Production of VEGF165 by Ewing's sarcoma cells induces vasculogenesis and the incorporation of CD34+ stem cells into the expanding tumor vasculature

The Ewing's sarcoma cell line TC71 overexpresses vascular endothelial growth factor isoform 165 (VEGF165), a potent proangiogenic molecule that induces endothelial cell proliferation, migration, and chemotaxis. CD34+ bone marrow stem cells can differentiate into endothelial and hematopoietic cells. We used a transplant model to determine whether CD34+ cells migrate from the bone marrow to Ewing's sarcoma tumors and participate in the neovascularization process that supports tumor growth. We also examined the role of VEGF165 in CD34+ cell migration. Human umbilical cord CD34+ cells were transplanted into sublethally irradiated severe combined immunodeficient mice. Seven days later, the mice were injected subcutaneously with TC71 tumor cells. Tumors were excised 2 weeks later and analyzed by immunohistochemistry. The tumor sections expressed both human VE-cadherin and mouse CD31, indicating involvement of donor-derived human cells in the tumor vessels. To determine the role of VEGF165 in the chemoattraction of CD34+ cells, we generated two VEGF165-deficient TC71 clones, a stable anti-sense VEGF165 cell line (Clone 17) and a VEGF165 siRNA-inhibited clone (TC/siVEGF(7-1)). The resulting VEGF165-deficient tumor cells had normal growth rates in vitro, but had delayed growth when implanted into mice. Immunohistochemical analysis revealed decreased infiltration of CD34+ cells into both VEGF165-deficient tumors. These data show that bone marrow stem cells contribute to the growing tumor vasculature in Ewing's sarcoma and that VEGF165 is critical for the migration of CD34+ cells from the bone marrow into the tumor.     

Sequential analysis of CD34+ and CD34- cell subsets in peripheral blood and leukapheresis products from breast cancer patients mobilized with SCF plus G-CSF and cyclophosphamide.

Administration of stem cell factor (SCF) has been proven to enhance cytokine-induced mobilization of CD34+ hematopoietic progenitor cells (HPC) into the peripheral blood (PB). The aim of the present study was to explore in a homogeneous group of 22 uniformly treated breast cancer patients: (1) the kinetics of mobilization into PB of both CD34+ and CD34- cell subsets, including dendritic cells, in sequential samples obtained from day +7 up to day +12 after mobilization; and (2) the composition of the CD34+ and CD34- cell subsets present in the two leukapheresis products obtained for each patient. The following CD34+ and CD34- subsets were analyzed: early CD34+ HPC, erythroid-, myeloid- and B-lymphoid-committed CD34+ precursor cells, mature T, B and NK cells, monocytes, neutrophils, eosinophils, basophils, and dendritic cells (DC) including three subsets of lin-/HLADR+DC (CD16+, CD33high and CD123high). Our results show that the absolute number of PB CD34+ HPC progressively increases from day +7 onwards. As far as the CD34- PB leukocyte subsets are concerned, monocytes (CD14+) displayed the earliest recovery after mobilization predicting neutrophil recovery 1 day in advance. The number of CD34+ HPC collected in a single leukapheresis product was always > or = 1.4 x 10(6) cells/kg body weight. No significant changes were observed between the two leukapheresis sessions either as regards their composition in CD34+ HPC subsets or their CD34- leukocyte populations except for a higher ratio of both CD34+ erythroid/CD34+ myeloid HPC (0.35 +/- 0.13 vs 0.30 +/- 0.13; P = 0.04) and neutrophils/monocytes (1.58 +/- 2.1 vs 0.69 +/- 0.27; P = 0.009) found for the first leukapheresis. Interestingly, the overall number of dendritic cells (DC) was higher in the second leukapheresis (1.06 +/- 0.56 vs 1.9 +/- 0.46; P = 0.02) due to a selective increase of the CD16+ antigen-presenting cells. In summary, our results show that the combination of cyclophosphamide, G-CSF and SCF is highly effective for stem cell mobilization, with differences observed in the mobilization kinetics of the different hematopoietic cell subsets analyzed.     

