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The systemic balance of angiogenic and anti-angiogenic factors has been proposed to play a key-role in primary tumor growth dependent growth suppression of secondary tumors. Despite the importance of the organ microenvironment to angiogenesis and microcirculation, the influence of a primary tumor on secondary bone tumors has not been investigated so far. Since breast cancer has a high propensity to spread to bone, we used an in vivo xenograft model to determine the impact of growing breast cancer cells (MCF-7) in the mammary fat pad on the microvascular properties of subsequently inoculated secondary breast cancer tumors in bone. Mice were either treated with a resection of the primary tumor (n = 10) or no surgery (n = 9) and intravital microscopy was performed over 25 days in bone tumors. Tumor growth in bone was temporarily suppressed by the primary tumor on days 10 and 14. While microvascular permeability and vascular diameter decreased in both groups over time, the presence of the primary tumor was accompanied by a decreased tumor perfusion on days 8 and 10 through a reduction in vessels with diameters between 5 and 20 µm. The results imply a potential benefit of a therapeutic regime in which the resection of the primary tumor is combined with an anti-angiogenic therapy in the perioperative or direct postoperative period. This might result in reduced progression of bone metastasis subsequent to excision of the primary tumor. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1251–1258, 2011

Microcirculatory properties of tumors play a pivotal role in tumor progression and inefficacy of therapies. It has been hypothesized that surgical interventions result in an accelerated tumor growth by increasing the level of pro-angiogenic cytokines with subsequent amplification of tumor angiogenesis. To characterize the microvascular properties of secondary bone tumors in vivo and determine the impact of minor surgery on the microcirculation, we performed intravital microscopy over 25 days using a xenograft model of breast cancer tumor growth (MCF-7) in bone. After engraftment of tumors the mice were treated with a mastectomy versus no surgery. Tumor growth was accompanied by angiogenic sprouting and decreased vascular diameters while blood flow rate and tumor perfusion remained constant. Mastectomy initially led to a significant reduction of functional vascular density, increased vascular diameter, and decreased blood flow velocities. However, neither tumor growth nor tissue perfusion was different between the groups. The presented study corroborates the assumption that tumor microcirculation in bone exhibits similar time-dependent alterations compared to soft tissue tumors. A minor surgical intervention did not change tumor growth kinetics however microcirculatory properties were altered. Whether major surgery has an impact on tumor growth in bone should be clarified in further studies.

The TGF-beta pathway controls a broad range of cellular behavior including cell proliferation, differentiation, and apoptosis of various cell types including tumor cells, endothelial cells, immune cells, and fibroblasts. Besides TGF-beta's direct effects on tumor growth and its involvement in neoangiogenesis have received recent attention. Germline mutations in TGF-beta receptors or coreceptors causing Hereditary Hemorrhagic Teleangiectasia and the Loeys-Dietz syndrome underline the involvement of TGF-beta in vessel formation and maturation. Several therapeutic approaches are evaluated at present targeting the TGF-beta pathway including utilization of antisense oligonucleotides against TGF-beta itself or antibodies or small molecule inhibitors of TGF-beta receptors. Some of these therapeutic agents have already entered the clinical arena including an antibody against the endothelium specific TGF-beta class I receptor ALK-1 targeting tumor vasculature. In conclusion, therapeutic manipulation of the TGF-beta pathway opens great opportunities in future cancer therapy.

Neoangiogenesis plays an important role in tumor growth and metastasis. Evaluation of new anti-angiogenic targets may broaden the armament for future therapeutic concepts. Focal adhesion kinase (FAK), expressed in endothelial and tumor cells, is essential for adhesion and mobility of adherent cells. In the current study we analyzed the anti-angiogenic properties of the FAK inhibitor TAE226 on the proliferation of blood outgrowth endothelial cell (OEC) and differentiation of endothelial progenitor cells (EPC), derived from peripheral blood CD133(+) cells, tube formation and on neovascularization in a HT29 xenotransplant model. The effects of TAE226 were compared to those of the rapamycin analogue RAD001. The combination of both drugs was also studied. We showed that HT29 tumor cells and OEC were most sensitive to the action of TAE226 compared to EPC in vitro. In contrast, RAD001 affected the proliferation of both types of endothelial cells stronger than that of HT29 cells. Furthermore we could show that TAE226 inhibited tube formation in a dose dependent manner. In a HT29 subcutaneous tumor model TAE226 and RAD001 diminished MVD at commonly employed doses to a similar degree. Combination of both compounds did not show synergy in vitro or in vivo. Since TAE226 has been shown to inhibit the PI3 kinase, Akt kinase, mTor pathway, addition of RAD001 may not increase this effect. In conclusion, we have shown that treatment with TAE leads to a reduction of neoangiogenesis in vitro and in a mouse model. The effects are mediated by inhibition of angiogenesis and vasculogenic OEC and EPC.

