Microvesicles (MVs) released by malignant cancer cells constitute an important part of the tumor microenvironment. They can transfer various messages to target cells and may be critical to disease progression. Here, we demonstrate that MVs circulating in plasma of B-cell chronic lymphocytic leukemia (CLL) patients exhibit a phenotypic shift from predominantly platelet derived in early stage to leukemic B-cell derived at advanced stage. Furthermore, the total MV level in CLL was significantly greater compared with healthy subjects. To understand the functional implication, we examined whether MVs can interact and modulate CLL bone marrow stromal cells (BMSCs) known to provide a "homing and nurturing" environment for CLL B cells. We found that CLL-MV can activate the AKT/mammalian target of rapamycin/p70S6K/hypoxiainducible factor-1␣ axis in CLL-BMSCs with production of vascular endothelial growth factor, a survival factor for CLL B cells. Moreover, MV-mediated AKT activation led to modulation of the ␤-catenin pathway and increased expression of cyclin D1 and c-myc in BMSCs. We found MV delivered phospho-receptor tyrosine kinase Axl directly to the BMSCs in association with AKT activation. This study demonstrates the existence of separate MV phenotypes during leukemic disease progression and underscores the important role of MVs in activation of the tumor microenvironment. (Blood. 2010;115:1755-1764) Introduction B-cell chronic lymphocytic leukemia (B-CLL) has been predominantly characterized as a clonal B-cell disorder 1 in which the defective apoptosis of CLL B cells is ascribed not only to intrinsic defects of the neoplastic cells but also to extrinsic factors that influence their behavior in the tissue microenvironment. The issue of CLL heterogeneity and the exact reasons for the clinical variety of disease progression are unknown. One important factor associated with disease progression is unfavorable prognostic features that may influence apoptotic resistance in the CLL B-cell clone but could be related to the ability of the clone to manipulate the microenvironment to its advantage. A recent study 2 demonstrated the importance of communication between tumor cells and their microenvironment through the shedding of membrane microvesicles (MVs), which can fuse to nearby cells within their circulatory pathways.MVs are shed from the cell surface of normal healthy or malignant cells and can "hijack" membrane components and engulf cytoplasmic contents from either type of cell. The shedding of membrane-derived MVs is a physiologic phenomenon that accompanies cell activation and growth. 3 MVs contain numerous proteins and lipids similar to those present in the membranes of the origination cells, and this likely facilitates their integration into cells they come in contact with during circulation. 2 The content of MVs and their impact on biologic function are dependent upon the cell of origin. 4 Thus, it is known that ovarian cancer MVs stimulate angiogenesis and that platelet-derived MVs promote tumor progression and...

IntroductionThe cancer microenvironment has been implicated as playing a critical role(s) in cancer initiation and progression. There are multiple cellular components present in cancer microenvironments, which in general include immune/inflammatory cells such as lymphocytes, monocytes/macrophages and neutrophils/mast cells, fibroblast-like cells such as cancer-associated fibroblasts, mesenchymal stromal cells (MSCs), and vascular cells such as endothelial cells, pericytes, and smooth muscle cells. 1 Chronic lymphocytic leukemia (CLL) has long been recognized as a cumulative disease resulting from a failure of apoptosis; however, recent evidence also points out that CLL is a dynamic disease of leukemic cell proliferation and circulation. 2,3 Indeed, proliferation centers in CLL have been identified in lymph nodes, bone marrow, and spleen. 4 In vitro experiments have demonstrated the importance of stromal cellular components including bone marrow stromal cells, 5,6 nurselike cells, 7 and T cells 8,9 in maintaining CLL cell survival and, to a lesser degree, leukemic cell proliferation. 9,10 However, the exact mechanism and nature of the interactions between diverse stromal cellular components and CLL leukemic cells remains to be clearly determined.MSCs comprise one cellular component in the cancer microenvironment, and these cells have recently been studied in terms of the immune regulatory function, cancer promoting function, and wound repair function in a variety of disease models. 11 We have established an in vitro culture system to generate bone marrow stromal cells and demonstrated that they are mesenchymal stem cell in nature. 10,12 When we examined the MSC-CLL B-cell interaction, we found active bidirectional crosstalk was present between the 2 cell types, and this crosstalk was able to activate both cell types. 10 This interaction was associated with both an increase in vascular endothelial growth factor (VEGF) and a decrease in thrombospondin-1 (TSP-1) expression, which taken together is representative of an angiogenic switch, 13 a phenomenon known to be associated with disease progression in malignancy. 13 Therefore, in our study, we were interested in defining the signals sent from leukemic cells to MSC and how these interactions modify MSC function. Our studies demonstrate that platelet-derived growth factor (PDGF), secreted by CLL cells, activated MSCs via its interaction with the PDGF receptor (PDGFR) present on the MSC membrane. This activation by PDGF was able to enhance MSC proliferation and induce AKT phosphorylation, which was necessary for MSC VEGF production. Methods Patient populationBlood and bone marrow biopsies were obtained from CLL patients who had provided written informed consent under a Mayo Clinic Institutional Review Board approved protocol in accordance with the Declaration of Helsinki. All CLL patients had a confirmed diagnosis using the National Cancer Institute Working Group definition. 14 Patients in this study cohort were from all Rai stages and had not been treated for at leas...