Lymphatic and vascular origin of Kaposi's sarcoma spindle cells during tumor development

The histogenesis of Kaposi's sarcoma (KS) tumor spindle cells (SC) remains controversial but several immunohistochemical studies favor a lymphatic origin. Twenty KS surgical biopsies were analyzed for the coexpression of LANA, CD34, LYVE-1, D2-40, VEGFR-2, VEGFR3 by using double or triple immunostaining. Most of the SC in both early and late KS expressed the lymphatic markers LYVE-1, D2-40 and VEGFR-3 and the blood vascular endothelial/endothelial precursor cell markers CD34 and endothelial stem cell marker VEGFR-2. All the LANA+ SC in early and late KS were LYVE-1+, but only 75% of these LANA+ cells were CD34(+). The CD34(+)/LANA+ cells increased from early- (68.8%) to late-stage KS (82.2%). However, approximately 18% of the LANA+ SC in early KS were CD34(-) but were LYVE-1+, suggesting that resident lymphatic endothelial cells (LEC) are targeted for primary infection by human herpesvirus-8. This LANA+/LYVE-1+/CD34(-) (resident LEC) cell population clearly decreased during the development of KS from early (18.7%) to late KS (2.9%). Thus, in late stages of KS, most SC were LANA+/CD34(+)/LYVE-1+. However, in both early- and late-stage KS, approximately 18% of the SC were CD34(+)/LANA-/LYVE-1 -- and could represent newly recruited endothelial precursor cells, which become infected in the lesion and eventually undergo a phenotype switch expressing LEC markers. Our study apparently indicates that KS represents a unique variant of tumor growth with continues recruitment of tumor precursor cells as well as proliferation and decreased apoptosis of SC.     

Antitumor activity of a monoclonal antibody against CD47 in xenograft models of human leukemia

The ligation of CD47 induces the apoptosis of leukemic cells in a caspase-independent manner. We generated a monoclonal antibody against CD47 (mAb-MABL) that possibly induced apoptosis from the ligation of CD47 in CCRF-CEM and JOK-1 cells in vitro. To confirm whether the ligation of CD47 caused cell death in vivo, we examined the antitumor activity of F(ab')2 of mAb-MABL in two xenograft models: The acute lymphoblastic leukemia (CCRF-CEM) and the B-cell chronic lymphocytic leukemia (JOK-1) cell line. Furthermore, in order to clarify the apoptotic activity selective for the tumor cells, we examined F(ab')2 of mAb-MABL apoptotic effects on CD34+ hematopoietic progenitor/stem and human endothelial cells. Male SCID mice were intravenously injected with CCRF-CEM (5 x 10(6) cells/mouse) or JOK-1 cells (5 x 10(6) cells/mouse) and intraperitoneally with JOK-1 cells (2 x 10(7) cells/mice). After the implantation of the cells, the mice were intravenously administered the vehicle or the F(ab')2 fragment of mAb-MABL at several doses and the length of survival was measured. F(ab')2 of mAb-MABL markedly prolonged the survival of mice transplanted with CCRF-CEM and JOK-1. Significantly, 40% of the mice intraperitoneally injected with JOK-1 cells became tumor-free when administered F(ab')2 of mAb-MABL, whereas even a high dose of fludarabine only slightly prolonged the median survival time. On the contrary, F(ab')2 of mAb-MABL showed no apoptotic effect on CD34+ hematopoietic progenitor/stem or human endothelial cells. Thus, monoclonal antibodies that cause cell death from the ligation of CD47 could be novel therapeutic agents for incurable leukemia after further optimization such as humanization or making single chain diabodies.     

CD44v10 in hematopoiesis and stem cell mobilization

Transplantation of hematopoietic progenitor cells provides in many instances of malignant tumors an ultimate chance of curative therapy, whereby the transfer of peripheral blood stem cells (PBSC) may even be advantageous as compared to bone marrow cells. Yet, the transfer of PBSC requires mobilization of stem cells into the periphery, which is mostly achieved via hematopoietic growth factors like G-CSF. Although G-CSF has been found to efficiently mobilize stem cells in most instances, some patients do not or insufficiently respond to G-CSF treatment In addition, G-CSF treatment may by accompanied by maturation of the most primitive progenitors and this may have an impact on stem cell homing and recovery of hemopoiesis. Therefore, additional approaches for stem cell mobilization have been searched for, in particular mobilization via a blockade of an adhesion molecule expressed by CD34-positive cells, like VLA-4 (CD49d) and the hematopoietic isoform of CD44 (CD44s). We recently described that in the mouse one of the CD44 variant isoforms, CD44v10, is expressed on a subpopulation of bone marrow cells, whereas a CD44v10 receptor-globulin only binds to stromal elements. These features appeared promising for anti-CD44v10 as a means of stem cell mobilization. Indeed, treatment with anti-CD44v10 revealed promising results concerning the recovery of multilineage colony forming units in the spleen and the peripheral blood. We here summarize features of expression and function of CD44 in hematopoiesis an provide further evidence for anti-CD44v10 as a means to mobilize hematopoietic progenitor cells.     