The existence of adult haemangioblasts with dual haematopoietic and endothelial developmental potential was confirmed after detection of Ph(+) vascular endothelial cells in chronic myeloid leukaemia (CML) patients. Blood outgrowth endothelial cells (OECs) from CML patients were found not to harbour the Philadelphia translocation and were thus not clonally related to BRC/ABL1(+) hematopoietic progenitors, but comprised a distinct subfraction of endothelial cells. Remarkably, the frequency of CML-derived OECs was 9-fold higher as compared to healthy donors (n = 19 and n = 300, respectively; P < 0.0001) and these cells showed increased proliferative potential, possibly reflecting the mobilisation of OEC progenitors by pro-angiogenic cytokines.

Expression of TGF-β receptor ALK-5 has a negative impact on outcome of patients with acute myeloid leukemia

The systemic balance of angiogenic and anti-angiogenic factors has been proposed to play a key-role in primary tumor growth dependent growth suppression of secondary tumors. Despite the importance of the organ microenvironment to angiogenesis and microcirculation, the influence of a primary tumor on secondary bone tumors has not been investigated so far. Since breast cancer has a high propensity to spread to bone, we used an in vivo xenograft model to determine the impact of growing breast cancer cells (MCF-7) in the mammary fat pad on the microvascular properties of subsequently inoculated secondary breast cancer tumors in bone. Mice were either treated with a resection of the primary tumor (n = 10) or no surgery (n = 9) and intravital microscopy was performed over 25 days in bone tumors. Tumor growth in bone was temporarily suppressed by the primary tumor on days 10 and 14. While microvascular permeability and vascular diameter decreased in both groups over time, the presence of the primary tumor was accompanied by a decreased tumor perfusion on days 8 and 10 through a reduction in vessels with diameters between 5 and 20 µm. The results imply a potential benefit of a therapeutic regime in which the resection of the primary tumor is combined with an anti-angiogenic therapy in the perioperative or direct postoperative period. This might result in reduced progression of bone metastasis subsequent to excision of the primary tumor.

Bone marrow derived hematopoietic stem cells can function as endothelial progenitor cells. They are recruited to malignant tumors and differentiate into endothelial cells. This mechanism of neovascularization termed vasculogenesis is distinct from proliferation of pre-existing vessels. To better understand vasculogenesis we developed a cell culture model with expansion and subsequent endothelial differentiation of human CD133(+) progenitor cells in vitro. alpha(v)beta(3)-integrins are expressed by endothelial cells and play a role in the attachment of endothelial cells to the extracellular matrix. We investigated the effect of Cilengitide, a peptide-like, high affinity inhibitor of alpha(v)beta(3)- and alpha(v)beta(5)-integrins in our in vitro system. We could show expression of alpha(v)beta(3)-integrin on 60+/-9% of non-adherent endothelial progenitors and on 91+/-7% of differentiated endothelial cells. alpha(v)beta(3)-integrin was absent on CD133(+) hematopoietic stem cells. Cilengitide inhibited proliferation of CD133(+) cells in a dose-dependent manner. The development of adherent endothelial cells from expanded CD133(+) cells was reduced even stronger by Cilengitide underlining its effect on integrin mediated cell adhesion. Expression of endothelial antigens CD144 and von Willebrand factor on differentiating endothelial precursors was decreased by Cilengitide. In summary, Cilengitide inhibits proliferation and differentiation of human endothelial precursor cells underlining its anti-angiogenic effects.

Abstract 1604 Poster Board I-630 Introduction Bone marrow neoangiogenesis plays an important role in acute myeloid leukaemia. TGF-β has been shown to be involved in angiogenesis. TGF-β receptors are comprised of class I, class II and accessory receptors. Class I TGF-β receptor ALK-1 is mainly expressed on endothelial cells whereas ALK-5 is found on endothelial cells and various cell types including tumor cells. Patients and Methods m-RNA and protein expression of ALK-1 and ALK-5 were studied in several leukemic cell lines by PCR and FACS analysis. Expression of both receptors and their respective ligands, bone morphogenic proteins (BMP) -9 and -10 and TGF-β1, were analyzed by real-time quantitative PCR in pretherapeutic samples from 93 acute myeloid leukaemia (AML) patients enrolled into the AMLSG 07-04 study of the Austrio-German AMLSG study group. To evaluate ALK-1 and ALK-5 receptor activation, expression of downstream target genes ID-1 and PAI-1 were also determined by quantitative PCR. Uni- and multivariate analyses were performed using clinical and gene expression variables. Results ALK-1 and ALK-5 were expressed by several leukemic cells lines including KG-1 and UKE-1 where endothelial cells (HUVECs and OECs) served as positive controls. Expression of ALK-1 and ALK-5 were found in the majority of AML patients (82 % and 88 %, respectively). All patients expressed TGF-β1, whereas the percentage of BMP-expressing patients was low (8 % for BMP-9, 13 % for BMP-10). Expression of downstream targets ID-1 and PAI-1 was detected in almost all patients (99 % and 100 %, respectively). The impact of gene expression on achievement of CR and event-free (EFS), relapse-free (RFS) and overall survival (OS), was analyzed. Due to low numbers of patients with BMP-9 and -10 expression and inconclusive PCR results for ALK-1 and PAI-1 in several patients, these variables were only studied by univariate analysis. No effect of ALK-1, PAI-1, BMP-9 or -10 expression on clinical outcome was detected. Patients with low vs. high ALK-5 expression achieved a CR rate 63 % vs. 27 %, respectively. Multivariate analysis included ALK-5, TGF-β1 and ID-1 gene expression as well as the baseline variables sex, age, karyotype and FLT3 mutation status. During the stepwise procedure of the Cox regression, several variables were eliminated. Only ALK-5 and the karyotype showed a significant impact on event-free, relapse-free and overall survival (ALK-5: p=0,001 for EFS, p Conclusion ALK-1 and ALK-5 expression is found in bone marrow and peripheral blood samples from the majority of AML patients. Both receptors are found on endothelial cells and on leukemic blasts. Both receptors are in an activated state as demonstrated by expression of downstream target genes. High ALK-5 expression results in a low CR rate which translates into reduced EFS, RFS and OS of AML patients. Therefore ALK-5 expression represents a new prognostic marker in AML. Recently published data (Shao, Blood. 2009 Jun 8. [Epub ahead of print]) demonstrate involvement of both ALK-1 and ALK-5 in the regulation of vascular endothelial growth factor which is believed to be the central growth factor in angiogenesis. Hence, the ALK-1/ALK-5 pathway might represent a novel therapeutic target in AML. Disclosures No relevant conflicts of interest to declare.

