PCR-Free Shallow Whole Genome Sequencing for Chromosomal Copy Number Detection from Plasma of Cancer Patients Is an Efficient Alternative to the Conventional PCR-Based Approach.

Somatic copy number alterations can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). PCR is typically included in library preparations, but a PCR-free method could serve as a high-throughput alternative. To evaluate a PCR-free method for research and diagnostics, archival peripheral blood or bone marrow plasma samples, collected in EDTA- or lithium-heparin-containing tubes, were collected from patients with non-small-cell lung cancer (n = 10 longitudinal samples; 4 patients), B-cell lymphoma (n = 31), and acute myeloid leukemia (n = 15), or from healthy donors (n = 14). sWGS was performed on PCR-free and PCR library preparations, and the mapping quality, percentage of unique reads, genome coverage, fragment lengths, and copy number profiles were compared. The percentage of unique reads was significantly higher for PCR-free method compared with PCR method, independent of the type of collection tube: EDTA PCR-free method, 96.4% (n = 35); EDTA PCR method, 85.1% (n = 32); heparin PCR-free method, 94.5% (n = 25); and heparin PCR method, 89.4% (n = 10). All other evaluated metrics were highly comparable for PCR-free and PCR library preparations. These results demonstrate the feasibility of somatic copy number alteration detection by PCR-free sWGS using cfDNA from plasma collected in EDTA- or lithium-heparin-containing tubes and pave the way for an automated cfDNA analysis workflow for samples from cancer patients.

IGFBP7 activates retinoid acid-induced responses in acute myeloid leukemia stem and progenitor cells.

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) in combination with low doses of arsenic trioxide or chemotherapy leads to exceptionally high cure rates (>90%). ATRA forces APL cells into differentiation and cell death. Unfortunately, ATRA-based therapy has not been effective among any other acute myeloid leukemia (AML) subtype, and long-term survival rates remain unacceptably low; only 30% of AML patients survive 5 years after diagnosis. Here, we identified insulin-like growth factor binding protein 7 (IGFBP7) as part of ATRA-induced responses in APL cells. Most importantly, we observed that addition of recombinant human IGFBP7 (rhIGFBP7) increased ATRA-driven responses in a subset of non-APL AML samples: those with high RARA expression. In nonpromyelocytic AML, rhIGFBP7 treatment induced a transcriptional program that sensitized AML cells for ATRA-induced differentiation, cell death, and inhibition of leukemic stem/progenitor cell survival. Furthermore, the engraftment of primary AML in mice was significantly reduced following treatment with the combination of rhIGFBP7 and ATRA. Mechanistically, we showed that the synergism of ATRA and rhIGFBP7 is due, at least in part, to reduction of the transcription factor GFI1. Together, these results suggest a potential clinical utility of IGFBP7 and ATRA combination treatment to eliminate primary AML (leukemic stem/progenitor) cells and reduce relapse in AML patients.

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 ß5 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.

Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis.

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividualex vivoapoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p= 0.0067 andp= 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p= 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.

Subendothelial heparan sulfate proteoglycans become major L-selectin and monocyte chemoattractant protein-1 ligands upon renal ischemia/reperfusion.

Leukocyte infiltration into inflamed tissues is considered to involve sequential steps of rolling over the endothelium, adhesion, and transmigration. In this model, the leukocyte adhesion molecule L-selectin and its ligands expressed on inflamed endothelial cells are involved in leukocyte rolling. We show that upon experimental and human renal ischemia/reperfusion, associated with severe endothelial damage, microvascular basement membrane (BM) heparan sulfate proteoglycans (HSPGs) are modified to bind L-selectin and monocyte chemoattractant protein-1. In an in vitro rolling and adhesion assay, L-selectin-binding HSPGs in artificial BM induced monocytic cell adhesion under reduced flow. We examined the in vivo relevance of BM HSPGs in renal ischemia/reperfusion using mice mutated for BM HSPGs perlecan (Hspg2(Delta3/Delta3)), collagen type XVIII (Col18a1(-/-)), or both (cross-bred Hspg2(Delta3/Delta3)xCol18a1(-/-)) and found that early monocyte/macrophage influx was impaired in Hspg2(Delta3/Delta3)xCol18a1(-/-) mice. Finally, we confirmed our observations in human renal allograft biopsies, showing that loss of endothelial expression of the extracellular endosulfatase HSulf-1 may be a likely mechanism underlying the induction of L-selectin- and monocyte chemoattractant protein-1-binding HSPGs associated with peritubular capillaries in human renal allograft rejection. Our results provide evidence for the concept that not only endothelial but also (microvascular) BM HSPGs can influence inflammatory responses.

Reduced supportive capacity of bone marrow stroma upon chemotherapy is mediated via changes in glycosaminoglycan profile.