超高CD34+采集的动员方案序贯二次自体造血干细胞移植治疗难治性 霍奇金淋巴瘤

目的:探讨超高CD34+采集的动员方案后序贯二次自体造血干细胞移植治疗难治性霍奇金淋巴瘤的疗效和安全性。方法:对1例经过多疗程一线、二线、新药、免疫等均难治的霍奇金淋巴瘤患者,予以IA+C方案化疗+G-CSF动员干细胞后采集出超高水平CD34+细胞,之后行自体造血干细胞移植,移植后获得完全缓解,再予序贯第二次自体造血干细胞移植进行巩固治疗。结果:总计输注单个核细胞数13.67×108/kg,CD34+细胞48.68×106/kg,第一次自体造血干细胞移植术后第7天造血功能恢复,复查全身PET-CT提示获得完全缓解,第二次自体造血干细胞移植术后第8天造血功能恢复,两次自体造血干细胞移植相关并发症均在可控范围内。结论:超高CD34+细胞采集的IA+C方案化疗+G-CSF动员可以让患者有机会进行多次自体造血干细胞移植,是临床动员的创新方案。对于难治性霍奇金淋巴瘤,序贯二次自体造血干细胞移植可达到更深层次缓解,且安全性较高,延长患者无疾病生存期及总生存期,为难治性霍奇金淋巴瘤治疗提供更多临床依据。     

人脑胶质瘤新生血管的细胞来源与血液流通功能的初步评价

目的 探讨胶质瘤中部分新生血管是否源于胶质瘤干细胞转分化,并初步评价其血液运输功能.方法 将SHG44人脑胶质瘤细胞系接种于30只NC裸小鼠皮下和20只NC裸小鼠脑内尾状核,将SU-2细胞接种于12只表达绿色荧光蛋白的NC/C57BL/6J-绿色荧光蛋白裸小鼠的尾状核内.取移植瘤组织,石蜡切片后,分别进行CD34-PAS双染及CD31、CD34、CD133、胶质纤维酸性蛋白(GFAP)、人类白细胞抗原(Ki67)、HLA免疫组化染色,并结合HLA免疫荧光共聚焦显微成像对移植瘤血管进行分类,对肿瘤血管起源细胞进行鉴定.每组取3只荷瘤鼠,以活性炭颗粒混悬液行心脏灌注,对移植瘤切片进行相应的免疫组化染色,并在光镜下观察炭颗粒在肿瘤血管中的分布.结果 裸小鼠移植瘤中,同时存在CD34-PAS双阳性的内皮细胞依赖型血管、CD34ˉPAS+的血管生成拟态和由肿瘤细胞与CD34+细胞嵌合而成的马赛克血管,炭颗粒随机分布在这些血管腔内.在人脑胶质瘤于细胞移植瘤中,CD31+细胞依附于血管壁腔侧面;CD34+细胞亦沿血管分布,但在形态上介于肿瘤细胞与内皮细胞之间;在血管壁上可见HLA+和人Ki67+细胞,提示构成血管壁的这些细胞来自人源细胞.HLA免疫荧光共聚焦显微成像可见,移植瘤中的血管以表达HLA(红色)的人源肿瘤细胞为主,兼有表达GFP(绿色)的宿主细胞,或人鼠融合细胞.结论 在移植性人脑胶质瘤组织中存在具有血液运输功能的内皮细胞依赖型血管、血管生成拟态和马赛克血管3类血管,人脑胶质瘤干祖细胞有通过分化和转分化机制自主形成后两类肿瘤血管的潜能.     

