Preclinical and clinical aspects of carboplatin and gemcitabine combined with whole-body hyperthermia for pancreatic adenocarcinoma.

UNLABELLED: Advanced pancreatic adenocarcinoma is usually treated with single-agent gemcitabine chemotherapy or combinations that include gemcitabine application that have palliative benefit but do not lead to survival benefits. We present the results of preclinical and clinical studies using combination chemotherapies that include 41.8 degrees C whole-body hyperthermia for pancreatic adenocarcinoma. MATERIALS AND METHODS: DAN-G pancreatic carcinoma cells were treated with carboplatin and gemcitabine in vitro under hyperthermic conditions of 37 degrees C, 39 degrees C, 41.8 degrees C and 43 degrees C and cytotoxic drug effects were measured under various conditions using crystal violet assays. Data on outcome and toxicity of a clinical study using gemcitabine and carboplatin with 41.8 degrees C whole-body hyperthermia in a compassionate manner in patients with advanced and heavily pretreated pancreatic adenocarcinoma are also shown. RESULTS: In vitro data showed the DAN-G cells did not show increased responses to gemcitabine with or without carboplatinum under hyperthermic culture conditions at 39 and 41.8 degrees C. Only temperatures of 43 degrees C led to increased hyperthermic damage. Clinical data showed that a therapy of whole-body hyperthermia at 41.8 degrees C with gemcitabine and carboplatin was well tolerated leading mainly to the expected hematological side-effects due to chemotherapy. The median overall survival after whole-body-hyperthemia was of 357 days, with a median progression-free survival of 140 days. CONCLUSION: Preclinical data indicate that hyperthermia does not increase the chemosensitivity of DAN-G pancreatic carcinoma cells to gemcitabine and carboplatin. Clinical data show that a treatment of pancreatic adenocarcinoma with C whole-body hyperthermia at 41.8 degrees with gemcitabine and carboplatin is feasible for patients with advanced disease.

Vasculogeneic maturation of E14 embryonic stem cells with evidence of early vascular endothelial growth factor independency.

Murine embryonic stem cells (ESC) provide a unique homogeneous cell system for studying early vasculogenic cell differentiation in vitro. In this report, we characterized endothelial development of cultured E14 ESCs and mapped the effects of vascular endothelial growth factor (VEGF) on these cells. After removal of leukemia inhibitory factor undifferentiated state ESCs were precultured for 6 days and then cultured for up to 30 days in differentiation culture medium, with or without supplemental VEGF. ELISA analysis was used to detect endogenous VEGF levels. Early vasculogenic development and expression of selected genes were characterized using flow cytometry for specific antigens and quantitative RT-PCR. ELISA analysis showed no endogenous VEGF after preculture and at day 2 in unsupplemented culture, therafter VEGF levels rise. Directly after preculture a high proportion (36%) of the ESCs showed positivity for endothelial CD31. We describe characteristic endothelial differentiation patterns in embryoid bodies (EB) kept in culture for up to 30 days. VEGF supplementation lead to qualitative changes in the EB vessels, specific activation of vasculogenesis-related genes (CD31, CD144, and ERG) and temporary down-regulation of the VEGF receptor gene flk-1. VEGF supplementation did not produce measurable changes in the endothelial cell fractions as judged by surface antigen presence. We conclude that early ESCs may undergo endothelial differentiation through VEGF-independent pathways, whereas endothelial cell patterns in EBs are cytokine dependent and fully stimulated by endogenous cytokine levels.

Bone marrow-derived endothelial cells contribute to angiogenesis in murine WEHI and JC tumors.