High dose chemotherapy and radiation have been found to impair the hematopoiesis-supportive capacity of bone marrow stroma. We now provide evidence for an important role of chemotherapy-induced alterations in stromal glycosaminoglycans (GAGs) in reduction of the supportive properties of stromal fibroblasts. Exposure to cytarabine resulted in a pronounced increase in hyaluronan, both in the cell/matrix (p<0.03) and supernatant fraction (p<0.05). Gene expression analysis showed a corresponding increase in gene expression of hyaluronan synthase 1, indicating that the increase in hyaluronan is at least partly under genetic control. Functionally, hyaluronan significantly inhibited the proliferation of early megakaryocytic progenitor cells in a dose dependent way (p=0.01). The increase in hyaluronan was confirmed in vivo by showing a >2-fold increase in bone marrow hyaluronan of patients after chemo- and/or radiotherapy as conditioning for an allogeneic stem cell transplantation, indicating physiologically relevance. Furthermore, there was a trend towards a decrease in the amount and sulfation of stromal heparan sulfate proteoglycans upon exposure to cytarabine, resulting in a 40% reduced binding of SDF1-alpha to stromal cells (p<0.05). In conclusion, there is a pronounced effect of cytarabine treatment on the expression of genes involved in GAG synthesis and degradation, affecting the synthesis and function of stromal GAGs. Our results indicate that chemotherapy-induced changes in stromal GAG profile are likely to affect normal hematopoiesis.

Identification of L-selectin binding heparan sulfates attached to collagen type XVIII.

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions.

Bone marrow stromal proteoglycans regulate megakaryocytic differentiation of human progenitor cells.

Adherence of hematopoietic progenitor cells (HPCs) to stroma is an important regulatory step in megakaryocytic differentiation. However, the mechanisms through which megakaryocytic progenitors are inhibited by stroma are poorly understood. We examined the role of sulfated glycoconjugates, such as proteoglycans (PGs), on human bone marrow stroma (hBMS). To this end, PG structure was altered by desulfation or enzymatic cleavage. PGs participated in adhesion of human HPC, as desulfation resulted in about 50% decline in adhesion to hBMS. Heparan sulfate proteoglycans (HSPGs) were found to be responsible by showing about 25% decline in adhesion after pre-incubation of HPC with heparin and about 15% decline in adhesion after enzymatic removal of HSPGs from hBMS. Furthermore, PGs were involved in binding cytokines. Both desulfation and enzymatic removal of stromal HSPGs increased release of megakaryocytopoiesis-inhibiting cytokines, that is, interleukin-8 (IL-8, 1.9-fold increase) and macrophage inflammatory protein-1alpha (MIP-1alpha, 1.4-fold increase). The megakaryocytic output of HPC grown in conditioned medium of desulfated stroma was decreased to 50% of the megakaryocytic output in CM of sulfated stroma. From these studies, it can be concluded that PGs in bone marrow, in particular HSPGs, are involved in binding HPC and megakaryocytopoiesis-inhibiting cytokines. Bone marrow stromal PGs thus reduce differentiation of HPC toward megakaryocytes.

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.

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.

Homing and clonogenic outgrowth of CD34(+) peripheral blood stem cells: a role for L-selectin?

OBJECTIVE: After transplantation of hematopoietic stem cells, adhesion molecules play a major role in the multistep process of engraftment in which L-selectin is suggested to be of relevance. A positive correlation previously was found between the number of reinfused L-selectin(+) stem cells and platelet recovery. In the present study, we determined the role of L-selectin in different engraftment steps, i.e., adhesion to endothelial cells, migration, and clonogenic outgrowth by in vitro assays that closely mimic the in vivo situation. MATERIALS AND METHODS: Flow adhesion and migration experiments were performed using the human bone marrow endothelial cell line 4LHBMEC and isolated peripheral CD34(+) cells with or without blocking of L-selectin-ligand interaction. Various clonogenic assays, including serum-free colony-forming unit-megakaryocytes (CFU-MK) and burst-forming unit-megakaryocytes (BFU-MK), were performed with sorted L-selectin(+)L-selectin(-) cells or in the presence of antibodies. RESULTS: Blocking of L-selectin on CD34(+) cells did not significantly affect rolling over and firm adhesion to 4LHBMEC. In addition, no role for L-selectin was found in transendothelial migration experiments. Finally, in clonogenic outgrowth of sorted or anti-L-selectin monoclonal antibody-incubated CD34(+) cells, no key role for L-selectin expression could be defined in BFU-MK and CFU-MK assays. CONCLUSION: Using in vitro assays for CD34(+) stem cell adhesion, migration, and clonogenic capacity, we were not able to define a major role for L-selectin.

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.