Floortje L. Kessler

Proteoglycans guide SDF-1-induced migration of hematopoietic progenitor cells

Stromal cell‐derived factor‐1 (SDF‐1) is a chemoattractant involved in hematopoietic progenitor cell (HPC) trafficking to the bone marrow. We studied the role of bone marrow endothelial proteoglycans (PGs) in SDF‐1‐mediated migration of HPC using a transwell assay. A subclone of progenitor cell line KG‐1 (KG‐1v) was used, displaying CXCR4‐dependent transmigration. Cell surface PGs on bone marrow endothelial cell line 4LHBMEC did not mediate SDF‐1‐induced transendothelial migration. In contrast, transwell filters precoated with various glycosaminoglycans (GAGs) enhanced migration toward SDF‐1. SDF‐1‐induced migration was reduced by degradation of heparan sulfate in subendothelial matrix produced by 4LHBMEC. The stimulating effect of GAGs was caused by the formation of a stable haptotactic SDF‐1 gradient, as SDF‐1 bound to immobilized GAGs and triggered migration. Soluble heparan sulfate enhanced SDF‐1‐induced migration dose‐dependently, suggesting that SDF‐1‐heparan sulfate complexes optimized SDF‐1 presentation. In conclusion, we provide evidence that PGs in the subendothelial matrix establish an SDF‐1 gradient guiding migrating HPC into the bone marrow.

Extensive early apoptosis in frozen–thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation

Stem cell doses necessary for engraftment after myelo-ablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze–thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen–thawed grafts, threshold numbers for adequate haematological recovery of 2.8–3.0 × 106 CD34+ cells/kg body weight determined for fresh grafts, now decreased to 1.2–1.3 × 106 CD34+ cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34+ cells were sufficient for recovery (0.3–0.4 × 106CD34+ cells/kg). In contrast to freeze–thawing of leucapheresis material, a high viability of CD34+ cells was preserved during storage for 3 days at 4°C, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen–thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34+ threshold doses for haematological recovery.Bone Marrow Transplantation (2002) 29, 249–255. doi:10.1038/sj.bmt.1703357

Early Apoptosis Largely Accounts for Functional Impairment of CD34+ Cells in Frozen-Thawed Stem Cell Grafts

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.

Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells

OBJECTIVE Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated. RESULTS Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin. CONCLUSIONS Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.

In vitro model for hematopoietic progenitor cell homing reveals endothelial heparan sulfate proteoglycans as direct adhesive ligands

Proteoglycans (PGs) play a dominant role within the bone marrow (BM), but their role in homing of transplanted hematopoietic progenitor cells (HPC) is unknown. In this study, the role of heparan sulfate (HS) PGs on BM endothelium as adhesive structures was investigated. HPC (primary CD34+ cells and cell line KG‐1a) were able to bind fractionated heparin, which could be competed by highly sulfated heparin/HS‐glycosaminoglycans (GAGs). Under flow conditions, HPC adhered to immobilized heparin after rolling over E‐selectin. Rolling of KG‐1a on BM endothelial cell (EC) line 4LHBMEC was completely E selectin‐dependent. Addition of heparin/HS‐GAGs, endothelial treatment with chlorate, or anti‐HS all partially inhibited firm adhesion. Moreover, enzymatic removal of endothelial HS‐GAGs reduced initial adhesion. Finally, HPC‐bound PGs isolated from 4LHBMEC, which was largely inhibited by enzymatic HS‐degradation. In summary, we identified sulfated structures on BM endothelium, most likely HSPGs, as a novel class of glycoconjugates involved in the multistep homing cascade of HPC.

The phenotypic profile of CD34-positive peripheral blood stem cells in different mobilization regimens