SummaryIt was hypothesized that contact between chronic lymphocytic leukaemia (CLL) B-cells and marrow stromal cells impact both cell types. To test this hypothesis, we utilized a long-term primary culture system from bone biopsies that reliably generates a mesenchymal stem cell (MSC). Co-culture of MSC with CLL B-cells protected the latter from both spontaneous apoptosis and drug-induced apoptosis. The CD38 expression in previously CD38 positive CLL B-cells was up-regulated with MSC co-culture. Upregulation of CD71, CD25, CD69 and CD70 in CLL B-cells was found in the co-culture. CD71 upregulation was more significantly associated with high-risk CLL, implicating CD71 regulation in the microenvironment predicting disease progression. In MSC, rapid ERK and AKT phosphorylation (within 30 min) were detected when CLL B-cells and MSC were separated by transwell; indicating that activation of MSC was mediated by soluble factors. These findings support a bi-directional activation between bone marrow stromal cells and CLL B-cells.

Acidovorax ebreus strain TPSY is the first anaerobic nitrate-dependent Fe(II) oxidizer for which there is a completed genome sequence. Preliminary protein annotation revealed an organism optimized for survival in a complex environmental system. Here, we briefly report the completed and annotated genome sequence of strain TPSY.

e22002 Background: Mesenchymal stem cells (MSC) residing in the marrow support hematopoiesis and protect cancer cells from undergoing cell death induced by chemotherapy. Recent reports have described clonal cytogenetic abnormalities in the MSC of acute myeloid leukemia and myelodysplastic syndrome patients. To determine if cytogenetic abnormalities are present in MSC from CLL patients, we analyzed karyotypes of MSC from 13 CLL patients and 5 normal subjects. Methods: Stromal cells from marrow core biopsies of 13 CLL patients and 5 normal control subjects were isolated, cultured and confirmed as MSC based on their immunophenotype and capacity to differentiate into three lineages. After 3–4 non-stimulated cell culture passages, the karyotype was analyzed in 5–40 metaphase cells from each subject Abnormalities were considered clonal using the accepted convention of the same chromosomal gain or rearrangement in 2 or more cells or loss in at least 3 cells. For each CLL patient, interphase FISH analysis to detect the common CLL-associated abnormalities was performed on freshly isolated peripheral blood mononuclear cells (PBMC). Results: Clonal cytogenetic abnormalities of the cultured MSC were observed in 6 of 13 CLL patients (46%) and 3 of 5 control subjects (60%). The 6 CLL MSC had different clonal abnormalities than the PBMC CLL FISH studies. One CLL MSC exhibited trisomy 5, one exhibited trisomy 8, and one had monosomy X clone. One MSC had a balanced t(1;16). One MSC had a balanced t(3;12) and a del(3p), and one had a complex karyotype with 4 unbalanced rearrangements. The clonal aberrations observed in the 3 controls included a del(3p) in two cases, and in one case an inv(5) and a del(10q). There was no correlation of the chromosomal abnormalities in CLL MSC with clinical stage. Conclusions: Marrow MSC derived from CLL patients and normal subjects do show an array of cytogenetic abnormalities including clonal chromosomal abnormalities. However the genetic abnormalities found in both CLL and normal MSC could represent acquired genomic instability associated with advanced age, rather than oncogenesis associated with CLL. [Table: see text]