Comparison of lenograstim vs filgrastim administration following chemotherapy for peripheral blood stem cell (PBSC) collection: a retrospective study of 126 patients

Mobilization techniques for peripheral blood stem cell (PBSC) collection include the administration of chemotherapy followed by hematopoietic growth factors or growth factors alone. Two forms of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are available for PBSC mobilization: lenograstim and filgrastim which are the glycosylated and non-glycosylated forms respectively. In order to determine the influence of the two forms of G-CSF following chemotherapy on PBSC collection, we conducted a retrospective study in 126 patients with various hematological malignancies: 65 and 61 for the lenograstim and filgrastim groups respectively. No significant differences between the two groups were observed in terms of sex, age and diagnosis. Prior therapies and PBSC mobilization regimen were also equivalent. No significant difference was observed between the groups for the median CD34+ cells harvested. The number of leukapheresis necessary to obtain a minimal number of 3 x 10(6) CD34+ cells/kg was equivalent for the two groups. The proportion of patients affected by a failure in PBSC collection was similar in the two groups. Our data suggest that lenograstim and filgrastim are equivalent for PBSC mobilization after chemotherapy.     

Isolation and transplantation of highly purified autologous peripheral CD34<Superscript>+</Superscript> progenitor cells: purging efficacy,hematopoietic reconstitution following high dose chemotherapy in patients with breast cancer: results of a feasibility study in Japan

BACKGROUND: High-dose chemotherapy with autologous stem cell support may have some therapeutic impact on certain groups of the patients with advanced breast cancer(BRCA). Since stem cell contamination by tumor cells might contribute to relapse, development of a tumor cell purging technique would improve the clinical outcome. The present study was undertaken to evaluate the purging efficacy of autologous mobilized CD34+peripheral stem cells in patients with breast cancer (BRCA) in an advanced stage or relapse. METHODS: CD34+cells were selected from autologous peripheral blood stem cells (PBSC) using a clinical scale of magnetic-activated cell sorting system (CliniMACS), followed by high-dose chemotherapy with transplantation of CD34+ selected cells. Amplification of cytokeratin 19 (CK19) and 20 (CK20) gene in leukapheresis products were measured to evaluate the performance of tumor cell elimination. RESULTS: Seven patients were entered into this study. After leukopheresis, 1 patient was dropped form this study due to poor mobilization. Among 6 patient, a total of 8 CD34+ selection was performed. The median purity and recovery rate of the CD34+ cells post selection was 85.1% (range 62.5-98.1%) and 74.2% (range 50.2-90.2%), respectively. After isolation of CD34+cells, the elimination rate in the logarithmic transformation of CK19 was 2.77 log, and that of CK20 were 2.43 log and 2.53 log. In 4 patients, high-dose chemotherapy was performed, followed by the transplantation of the isolated CD34+cells. Rapid neutrophil recovery, as well as platelet recovery was seen with a median time to reach 0.5 x 109/l neutrophils of 9 days(range 8-9), and 20 x 109/l platelets of 12 days (range 10-13). There was no treatment related death and no serious adverse events directly associated with the selection procedure or infusion of selected cells. CONCLUSIONS: The present study demonstrated that the CliniMACS system is a highly effective positive selection method and that a high purging efficacy could be obtained without compromising the hematopoietic reconstitution capacity of the graft in BRCA patients undergoing high-dose chemotherapy.     

Age has no influence on mobilization of peripheral blood stem cells in acute myeloid leukemia

The upper age limit for autologous stem cell transplantation (ASCT) in acute myeloid leukemia (AML) is increasing and peripheral blood (PB) represents the standard source of stem cell (SC). However, no data are available on the impact of age on SC mobilization in AML. We analyzed a cohort of 150 consecutive AML patients in first complete remission in order to make a comparison between patients up to 60 years and above 60 years, by evaluating CD34+ cells mobilization into PB and the number of leukapheresis needed to collect at least one single SC graft. The successful mobilization rate (>2 x 10(6) CD34+ cells/kg) was comparable between the two groups (87% vs. 80%, p = 0.29). In addition, no statistically significant difference was found in terms of either median number of CD34+ cells collected (p = 0.54) or CD34+ cells peak in PB (p = 0.70). Both groups of patients needed a median of two apheresis and no difference was found in the median number of CD34+ cells collected per single apheresis (p = 0.67). Finally, no correlation was found between age and total number of CD34+ cells collected (r = 0.003, p = 0.58). We conclude that age has no impact on mobilization of PBSCs in AML.     

