Christian Beltinger

Endothelial progenitor cell culture and differentiation in vitro: a methodological comparison using human umbilical cord blood.

OBJECTIVEnEndothelial progenitor cells (EPC) can contribute to vascular repair and targeted tumour therapy. Little is known about generating EPC from human umbilical cord blood. We therefore compared methods for purification of EPC from human umbilical cord blood.nnnMETHODSnMononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation and used either unselected or after CD34 preselection. Unselected mononuclear cells were cultured for 9 days. Culture-dish-adherent (CDAC) and non-adherent (CDNAC) CD34+ cells were cultured separately for 4 weeks. Surface markers were assessed by immunofluorescence staining and FACS analysis.nnnRESULTSnIn unselected mononuclear cells, VEGF-R2 and VE-cadherin expression increased up to day 6. They stained positive with UEA-1 and took up acetylated LDL. Expression of CD45 and CD14 decreased over time, but remained strong. CD133 and CD34 were not expressed. CD34+-CDNAC acquired an endothelial phenotype over time with an increase of VEGFR-2 and von Willebrand factor (vWF). CD45 and CD14 decreased, while CD34 and the progenitor-cell marker CD133 remained strongly expressed. CD34(+)-CDAC showed a strong increase in VEGFR-2, CD133, CD34 and vWF, while CD14 decreased, and CD45 did not change.nnnCONCLUSIONnPutative EPC can be obtained from human umbilical cord blood. When selected for CD34, cells can be differentiated in culture to express markers of mature endothelial cells, while keeping progenitor markers. In contrast, short-term culture of unselected mononuclear cells leads to an endothelioid-monocytoid phenotype devoid of progenitor markers. Thus, the outgrowth from CD34-selected cells appears to be superior to short-term culture of unselected mononuclear cells with regard to endothelial cell-lineage specific differentiation of cells with a progenitor marker profile.

Embryonic endothelial progenitor cells armed with a suicide gene target hypoxic lung metastases after intravenous delivery

We show that mouse embryonic endothelial progenitor cells (eEPCs) home preferentially to hypoxic lung metastases when administered intravenously. This specificity is inversely related to the degree of perfusion and vascular density in the metastasis and directly related to local levels of hypoxia and VEGF. Ex vivo expanded eEPCs that were genetically modified with a suicide gene specifically and efficiently eradicated lung metastases with scant patent blood vessels. eEPCs do not express MHC I proteins, are resistant to natural killer cell-mediated cytolysis, and can contribute to tumor vessel formation also in nonsyngeneic mice. These results indicate that eEPCs can be used in an allogeneic setting to treat hypoxic metastases that are known to be resistant to conventional therapeutic regimes.

Caspase-8L expression protects CD34+ hematopoietic progenitor cells and leukemic cells from CD95-mediated apoptosis

Regulation of sensitivity or resistance for apoptosis by death receptor ligand systems is a key control mechanism in the hematopoietic system. Dysfunctional or deregulated apoptosis can potentially contribute to the development of immune deficiencies, autoimmune diseases, and leukemia. Control of homeostasis starts at the level of hematopoietic stem cells (HSC). To this end, we found that CD34+ hematopoietic progenitor cells are constitutively resistant to CD95-mediated apoptosis and cannot be sensitized during short-term culture to death receptor-mediated apoptosis by cytokines. Detailed analysis of the death machinery revealed that CD34+ cells do not express caspase-8a/b, a crucial constituent of the death-inducing signaling complex (DISC) of death receptors. Instead, we found a smaller splice variant termed caspase-8L to be present in HSC. Forced expression of caspase-8L using a recombinant lentiviral vector was able to protect hematopoietic cells from death receptor-induced apoptosis even in the presence of caspase-8a/b. Furthermore, we found that caspase-8L is recruited to the DISC after CD95 triggering, thereby preventing CD95 from connecting to the caspase cascade. These results demonstrate an antiapoptotic function of caspase-8L and suggest a critical role as apoptosis regulator in HSC. Similar to CD34+ HSC, stem cell-derived leukemic blasts from AML(M0) patients only expressed caspase-8L. Additionally we found, caspase-8L expression in several AML and ALL samples. Thus, caspase-8L expression might explain constitutive resistance to CD95-mediated apoptosis in CD34+ progenitor cells and might participate in the development of stem cell-derived and other leukemias by providing protection from regulatory apoptosis.