The goal of this study was to determine the contribution of bone marrow-derived circulating endothelial progenitor cells to the formation of endothelial cell linings of tumor vessel walls. The proportion of male endothelial cells in female JC and WEHI tumors was measured in male BALB/c mice and in female mice displaying complete marrow chimerism, after receiving male bone marrow cells. The gender origin of the perivascular endothelial cells was determined by fluorescent in situ hybridization (FISH) analysis of adjacent cuts of the tumors, using CD31 and Y-chromosomes as markers. High proportions of male cells were detected in the perivascular endothelial cell linings of the JC (60 +/- 4%) and WEHI (67 +/- 4%) tumors after implantation into normal male mice. Furthermore, in marrow chimeric female mice, very high levels of male cells were observed in the endothelilal cell linings of the tumor vessel walls of both tumor types, after bone marrow transplantation. We conclude that JC and WEHI tumors can serve as a murine experimental model and that bone marrow cells from these can be manipulated and cultured in vitro for use in studies of tumor vessel walls after transplantation into myeloablated recipients.

Low-dose oral metronomic chemotherapy prevents mobilization of endothelial progenitor cells into the blood of cancer patients.

UNLABELLED: Circulating endothelial progenitor cells (EPCs) actively supply cells that may participate in tumor angiogenesis. The differing effects of low-dose metronomic trofosfamide as opposed to conventional dose-dense chemotherapy on plasma levels of vascular endothelial growth factor (VEGF) and the numbers of circulating EPC are reported. PATIENTS AND METHODS: Blood samples were obtained from cancer patients, 18 receiving oral metronomic chemotherapy of trofosfamide with or without celecoxib, and 24 receiving conventional dose-dense chemotherapy, eight of them in adjuvant intention. Mononuclear cells were analyzed by flow cytometry for CD34, CD45 and vascular endothelial growth factor-receptor 2 (VEGF-R2) coexpression, defining EPCs, and for plasma levels of VEGF by ELISA at day 0, 10 and 21 of therapy. RESULTS: After conventional dose-dense chemotherapy, the numbers of circulating EPCs and the VEGF plasma concentrations increased sharply, doubling pretherapeutic levels at day 21. In contrast, under low-dose metronomic chemotherapy, the numbers of circulating EPCs decreased significantly and VEGF plasma concentrations remained unchanged. CONCLUSION: These observations provide evidence that conventional dose-dense chemotherapy leads to rebound EPC mobilization even when given with adjuvant intention, while low-dose metronomic scheduling of cytotoxic substances such as trofosfamide may sharply reduce EPC release into the circulation.

Chloroacetaldehyde: mode of antitumor action of the ifosfamide metabolite.

BACKGROUND: The ifosfamide metabolite chloroacetaldehyde had been made responsible for side effects only. We found in previous studies a strong cytotoxicity on human MX-1 tumor cells and xenografts in nude mice. Chloroacetaldehyde is supposed to act via alkylation or by inhibition of mitochondrial oxidative phosphorylation with decrease of ATP. The aim of this study was to further elucidate chloroacetaldehyde's mode of action. METHODS: MX-1 breast carcinoma cells were measured for ATP-content after exposure to chloroacetaldehyde. Further, the effect of chloroacetaldehyde on DNA-synthesis and its potency of causing strand-breaks or cross-links were investigated by bromodeoxyuridine-incorporation, comet-assay and a DNA interstrand cross-linking-assay. RESULTS: Chloroacetaldehyde in high concentrations induces a reduction of ATP-levels when anaerobic glycolysis is blocked by oxamate and reduces the bromodeoxyuridine-incorporation to 46.3% after 4 h when used in IC(50) concentrations (7.49 mumol/l). In addition we observed DNA single strand-breaks in MX-1 cells treated with chloroacetaldehyde visible in the Comet assay, but no DNA-cross-linking by comet assay and cross-linking assay. CONCLUSION: In summary, our results show that chloroacetaldehyde influences the oxidative phosphorylation in mitochondria, however, this is observed only in high concentrations and is not of clinical relevance because the tumor cells regenerate ATP by anaerobic glycolysis. Nevertheless, chloroacetaldehyde causes DNA-strand-breaks and strong inhibition of DNA-synthesis.

Hematopoietic progenitor cells residing in muscle engraft into bone marrow following transplantation.