7062 Background: As main actors in the generation of new blood vessels, endothelial progenitor cells have been intensely investigated during the past years. The detection of Philadelphia chromosome...

Angiogenesis and lymphangiogenesis promote tumor growth and metastasis. In addition to vessel sprouting circulating bone marrow derived endothelial progenitor (ECPs) and endothelial cells (CECs) contribute to neoangiogenesis in a process termed vasculogenesis. CECs can be detected in the blood of cancer patients and increased numbers are related to tumor progression. CECs and EPCs express endothelial cell surface markers as CD144 (VE-Cadherin) and VEGFR-2. Sprouting of new lymphatics from the preexisting lymphatic vessels has been described in solid tumors. Circulating lymphatic endothelial progenitor cells expressing CD133 and VEGFR3 were shown to differentiate into LYVE+ lymphatic endothelial cells (LECs) in vitro. Recently integration of circulating lymphatic endothelial progenitor cells in lymphatic neovessels was demonstrated in vivo. Therfore a distinct mechanism similar to vasculogenesis exists in lymphangiogenesis. Goal of our study was to identify and characterize circulating lymphatic endothelial cells in cancer patients. Presence of lymphatic endothelial cells in the peripheral blood was investigated in 26 patients with metastatic cancer and 10 healthy individuals. Additionally PBMNCs from G-CSF mobilised peripheral blood were analyzed (n=5). By developing a sensitive immunocytochemical approach we could identify a novel population of circulating lymphatic endothelial cells (CLECs) in patients with metastatic cancer. CLECs were LYVE+ and could be detected in 16 of 26 patients (62%) with metastatic cancer and in 4 of 10 healthy subjects (40%). The mean number of lymphendothelial cells was significantly higher in cancer patients (8.8 cells/1x106 PBMNCs [range 0–131] vs. 0.3 cells/1x106 PBMNCs [range 0–2]; p=0.03). We additionally investigated if CLECs were mobilised from bone marrow upon stimulation with G-CSF. 60% of analysed PBMNCs from leukapheresis products contained low numbers of circulating lymphatic endothelial cells comparable to normal peripheral blood (0.3 cells/1x106 PBMNCs [range 0–1]). Consequently CLECs unlike CEPs are not mobilised after cytokine stimulation from bone marrow. To better characterize CLECs, co-expression of VEGFR3 and LYVE was analyzed. We found that 90% of LYVE+ cells were positive for VEGFR3 which is consistent with a LEC phenotype previously described. Coexpression of vascular endothelial specific cell adhesion molecule CD144 (VE-Cadherin) was determined on LYVE+ cells by two-colour immunofluorescence. LYVE+ cells were negative for VE-Cadherin consistent with their lymphatic nature. We investigated whether LYVE+ cells represented a differentiated or a progenitor phenotype by double staining for LYVE and CD34 (n=3). All LYVE+ cells were negative for the progenitor marker CD34 indicating a differentiated lymphendothelial phenotype. Macrophages posess the capacity to transdifferentiate into lymphatic endothelial cells. We investigated whether circulating LYVE+ cells expressed CD11b and found a subfraction with a mean of 19% expressing LYVE and CD11b probably representing this macrophage subpopulation (range 13–25%, n=3). In summary our data show significantly higher levels of LYVE+ circulating lymphendothelial cells in patients with metastatic cancer compared to healthy subjects. Further studies are needed to clarify the origin of the circulating LECs and to analyse their potential as surrogate marker for lymphangiogenesis in cancer patients.