Murine models for experimental therapy of pediatric solid tumors with poor prognosis

Novel therapeutic strategies are required for pediatric solid tumors with poor prognosis such as metastasizing neuroblastoma, rhabdomyosarcoma and Ewings sarcoma. A prerequisite for the development of such new therapies is the availability of murine models. To be useful for therapeutic studies, these models should not only recapitulate the genetic alterations characteristic of the human disease but should also mimic the metastatic process and the response to current therapy, both of which ultimately determine the fate of children with these tumors. This review scrutinizes the utility of existing murine models of neuroblastoma, rhabdomyosarcoma and Ewings sarcoma for investigating novel therapies. Much experience has been gained with both syngeneic and xenogeneic transplantable models of these tumors, while transgenic and knockout mice are just beginning to be available for therapeutic investigations. Modeling the genetic aberrations characterizing these tumors may provide faithful models for therapeutic studies in the future.

N-myc augments death and attenuates protective effects of Bcl-2 in trophically stressed neuroblastoma cells.

N-myc has proapoptotic functions, yet it acts as an oncogene in neuroblastoma. Thus, antiapoptotic mechanisms have to be operative in neuroblastoma cells that antagonize the proapoptotic effects of N-myc. We conditionally activated N-myc in SH-EP neuroblastoma cells subjected to the trophic stress of serum or nutrient deprivation while changing the expression of Bcl-2, survivin and FLIPL, antiapoptotic molecules often overexpressed in poor prognosis neuroblastomas. Bcl-2 protected SH-EP cells from death during nutritional deprivation by activating energetically advantageous oxidative phosphorylation. N-myc overrode the metabolic protection provided by Bcl-2-induced oxidative phosphorylation by reestablishing the glycolytic phenotype and attenuated the antiapoptotic effect of Bcl-2 during metabolic stress. Survivin partially antagonized the growth suppressive function of N-myc in SH-EP neuroblastoma cells during serum deprivation whereas FLIPL did not. These findings advance our understanding of the functions of N-myc in neuroblastoma cells.

Growth inhibition of murine neuroblastoma cells by c-myc with cell cycle arrest in G2/M.

c-myc and N-myc belong to the myc family of proteins that plays an important role in cell proliferation, differentiation and apoptosis. The N-myc gene is amplified in aggressive neuroblastoma and c-myc is overexpressed in many lymphomas and cancers. However, c-myc has not been implied in tumorigenesis or progression of neuroblastoma. We therefore investigated the so far unknown effects of c-myc overexpression on the aggressiveness of neuroblastoma cells with single copy N-myc. c-myc overexpression in serum-deprived murine NXS2 neuroblastoma cells led to cell cycle progression and massive apoptosis, causing a net decrease of viable cells. In serum-replete medium c-myc caused NXS2 cells to arrest in G2/M. Furthermore, c-myc decreased clonogenic growth of neuroblastoma cells. Taken together, these data suggest that c-myc attenuates the malignant phenotype of NXS2 neuroblastoma cells. Thus, although c-myc increased NXS2 tumor mass in vivo, c-myc appears to have decreased malignant potency in neuroblastoma cells compared to N-myc. This may be one reason why c-myc does not play a role in neuroblastomagenesis.

Enriching suicide gene bearing tumor cells for an increased bystander effect.

