Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

Medical university of innsbruck

Tyrolean Cancer Research Institute (TKFI)

The objective of this study was to analyze the mobilization kinetics of normal (BCR-ABL(neg)) and malignant (BCR-ABL(pos)) progenitor cells using a new, low toxic, out-patient-based mobilization regimen for Philadelphia chromosome-positive (Ph(pos)) chronic myelogenous leukemia (CML) patients. High doses of hydroxyurea (HD-HU, 3.5 g/m(2) per day, orally for 7 days) followed by granulocyte colony-stimulating factor (G-CSF) (10 microg/kg subcutaneously) were administered to 11 newly diagnosed CML patients. Each apheresis product (n = 30) was individually analyzed for the number and genotype of mature colony-forming cells (CFC) and primitive long-term culture initiating cells (LTC-IC), respectively, by reverse transcription polymerase chain reaction (RT-PCR) of individual colonies. Sufficient numbers of CD34(+) cells/kg bodyweight (BW) could easily be obtained in all patients (median, 15 x 10(6)/kg BW per patient) with a median number of three aphereses performed per patient (range 2-4). Almost each apheresis itself (25/30) contained > or =2 x 10(6) CD34(+) cells/kg BW. All patients with low and intermediate Sokal risk indices (9/11) mobilized primarily BCR-ABL(neg) LTC-IC (median 92%, range 47-100) and CFC (median 89%, range 57-100). Moreover, the mean percentage of BCR-ABL(neg) CFC and LTC-IC in the various apheresis products in these patients did not change throughout the entire time of hematopoietic regeneration. The toxicity of the mobilization procedure was low. Side effects were mild erythema in 8/11 and oral mucositis in 3/11 patients. Overall, the low toxicity of this regimen, together with the fact that sufficient BCR-ABL(neg) progenitors can be collected throughout the entire period of hematopoietic regeneration, renders this mobilization regimen particularly attractive for the collection of BCR-ABL(neg) progenitors in early chronic phase of Ph(pos) CML.

High-dose hydroxyurea plus G-CSF mobilize BCR-ABL-negative progenitor cells (CFC, LTC-IC) into the blood of newly diagnosed CML patients at any time of hematopoietic regeneration.

Hematopoietic stem cell grafts from unrelated donors are commonly transported by aircraft. They must not be subjected to x-rays during security checks, which may cause inconvenient discussions between the courier and the airport security staff. We exposed hematopoietic stem cells from mobilized peripheral blood to a widely used x-ray hand-luggage control system. Cell viability as well as growth in vitro of mature progenitor cells (colony-forming cells), primitive progenitor cells (long-term culture-initiating cells), and lymphocytes were not altered even after 10 passages through the hand-luggage control system. Thus, repeated exposure to the low radiation dose of hand-luggage control systems (1.5 +/- 0.6 microSv per exposure) seems to be harmless for hematopoietic stem cells, which should simplify the international transport of stem cell grafts.

Breaking the rules? X-ray examination of hematopoietic stem cell grafts at international airports.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions supporting the expansion of transplantable hematopoietic stem cells. We assessed the expansion potential of CD34+CD71-CD45- cells derived from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood under recently defined serum-free culture conditions. The CD34+CD71-CD45- cells in mobilized peripheral blood were found to contain the majority (92%+/-5.6) of primitive long-term culture initiating cells (LTCIC) and 53.5%+/-16.7 of the more committed colony-forming cells (CFC). Furthermore, this population represents 23.3%+/-4.1 of the total CD34+ cells and allows reduction of the cell density important for maintenance/expansion of primitive progenitor cells. CD34+ CD71- CD45- cells were cultured in defined serum-free media supplemented with 300 ng each of Flt-3 ligand and stem cell factor (SCF), 60 ng of interleukin (IL)-3, and 20 ng each of IL-6 and G-CSF. Mononuclear cells (MNC) and CFC were expanded 50-fold and 200-fold, respectively; primitive progenitor cells (LTC-IC) were maintained at input values after a total of 10 days of expansion. The addition of IL-15 to our cytokine cocktail expanded LTC-IC 2- to 3-fold and CFC to >500-fold. The data presented should allow clinical manipulation (purging) and expansion procedures with mobilized PBPC harvests without the loss of primitive progenitor cells and could be made applicable for large-scale clinical expansion.

Expansion of mobilized peripheral blood progenitor cells under defined culture conditions rsing CD34+CD71-CD45- cells as a starting population.

Induction of peripheral tolerance can be facilitated when the antigen is linked to the B subunit of cholera toxin (CTB), an efficient mucosal carrier. In the present study, a genetic fusion molecule of Bet v 1 and CTB was produced to test whether mucosal application of this construct would lead to suppression of Th2 responses. Intranasal pretreatment of BALB/c mice with rCTB-Bet v 1 prior to allergic sensitisation with the allergen significantly decreased IgE but markedly increased allergen-specific IgG2a levels in sera as well as IFN-gamma production of splenocytes. This Th1 shift was supported by an increased T-bet/GATA3 mRNA ratio. IL-5 production within the airways was suppressed after the pretreatment with rCTB-Bet v 1, while local allergen-specific IgA antibodies were markedly enhanced by pretreatment with the construct. Upregulation of Foxp3, IL-10 and TGF-beta mRNA expression was detected in splenocytes after pretreatment with unconjugated allergen but not with the fusion molecule, indicating that antigen conjugation to a mucosal carrier modifies the immunomodulating properties of an antigen/allergen.