Background: This is an initial report of a correlative laboratory study of an ECOG clinical trial, E2903, to investigate an array of prognostic factors that are potentially associated with the clinical outcome in previously treated, high risk Chronic Lymphocytic Leukemia (CLL) patients. The ultimate goal of this study is to develop a prognostic model for this group of patients. Method: We have previously reported (ASH, 2007) on initial clinical and toxicity results using a combination of PCR followed by Campath. To date we have clinical response data on 37 patients and have conducted correlative prognostic factor analysis on these patients at entry to the study. The prognostic parameters assessed were fluorescence interphase hybridization (FISH) panel, CD38, ZAP-70 and IGVH (mutated vs. unmutated) status and plasma levels of pro- and anti-angiogenic cytokines. In addition, features such as clinical stage, age, sex and laboratory parameters including total white blood count, absolute lymphocyte count (ALC) and beta 2 microglobulin (b2m) were studied as predictors of response to PCR. Results: As of July 2008, the response data for the first 41 (the accrual goal is 110) patients who started the induction treatment are available. Of these 41, 3 patients are ineligible and one patient had no response data. The median age was 63 (range 51–76) with 29 males and 8 females, median b2m was 4 (range 2–28), 11 (31%) were Rai stage 0–2, and 26 (69%) were Rai 3–4. The median number of prior treatments was 2 (range 1–5); 15 of 23 (65%) patients tested for IGVH were unmutated, while on FISH analysis 4 had del 6q, 8 had del 11q, 4 had del 17p and 30 patients had complex FISH defects (>1 defect). Despite the prevalence of high risk features in this cohort of patients, 17 out of 37 responded (best response=partial response), 18 were stable and 2 had progressive disease with an overall response rate of 46%. Prognostic parameters most strongly associated with a clinical response included CD38 negative (p=0.008), ZAP-70 negative status (p=0.01) and mutated status (p=0.03). Responses were independent of age, sex, b2m, ALC, number of prior therapy or FISH defects. Responders showed a prolonged progression-free survival (PFS) compared to the PFS of non-responders (i.e., 2-yr PFS 29% vs. 0%, respectively, p<0.0001). Prognostic parameters most strongly associated with PFS included being both ZAP-70 negative (p=0.0009) and CD38 negative (p=0.004). Also, ZAP-70 negative and CD38 negative status was a strong predictor of favorable PFS (p=0.002). Mutated status was borderline significant (p=0.07) in univariate analysis and there was no obvious correlation of baseline angiogenic cytokine plasma levels with PFS. These results also hold in a multivariable Cox model. In the Cox model, mutated status was significantly associated with PFS (HR=0.2, p=0.01). Conclusion: We believe that the PCR regimen is highly effective at inducing responses in previously treated relapsed or refractory CLL patients who carry high risk prognostic markers. Thus, while no CRs were seen all but 2 patients had a PR or were stable after 6 cycles of PCR. The prognostic parameters most strongly associated with predictors of clinical outcome appear to be ZAP-70, CD38 and mutation status. The ability to attain a response also appears to be important for achieving a prolonged PFS. In summary, we believe that the strong clinical activity of PCR in a high risk group of CLL patients provides more evidence for pursuing the regimen as a platform for other combinations. In addition, we have generated a first level prognostic model that will continue to be refined as we proceed to complete this trial.