The success of cancer gene therapies requiring in vivo gene transfer is severely hampered by the low efficacy of gene transfer, which has been difficult to improve. We therefore established a novel strategy to increase the share of transduced cells post gene transfer. We hypothesized that in vivo selection of tumor cells transduced with a suicide gene effectively enriches these cells within a tumor, thus allowing for an increased bystander effect after the prodrug is given, leading to enhanced eradication of tumor cells. We reasoned that in vivo enrichment should be achieved by exploiting the metabolism of the suicide gene product. For this ‘enrichment–eradication’ strategy we chose a fusion gene of cytosine deaminase and uracil phosphoribosyl transferase. Positive selection (enrichment) was to be achieved by concurrently giving N-(phosphonacetyl)-L-aspartate, an inhibitor of pyrimidine de novo synthesis, which leads to pyrimidine depletion-mediated death of non-transduced cells, and cytosine, to rescue fusion gene expressing cells via the pyrimidine salvage pathway. Negative selection (eradication) was to be induced by giving the prodrug 5-fluorocytosine. Indeed, murine NXS2 neuroblastoma cells transduced with the fusion gene were effectively enriched in vitro, leading to a near-complete bystander effect. In vivo enrichment–eradication of NXS2 cells led to decreased tumor growth. This proof-of-principle study shows that enrichment–eradication may compensate the effects of low in vivo gene transfer efficacy, a major obstacle in cancer gene therapy.

Cytosine deaminase/5-fluorocytosine gene therapy and Apo2L/TRAIL cooperate to kill TRAIL-resistant tumor cells

The death ligand Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand) eradicates many tumor types while sparing most normal tissues. However, some tumors are resistant to TRAIL. We therefore determined if TRAIL cooperates with cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and investigated the mechanisms involved. Transfection of human LAN-5 neuroblastoma cells with CD rendered the cells (LAN-5-CD) sensitive to 5-FC-induced, caspase-dependent apoptosis. Mediated by caspase-3, CD/5-FC and TRAIL cooperated to induce apoptosis in these TRAIL-resistant cells and to cleave X-linked inhibitor of apoptosis protein (XIAP). In established LAN-5-CD tumors growing subcutaneously in mice, intratumorally applied TRAIL did not decrease tumor growth and systemically administered 5-FC only attenuated tumor growth. In contrast, 5-FC together with TRAIL dramatically decreased tumor growth and eradicated a tumor. Assuming sufficient gene transfer of CD in situ, CD/5-FC with TRAIL may be useful for the therapy of tumors resistant to TRAIL.

Apoptosis-Inducing Cellular Vehicles for Cancer Gene Therapy

Endothelial progenitor cells (EPCs) and neural progenitor cells (NPCs) are promising for cancer therapy because they specifically target tumors. They have the capacity to home to, invade, migrate within, and incorporate into tumor structures. They are easily expanded and can be armed with therapeutic payloads protected within the progenitor cells. Once in the tumor, armed progenitors can be triggered to induce apoptosis in surrounding tumor cells. Pro-and antiapoptotic mechanisms are pivotal to effectively kill tumor cells while simultaneously protecting the cellular vehicles from premature demise. Increasing the ratio of tumor cell apoptosis to progenitor apoptosis will be crucial among other efforts to enhance the efficacy of endothelial and neural progenitor cells to a level sufficient for clinical application.

Abstract 3512: MYCN and survivin cooperatively contribute to malignant transformation of fibroblasts

The oncogenes MYCN and survivin (BIRC5) maintain aggressiveness of diverse cancers including sarcomas. To investigate whether these oncogenes cooperate in initial malignant transformation, we transduced them into Rat-1 fibroblasts. Indeed, survivin enhanced MYCN-driven contact-uninhibited and anchorage-independent growth in vitro. Importantly, upon subcutaneous transplantation into mice, cells overexpressing both instead of either one of the oncogenes generated tumors with shortened latency, marked anaplasia and an increased proliferation-to-apoptosis ratio resulting in accelerated growth. Mechanistically, the increased tumorigenicity was associated with an enhanced Warburg effect and HIF1α-linked vascular remodeling. This cooperation between MYCN and survivin may be important in the genesis of several cancers. Citation Format: Nora Hipp, Lisa Christner, Thomas Wirth, Wolfgang Mueller-Klieser, Stefan Walenta, Evelin Schrock, Klaus-Michael Debatin, Christian Beltinger. MYCN and survivin cooperatively contribute to malignant transformation of fibroblasts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3512. doi:10.1158/1538-7445.AM2014-3512