Immune system augmentation via humanization using stem/progenitor cells and bioengineering in a breast cancer model study

Despite significant advances, most current in vivo models fail to fully recapitulate the biological processes that occur in humans. Here we aimed to develop an advanced humanized model with features of an organ bone by providing different bone tissue cellular compartments including preosteoblasts, mesenchymal stem/stromal (MSCs), endothelial and hematopoietic cells in an engineered microenvironment. The bone compartment was generated by culturing the human MSCs, umbilical vein endothelial cells with gelatin methacryloyl hydrogels in the center of a melt‐electrospun polycaprolactone tubular scaffolds, which were seeded with human preosteoblasts. The tissue engineered bone (TEB) was subcutaneously implanted into the NSG mice and formed a morphologically and functionally organ bone. Mice were further humanized through the tail vein injection of human cord blood derived CD34+ cells, which then populated in the mouse bone marrow, spleen and humanized TEB (hTEB). 11 weeks after CD34+ transplantation, metastatic breast cancer cells (MDA‐MB‐231BO) were orthotopically injected. Cancer cell injection resulted in the formation of a primary tumor and metastasis to the hTEB and mouse organs. Less frequent metastasis and lower tumor burden were observed in hematochimeric mice, suggesting an immune‐mediated response against the breast cancer cells. Overall, our results demonstrate the efficacy of tissue engineering approaches to study species‐specific cancer‐bone interactions. Further studies using genetically modified hematopoietic stem cells and bioengineered microenvironments will enable us to address the specific roles of signaling molecules regulating hematopoietic niches and cancer metastasis in vivo.     

rHuG-CSF动员健康供者外周血造血干细胞的效果观察

目的:观察重组人粒细胞集落刺激因子(recombinant human granulocyte-colony stimulating factor,rHuG-CSF)动员健康供者外周血造血干细胞的效果及影响因素。方法:将本研究中心异基因造血干细胞移植健康供者163例,分别采用3种不同厂家生产的rHuG-CSF进行外周血造血干细胞动员,对其动员效果、受者移植后造血重建等情况进行比较。结果:不同种rHuG-CSF动员后采集的单个核细胞(MNC)及CD34 细胞均能满足临床造血干细胞移植的需要,130例人类白细胞抗原(human leucocyte antigens,HLA)配型相合的同胞受者移植后均获得顺利植入。动员的单个核细胞数及CD34 细胞数与性别无关,而CD34 细胞数在41~60岁年龄组中明显减少(P<0.05);对采集时机的分析显示:第5天采集的单个核细胞数及CD34 细胞数最高(P<0.05),此后逐渐下降。结论:糖基化的及两个非糖基化的rHuG-CSF制剂均能有效地在异基因移植中作为外周血造血干细胞动员剂。     

Expression of G-CSF receptor on myeloid progenitors

Although granulocyte colony-stimulating factor (G-CSF) has been reported to act on cells of neutrophilic lineage, the expression of receptors for G-CSF (G-CSFR) on human hematopoietic progenitor cells has been unclear. We then analyzed the expression of G-CSFR on human bone marrow and G-CSF mobilized peripheral blood CD34+ cells, and examined the proliferation and differentiation capabilities of sorted CD34+ G-CSFR+ and CD34+ G-CSFR- cells using methylcellulose clonal culture. These results indicate that the expression of G-CSFR on CD34+ cells is restricted to myeloid progenitors, suggesting that the specific activity of G-CSF on myelopoiesis depends on the exclusive expression of its receptor on myeloid progenitors, and that the mobilization of various hematopoietic stem/progenitor cells is not a direct effect of G-CSF in humans.     