The type of regimen used might result in mobilization of phenotypically and functionally different CD34+ cells. We compared the phenotype of CD34+ cells in leukapheresis products of three homogeneous groups: I, healthy individuals treated with granulocyte colony‐stimulating factor (G‐CSF) alone (n = 13); II, patients mobilized with G‐CSF following chemotherapy (n = 16); and III, patients mobilized with G‐CSF after high‐dose chemotherapeutic pretreatment (n = 24). Multiparameter flow cytometry was performed for CD34+ subpopulation analysis and focused on adhesion molecules, differentiation markers and megakaryocytic markers relevant for stem cell homing, with special reference to the importance of L‐selectin expression. Regimens I and II led to higher numbers of mobilized CD34+ cells (mean 468 × 106 and 491 × 106 CD34+ cells per leukapheresis procedure respectively) than regimen III (mean 41 × 106 CD34+ cells per leukapheresis procedure). Both the expression of L‐selectin and CD54 on CD34+ cells was significantly lower in group III, as was the percentage of megakaryocytic (CD41+) progenitors. A higher percentage of primitive (CD38− and/or HLA−DR−) CD34+ cells was found in group III, correlating with a higher clonogenicity of the CD34+ cells. However, when comparing the CD34+ subpopulations that were also positive for L‐selectin, there was no significant difference between the three regimens. A similar approach for the megakaryocytic CD34+ population resulted in an even worse quality of regimen III: 5·1% of CD34+ being CD41+/L‐selectin+ compared with 9·2% and 8·9% in regimens I and II respectively. We concluded that the phenotypes of the CD34+ cells in the G‐CSF (group I) and G‐CSF–chemotherapy (group II) regimens are similar, whereas the phenotype of the CD34+ cells mobilized in the high‐dose regimen (group III) displayed features that might negatively influence homing of the cells. Future studies will be directed towards regimens that will lead to the mobilization of a higher amount of CD34+ cells with a phenotypically favourable phenotype.

Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors

PSMB5 mutations and upregulation of the β5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress.

Abstract 4764: Transfer of regulatory protein networks via extracellular vesicles as a candidate mechanism of apoptosis-resistance in acute myeloid leukemia

Defects in apoptosis regulation are known to impact chemotherapy resistance and consequently refractoriness and relapse of acute myeloid leukemia (AML). We previously showed that apoptosis-resistant protein profile of AML blasts at diagnosis is associated with shorter disease-free survival. Specifically, by flow cytometry, we measured the expression of Bcl-2, Bcl-xL, Mcl-1 and Bax in leukemic cells and combined these parameters to define their anti-apoptosis index (AAI). Interestingly, the AAI of normal lymphocytes in the AML patients corresponded to the AAI of AML blasts obtained from the same patient, reaching values far outside the normal AAI range of lymphocytes. In addition, the AAI in both cell types displayed parallel changes during the course of therapy. This points to a role of microenvironment in regulation of apoptosis in bone marrow cells of AML patients. Therefore, the aim of the current study was to assess if apoptosis-resistant AML cells are able to regulate the AAI of apoptosis-sensitive cells by influencing the microenvironment, as well as to perform molecular dissection of microenvironment, to identify novel proteins that regulate apoptosis. First, we showed that apoptosis-resistant AML blasts (high AAI) release factors that modulate sensitive AML blasts (low AAI) to upregulate Bcl-2 and become apoptosis-resistant. In the majority of cases (10 out of 14), Bcl-2 expression was significantly increased in apoptosis-sensitive AML blasts upon contact culture with apoptosis-resistant AML blasts (1.7-fold; p=0.0067). To characterize the AML microenvironment, conditioned medium (18 hrs) from patient samples displaying either apoptosis-resistant (n=5) or apoptosis-sensitive profile (n=6) were collected. Using mass spectrometry-based proteomics, comparative analysis was performed on these secretomes. Strikingly, we found that the major functional protein clusters upregulated in secretomes of the apoptosis-resistant AML were involved in mRNA splicing, protein translation and chromatin remodeling/chromosome organization. We further compared protein profiles of the soluble secretome and the extracellular vesicle fraction of a high AAI patient to those of a low AAI patient. Proteomic analysis of these fractions of the conditioned medium showed that the functional protein networks found in the whole secretome are well-represented in extracellular vesicles that are enriched for exosome markers. Transfer of functional proteins between cells by extracellular vesicles is a well documented phenomenon. Therefore, it is conceivable that the regulatory protein networks detected in the vesicles excreted by AML blasts are involved in regulation of apoptosis-related proteins in recipient AML blasts and other cells residing in the bone marrow, thereby contributing to therapy resistance. Funded by STR and KiKa - Children cancer-free Citation Format: Anna Wojtuszkiewicz, Jacqueline Cloos, Floortje L. Kessler, Sander Piersma, Jako Knol, Gerrit Jansen, Yehuda G. Assaraf, Gertjan L. Kaspers, Sonja Zweegman, Gerrit J. Schuurhuis, Connie R. Jimenez. Transfer of regulatory protein networks via extracellular vesicles as a candidate mechanism of apoptosis-resistance in acute myeloid leukemia. [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 4764. doi:10.1158/1538-7445.AM2014-4764