804 Background: We have demonstrated that crosstalk between CLL B-cells and marrow-derived mesenchymal stem cells (MSC) can modulate the activation of both. Soluble factors in the CLL-condition media (CM) regulate the migration and proliferation of MSC. We previously found that platelet-derived growth factor (PDGF) receptors were selectively activated in MSC by CLL-CM and are necessary for the downstream Akt activation in MSC. PDGF is capable of promoting vascular endothelial growth factor (VEGF) production in MSC; however, several questions remain unanswered. Can CLL B-cells produce PDGF; is PDGF the specific mediator in CLL-CM responsible for PDGFR activation in MSC; what is the mechanism of PDGF mediated VEGF production in MSC; and is there any clinical significance of PDGF mediated VEGF production by MSC for CLL patients? Methods: MSC were generated from CLL patients and controls as previously described. To determine the specific ligand responsible for PDGFR activation mediated by CLL-CM blocking antibody to PDGF or VEGF or both was used to neutralize the binding activity of PDGF or VEGF in the CLL-CM. Subsequently, the pattern of membrane receptor activation was evaluated using a commercially available receptor tyrosine kinase (RTK) array assay. ELISA was performed to measure PDGF and VEGF levels from plasma and CM of CLL patients and normals. The protein expression of PDGF isoforms in CLL and normal B-cells was assessed using Western blot. To assess the mechanism of PDGF mediated VEGF production by MSC, we used either a PI3K inhibitor, LY294002 (20 mM), or a p38 MAPK inhibitor, SB202190 (100 nM), to block these pathways in MSC. Results: The known ligands for PDGF receptors, PDGF and VEGF, were detected in the CLL-CM (PDGF, 113.7 ± 23.6 pg/ml, VEGF, 186.94 ± 54.57 ng/ml; n = 12). We also found that PDGF is present in CLL cells by Western blot analysis and expression of PDGF in CLL cells was up regulated by hypoxia. By using PDGF- or VEGF-blocking antibody or both to neutralize the binding activity of PDGF or VEGF in CLL-CM, we found only PDGF-blocking antibody was able to neutralize the majority of PDGFRa activation, indicating that PDGF in the CM is the predominant ligand for PDGFR activation in MSC. CLL MSC was exposed to either pooled CLL (n = 5) or pooled normal plasma (n = 5). Interestingly, PDGFR was more potently activated by CLL plasma than the normal plasma, suggesting PDGF levels were elevated in the CLL plasma. Indeed, PDGF in the normal plasma appeared to be much lower compared to the CLL plasma [normal (n = 11): 554.43 ± 88.55 pg/ml; CLL (n=43): 3115.36 ± 428.79 pg/ml. p < 0.0001]. In addition, the plasma PDGF level was found to be weakly correlated with VEGF level in 43 CLL patients assessed using Spearman correlation analysis (r = 0.3, p = 0.001). However, when CLL patients were grouped by clinical prognostic factors, the plasma PDGF level was found to be strongly correlated with plasma VEGF level in CLL patients with high-risk features including more advanced Rai stage 2-4 (r = 0.87, p < 0.0001), ZAP-70 positive (r = 0.8, p =0.04) and CLL patients who had required treatment (r = 0.8, p = 0.0008). To examine the mechanism for PDGF augmentation of VEGF by MSC, we found that PI3K pathway interruption using LY294002 (n = 6, p = 0.04), but not p38 MAPK inhibition (n = 6, p = 0.5), was able to abrogate the PDGF mediated VEGF production in MSC. Importantly, we also found that the secreted VEGF level in the CM of unstimulated CLL MSC appeared to be significantly higher compared to the level from culture media of unstimulated normal MSC [CLL (n = 14) vs. normal (n = 5), 298.1± 61.2 vs. 109.9 ± 52.3, p = 0.03]. Conclusions: These results indicate that PDGF secreted by CLL B-cells is necessary for PDGFR activation in MSC and can lead to increased VEGF production by MSC via a PI3K-Akt dependent mechanism. These findings have clinical implications as plasma PDGF levels are strongly correlated with plasma VEGF levels in high risk CLL. The finding that CLL MSC are more prone to secrete VEGF than normal MSC suggests the stromal microenvironment may function to facilitate CLL disease progression. Finally, as VEGF is known to enhance CLL B-cell survival and drug resistance, interrogation of the PDGF-VEGF regulation in the microenvironment of CLL patients should yield important information that can be used for therapeutic strategies in