Biological purging of breast cancer cells using an attenuated replication-competent herpes simplex virus in human hematopoietic stem cell transplantation

Autologous hematopoietic stem cell transplantation after myelosuppressive chemotherapy is used for the treatment of high-risk breast cancer and other solid tumors. However, contamination of the autologous graft with tumor cells may adversely affect outcomes. Human hematopoietic bone marrow cells are resistant to herpes simplex virus type 1 (HSV-1) replication, whereas human breast cancer cells are sensitive to HSV-1 cytotoxicity. Therefore, we examined the utility of G207, a safe replication-competent multimutated HSV-1 vector, as a biological purging agent for breast cancer in the setting of stem cell transplantation. G207 infection of human bone marrow cells had no effect on the proportion or clonogenic capacity of CD34+ cells but did enhance the proliferation of bone marrow cells in culture and the proportion of CD14+ and CD38+ cells. On the other hand, G207 at a multiplicity of infection of 0.1 was able to purge bone marrow of contaminating human breast cancer cells. Because G207 also stimulates the proliferation of human hematopoietic cells, it overcomes a limitation of other purging methods that result in delayed reconstitution of hematopoiesis. The efficient infection of human bone marrow cells in the absence of detected toxicity suggests that HSV vectors may also prove useful for gene therapy to hematopoietic progenitor cells.     

Fresh peripheral blood mononuclear cell preparations are a better starting material than bone marrow after cryopreservation for immunomagnetic harvesting of CD34(+) hematopoietic cells

Immunomagnetic separation using anti-CD34 monoclonal antibodies and paramagnetic microspheres has been used to enrich hematopoietic stem cells from human bone marrow, whole cord blood, or mobilized peripheral blood mononuclear cell collections. This method has been reported to achieve high separation purity of CD34+ cells in small scale experiments with fresh material. The aim of the present study was to compare the efficacy of the CD34+ cell selection technique, when thawed bone marrow or fresh peripheral blood mononuclear cells were enriched. Starting with thawed bone marrow containing 2.9% CD34+ cells the final product purity was 67.7% with a 6% CD34+ cell yield (enrichment factor 25.7), and a 85-fold CFU-GM enrichment. Using fresh mobilized peripheral blood mononuclear cells the released cells contained 77.6% CD34+ cells with a 47% yield (enrichment 86.5-fold), and a 46-fold CFU-GM enrichment. These results indicate that CD34+ cells can be selected from cryopreserved bone marrow using immunomagnetic procedures. However, fresh leukapheresis products seem to be a much better material for a positive immunomagnetic stem cell selection technique in terms of purity, yield and enrichment.     

Functional cloning of IGFBP-3 from human microvascular endothelial cells reveals its novel role in promoting proliferation of primitive CD34+CD38- hematopoietic cells in vitro

Ex vivo expansion of hematopoietic stem cells (HSCs) has been investigated as a means of enhancing engraftment of transplantation therapies, but current ex vivo expansion methods typically result in a loss of functional stem cell activity. Factors that can selectively expand human HSCs remain elusive. Recently we have isolated three functionally distinct human brain microvascular endothelial cells (HBMVECs) that differ greatly in their ability to support in vitro proliferation of human umbilical cord blood (UBC) CD34+CD38-cells. Using these distinct HBMVEC populations, we have devised a cell-based functional cloning assay to identify a molecule(s) capable of facilitating expansion of HSCs in vitro. A gene encoded for IGFBP-3 (insulin-like growth factor binding protein-3) has been identified. IGFBP-3 mRNA and protein are differentially expressed in distinct HBMVEC populations. In vitro cell proliferation assay and CD34+CD38- immunophenotype analysis showed that the addition of an exogenous IGFBP-3 to cultures of purified CD34+/-CD38-Lin- cells (CD2/CD3/CD14/CD16/CD19/CD24/CD56/CD66b/GlyA depleted) enhanced proliferation of primitive hematopoietic cells with CD34+CD38- phenotype, suggesting that IGFBP-3 is capable of expanding primitive human blood cells. These expanded primitive blood cells were illustrated to maintain ability to generate functional progenitors. IGFBP-3 belongs to a family of high-affinity IGFBPs, which binds to IGFs and modulates their actions. IGFBP-3 appears to have intrinsic bioactivity that is independent of IGF binding. We are currently exploring the underlying mechanism by which IGFBP-3 modulates proliferation of primitive hematopoietic cells, and the potential of IGFBP-3 to expand pluripotent human repopulating cells capable of hematopoietic reconstitution of irradiated NOD/SCID recipients.     