Use of a genetic cholera toxin B subunit/allergen fusion molecule as mucosal delivery system with immunosuppressive activity against Th2 immune responses.

The armamentarium of anti-leukemic drugs has increased substantially since anti-leukemic activities were recently found for a variety of non-classical cytostatic drugs, among them the histone deacetylase (HDAC) inhibitor valproic acid (VPA). This study investigated the effect of VPA on proliferation and apoptosis of human Philadelphia chromosome-positive (Ph+) acute lymphatic (ALL) and chronic myeloid leukemia (CML) cells and on colony formation of human chronic-phase CML progenitor cells. Strong anti-proliferative and pro-apoptotic effects of VPA were observed on human ALL and CML cell lines at concentrations achievable in vivo. These effects were most pronounced in ALL cell lines as well as in primary ALL cells. Notably, VPA revealed enhanced activity with imatinib mesylate, nilotinib, the farnesyl transferase inhibitor SCH66336, interferon-alpha and cytosine arabinoside. VPA inhibited the growth of colony-forming cells from 12 Ph+ chronic-phase CML patients but also of those from normal healthy controls in a dose-dependent fashion. HDAC-inhibiting activity of VPA was confirmed on ALL and CML cells. In conclusion, VPA, whether alone or in combination with other non-classical anti-leukemic compounds, exerts significant anti-leukemic effects on human ALL and CML cells.

Anti-leukemic activity of valproic acid and imatinib mesylate on human Ph+ ALL and CML cells in vitro.

The IgE-mediated and Th2-dependent late-phase reaction remains a mechanistically enigmatic and daunting element of human allergic inflammation. In this study, we uncover the FcεRI on dendritic cells (DCs) as a key in vivo component of this form of allergy. Because rodent, unlike human, DCs lack FcεRI, this mechanism could be revealed only by using a new transgenic mouse model with human-like FcεRI expression on DCs. In the presence of IgE and allergen, FcεRI(+) DCs instructed naive T cells to differentiate into Th2 cells in vitro and boosted allergen-specific Th2 responses and Th2-dependent eosinophilia at the site of allergen exposure in vivo. Thus, FcεRI on DCs drives the cascade of pathogenic reactions linking the initial allergen capture by IgE with subsequent Th2-dominated T cell responses and the development of late-phase allergic tissue inflammation.

High-affinity IgE receptors on dendritic cells exacerbate Th2-dependent inflammation.

Among birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery) or Dau c 1 (carrot), termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy.

Prevention of birch pollen-related food allergy by mucosal treatment with multi-allergen-chimers in mice.

The hygiene hypothesis implies that microbial agents including probiotic bacteria may modulate foetal/neonatal immune programming and hence offer effective strategies for primary allergy prevention; however their mechanisms of action are poorly understood. We investigated whether oral administration of Lactobacillus paracasei NCC 2461 to mothers during gestation/lactation can protect against airway inflammation in offspring in a mouse model of birch pollen allergy, and examined the immune mechanisms involved.

Perinatal maternal administration of Lactobacillus paracasei NCC 2461 prevents allergic inflammation in a mouse model of birch pollen allergy.

The Indianmeal moth Plodia interpunctella is a highly prevalent food pest in human dwellings, and has been shown to contain a number of allergens. So far, only one of these, the arginine kinase (Plo i 1) has been identified.

Thioredoxin from the Indianmeal moth Plodia interpunctella: cloning and test of the allergenic potential in mice.

One third of the human population is currently infected by one or more species of parasitic helminths. Certain helminths establish long-term chronic infections resulting in a modulation of the host's immune system with attenuated responsiveness to "bystander" antigens such as allergens or vaccines. In this study we investigated whether parasite-derived products suppress the development of allergic inflammation in a mouse model. We show that extract derived from adult male Oesophagostomum dentatum (eMOD) induced Th2 and regulatory responses in BALB/c mice. Stimulation of bone marrow-derived dendritic cells induced production of regulatory cytokines IL-10 and TGF-beta. In a mouse model of birch pollen allergy, co-administration of eMOD with sensitizing allergen Bet v 1 markedly reduced the production of allergen-specific antibodies in serum as well as IgE-dependent basophil degranulation. Furthermore, eMOD prevented the development of airway inflammation, as demonstrated by attenuation of bronchoalveolar lavages eosinophil influx, peribronchial inflammatory infiltrate, and mucus secretion in lungs and IL-4 and IL-5 levels in lung cell cultures. Reduced secretion of Th2-related cytokines by birch pollen-re-stimulated splenocytes and mesenteric lymph node cells was observed in eMOD-treated/sensitized and challenged mice in comparison to sensitized and challenged controls. The suppressive effects of eMOD were heat-stable. Immunization with model antigens in the presence of eMOD reduced production of antibodies to thymus-dependent but not to thymus-independent antigen, suggesting that suppression of the immune responses by eMOD was mediated by interference with antigen presenting cell or T helper cell function but did not directly suppress B cell function. In conclusion, we have shown that eMOD possesses immunomodulatory properties and that heat-stable factors in eMOD are responsible for the dramatic suppression of allergic responses in a mouse model of type I allergy. The identification and characterization of parasite-derived immune-modulating molecules might have potential for designing novel prophylactic/therapeutic strategies for immune-mediated diseases.

Elisabeth Hoflehner

Internal Medicine V

Medical university of innsbruck

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Elisabeth Hoflehner

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Internal Medicine V

Medical university of innsbruck

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Internal Medicine V