CLL. Disclosures: Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Background: It is believed that malignant cells can “condition” microenvironment to facilitate tumor cell survival. Our previous finding demonstrated that the culture of chronic lymphocytic leukemic (CLL) B-cells and marrow-derived mesenchymal stem cells (MSC) impacts both cell types bi-directionally (ASH 2007, Blood, 110: 337). Thus, MSC are capable of promoting CLL B-cell activation and proliferation while soluble factors secreted from CLL B cells were found to induce MSC activation in terms of both Erk and Akt activation. However, the exact mechanism and extent of this leukemic cell-MSC crosstalk has not been explored yet in CLL. Methods: To investigate if factors secreted from the CLL B-cells can modulate the migration and activation of marrow derived MSC, the conditioned medium (CM) of CLL B-cells was generated by collecting the supernatant of freshly isolated PBMC from CLL patients cultured for approximately 4 days at a concentration of 5 × 106 per ml in AIM-V medium. The migration and proliferation capacities of MSC were measured when they were cultured with or without the CM of CLL cells. Subsequently, the membrane receptors activated in MSC after 30 minutes of exposure to the CM of CLL cells were tested using a receptor tyrosine kinase array assay. In addition, we studied the downstream signal pathways of MSC by immunoblot approaches pre and post exposure to CLL CM and in some experiments we added specific, commercially available inhibitors for individual signaling pathways of interest. ELISA was used to measure angiogenic cytokines including PDGF, VEGF, BFGF and TSP-1 levels from CLL plasma as well as from CM of CLL B cells. Results: To test if CLL B-cells were capable of secreting soluble factors that activate or signal MSC, we examined the migration and proliferation capacities of CLL-MSC when they were exposed to CM generated from cultured CLL B-cells compared to AIM-V medium. Bone marrow MSC of CLL patients exhibited an increased migration as measured by a modified Boyden chamber assay (n = 4, mean increase of migration: 15%, p = 0.01) and proliferation as measured by direct cell counting with trypan blue staining (n =4, mean increase of total cell levels: 3.2 fold, p = 0.02) when cultured with CM of CLL B-cells compared to AIM-V medium. Using a receptor tyrosine kinase array assay (R&D system), we found that the sole growth factor receptor activated on CLL –MSC was PDGFRα when MSC were exposed to CM of cultured CLL B-cells for 30 minutes. This finding was further confirmed by demonstrating that PDGFRα was phosphorylated by immunoprecipitating MSC lysates with anti-PDGFRα followed by immunoblot with anti-phosphotyrosine antibody. We subsequently found that both PDGFRα and Akt were activated within 10 minutes of exposure by CM of CLL B-cells. When CLL-MSC were pretreated with a PDGFR inhibitor (PDGFR tyrosine kinase inhibitor III, Calbiochem), an inhibitor known to block the ATP binding site of PDGFR, neither PDGFRα nor Akt activation was detectable when MSC were exposed to CM from CLL B-cells. These results imply that Akt is likely activated downstream of the PDGFR signal pathway in CLL-MSC. We next found that PDGF was secreted by CLL B-cells by detecting its presence using ELISA in both the CM (n = 12,113.7 ± 23.6 pg/ml, mean ± sem) and plasma of CLL patients (n = 21, 3296.7 ± 800.1 pg/ml). Since we have previously found that a switch in pro- vs. anti-angiogenic cytokines can occur when CLL B cells are added to MSC, we tested if PDGF can upregulate MSC angiogenic cytokine levels. When PDGF (5ng/ml, R&D system) was introduced to the CLL-MSC, we found that VEGF production, but not TSP-1 or BFGF significantly increased over control levels (n = 3, mean increase: 3.3 fold, p = 0.05) in the CM of the MSC harvested after a 72 h culture period. Conclusions: These results indicate that CLL B-cells are capable of activating MSC function by increasing their migration and proliferation capacity likely via signaling through the PDGF receptor. The PDGF activated MSC results in downstream Akt activation and the increased secretion of VEGF, a cytokine known to enhance CLL B-cellsurvival and drug resistance. Further interrogation of the mechanism(s) that regulate the interaction between leukemic CLL B cells and stromal cells should yield important information that can be used for therapeutic strategies in CLL.