Francisco M. Barriga

The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse

A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse.

Isolation and in vitro expansion of human colonic stem cells.

Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.

Colon cancer cells colonize the lung from established liver metastases through p38 MAPK signalling and PTHLH

The mechanisms that allow colon cancer cells to form liver and lung metastases, and whether KRAS mutation influences where and when metastasis occurs, are unknown. We provide clinical and molecular evidence showing that different MAPK signalling pathways are implicated in this process. Whereas ERK2 activation provides colon cancer cells with the ability to seed and colonize the liver, reduced p38 MAPK signalling endows cancer cells with the ability to form lung metastasis from previously established liver lesions. Downregulation of p38 MAPK signalling results in increased expression of the cytokine PTHLH, which contributes to colon cancer cell extravasation to the lung by inducing caspase-independent death in endothelial cells of the lung microvasculature. The concerted acquisition of metastatic traits in the colon cancer cells together with the sequential colonization of liver and lung highlights the importance of metastatic lesions as a platform for further dissemination.

Isolation of Human Colon Stem Cells Using Surface Expression of PTK7

Summary Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs).

Conserved Mechanisms of Tumorigenesis in the Drosophila Adult Midgut

Whereas the series of genetic events leading to colorectal cancer (CRC) have been well established, the precise functions that these alterations play in tumor progression and how they disrupt intestinal homeostasis remain poorly characterized. Activation of the Wnt/Wg signaling pathway by a mutation in the gene APC is the most common trigger for CRC, inducing benign lesions that progress to carcinomas due to the accumulation of other genetic alterations. Among those, Ras mutations drive tumour progression in CRC, as well as in most epithelial cancers. As mammalian and Drosophilas intestines share many similarities, we decided to explore the alterations induced in the Drosophila midgut by the combined activation of the Wnt signaling pathway with gain of function of Ras signaling in the intestinal stem cells. Here we show that compound Apc-Ras clones, but not clones bearing the individual mutations, expand as aggressive intestinal tumor-like outgrowths. These lesions reproduce many of the human CRC hallmarks such as increased proliferation, blockade of cell differentiation and cell polarity and disrupted organ architecture. This process is followed by expression of tumoral markers present in human lesions. Finally, a metabolic behavioral assay shows that these flies suffer a progressive deterioration in intestinal homeostasis, providing a simple readout that could be used in screens for tumor modifiers or therapeutic compounds. Taken together, our results illustrate the conservation of the mechanisms of CRC tumorigenesis in Drosophila, providing an excellent model system to unravel the events that, upon mutation in Apc and Ras, lead to CRC initiation and progression.

Bone marrow stromal cells modulate mouse ent1 activity and protect leukemia cells from cytarabine induced apoptosis

Background Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells. Methods Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of 3H-adenosine. Results Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used. Conclusion The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML.

Hematopoietic stem cell transplantation: clinical use and perspectives

Hematopoietic stem cell transplantation is the accepted therapy of choice for a variety of malignant and non-malignant diseases in children and adults. Initially developed as rescue therapy for a patient with cancer after high doses of chemotherapy and radiation as well as the correction of severe deficiencies in the hematopoietic system, it has evolved into an adoptive immune therapy for malignancies and autoimmune disorders. The procedure has helped to obtain key information about the bone marrow environment, the biology of hematopoietic stem cells and histocompatibility. The development of this new discipline has allowed numerous groups working around the world to cure patients of diseases previously considered lethal. Together with the ever growing list of volunteer donors and umbilical cord blood banks, this has resulted in life saving therapy for thousands of patients yearly. We present an overview of the procedure from its cradle to the most novel applications, as well as the results of the HSC transplant program developed at our institution since 1989.

Iro/IRX transcription factors negatively regulate Dpp/TGF‐β pathway activity during intestinal tumorigenesis

Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal co‐activation of these pathways in the adult Drosophila midgut induces “tumor‐like” overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF‐β acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX‐family‐protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF‐β. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF‐β. In addition, Irx5 expression confers a growth advantage in the presence of TGF‐β, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF‐β activity. We propose that during the characteristic adenoma‐to‐carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF‐β, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF‐β pathway components.

Uso de desmopresina en niños con disfunción plaquetaria congénita sometidos a adeno y/o amigdalectomía

INTRODUCTION AND GOALS Adenotonsillar surgery represents a major haemostatic challenge in paediatric patients with mild inherited platelet dysfunction. While there are recommendations for perioperative haemostatic management, there are no reports of the outcomes with the different recommendations in these children when undergoing adenotonsillectomy. Our objective was to evaluate the management of perioperative bleeding with desmopressin in children with mild platelet dysfunctions who underwent adenotonsillar surgery in our hospital. METHODS We performed a retrospective study aimed at determining the perioperative bleeding and complication rate in children with mild inherited platelet dysfunction in whom desmopressin was used while undergoing adenotonsillar procedures. RESULTS Between 2004 and 2010, 27 children with mild inherited platelet dysfunction underwent adenotonsillar procedures in our hospital and were treated with desmopressin. One patient developed perioperative bleeding (3.7%) and there was 1 child (3.7%) who presented transitory hypotension as a side effect of desmopressin. CONCLUSIONS The use of desmopressin allowed adequate perioperative bleeding prophylaxis management in children with mild inherited platelet dysfunction who underwent adenotonsillar procedures without presenting severe complications.

t(1;5)(q23;q33) in a patient with high-risk B-lineage acute lymphoblastic leukemia

The t(1;5)(q23;q33) is a rare genetic anomaly that was reported previously in two infants with a myeloproliferative disorder and eosinophilia and in one adult patient with acute nonlymphocytic leukemia (ANLL). A 13-year-old boy with high-risk early pre-B acute lymphoblastic leukemia (ALL) who presented to our institution carried the t(1;5)(q23;q33). He had an initial blast count of 230 X 10(9)/L and responded poorly to prednisone. Complete remission (CR) was achieved, and he had a bone marrow (BM) relapse 3 months after despite intensive consolidation therapy. He underwent allogeneic BM transplantation (BMT) from a human leukocyte antigen (HLA)-identical siblings in early relapse with total body irradiation (TBI) and cyclophosphamide conditioning. He had a short second CR with a central nervous system (CNS) relapse on day + 106 after BMT. Two of the previously reported patients also did not respond to chemotherapy. The t(1;5)(q23;q33) appears to be a rare lineage nonspecific anomaly related to hematologic malignancies that are resistant to current therapy.