafting (100% grafting as compared to 60% for DMSO treated control group). Moreover, these TMZ-treated non-CSC xenografts demonstrate extremely invasive characteristics, which is similar to the CSC enriched xenografts. In addition, they express elevated levels of HIF2α, a critical factor for maintaining the CSC pool. These findings strongly corroborate that chemotherapy-induced selection pressure can facilitate a CSC specific ‘niche’, which may provide the necessary signals to induce an interconversion between differentiated tumor cells and cancer “stem like” cells, leading to a more infiltrative glioma phenotype thereby resulting in therapeutic relapse and may frustrate our attempts to develop effective anti-glioma therapy. Citation Format: Atique U. Ahmed, Brenda Auffinger, Alex Tobias, Bart Thaci, Yu Han, Maciej S. Lesniak. Temozolomide-induced conversion of glioma cells into glioma “stem-like” cells with enhanced invasion properties. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5021. doi:10.1158/1538-7445.AM2013-5021