Hematopoietic and mesenchymal stem cells can potentially be the same cell type or adhere simultaneously in both bone marrow (BM) and muscle. In this study, we asked whether murine BM-derived cells could be tracked in muscle tissue after BM transplantation and whether muscle-derived cells have hematopoietic potential. To answer the first question, we transplanted BM from male BALB/c mice into irradiated female recipients and analyzed for engraftment. We used quantitative polymerase chain reaction (PCR) and fluorescent in situ hybridization techniques for Y chromosome-specific gene probes. A high number of BM-derived cells were located in both the intravascular and extravascular spaces in muscle tissue after BM transplantation. To answer the second question, we analyzed colony-forming potential in vitro with soft-agar assays and the competitive engraftment potential in vivo of muscle-derived cells. Engraftment levels of male cell populations were tested by quantitative PCR. The long-term engraftment potential of muscle-derived cells was low compared with that of BM. We conclude that there is intensive cellular trafficking between BM and muscle tissue. The engraftment potential of muscle-derived stem cells into BM is low and corresponds to the low amounts of hematopoietic colony-forming cells found in muscle tissue.

Cytokine modulation of murine stem cell engraftment: the role of adherence to plastic surfaces.

Murine marrow stem cells acquire an engraftment defect when cultured for 48 hours in cytokines, whereas the number of progenitor cells expands. Stem or progenitor cells have been noted to adhere to various surfaces, including plastic. Despite vigorous harvesting by cell scraping, the possibility existed that cytokines might induce selective adhesion of the rare engraftable stem cells to plastic surfaces. We have evaluated whether loss of engraftability by cytokine-treated marrow cells could be due to adhesion to plastic culture vessels. BALB/c marrow cells were cultured in the presence of interleukin 3 (IL-3), IL-6, IL-11, and steel factor for 48 hours in plastic tissue culture flasks from which cells were harvested by standard scraping and washing or after 5 or 10 minutes of additional exposure to trypsin (0.25%), or they were cultured with the same cytokines in nonadherent polytetrafluoroethylene (Teflon) culture bottles. Harvested cultured or fresh-starting male cells were then engrafted into nonirradiated female hosts or were placed in competition with fresh female BALB/c marrow in lethally irradiated female hosts. Defective engraftment was seen in nonmyeloablated or irradiated female hosts 7 to 24 weeks after marrow infusion in all cultured cell groups. These data indicate that cytokine-treated engraftable stem cells do not show significant adherence to plastic surfaces.

Stem cell toxicity of oxazaphosphorine metabolites in comparison to their antileukemic activity.

The oxazaphosphorine agent cyclophosphamide (CP) is an alkylating agent with a relative low stem cell toxicity. The aim of this study was to further evaluate the stem cell toxicity of the active metabolites of CP and its structural analogue ifosfamide (IFO) in comparison to their antileukemic efficacy. Cells of different malignant hematologic disorders (HL-60, HS-Sultan and THP-1) and CD34+ stem cells were treated with cytotoxic CP-metabolite mafosfamide (MAFO) and IFO-metabolites 4-hydroxy-IFO (4-OH-IFO) and chloroacetaldehyde. The clonogenity of the cells was investigated by using a colony-forming assay. All metabolites reduced the formation of both tumor-derived colonies and stem cell-derived CFU-GMs in a concentration-dependent manner. Our data showed a relative tumor-specific, stem cell protecting action of the substances tested with a higher toxicity against tumor cells (IC(50) against HS-Sultan: MAFO 1.1 microM; 4-OH-IFO 1.3 microM; CAA 3 microM) than against stem cells (IC(50) MAFO 14.8 microM; 4-OH-IFO 16.9 microM; CAA 14 microM). However, while the cytotoxic action of 4-OH-IFO corresponded to MAFOs activity, CAAs cytotoxic effect against the hematologic tumor cells was lower. In conclusion, the results confirm the observed cytotoxicity of CAA against solid tumors for cells of malignant hematologic disorders. Although the relative cytotoxic specificity of CAA is lower than for 4-OH-IFO and MAFO, also CAA, like 4-OH-IFO and MAFO, was found to be in part a tumoricidal, stem cell protecting substance.