Paclitaxel and filgrastim for hematopoietic progenitor cell mobilization in patients with hematologic malignancies after failure of a prior mobilization regimen

Paclitaxel and G-CSF have been evaluated for HPC mobilization in breast cancer and found to have tolerable toxicity with a predictable time to initiate leukapheresis. However, this approach has not been reported in patients with hematologic malignancies failing prior mobilization. We report a case-series of 26 adults given paclitaxel and G-CSF for HPC mobilization after failure of an initial mobilization. Patients received paclitaxel 250 mg/m(2) followed by G-CSF 10-16 mcg/kg/day. Compared to the initial regimen, paclitaxel mobilization produced greater CD34+ cell yields (median 1.53 x 10(6) CD34+ cells/kg vs. 0.79 x 10(6) CD34+ cells/kg, p = 0.004). Seventy-six percent of patients initiated leukapheresis on day 8, the remainder on day 9 or 10. Three patients developed febrile neutropenia resulting in one death prior to leukapheresis. Overall, 73% of patients proceeded with autologous HPC transplant. This case-series suggests paclitaxel may be an option for HPC mobilization in patients failing prior regimens.     

Endothelial cells (EC) and endothelial precursor cells (EPC) kinetics in hematological patients undergoing chemotherapy or autologous stem cell transplantation (ASCT)

Our objective was to study the kinetics of circulating endothelial cells (EC) and endothelial precursor cells (EPC) in hematological patients during chemotherapy and autologous stem cell transplantion (ASCT). Eighteen newly diagnosed patients and 17 patients undergoing ASCT were studied and compared to healthy controls. ECs were evaluated as CD146+CD31+Lin- cells, while EPCs were evaluated as CD34+CD133+Lin-, or CD34+VEGFR2+Lin- cells, or CFU-En colony forming units. Numbers of these cells were evaluated before and after treatment, and, in patients treated with ASCT, during mobilization of hematopoietic progenitors. Both newly diagnosed patients and patients before ASCT had significantly higher number of CD146+CD31+Lin- cells and significantly lower number of CFU-En colonies than healthy controls. These parameters did not return to normal for at least 3 months after chemotherapy or ASCT. Numbers of CFU-En did not correlate either with numbers of CD34+CD133+Lin- cells or with numbers of CD34+VEGFR2+Lin- cells but they did correlate with numbers of CD4+ lymphocytes and NK cells. In conclusion, we have found that hematological patients have higher number of EC and lower numbers of CFU-En than healthy controls and that these parameters do not return to normal after short-term follow-up. Furthermore, our observations support emerging data that CFU-En represent cell population different from flowcytometrically defined EC and endothelial precursors and that their development requires cooperation of monocytes and CD4+ lymphocytes. However, cells forming CFU-En express endothelial surface markers and can contribute to proper endothelial function by NO production.     

Genetically modified CD34+ cells exert a cytotoxic bystander effect on human endothelial and cancer cells.

We and others have proposed mammalian cells as gene delivery vehicles with the potential for overcoming physiological barriers to viral vectors. To that end, we previously have shown the potential of CD34+ endothelial progenitors for systemic gene delivery in a primate angiogenesis model. Here we seek to explore the utility of CD34+ cells of human origin as vehicles for toxin genes and, in particular, to measure their capacity to effect a cytotoxic bystander effect in human endothelium and tumor cells. To this end, CD34+ cells were transduced with TOZ.1, a nonreplicative herpes simplex vector encoding thymidine kinase. To test the capacity of CD34+ cells to induce a cytotoxic bystander effect in target cells, we performed mixing experiments, whereby TOZ.1-transduced CD34+ cells were mixed with either human vascular endothelial cells or human ovarian tumor cells (SKOV3.ip1). Cell viability was measured by the MTS assay. Lastly, mixtures of TOZ.1-transduced CD34+ cells and SKOV3.ip1 tumor cells were injected s.c. to evaluate the bystander effect in vivo. After transduction of CD34+ cells with TOZ.1, treatment with ganciclovir induced the killing of 99% of cells. In cell-mixing experiments, a linear correlation was observed between the percentages of TOZ.1-transduced CD34+ cells and total cell killing. For example, when 50% of CD34+ transduced cells were mixed with nontransduced SKOV3.ip1, >70% of all cells died. Similarly, when the same percentage was mixed with human vascular endothelial cells, >80% of the total number of cells died. In vivo studies showed an abrogation of tumor formation when TOZ.1-transduced CD34+ cells and ganciclovir were administered. Our observations establish the feasibility of a method for cell-based toxin gene delivery into disseminated areas of tumor angiogenesis.